Monday Abstracts

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Abstracts / Experimental Hematology 30 (2002) 37-146

High expression of stanniocalcin-1 in megakaryocytes 269 and platelets M. Serlachius , R. Alitalo , H. Olsen , *L. Andersson ,

Autologous Stem Cell Transplantation in Non-Hodgkin’s 271 Lymphomas *N. Gorin,

Stanniocalcin (STC) is a 28kDa glycoprotein originally found in bony fish. STC functions as a regulator of the calcium/phosphate homeostasis and protects the fish against toxic hypercalcemia. The recently characterized mammalian STC-1 shows over 70% identity with fish STC indicating a high conservation of STC through evolution. We reported earlier a high expression of STC-1 in terminally differentiated human and rodent brain neurons while STC-1 was not detected in fetal, still proliferating nerve cells. STC appears to contribute to maintain the integrity of the terminally differentiated neuronal cells. We now report that mature megakaryocytes and platelets display high contents of STC-1. K562 leukemia cells, induced to megakaryocytoid differentiation in vitro also acquire expression of STC-1, which is not seen in K562 cells induced to erythroid differentiation.

Abstract not received at time of printing.

Non-myeloablative Allogeneic Transplantation for 270 HematologicMalignancy *S. Forman,

Extrathymic T Cell Development 272 *C. Perreault, M. Blais, G. Gérard, I. Louis, R. Terra,

1 2 3 1 1 University of Helsinki, Finland, 2HUCH Stem cell Laboratory, Finland, 3Human Genome Science Inc., U.S.A.

Hematologic Neoplasia Program, U.S.A.

Hopital Saint-Antoine, Service Maladies du Sang, France

Guy-Bernier

Research Ctr. & University of Montreal, Canada

It has long been recognized that the cures achieved by allogeneic transplantation are due to the high dose chemotherapy regimen as well as the anti-tumor effect mediated by the donor cells. Evidence for a graft-versustumor effect in man have come from studies of twin transplants, autologous transplants, allogeneic transplants with and without graft-versus-host disease, all of which suggest that the allogeneic donor-derived effect is important in elimination of residual disease and preventing relapse. Although historically it was thought that high dose myeloablative therapy was required to facilitate engraftment and mediate an anti-tumor response, a large number of studies have been conducted around the world which now show that less intensive regimens which achieve only moderate ablation or no myeloablation are effective in facilitating engraftment and that the graft can facilitate a graft-versus-tumor effect with achievement of remission. It appears that those diseases that have a more indolent course such as multiple myeloma, low grade lymphoma and CML are the most sensitive to this approach. Those patients with higher grade malignancies and those in relapse do not do as well and may need some degree of myeloablation to allow time for the graft-versus-tumor effect to develop. The overall approach is important given that a large number of diseases that can be cured by allogeneic transplantation are more common in older patients, a population that, in general, has not been the beneficiary of the potential curative role of transplantation. Such an approach has not only improved the safety profile of allogeneic transplantation, but can now be extended to patients who have not traditionally been considered for transplant. In addition, the nonmyeloablative approach can be a platform for the development of novel immune based therapies to augment the immune response after transplant. In the future it may be possible even to use non-myeloablative approaches to achieve cures in younger patients while retaining the potential for fertility and decreasing short and long-term complications.

Separation between primary and secondary lymphoid organs is a universal feature in jawed vertebrates. Strikingly, Oncostatin M (OM)-transgenic mice present massive extrathymic T cell development, localized exclusively in the lymph nodes (LN). According to the prevailing paradigm, the thymus is the main source of T lymphocytes in gnathostomes mainly because thymic epithelial cells have a unique ability to support early steps in T cell development. It is therefore remarkable that we found massive and productive T cell development in the OM+ LN despite the absence of epithelial cells. Our recent studies showed that in the OM+ LN, (a) T cell development is regulated by a COX-2 dependent neoangiogenesis involving high endothelial venules, (b) MHC class I expression strictly on hematopoietic cells is sufficient to support the development of a diversified repertoire of CD8 T cells, (c) the efficiency of positive selection of specific TCR transgenic T cells differs from what is found in the thymus, (d) negative selection is very effective despite the lack of an organized thymic-like medulla. In addition, extrathymic T lymphocytes developing in the OM+ LN undergo extensive post-selection expansion and exhibit functional features (proliferation, cytokine production) typical of memory cells. The memory phenotype and high turnover rate of these extrathymic T cells result from homeostatic expansion induced by chronic exposure to the LN MHC/peptide mixture that entailed their positive selection. Furthermore, the mature progeny of the OM-regulated pathway is able to generate specific immune response against the model pathogen LCMV. This work illustrates how the division of labor between primary and secondary lymphoid organs influences the repertoire and homeostasis of T lymphocytes. Furthermore, these findings have profound implications as to the essence of a primary T lymphoid organ and could potentially be used in the generation of thymic substitutes.

Abstracts / Experimental Hematology 30 (2002) 37-146

Potentials for tissue repair by hematopoietic stem cell 273 transplant *S. Sharkis,

Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, U.S.A.

105

Factors inherent to the cord blood stem cell graft 275 determine telomere dynamics after transplantation to NOD/SCID mice H. Roelofs1, W. Noort1, A. Zwinderman1, R. Willemze1, *W. Fibbe2, 1

Leiden University Medical Ctr, The Netherlands, 2Leiden University Hospital, The Netherlands

Purification of rare Hematopoietic Stem Cell(s) (HSC) to homogeneity is required to study their self-renewal, differentiation, phenotype, and homing. Long-term repopulation (LTR) of irradiated hosts and serial transplantation to secondary hosts are the gold standard for demonstrating self renewal and differentiation, the defining properties of HSC. We show that rare cells that home to bone marrow can LTR primary and secondary recipients. During the homing, CD34 and SCA-1 expression increases uniquely on cells that home to marrow. These adult bone marrow cells have tremendous differentiative capacity as they can also differentiate into epithelial cells of the liver, lung, GI tract, and skin. This finding may contribute to clinical treatment of genetic disease or tissue repair

Telomere length dynamics after stem cell transplantation is determined by multiple factors. Induction of telomerase or the recruitment of immature precursors with longer telomeres results in an increase of telomere length while the excessive expansion of stem cells following transplantation might cause telomere shortening. The balance between these factors and their result might be dependent on intrinsic properties of the graft as well as on extrinsic factors inherent to the recipient environment. To differentiate between such intrinsic and extrinsic factors, we performed xenotransplantations of human progenitor cells into NOD/SCID mice. Hematopoietic progenitor cells (CD34+) were isolated from 4 umbilical cord blood (UCB) samples and transplanted into cohorts of 3 – 4 recipient NOD/SCID mice. Six to eight weeks after transplantation, the mice were sacrificed and human progenitor cells (CD45+ /34+) as well as B cells (CD45+/19+) were isolated from the bone marrow. Using FlowFish analysis, we compared the telomere length of these expanded cell populations with the telomere length of the transplanted CD34+ cells. The expanded B cell populations invariably showed longer telomeres than the transplanted CD34+ cells. However, transplantation of CD34+ cells from 3 out of 4 different UCB sources resulted in longer telomeres in the expanded CD34+ cells, while transplantation of CD34+ cells from 1 UCB source resulted in shorter telomeres in all 4 recipients. Our data, therefore, indicate that the change in telomere length during the expansion period after transplantation in the recipient mice depends on the specific progenitor cell source. We considered that the increase in telomere length, observed in the majority of the expanded CD34+ and B cell populations, was due to the recruitment of immature precursors (CD34+/38-) with longer telomeres. However, in a direct comparison between the CD34+/38- and CD34+/38+ umbilical cord progenitor cells no difference in telomere length was found. Since the grafts contained similar numbers of CD34+ cells, the heterogeneity in telomere length dynamics is unlikely explained by differences in graft size. Our data indicate that factors that are intrinsic to the graft, e.g. the capacity of the transplanted cells to engraft and upregulate telomerase expression, influence the telomere dynamics following transplantation.

Visualization of tumor and effector cell trafficking 274 in hematologic malignancies *R. Negrin, M. Edinger, M. Verneris, Y.-A. Cao, M. Bachmann,

Increased time interval between radiation and 276 transplantation negatively impacts homing and engraftment of primitive hematopoietic progenitor cells

C. Contag, Stanford University, U.S.A.

P. Plett1, *C. Orschell2, 1Indiana University, U.S.A., 2Indiana University School of Medicine, U.S.A.

Cancer therapeutics have achieved success in the treatment of a variety of malignancies, however, disease relapse remains a major obstacle. Animal models capable of non-invasively detecting minimal tumor burden in a quantitative fashion will be extremely valuable in evaluating strategies to improve outcomes. We have developed a bifunctional imaging strategy combining fluorescence with either the green or yellow fluorscence proteins and bioluminescence with luciferase capable of visualizing as few as 1,000 tumor cells non-invasively and quantitatively over at least 5 logs of tumor burden. Utilizing this sensitive system, we have evaluated therapies including radiation, chemotherapy and immunebased strategies with ex vivo expanded CD8+ NK-T cells. By incorporating the bifunctional marker into the effector NK-T cells, the time course of tumor site homing and disease eradication could be directly visualized. These studies demonstrate the ability to directly visualize the entire course of disease including initial seeding, expansion, metastasis and effective treatment.

We previously demonstrated that primitive hematopoietic progenitor cells (HPC) with capacity to sustain long-term hematopoiesis for 2 generations home more efficiently to the bone marrow (BM) microenvironment of non-irradiated recipients than to that of irradiated. It has been reported that disruption of endothelium and stromal cells following lethal irradiation begins within 6 hours of radiation exposure. We rationalized that since homing and recovery of long-term repopulating cells is higher in non-irradiated recipients, the same would be true in lethally-irradiated recipients transplanted immediately following radiation when endothelial damage is minimal. To this end, we examined homing patterns and possible homing mechanisms of primitive HPC transplanted into mice lethally irradiated (950cGy) 24hr prior (d-1) or 2hr prior (d0) to transplantation. Percent recovery of total cells in BM 20hr after transplantation of BM mononuclear cells was 2.7-fold higher in d0 mice compared to d-1 (6.1±0.3% vs. 2.3±0.5%, respectively, p<0.05). Percent recovery of donor Sca-1+c-kit+ cells was 7-fold higher in d0 mice, suggestive of more efficient homing of primitive HPC to recently irradiated marrow. We next examined whether scheduling of radiation affects subsequent engraftment of transplanted cells in competitive repopulation assays. Long-term donor-derived chimerism in d0 mice was 1.7-fold higher than in d-1 mice, suggesting a possible clinical benefit to close timing of radiation and transplantation. To investigate possible mechanisms behind the increased homing in non-irradiated or d0-irradiated mice, expression of adhesion molecules implicated in homing of HPC was examined on BM cells harvested from nonirradiated mice, or mice irradiated d0 or d-2 prior to analysis. Interestingly, expression of PECAM, ICAM, and VCAM was decreased 30-50% on d-2 BM relative to d0 or non-irradiated BM. Furthermore, preliminary data suggest the existence of a soluble factor in irradiated marrow which adversely affects trafficking of transplanted primitive HPC to BM. Collectively, these data suggest the presence of radiation-sensitive BM microenvironmental cues in controlling homing, survival, or proliferation of transplanted primitive HPC, which may have important clinical implications in the design of BM transplantation protocols.

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Abstracts / Experimental Hematology 30 (2002) 37-146

Hemangioblasts derived from embryonic stem (ES) cells 277 can reconstitutehematopoiesis in vivo in SCID mice *T. Miyagi , M. Takeno , M. Takahashi , N. Suzuki ,

1 2 2 2 1 St. Marianna University School of Medicine, Japan, 2St. Marianna University, Japan

Polymorphonuclear Neutrophil (PMN) Dependent 279 and IndependentMobilization of Hematopoietic Stem and Progenitor Cells into Peripheral Blood in Mice *L. Pelus, H.-M. Bian, S. Fukuda, Indiana Univ School of Medicine, U.S.A.

ES cells are pluripotent stem cells and can theoretically differentiate into any cell types in vitro. We induced differentiation of ES cells into hematopoietic lineage cells (HLC) by using semi solid media containing methylcellulose. While the undifferentiated ES cells hardly expressed fetal liver kinase 1 (Flk1), a putative marker of hemangioblasts, the HLC expressed Flk1 up to 80%. Podocalyxinlike protein 1 (PCLP1) is another marker of hemangioblasts located in aorta-gonad-mesonephros (AGM) region of mouse embryo at day 11.5. PCLP1 is expressed on a majority of the Flk1+ cells in the ESderived HLC, suggesting that the HLC contained hemangioblasts. Cells expressing c-Kit and Sca1, both of which are markers of hematopoietic stem cells, were highly enriched in the HLC. The HLC were IV injected into irradiated SCID mice. The survival rate and peripheral blood WBC counts of the HLC transplanted mice predominated over those of sham treated mice. The HLC were found in bone marrow and spleen of the transplanted mice. These data suggest that the differentiation process of ES cells into the HLC in vitro closely parallels the differentiation from primitive hematopoiesis to definitive hematopoiesis in normal mouse ontogeny. Furthermore, the HLC derived from ES cells have potency to repopulate in bone marrow. It is expected that hemangioblasts induced from ES cells can be used as new source of hematopoietic stem cell transplantation.

The role of PMN in regulating peripheral blood stem cell (PBSC) mobilization by G-CSF or the CXCR2 chemokine receptor selective CXC chemokine GRO-beta was investigated in BALB/c mice following administration of a monoclonal antibody (clone RB6-8C5) against the murine GR1 (Ly-6G) antigen. Administration of 150 ug/mouse of anti-GR1, IP, resulted in a selective and complete elimination of circulating PMN between 24 and 96 hrs. During this period, PBSC mobilization induced by GRO-beta or G-CSF was completely lost. No effect was observed in mice receiving isotype control antibody. Anti-GR1 treatment had no effect on the number of CFU-GM per femur, but reduced total nucleated cell counts in femurs by 30%, selectively depleting early and late neutrophilic cells. At 120 hrs post administration of anti-GR1, a rebound in PMN counts was observed that was coincident with the return of the ability of GRO-beta or G-CSF to mobilize PBSC. These data suggest that the PMN is a critical cell for PBSC mobilization induced by these two proteins. Analysis of PBSM mobilization in antibody treated mice indicated that anti-GR1 administration resulted in mobilization of CFU-GM in a time dependent fashion, reaching >25fold above baseline (isotype control 68+/-11; anti-GR1 1740+/-389 CFU-GM/ml blood; P<0.001) at 96 hrs, when circulating PMN were undetectable. Peripheral blood low-density mononuclear cells (LDMC) were isolated from mice at 96 hrs after anti-GR1 treatment and injected into lethally irradiated syngeneic mice. Administration of 2x10E6 and 0.5x10E6 LDMC from anti-GR1 treated mice resulted in 100% and 80% survival of lethally irradiated mice, respectively at >65 days. Administration of 2x10E6 and 0.5x10E6 LDMC from mice mobilized with G-CSF (100 ug/kg/day, BID, x4 days) protected 80% and 10% of irradiated mice, respectively. The superior radioprotective effect of anti-GR1 mobilized LDMC was not due to elevated levels of short term repopulating cells (CFU-GM per 2x10E6 LDMC: PBS 20+/-6; G-CSF 1090+/-46; anti-GR1 463+/-24). Anti-GR1 mediated PBSC mobilization did not correlate with plasma protease levels that are associated with GRO-beta and G-CSF induced mobilization. These data indicate that while PMN are required for PBSC mobilization by GRO-beta and G-CSF, severe neutropenia itself is associated with mobilization of PBSC.

Flow cytometric assay for counting of dendritic cells type278 1 (DC1) and dendritic cells type-2 (DC2) in whole blood M. Zysk, A. Dzionek, *J. Schmitz,

Expansion and Exhaustion of Hematopoietic Stem Cells 280 by Serial Transplantation *L. Kamminga, E. Dontje, G. de Haan,

Miltenyi Biotec GmbH, Germany

University of Groningen,

The Netherlands

The role of type-1 (DC1) and type-2 (DC2) dendritic cells in regulating immune responses, GvHD, and GvT effects after allogeneic stem cell transplantation has become an area of increasing interest. DC1 and DC2 are currently identified in blood based on a multitude of immunophenotypic criteria, such as the absence of a panel of leukocyte lineage-specific antigens (CD3, CD14, CD16, CD19, CD20 and CD56) and the presence of HLA-DR. DC1, which are also known as myeloid DC (MDC) and DC2, which are also known as plasmacytoid DC (PDC) are often distinguished among HLA-DR+ lineage- DC based on expression of CD11c on DC1 and CD123 on DC2. Recently, we have generated monoclonal antibodies that identify novel markers of DC in blood: 1) BDCA-2 and BDCA-4 for DC2 and 2) BDCA-1 (CD1c) and BDCA-3 for two distinct subsets of DC1 (Dzionek et al., J. Immunol., 2000, 165:6037-6046 and Dzionek et al., J. Exp. Med., 2001, 194:1823-1834). Using these markers, we have developed a new flow cytometric assay for counting of DC in blood. The three DC populations are identified based on staining with anti BDCA-2-FITC, anti BDCA-1-PE and anti BDCA-3-APC, respectively. B cells, which also express BDCA-1, and monocytes, which express BDCA-3 at a low level, are excluded from the analysis based on staining with CD19-PE-Cy5 and CD14-PE-Cy5, respectively. Dead cells are excluded based on staining with ethidium monoazide bromide (EMA), a cell-impermeant dye that, after photolysis, binds covalently to nucleic acids in cells with compromised membranes (dead cells). After staining and erythrocyte lysis, leukocytes are fixed, counted using a hemocytometer, and finally analyzed using a flow cytometer. Median frequencies of DC among leukocytes (F) and median absolute numbers of DC per liter of blood (N°) in normal donors as determined with this assay were as follows (n=15; range in parenthesis): 1) BDCA-2+ DC2: F=0.228% (0.1160.347%) and N°=1.10E7 (0.73-1.88E7); 2) BDCA-1+ DC1: F=0.141% (0.0710.255%) and N°=0.73E7 (0.45-1.15E7); 3) BDCA-3+ DC1: F=0.015% (0.00470.037%) and N°=0.83E6 (0.26-1.80E6). The assay is rapid, reproducible, allows fixation, allows exclusion of dead cells, requires only 300 µl of blood, and can be readily used by a clinical immunology laboratory.

Hematopoietic stem cells (HSC) from DBA/2 (D2) and C57BL/6 (B6) mouse strains were challenged to maximal proliferation by serial bone marrow transplantation, in order to study genetic determinants of replicative exhaustion. D2 stem cells have been shown to be more actively proliferating than B6 cells, leading us to predict that exhaustion will occur earlier in D2 mice. Serial transplantations were performed in lethally irradiated D2 and B6 mice. To be able to quantify expansion and observe exhaustion of the stem cell compartment, we determined HSC numbers prior to each transplantation, and four months post transplantation when bone marrow was transplanted in a new group of D2 and B6 mice using the cobblestone area forming cell assay. After three consecutive transplantations peripheral blood cell count, hematocrit and bone marrow cellularity were normal. No detrimental effect was observed on the number of progenitors. The fold-expansion of B6 stem cells increased after each transplantation (from ~23-fold to ~47fold), whereas this parameter dropped in D2 mice (from ~13-fold to ~9fold). Although the number of transplanted stem cells expanded, the total stem cell pool present in the recipients declined with each transplantation, most notably in D2 mice. After two transplantations the absolute number of HSC dropped to 14% of normal in B6 and to 5% of normal in D2. We conclude that D2 HSC are more rapidly exhausted compared to B6 HSC. We propose that D2 HSC are more sensitive to the repopulating stress accompanying transplantation as a result of their intrinsic higher proliferation rate.

Abstracts / Experimental Hematology 30 (2002) 37-146

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Successful engraftment of Chronic Myelomonocytic 281 Leukemia cells in NOD/SCID mice requires granulocytemacrophage colony-stimulating factor (GM-CSF)

The SCL-complex regulates c-kit expression 283 in hematopoietic cells throughfunctional interaction with Sp1

*H. Ramshaw1, P. Bardy2, A. Lopez2, 1Hanson Center for Cancer

*E. Lecuyer1, S. Herblot1, M. St-Denis1, R. Martin1, C. Begley2, C. Porcher3, S. Orkin4, T. Hoang1, 1IRCM, Canada, 2TVW Telethon

2

Research, Australia, IMVS, Australia

Chronic myelomonocytic leukaemia (CMML) is a malignancy that has no effective treatment or cure. Several growth factors have been implicated in the pathogenesis of this disease and we, and others, have found that CMML cells form spontaneous colonies in culture. These colonies that form in the absence of added cytokines are inhibited by up to 95% by the addition of the GM-CSF antagonist E21R (at 1-10 µg/ml) in the assay. To determine whether this in vitro dependence of CMML cells to GM-CSF also occurred in vivo we constructed mice transgenic for human GM-CSF and transplanted them with peripheral blood (PB) cells from normal individuals or from CMML patients. Transgenic mice that produce human GMCSF, but not normal NOD/SCID littermates, allowed the engraftment of cells from 8 patients. Cells were injected at a range of concentrations, and the optimal dose of cells was shown to be 0.5-1 x 10e8 PB mononuclear cells per mouse. Analysis at a range of time points showed that 6 weeks post injection was the optimal time for engraftment. No normal PB cells engrafted under these conditions. Thus, we report the first successful engraftment of CMML cells in NOD/SCID mice and have shown that the engraftment is dependent on the presence of human GM-CSF. This in vivo CMML model may be useful to investigate the molecular defects in CMML and to examine potential therapeutics.

Institute, Australia, 3Weatherall Institute, U.K., 4Harvard Medical School, U.S.A.

SCL/TAL1 is a tissue-specific bHLH factor that plays a central function during hematopoietic development, although its target genes and molecular mode of action remain to be elucidated. Here we show that SCL and c-Kit are co-expressed in hematopoietic progenitors at the single cell level, and that SCL induces c-kit in chromatin, as ectopic SCL expression in transgenic mice sustains ckit transcription in developing B lymphocytes in which both genes are normally down-regulated. Through transient transfection assays and co-immunoprecipitation of endogenous proteins, we define the role of SCL as a nucleation factor for a multi-factorial complex (SCL-complex) that specifically enhances c-kit promoter activity without affecting the activity of two myelo-monocytic promoters, the G-CSFR and c-fms promoters. This complex, containing hematopoietic-specific (SCL, GATA 1/2) and ubiquitous (E2A/HEB, LMO1/2, Ldb-1) factors, is tethered to DNA via an Sp1 motif, through multiple physical interactions between elements of the SCL pentameric complex and the Sp1 zinc-finger protein. Finally, by chromatin immunoprecipitation, we demonstrate that SCL, E2A and Sp1 specifically co-occupy the c-kit promoter in vivo. Together, our results reveal that c-kit is a direct target of the SCLcomplex and illustrate how SCL governs hematopoietic cell fate, by recruiting its partners onto target genes that are essential for hematopoiesis.

Stroma cell-dependent transdifferentiation of bone 282 marrow stem cells in uterus *S. Okamoto , H. Tamura , Y. Futamata , K. Tamura , T. Hara ,

Notch signalling directly upregulates PU.1 expression 284 and induces multilineage myeloid differentiation T. Schroeder, H. Kohlhof, N. Rieber, *U. Just,

1

The Tokyo Metropolitan Institute of Medical Science, Japan, 2Nippon Becton Dickinson, Japan, 3Tokyo University of Pharmacy, Japan

Molecular Biology, Germany

Recent studies have suggested that bone marrow (BM) hematopoietic stem cells are transdifferentiated to various types of non-hematopoietic cells in mice carrying a damage of specific organ or tissue. To know whether it is true for the physiological tissue remodelling process, we neonatally transplanted GFP transgenic adult BM cells into busulfan-treated mice and analyzed the fate of donor cells in uterus during pregnancy. BM cells not only generated peripheral blood cells but also stroma-like cells in the desidual region in these mice on pregnant day 7.5. On day 14.5, frequency of donor-derived stroma-like cells decreased and GFPpositive/PECAM1-positive endothelial cells appeared in the peripheral maternal vessels. After delivery, however, a majority of GFP-positive cells were again localized in the stromal layer of uterus. Therefore, differentiation of BM stem cells in uterus are regulated depending on pregnant stage. We next established novel stroma cell lines from mouse uterus and cocultured with CD45positive/CD34-negative/side population (SP) of GFP transgenic BM cells. After 9 days in culture in the presence of IL-3, IL-6, and stem cell factor, 5-10 % of whole BM-derived cells became CD45negative. Such conversion occurred in a stroma cell-dependent fashion. These results implicated that BM SP cells are mobilized to desidual region of uterus and transdifferentiated to nonhematopoietic cells by some factors provided by uterus stroma cells.

Hematopoietic commitment is initiated by and depends on activation of transcription factors. However, it is unclear if activation of lineage-affiliated transcription factors is extrinsically regulated by thus far unknown agents or is the result of a cell-autonomous programme. Here we show that signalling by the Notch1 transmembrane receptor directly upregulates expression of the transcription factor PU.1 and instructively induces myeloid differentiation of multipotent hematopoietic progenitor cells. Transient activation of Notch1 signalling is sufficient to irreversibly reduce self renewal of multipotent progenitor cells accompanied by increased and accelerated differentiation along the granulocyte, macrophage and dendritic cell lineage. Activated Notch1 has no influence on apoptosis of multipotent progenitor cells, shows a weak inhibition of proliferation and does not substitute for survival and proliferation signals provided by cytokines. Activated Notch1 directly increases PU.1 expression leading to a high concentration of PU.1 protein which has been shown to direct myeloid differentiation. These findings identify Notch as an extrinsic regulator of myeloid commitment and the lineage-affiliated transcription factor PU.1 as a specific direct target gene of Notch.

1

1

2

3

1

Institute of Clinical

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Abstracts / Experimental Hematology 30 (2002) 37-146

Runx1 dependent IGFBP-3 suppression in the cell line 285 derived from the Aorta-Gonad-Mesonephros region of mouse embryo

Kit-ligand inactivates the Forkhead transcription 287 factors FKHR and FKHRL1 via phosphoinositide 3-kinase/PKB(Akt) in multipotent progenitor cells

*K. Iwatsuki1, S. Takahashi2, Y. Futamata3, T. Hara1, 1The Tokyo

*M. Engstrom, R. Karlsson, J. I. Jonsson, Lund University, Sweden

Metropolitan Institute of Medical Science, Japan, 2University of Tokyo, Japan, 3Becton Dickinson, Japan RUNX1/AML1/PEBP2∂B is a transcription factor whose gene is commonly translocated in patients with acute myeloid leukemia. Gene disruption studies have revealed that functions exhibited by RUNX1 are essential for the development of hematopoietic stem cells (HSCs) in the Aorta-GonadMesonephros (AGM) region of mouse embryo at E10.5-11.5. However, down stream effector molecules of RUNX1 in the AGM region remain to be elucidated. To identify a critical role played by RUNX1, we established a new cell li ne from the AGM region of E11.5 RUNX1 null mouse by introducing temperature sensitive mutant of SV40 large T antigen. Flow cytometric analysis demonstrated that this cell line expressed Sca1, podocalyxin-like protein 1 and oncostatin M receptor. In addition, it incorporated DiI-labeled acetylated low density lipoprotein and displayed endothelial-like morphology, indicating that this cell line may retain some of properties as hemangioblasts, common precursor cells for hematopoietic and vascular endothelial cells. In order to examine gene expression affected by RUNX1, we reintroduced RUNX1 cDNA into the RUNX1(-/-) cell line using the tetracycline inducible gene expression system. When these cells were treated with doxycycline, overexpression of bo th RUNX1 mRNA and protein was observed. Next, population of mRNAs existed in the RUNX1(-/-) cells were compared with those after the doxycycline stimulation by representational difference analysis (RDA). We found that mRNA for insulin-like growth factor binding protein 3 (IGFBP-3) was negatively regulated by RUNX1. Among IGFBP family examined, only IGFBP-3 and IGFBP-6 were expressed in this cell line. Suppression of the IGFBP-3 mRNA was also observed in the RUNX(-/-) cell line when adeno viral vector carrying RUNX1 was infected. These results suggest that IGFBP-3 is a down stream gene regulated by RUNX1. Interestingly, IGFBP-6 mRNA was also suppressed by RUNX1 expression. In colony assays using side population fraction of adult bone marrow cells, addition of IGFBP-3 decreased the colony number of granulocyte/macrophage lineages. Our data suggest the possibility that IGFBP-3 production in the AGM region is regulated by RUNX1 to control the differentiation of HSCs.

Proliferation, differentiation and survival of hematopoietic stem cells (HSCs) and multipotent progenitor cells are regulated by cytokines and cell-cell interactions. The function of cytokines on early hematopoietic cells seems to be mainly to protect them from apoptosis. Kit ligand (KL) has been shown to have a maintaining ability on HSCs without affecting cell division. Many studies have demonstrated that anti-apoptotic Bcl-2 family members are expressed in HSCs and hematopoietic progenitors, however it has not been demonstrated whether the survival effects of KL involve members of the Bcl2 family or other mechanisms. We demonstrate that KL does not lead to upregulation of Bcl-2 or Bcl-XL expression in the multipotent progenitor cell line FDCP-mix, instead another signal seems to mediate the survival of these cells. In our model KL mediates survival via phosphorylating the serinethreonine kinase Akt/PKB in a PI 3-kinase dependent manner. Phosphorylation of Akt/PKB leads to activation of its kinase activity leading to phosphorylation and thereby inactivation of members of the Forkhead family of transcription factors, FKHR and FKHRL1. In bone marrow derived Lin- progenitor cells the main target seems to be FKHRL1. The phosphorylation of FKHRL1 leads to a PI 3-kinase dependent translocation of the protein from the nucleus to the cytosol. We also demonstrate that cells overexpressing FKHRL1 are unable to be rescued from apoptosis via KL. Taken together with other reports our results suggest that survival of HSCs and progenitors depends on two antiapoptotic signals: some cytokines initiate their effects mainly via the Bcl-2 family, others such as KL mediates survival via Forkhead proteins. FKHRL1 has been shown to transcriptionally activate proapoptotic genes such as Bid and FasL but also cell cycle regulators such as p27kip1 and cyclin B. Thus inactivation of Forkhead proteins may not only block apoptosis but may also prepare the cells for cell cycle entry. Both signals combined may serve an important function necessary for survival and proliferation.

Enforced expression of HoxB4 in human primitive 286 hematopoietic progenitors determines cell fate in a concentration dependent manner

Regulation of a sub-set of HOX genes by ATRA in the 288 Acute Promyelocytic Leukemia cell line, NB4 *C. O’Neill, M. McMullin, T. Lappin, A. Thompson,

*A. Brun1, X. Fan2, J. Bjornsson2, K. Humphries3, S. Karlsson2,

University Belfast, U.K.

1 Lund University Hospital, Sweden, 2Lund University Hospital, Molecular Medicine & Gene Therapy, Sweden, 3Terry Fox Laboratory, British Columbia Research Agency, Canada

Retroviral overexpression of the transcription factor HoxB4 causes increased proliferation of murine hematopoietic cells (HSC) in vivo. We asked whether transient overexpression of HoxB4 and eGFP (AdHoxB4 vector) generated in CD34+ umbilical cord blood (UCB) cells by recombinant adenovirus would increase proliferation of human primitive hematopoietic progenitors. Cells were transduced with the AdHoxB4 vector and the AdeGFP control vector and selected for GFP expression by FACS before studies in biological assays. Cells were cultured in serum free medium with cytokines mainly supporting primitive HSC growth, as well as LTC-IC and 6 week liquid culture. Adenoviral vector overexpression of HoxB4 in UCB CD34+ cells lead to reduced proliferation recruitment of primitive hematopoietic progenitors with single cytokine support in Tpo (p< 0.05), reduced colony formation of clonogenic progenitors (50-70% reduction, p< 0.05), reduction in LTCIC CFC’s ( p< 0.05), and reduced CFC after 6 week liquid culture. In contrast to retroviral overexpression, AdHoxB4 lead to increased myeloid differentiation of primitive hematopoietic progenitors when cultured in SCF, FL, Tpo and Epo for 7-9 days (p< 0.05). Quantitative PCR analysis of transgene expression after retroviral gene transfer showed a two-fold increase of HoxB4 mRNA, while the level of transgene expression in AdHoxB4 transduced cells was 10-15 fold higher compared to mock transduced cells. We conclude that the high levels of HoxB4 expression generated by AdHoxB4 in human CD34+ UCB cells lead to differentiation rather than proliferation demonstrating that the fate of these transduced primitive hematopoietic cells is determined by the concentration of HoxB4.

Queen’s

Acute Promyelocytic Leukemia (APL) is characterized by the karyotypic marker t(15;17) and results in the alteration of normal retinoic acid receptor-a (RARa) function. Morphologically APL shows maturation-arrest at the promyelocyte stage, which may be overcome by all-trans retinoic acid (ATRA) treatment. The well established t(15;17) NB4 cell line model retains the ATRA-response leading to granulocytic maturation. HOX gene products act in concert as master regulators of developmental processes, such as hematopoiesis. ATRA has been shown to regulate HOX gene expression in embryogenesis and organogenesis. We have developed and validated a specific Real Time PCR platform for the simultaneous measurement of 27 human HOX genes, using TaqmanÔ probe based chemistry (ABI 7700). NB4 cells were monitored by morphological analysis and CD11b expression following treatment with a pharmacological dose of ATRA for up to 96 hours. Untreated NB4 cells showed high expression of HOXB9 and HOXD13, at levels comparable to GAPDH. The expression of the majority of HOX genes did not change significantly following ATRA-treatment. However HOXA1 showed a moderate increase in expression four hours post ATRA-treatment that became more significant (5-6 fold) at the 48-hour time point. An initial decrease in expression (4-6 fold) of HOXA11 and HOXD13 was retained at later time points. HOXC12 showed a significant early increase in expression (up to 20 fold) that was also maintained at 48 hours. We conclude that ATRA regulates a subset of HOX genes in this APL model. The significance of these findings will require further investigation in vivo.

Abstracts / Experimental Hematology 30 (2002) 37-146

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Acquired mutations in GATA1 in the megakaryoblastic 289 leukemia of Down syndrome J. Wechsler , M. Greene , M. McDevitt , J. Anastasi , J. Karp ,

NF-kB transcriptional factor is involved in the regulation 291 of the Wilms’tumor gene expression *D. Cilloni, F. Messa, A. Morotti, E. Messa, M. Fava, E. Gottardi,

M. Le Beau1, *J. Crispino1, 1University of Chicago, U.S.A., 2John

G. Saglio, Universita di Torino, Italy

1

1

2

1

3

Hopkins, U.S.A., 3University of Maryland, U.S.A.

Children with Down syndrome (DS) have a 10-20 fold increased risk of developing leukemia, particularly acute megakaryoblastic leukemia (AMKL). While a subset of pediatric AMKLs are associated with the 1 ;22 translocation and the expression of a novel fusion protein, the genetic alterations that promote DS-AMKL have remained elusive. In this study we demonstrate that leukemic cells from all DS-AMKL patients examined (n=6) harbor mutations in the essential hematopoietic transcription factor gene GATA1. These mutations are specific for this subtype of leukemia, as we did not find GATAl mutations in DNAs from more than 70 patients with other types of acute myeloid leukemia. In every instance, the mutation results in the introduction of a premature stop codon within the gene sequences encoding the N-terminal activation domain. These mutations prevent the synthesis of the 50-kD full length GATA-l, but not of a 40-kD protein initiated further downstream. We show that this 40-kD protein, which lacks the N-terminal activation domain, binds DNA and interacts with its essential cofactor FOG-l (Friend of GATA-l) to the same degree as full length GATA-l, but exhibits a reduced trans activation potential. While previous reports suggest that the activation domain is dispensable in cell culture models of hematopoiesis, a recent study has demonstrated that it is required for normal development in vivo. These findings therefore argue that loss of wild-type GATA-l is one step in the pathogenesis of acute megakaryoblastic leukemia in Down syndrome.

WT1 is a tumor suppressor gene coding for a zinc-finger transcriptional factor. WT1 is expressed at low level in normal hematopoiesis while it is overexpressed in leukemic blast cells. Although at present the significance of WT1 overexpression in haematological malignancies is still unknown, its activation may represent an important event in the leukemic process. A conserved NF-kB site has been identified in the WT1 promoter, suggesting a possible role of NF-kB in WT1 regulation. The aim of the study was to establish the role of NF-kB in the activation of WT1 transcription. We tested the effects on WT1 expression of an irreversible inhibitor of IkB phosphorylation named Bay 11-7082 (Alexis Biochemicals), which completely and specifically abrogates the NF-kB translocation to the nucleus. We incubated several cell lines including K562 and HL-60 and PB and BM samples from acute and chronic leukemia patients with Bay 11-7082 (2,5 µM, 18 h, 37°C). WT1 transcript levels were analyzed by the quantitative RTQ-PCR (Taqman) assay. In addition we co-transfected K562 cell line with SR-IkBa2A and neomicine resistance gene to obtain a cell line in which NFkB results constitutively inhibited. WT1 expression was tested in both parental and transfected cell lines. We found that WT1 expression is strongly inhibited in Bay treated cell lines with respect to controls. We observed a decrease of 6 and 7 fold of WT1 copy number in K562 and HL-60 respectively. Similar results were obtained in PB and BM samples from leukemia patients. We found a 5 and 3 fold reduction of WT1 expression in BM and PB samples from AML patients and 4 and 3 fold reduction in BM and PB sample from CML patients. In agreement with these data, in SR-IkBa2A transfected cells we detected a significant inhibition of WT1 expression with respect to the parental cell line. Taken together, these results suggest that NFkB transcription factor is implicated in the modulation of WT1 gene expression in leukemic hematopoiesis, further supporting the role of NF-kB inhibitors as agents for molecularly-targeted therapy.

Interaction and Cooperation of MITF and MAZR for 290 Transcription of Mouse Mast Cell Protease 6 Gene *E. Morii , Y. Kitamura ,

Molecular Cloning and Characterization of a Novel 292 Hematopoietic Marker Expressed in Early Embryonic Stem Cells Through Mature NK Cells

2

*M. Kirszenbaum1, S. Prost1, R. Maddad1, J. Gluckman2, M. Le Discorde1, B. Canque2, 1Commissariat à l’Energie Atomique,

1

2 1

Osaka University Medical School , Japan,

Osaka University, Japan

France, 2EPHE-INSERM, France Development of hematopoietic cells is achieved by regulated expression of cell type-specific genes, in which transcription factors play important roles. The mi transcription factor (MITF) is a basic-helix-loop-helix leucine zipper transcription factor that is encoded at the mi locus. Mutant mice at the mi locus show the abnormality in the phenotype of mast cells, indicating that MITF is important for the development of mast cells. Most transcription factors function in cooperation with other factors by protein-protein interactions, and these interactions are necessary for the cell-type specific expressions. In the present study, we searched proteins interacting with MITF. A yeast two-hybrid screen was done, and mycassociated zinc-finger protein related factor (MAZR) was isolated as a partner of MITF. When expressed with MITF in NIH/3T3 cells, MAZR was colocalized with MITF. The association of MAZR with MITF was further confirmed by a co-immunoprecipitation study and in vitro binding assay. MAZR was expressed in cultured mast cells and MST mastocytoma cells containing mouse mast cell protease (mMCP)-6 transcript abundantly. The overexpression of dominant negative MAZR in MST mastocytoma cells reduced the amount of mMCP-6 mRNA. The simultaneous transfection of MAZR and MITF significantly increased the promoter activity of the mMCP-6 gene, indicating that the MAZR and MITF synergistically transactivated the mMCP-6 gene. MAZR appeared to play important roles in the normal phenotypic expression of mast cells in association with MITF.Æ

In order to understand the molecular mechanisms involved in human embryonic hematopoiesis, we have compared the Yolk-Sac, AGM region, embryonic liver, and UCB RNAs using RNA Differential Display technique. We have identified a new gene KLIP-l (Killer Lineage frotein) encoding a 37-38 kDa molecule that consisting of 350 amino acids and containing five-transmembrane domains. We found intracellular KLIP-l protein to be expressed by all nucleated hematopoietic cells from early embryonic stem cells to mature adult blood lineages, whereas membrane KLIP-l expression displayed a more restricted pattern suggesting that the KLIP-l molecule is subjected to post-translational regulation. In day-30/32 human embryo sections, KLIP-l protein expression is restricted to circulating hematopoietic cells at hematopoiesis sites. Membrane KLIP-l is expressed by fetal and adult GP-A + erythroblasts, fetal liver CD34+ subset, fetal spleen, and adult bone marrow CD56+ NK and CD 19+ B cells. Among mature peripheral blood, membrane KLIP-l expression is restricted to CD56+ NK cells, indicating KLIP- 1 to be a novel marker of this population. The antiKLIP-l antibody has no influence on cytotoxicity against NK-sensitive K562 cells. Altogether, these results indicate that the KLIP-l protein has at least two functions: membrane and cytoplasmic molecule. The high degree of conservation among mammals of the KLIP-l protein sequence strongly suggests that the KLIP-l protein plays an important role during various stages of hematopoiesis and may exercise similar functions in human and mouse blood cells. The KLIP-l molecule may therefore constitute a powerful tool for improving knowledge of both human hematopoiesis and NK cell ontogeny.

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Abstracts / Experimental Hematology 30 (2002) 37-146

Autologous stem cell transplantation followed by a dose293 reduced allograft induces high complete remission rate in multiple myeloma

Lymphocyte recovery and age: determinant factors 295 of survival after autologous peripheral stem cell transplantation in hematologic malignancies.

*N. Kröger1, R. Schwerdtfeger2, M. Kiehl3, H. Sayer4, T. Zabelina5, B. Fehse5, R. Kuse6, G. Wittkowsky6, A. Zander5, 1Bone Marrow

*A.-L. Herr1, M. Edwardes2, S. Lachance2, P. Laneuville2, J. Routy2,

Transplantation, Germany, 2BMT Wiesbaden, Germany, 3BMT IdarOberstein, Germany, 4University Hospital Jena, Germany, 5 University Hospital Hamburg, Germany, 6A.K. St.Georg Hamburg, Germany

1

Montreal General Hospital, McGill University, Canada, 2McGill University, Canada

We evaluated toxicity, engraftment, chimerism, graft-versus host disease (GvHD) and response to a dose-reduced allograft after cytoreductive autograft in 17 patients with advanced stage II/III multiple myeloma (MM). After autografting with melphalan (200 mg/m2) the patients received after a median interval of 119 days (range 60-210) a dose-reduced regimen consisting of fludarabine (180 mg/m2), melphalan (100 mg/m2) and antithymocyte globulin (3x10mg/kg) followed by allografting from related (n=7), mismatched related (n=2), or unrelated (n=8) donors to induce a graft versus myeloma effect. After dose-reduced allografting all patients became neutropenic ( < 0.2 x109/L) for at least 8 days. All patients engrafted with a median time for leukocytes (>1 x 109/L) and platelets (>20 x 109/L) count of 16 (range 11-24) and 23 days (range 12-43), respectively. Complete donor chimerism was detected after a median of 30 days (range 19 – 38). Acute GvHD II-IV occured in 4 patients (25%), grade III GvHD in two patients (13%). 40% developed chronic GvHD, but only one patient experienced extensive chronic GvHD requiring further immunosuppressive therapy. Two patients died of alveolar hemorrhage and of pneumonia, respectively, resulting in a day 100 mortality of 11%. The rate of complete remission (CR) with negative immunofixation increased from 18% after autografting to 73% after allografting. After a median follow-up of 17 months after autologous and 13 months after allogeneic transplantation 13 are alive and 12 of them free of relapse- or progression. The tandem auto-allo-transplant-protocol is highly active and provides rapid engraftment with complete donor chimerism and tolerable toxicity.

Early lymphocyte recovery may predict superior survival after autologous peripheral stem cell transplantation (APSCT) in hematological malignancies and breast cancer. Objective: To assess the prognostic value of early lymphocyte recovery on disease-free survival (DFS) and overall survival (OS); and to test whether characteristics such as age, disease duration, baseline lymphocyte count, stem cell dose and prior radiotherapy may influence lymphocyte recovery. Methods: We conducted a retrospective study of 94 patients (13 Hodgkin’s Lymphoma, 46 Non-Hodgkin’s Lymphoma and 35 Multiple Myeloma) who underwent APSCT between 1996 and 2001 at the MUHC. Lymphocyte recovery was defined as “early” if the absolute lymphocyte count was ≥0.5 x 10 9/L at day 15 or before, and “late” if not. Chi-square, Fisher’s Exact tests and multivariate logistic regression were used to determine relations between baseline characteristics and lymphocyte recovery. Cox regression analysis was performed to study the prognostic value of early lymphocyte recovery on DFS and OS. Results: Median age was 51, disease duration before APSCT was 25 months, baseline lymphocyte count was 0.7 x 10 9/L, CD34 and CFU-GM doses infused were 4.96 x 10 6/kg and 386 x 10 3/kg respectively. 29 patients had an early lymphocyte recovery. 34 relapses and 24 deaths were observed after a median follow-up of 21 months (1.7 to 69 months). Shorter disease duration correlated with an earlier lymphocyte recovery. For patients younger than 50 years of age, early lymphocyte recovery was significantly associated with a prolonged DFS and OS: risk ratio for DFS 0.25 (95% CI 0.07-0.92; p=0.038) and for OS 0.11 (95% CI 0.01-0.90; p=0.040). This relationship did not change after controlling for any other baseline characteristic, including CD34 dose. Causes of death were similar for patients below and above 50. Conclusion: Early lymphocyte recovery after APSCT strongly predicts survival for patients younger than 50, even after controlling for infused CD34 dose. Immune function may play a role in the control of residual disease after APSCT.

Allogeneic stem cell transplantation from unrelated 294 donors after a dose-reduced conditioning in patients with advanced multiple myeloma

Universal immunotherapeutic approach for patients 296 with refractory malignancies: HLA-haploidentical transplants in 100 cGy-conditioned hosts

*N. Kröger1, R. Schwerdtfeger2, A. Nagler3, M. Kiehl4, H. Sayer5, H. Renges6, T. Zabelina6, B. Fehse6, A. Zander6, 1Bone Marrow

*G. Colvin, J.-F. Lambert, L. Lum, R. Rathore, P. Quesenberry, G. Elfenbein, Roger Williams Medical Center, U.S.A.

2

3

Transplantation, Germany, BMT Wiesbaden, Germany, BMT TelHashomer, Israel, 4BMT Idar-Oberstein, Germany, 5University Hospital Jena, Germany, 6University Hospital Hamburg, Germany We evaluated the feasibility of allogeneic stem cell transplantation from matched unrelated donors after a dose-reduced conditioning in 17 patients with advanced stage II/III multiple myeloma (MM). The conditiong regimen consisted of fludarabine (180 mg/m2), melphalan (100-140 mg/m2) and antithymocyte globulin (3x10-20mg/kg) followed by allografting from HLAmatched (n=15) or mismatched (n=2) unrelated donor. All patients had received prior autologous transplantation. 11 pat relapsed after autografting and 6 received an autograft for cytoreduction prior to dose-reduced allografting. Remission status prior allograft was CR (1), PR (6), NC (3) or PD (7). After dose-reduced allografting all patients became neutropenic ( < 0.2 x109/L) for at least 8 days. All patients engrafted with a median time for leukocytes (>1 x 109/L) and platelets (>20 x 109/L) count of 16 (range 1124) and 22 days (range 11-36), respectively. Complete donor chimerism was detected in all patients at day 40. Acute GvHD II-IV occured in 6 patients (35%), grade III/IV GvHD in three patients (18%). 50% developed chronic GvHD, but only two patient experienced extensive chronic GvHD. Two patients died of GvHD and sepsis, one due to aspergillus and one with sudden cardiac arrest, resulting in a treatment related mortality of 23%. After allografting all patients responded with PR (50%) or CR (50%). After a median follow-up of 10 months the 1year pobability of OS is 74% (95% CI:53-95%) and the 1 year probabilty of progression free survival is 65% (95% CI: 40-90%) Dose reduced allograft with unrelated donor is a feasible approach in highly pretreated myeloma patients and provides rapid engraftment with complete donor chimerism and tolerable toxicity.

Only ~25% of eligible patients for allogeneic transplantation have an HLAidentical sibling donor, however nearly 100% do have HLA-haploidentical donors. We evaluated CD3+ cell dose escalation with haploidentical transplantation in patients with refractory malignancy. We performed 29 transplants, 27 haploidentical, one 4/6 and one 6/6 HLA-matched. The haploidentical CD3+ cell dose ranged from lxl0 6 - 2x10 8 cells/kg infused with a fixed dose 4x106 CD34+ cells/kg in all patients. G-CSF primed PBSC with a conditioning regime of low dose day 0 TBI of 100cGy was used. Median age was 58 (range 16-82). Diagnoses include: NHL-(6), MM-(4), AML-(3), bladder-(3), breast-(3), renal-(3), Ewing’s-(2), MDS-(2), Hodgkin’s-(I), lung-(1), melanoma(l), prostate-(l). Chimerism was measured Q2 wks. One treatment related death (3%) occurred from grade IV AGVHD (bowel perforation) in a haplo-patient with 100% chimerism. We observed a post-infusion syndrome coined “haploimmunostorm” in which hyperpyrexia, morbilliform rash, malaise +- elevated liver enzyme occurred within 6-24 hrs after infusion. The haplo-immunostorm occurred in 11 of 13 (85%) patients at a CD3+ dose>=1x10 8 cells/kg. This was a limited syndrome, not consistent with classic AGVHD. A brief steroid use abated the syndrome if not self resolved. Patients had mild pancytopenia lasting a median of 18 days with a nadir at ~4 wks requiring on average 2 PRBC and 2 platelet transfusions. There have been only 2 febrile neutropenia admissions (7%). Three major clinical responses occurred, all in the absence of measurable donor chimerism ( <5%). Two AML patients that developed in the setting of MDS are free of blasts (144+, 190+ days). One lost 2 of3 cytogenetic clones with residual5q- and the other patient lost all megakaryopoiesis and had a second transplant day 173+. One breast cancer patient has non-progressive bone disease day 245+ off all treatment (persistent CT changes may be necrotic tissue). In summary: 1) very low dose TBI of 100cGy followed by haploidentical transplant can eradicate evidence of disease 2) donor macrochimerism does not need to be present for tumor response 3) this is a well tolerated outpatient treatment that produced minimal toxicity for the majority of patients 4) this is the first report of successful outpatient haploidentical transplant achieving several clinical responses for patients with end stage, refractory malignancies.

Abstracts / Experimental Hematology 30 (2002) 37-146

111

Dendritic cells numbers and their subsets during the 297 treatment in multiple myeloma *L. Kovarova , A. Svobodnik , T. Büchler , M. Klabusay , R. Hajek ,

Intramyocardial inoculation of autologous bone marrow 299 cells in patients with refractory myocardial ischemia *A. Porcellini , B. Reimers , G. Azzarello , P. Pascotto , O. Vinante ,

1

Masaryk University Hospital, Czech Republic, 2Masaryk University Brno, Czech Republic, 3Masaryk University Hospital - Brno, Czech Republic

1

Backgrounds : Imunotherapeutic application of the peripheral blood dendritic cells (DC) loaded with antigen may lead to better survival of patients with multiple myeloma (MM). In this study we evaluated the proportions of dendritic cells and their subsets – DC1, DC2 – in peripheral blood of patients with MM during treatment. Methods : Flow cytometric identification and analysis of DC numbers was performed on the leukocytes of peripheral blood in a reference population of healthy adults as well as in population of patients with MM. Expression of surface antigens characteristic for the DC – CD83 in combination with HLADR and CD11c or CD123 was determined. Results : Numbers of DC were variable with intraindividual discrepancies. We found no signifikant count changes in initial values between the group of healthy volunteers (n = 15; mean count of CD83+ cells was 0,62 ± 0,06 % and absolutely DC count was 3,26 ± 0,57 x 109/l; ratio DC1/DC2 = 2,5) and the group of patients before treatment (n = 15; 0,59 ± 0,13 %; 3,22 ± 1,15 x 109/l; DC1/DC2 = 2,17). In the group of patients (n = 10), after induction treatment with four courses of the VAD regimen (vincristin, adriamycin, dexamethason), mean percentage of DC (0,84 ± 0,4 % CD83+ cells) and their subtypes (DC1/DC2 = 1,59) was higher than initial values. Administration of the G-CSF reduced the total DC numbers (0,66 ± 0,6 %; DC1/DC2 = 3,71). The lowest percentage of the total DC as well as the DC1 and DC2 numbers were in peripheral blood stem cells (PBSC) collections (0,36 ± 0,2 %; DC1/DC2 = 4,75). Administration of the GM-CSF increased DC numbers (0,56 ± 0,3 %; DC1/DC2 = 1,59) and the normal pre-treatment DC values were founded in peripheral blood in the sixth month after transplantation (0,83 ± 0,6 %; DC1/DC2 = 3,92). Conclusions : The highest number of the total DC was found after induction treatment. Ratio DC1/DC2 showed the relative supremacy of DC1 subtype during the treatment, the highest ratio was found in the autologous PBSC collection and in the sixth month after transplantation.

Recent studies in the animal model have demonstrated that bone marrow cells inoculated in ischemic hearths enhance angiogenesis and can generate de novo myocardium. The purpose of our study was to assess the feasibility and safety of direct intramyocardial inoculation of filtered whole autologous bone marrow (ABM) in five patients with chronic refractory myocardial ischemia non suitable for the conventional revascularization strategies. Five male pts (mean age 68+/-10 years) with severe refractory angina were included. Catheter–based electromechanical mapping of the left ventricle (NOGA) was performed to guide intramyocardial ABM inoculations using the Myostar catheter (J&J Biosense). Eight to ten 1ml inoculations of ABM into the target ischemic area were performed. Myocardial perfusion was assessed at baseline and 1 month after the procedure with NH3 positron emission tomography (PET). Procedural or 30 day adverse events were not observed. At each injection site a mean of 27.8 x 10e6/ml of ABM nucleated cells (range 17.3 to 50.8 x 10e6/ml) were injected. The mean percentage value of CD34+ cells and the CD34- CD117+ CD45+/-CD4+/subset in the mononuclear fraction were respectively 2.9 (range 1.96-4.33) and 0.21 (range 0.04-0.49). PET evaluation at 1 month was available in 4 patients and showed improvement of perfusion in the target area in 2 patients. This preliminary intramyocardial catheter injection experience proved safe and feasible.

GvH-prophylaxis with ATG (Fresenius) in recipients 298 of matched and mismatched unrelated donor transplants for chronic myelogenous leukemia: A retrospective analysis

Peripheral T Cell Levels Correlate with Outcome 300 in Patients Undergoing Autologous Stem Cell Transplant *S. Rosinski , I. McNiece , E. Shpall , N. Clough , P. Russel ,

*A. R. Zander1, M. Schleuning2, N. Kroeger1, J. Finke3, T. Zabelina1, J. Berger1, D. Beelen4, R. Trenschel5, R. Schwerdtfeger6, H. Baurmann6, M. Bornhaeuser7, G. Ehninger7, A. Fauser8, M. Kiehl8, H. Kolb2, U. Schaefer5, 1University Hospital Hamburg, Germany,

J. McDermott2, Y. Nieto2, 1University of Colorado, Health Science

1

2

1

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3

University Hospital Munich, Germany, 3University Hospital Freiburg, Germany, 4University Hospital Saarland, Germany, 5 University Hospital Essen, Germany, 6German Clinic for Diagnostic Wiesbaden, Germany, 7University Hospital Dresden, Germany, 8 BMT-Clinic Idar-Oberstein, Germany Matched unrelated donor transplant has an increased risk of severe graft versus host disease and transplant related mortality (TRM). A TG has been introduced to decrease GvHD and to facilitate engraftment. We conducted a retrospective analysis of Fresenius ATG, n = 145, average = 90 mg/kg bw, range 40- 90 mg/kg bw compared with a no-ATG group, n = 188, age, sex, HLA-matched versus mismatched were comparable. Cell source (bone marrow vs. peripheral blood stem cell) was significantly different in the 2 groups. Results:

Conclusion: A TG Fresenius decreases the incidence of acute GvHD and TRM and leads to a significant better overall survival. A prospective randomized study is needed to evauate the definite role of ATG in HSCT.

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Section of Hematology, Italy, 2Dpt of Cardiology, Italy, 3Dpt Oncology/Hematology, Italy

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Center, U.S.A., 2University of Colorado, U.S.A. An intrinsic anti-tumor effect in the non-allogeneic setting remains unproven for breast cancer. Observations suggesting an anti-tumor effect for breast cancer (BC) include: majority of breast adenocarcinomas demonstrate a lymphocyte infiltration, and both an in-vitro cellular immune response, as well as in-vivo delayed type hypersensitivity response have been demonstrated to a number of different breast cancer antigens. By studying components of immune recovery following autologous stem transplantation (SCT) we acquired absolute memory and naïve T cell levels before and sequentially (D+30, D60, D+90, and D+180) post transplant. These T cell levels were acquired from 61 patients treated for breast cancer (BC) (N=46), nonHodgkin’s lymphoma (NHL) (N=6), myeloma (N=6), and Hodgkin (HD) (N=3). These data demonstrated that prior to transplant, CD4+ T cell levels correlated with relapse free survival (RFS) (log-rank p=.003) and overall survival (OS) (p=0.008). Correlation between CD4+ T cells and RFS remained apparent when patients were separated into BC, advanced (Stage III-IV) BC (ABC) and metastatic BC (MBC) (p=0.03, p=0.04 and p=0.03, respectively). Additionally, post-transplant CD4 counts on day+30 correlated with RFS in the whole group (p=0.03) and in BC (p=0.04). In further delineating this correlation we determined which memory and naïve T cell subsets were significant. The CD4+, CD45RA-, CD62L- memory phenotype correlated with RFS for all autologous recipients (p=.01), BC (p=.01), ABC (p=.01), and MBC (p=.02). In contrast, we did not observe an association with outcome for the other memory CD4, naïve CD4, memory CD8, naive CD8, or even the total CD3 counts. Finally, the absolute levels of CD4+ and CD4+, CD45RA-, CD62L- T cells showed to be independent predictors of RFS for all autologous recipients and for the BC group, in multivariate analyses including the following parameters: disease diagnosis, other lymphocyte subsets, the stage of disease, the refractoriness to chemotherapy, and HER2-neu status, and number of tumor sites. These results show an independent prognostic impact on outcome of CD4+ and more precisely CD4+, CD45RA-, CD62L- T cells, before and after transplant. Furthermore, our results would suggest that T cells may play a role in eliminating residual disease post transplant.

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Abstracts / Experimental Hematology 30 (2002) 37-146

Allogeneic immunotherapy in 46 patients suffering from 301 advanced solid tumors (ST) *D. Blaise , J. Bay , M. Michallet , J. Boiron , J. Cahn , N. Ifrah , 1

2

3

4

5

5

J. Sotto6, N. Gratecos7, C. Faucher1, J. Blay3, D. Olive8, D. Maraninchi1, P. Viens1, 1Institut Paoli Calmettes, France, 2Centre

Jean Perrin, France, 3Hopital Edouard Herriot, France, 4H Haut Lévèque, France, 5CHU, France, 6CHU Michallon, France, 7CHU Cimiez, France, 8Inserm U119, France Allo SCT has been shown to eradicate malignant hemopathies while a large number of pts always dies from ST evolution. We started in 1996 a program assessing ASCT in ST. We treated 46 patients. After using myeloablative (MA) regimen (Bus-Mel) for the 6 first pts, we moved to non MA regimen . NMA included Fludarabine (30 mg/m2x6), Busulfan (1mg/kgx8) and ATG (Thymoglobuline: 2.5 mg/kg/d: 4days:8;32days:8; 1day:24). Age: 44 (28-60) and M/F: 20/46; BM: 20; PBSC: 26). Renal (16); Breast (10); Melanoma (7); Ovarian (6); others (7). Among the 6 MA pts, 2 experienced grade ≥ 2 AGVHD and 2 died from TRM. 1 pt had PR (Ovarian). Among the 40 NMA pts, overall grade >= 2 GVHD was 31 %. TRM occurred in one patient Response evaluation is still under study but at least 5 had an OR(renal (2) and ovarian (3)) and 2 patients (breast) are experiencing a disease stabilization under GVHD. Complete evaluation of transplant course and response will be presented with a minimal follow-up of 6 months. However first conclusions can be drawn: 1) TRM is minimal with NMA 2) All responses but 1 have been documented with 1 day of ATG. 3) Pts with very active disease are unlikely to be controlled: pre graft tumor kinetic must be slowed down 4) Response is associated with clinical expression of GVHD 5) Early post graft immuno-modulation is crucial to reach rapid antitumor alloreactivity. 6) Not all diseases have the same level of sensibility (high: Renal, ovarian; low: melanoma)

Isolation and rapid mapping of recessive mutations 303 affecting murine hemopoiesis *B. Kile , A. Bradley , R. Behringer , M. Justice ,

1 1 2 1 1 Baylor College of Medicine, U.S.A., 2MD Anderson Cancer Center, U.S.A.

The enormous sequence resources now available, coupled with technical advances in positional cloning of genes, make feasible large-scale mutagenesis in the mouse, the primary experimental model for human diseases. As part of an ongoing mutagenesis screen utilizing the powerful mutagen N-ethyl-N-nitrosurea (ENU), we are attempting to identify genetic lesions causing hemopoietic abnormalities and malignancies. This screen represents the first use of an engineered mouse balancer chromosome to simultaneously isolate and map ENU-induced mutations. The balancer chromosome contains a 24 cM inversion between the p5J and WntJ loci on chromosome 11, the effect of which is to completely suppress recombination across this gene-rich interval. The inversion is tagged by the K14-Agouti transgene, thereby allowing heterozygous animals to be distinguished from non-inversion littermates on the basis of ear and tail pigmentation. Mice homozygous for the inversion die in utero due to the WntJ disruption. The three-generation mutagenesis screen employed in this laboratory utilizes all these features of the balancer to isolate ENU-induced recessive mutations that localize to, or are closely linked to, the p5J-WntJ interval. In addition to providing an immediate chromosomal location for new mutations, the inversion allows lethal or semi-viable mutations to be easily maintained in the heterozygous state. Thus far, we have isolated mutations causing micro and macrocytic anemias, neutrophilias and thrombocytopenias. The genetic lesions underlying these mutations are being pursued using positional cloning and candidate gene approaches.

Comparison of 2 different high-dose protocols in patients 302 with breast cancer (BC) undergoing autologouse stem cell transplantation (ASCT)

Genetic Regulation of Hematopoietic Stem Cell Telomere 304 Length in Mice *E. Manning , J. True , E. Henckaerts , H. Snoeck , H. Geiger ,

*A. Uss1, V. Zmatchinski2, E. Zhavrid3, A. Putirski3, 1Bone Marrow Transplantation Center, Belarus, 2Hematology-BMT Center, Belarus, 3Oncology Center, Belarus

G. de Haan3, L. Lu4, R. Williams4, G. Van Zant1, 1University of

Background: We evaluated 2 high-dose regimens (cisplatin/cyclophosphamide/BCNU and mitoxantrone/ cyclophosphamide/ melphalan) in respect to outcome in 52 patients with BC. Methods: From November 1997 to February 2002, 52 patients with histologically proven BC, stage II or III and 10 or more positive lymph nodes underwent mastectomy with axillary node dissection, radiotherapy and were treated with three cycles of standard adjuvant chemotherapy FEC. Peripheral blood stem cells were collected following administration of last course FEC with G-CSF 5 mcg/kg/day. Than patients were undergone to different high- dose regimens either cyclophosphamide 1875 mg/m2 and cisplatin 55 mg/m2 from days -6 to -4 and BCNU 450 mg/m2 on day -3 for group I, 19 patients, median age -42(range 25-53) or mitoxantrone 45 mg/m2 on day -6, cyclophosphamide 60 mg/kg from days -5 to -4, melphalan 140 mg/m2 on day -2 for group 2, 33 patients, median age -44(range 31-54), followed by ASCT on day 0. Median number of positive CD34+ cells infused were 5,9 6 6 (range 1,2-19,4)x10 /kg for group 1 patients and 7,9 (range 2,0-72,6)x10 /kg for group 2 patients Results: All patients suffered from nausea and vomiting and had one or more episodes of febril neutropenia. Engraftment after ASCT infusion for neutrophils > 0.5x109/1 and platelets > 20x109/1 for group 1 patients was carried out a median of 11 days (range 8-19) and 10 days (range 7-18) respectively and for group 2 patients -median of 13 days (range 9-21) and 10 days (range 3-19) respectively. Transplant-related mortality was encountered in one patient (5,2%) from group 1 and one (3%) patient from group 2 due to gram-negative septicemia and multiple organ failure. Median follow up period is 25,2 months ( 4-48) for group 1 patients and 25,3 months (4-51 ) for group 2 patients. To date 4 years DFS is 81% for group 1 patients and 73,5% for group 2 patients (p>0,05) and OS is 76,7% for group 1 patients and 76,5% for group 2 patients (p>0,05). Conclusion: These results confirm that high-dose chemotherapy can be safely administered to patients with high-risk breast cancer. Further studies are necessary to clarify the role of different high dose protocols in high-risk breast cancer.

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Kentucky, U.S.A., 2Mt. Sinai School of Medicine, U.S.A., 3University of Groningen, The Netherlands, 4Univ of Tennessee Memphis, U.S.A. To study the genetic regulation of telomere length in hematopoietic stem cells, we used (a) a Flow FISH technique for measuring telomere length and (b) quantitative trait loci (QTL) mapping in recombinant inbred mice. We first measured telomere length in both Lin-, Sca-1+, c-kit+ (LSK) and peripheral blood leukocytes (PBL) of two inbred mouse strains, DBA/2 (D) and C57BL/6 (B6), to demonstrate that PBL telomere length could be used as a surrogate assay for telomere length in more primitive cells. PBL telomere length accurately reflected that of LSK cells, and a large heritable difference was found between D and B6. Therefore we phenotyped, by Flow FISH analysis of PBL, both a middle age (8-15 months) and an old age (20-21 months) set of BXD recombinant inbred mice. Using Map Manager software, we mapped QTL for telomere length based on the strain distribution patterns of the BXD sets. A genomic interval from 43-58 cM on Chromosome 11 was suggestive based on permutation analysis in both age sets. Candidate genes in this region include the Rad51 paralogs, Rad51D (48.5 cM) and Rad51C (49 cM), which are involved in mediating DNA strand pairing and strand transfer in homologous recombinational repair (HRR) following DNA double-strand breaks. Homologous recombination is thought to be important in mediating telomere elongation by the telomerase independent Alternative Lengthening of Telomeres (ALT) pathway. Our analysis also uncovered a locus of interest on Chromosome 10 (21 cM) in the old, but not in the middle age BXD set. We validated the Chr. 11 locus by measuring PBL telomere length in congenic mice containing a region of D Chr. 11 (17-80 cM) on a B6 genetic background. These congenic mice displayed PBL telomere length significantly longer than that of the B6 background strain. In order to fine map the QTL, we phenotyped a cohort of 197 tenth generation advanced intercross mice generated from B6 x D mice. Surprisingly, 91% of the mice had a D allele at the Chr. 11 QTL, and 94% had a B allele at the Chr. 10 QTL, suggesting strong allelic preferences at these loci.

Abstracts / Experimental Hematology 30 (2002) 37-146

Complex clustered gene expression patterns in 305 hematopoietic stem cells. *L. Bystrykh, E. Weersing, B. Dontje, E. Vellenga, G. de Haan,

State

113

The Polycomb-Group (PcG) gene eed is a negative 307 regulator of bone marrow progenitor cell proliferation and suppresses radiation-induced lymphomagenesis *J. Lessard1, S. Girard1, A. Schumacher2, G. Sauvageau2, 1Clinical

University of Groningen, The Netherlands

Research Institute of Montreal, Canada, 2Baylor College of Medicine, TX, U.S.A.

Previously we have identified a locus (Scp2) that controls variation in cycling activity of hematopoietic stem cells (HSC) in C57BL/6 and DBA/2 strains of mice, and which maps to chromosome 11, at 41-49 cM. Here we present data on differential gene expression patterns of a small region of the Scp2 locus. This gene-dense region includes Defcap and Rab5ep, and spans an interval of 250 kb. We have compared the levels of expression and the extent of sequence polymorphisms of 3 definite genes (Rab5ep, Prei2, C1qbp) and 8 ESTs (2400006NO3Rik, AK14230, BC011309, BC005682, 2510025F08Rik, AK007359, AK005200) in this interval in B6, DBA, and B6.DBA.chr.11 congenic cDNA samples. The results indicate that the entire cluster shows a high level of sequence polymorphism and, strikingly, all tested transcripts were differently expressed when B6, DBA and congenic cells were tested. This demonstrates the presence of clusters of closely linked genes in genomic regions that show a high degree of sequence polymorphisms which are accompanied by extensive differences in gene expression levels. In addition, our data suggest the presence of a multiple locus control regions affecting the expression of a wide variety of stem cell transcripts.

The mouse Polycomb-group (PcG) gene embryonic ectoderm development (eed) is a component of a multimeric protein complex that governs anteriorposterior patterning of the axial skeleton by regulating Hox gene expression. Beyond its role in embryonic development, eed is also involved in regulating hemopoiesis. Heterozygosity for a null allele of eed (eed3354/+) causes marked myelo- and lympho-proliferative defects in mice (3-fold increase in primitive (LTC-IC and WW-IC) and 19-fold increase in late (myeloid and pre-B CFC) bone marrow progenitor cell numbers on average), indicating that eed plays a crucial role in the negative regulation of the proliferative capacity of both lymphoid and myeloid progenitor cells (Lessard et al., Genes & Dev., 1999). To investigate whether Eed performs a tumor-suppressive function in hemopoietic cells, a cohort of mice homozygous for a hypomorphic allele of eed (eed1989/1989) or heterozygous for a null allele of eed (eed3354/+) were given a single dose of ionizing radiation (600 RAD) at 5 weeks of age and monitored for the development of disease. Homozygous eed1989/1989 mutant mice developed a high incidence of B or T cell lymphomas with shorter latency compared to wild-type littermates (n=7/13 vs n=1/16; 16±4 wks vs 34±0 wks post-irradiation, respectively). Heterozygous eed3354/+ null mice were also more susceptible to radiation-induced B and T cell lymphomas than control littermates (n=11/15 vs n=4/15), although with a much longer latency period than homozygous eed1989/1989 mice (27±9 wks vs 16±4 wks, respectively), indicating that eed performs tumor-suppressive function in lymphoid cells. Together our data demonstrate that eed controls the proliferative potential of hemopoietic progenitor cells by acting as a growth and tumor suppressor.

HoxB4 and HoxB3 control the proliferative capacity 306 of hematopoietic stem cells *J. Bjornsson , N. Larsson , A. Brun , M. Magnusson ,

Haploinsufficiency of the Nf1 Tumor Suppressor Gene 308 Enhances the Migration of Mast Cells Via Increased Adhesion to VLA-4 and Hyperactivation of the Ras-PI-3K-Rac2

K. Humphries3, S. Karlsson2, 1Inst. for Lab. Medicine, University

Pathway *D. Ingram1, S. Chen2, L. Fisher2, M. Wenning2, H. Huddleston2, S. Atkinson2, D. Williams2, R. Kapur2, D. Clapp2, 1Indiana

1

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of Lund, Sweden 2Lund University, Sweden, 3Terry Fox Laboratory, Canada HoxB3 and HoxB4 are expressed at early stages of hematopoiesis and then downregulated, indicating a regulatory role for these genes in hematopoiesis. In addition, constitutively engineered expression of these genes clearly affects both proliferation and maturation of hematopoietic cells. To further investigate the role of these transcription factors, we generated mice deficient of both HoxB3 and HoxB4 (B3/B4-/-) which are born viable at normal Mendelian ratio. However, the cellularity of bone marrow and spleen were reduced in the B3/B4-/- mice (p<0.01). Furthermore, peripheral red blood cell counts and hemoglobin values were also reduced (p=0.05). Lineage distribution of hematopoietic cells was normal in these organs indicating a more general reduction in cellularity rather then abnormalities in any specific lineage. Frequency of colony forming cells and Lin-Sca1+ckit+ (LSK) cells was the same in B3/B4-/- mice as in controls, resulting in lower absolute numbers of these populations. In d14.5 fetal liver the frequency and absolute number of B3/B4-/- Sca1+AA4.1+Lin- cells was also reduced. B3/B4-/LSK cells display normal cell cycle distribution in steady state hematopoiesis, however the proliferation recruitment of single sorted LSK cells was two fold lower (p=0.01) for the B3/B4-/- cells when highly stimulated in a six cytokine cocktail. To analyze whether this observed reduction in proliferation affected repopulation capability, we performed a competitive transplantation study. At 6 weeks post transplant the B3/B4-/- derived cells displayed lower engraftment than B3/B4+/+ cells (p=0.01), but this difference was not significant at 17 weeks (p=0.1). In secondary recipients, however, the overall reconstitution of B4/B3-/cells was significantly lower than their B3/B4+/+ counterparts (p<0.05), indicating a reduced proliferation capacity of B3/B3-/- stem cells. A double treatment with 5-fluorouracil (5-FU) prior to transplantation also showed that B3/B4-/- cells have a higher tolerance to the drug, since those cells post-treatment, showed significantly higher engraftment capability compared to normal control cells (p<0.01). We conclude that HoxB3/B4 deficiency negatively affects the proliferation of primitive hematopoietic progenitors without affecting differentiation and furthermore, that these transcription factors are important in enhancing proliferation of primitive hematopoietic cells under stressful conditions that require rapid proliferation of stem cells.

University, Cancer Research Inst., U.S.A., 2Indiana University, U.S.A. Mutations in NF1 cause neurofibromatosis type I. Individuals with NF1 develop neurofibromas, which are infiltrated with mast cells associated with increased mRNA transcripts for stem cell factor (SCF), a chemoattractant for these cells. Recruitment of mast cells is a potentially critical event in tumor formation as recently shown in genetically engineered Nf1 deficient mice. Neurofibromin, the protein encoded by NF1, functions by negatively regulating Ras activity in mammalian cells. Specifically Nf1 +/- mast cells have increased survival and proliferation in response to SCF compared to wildtype (WT) cells (Ingram, JEM 2000 and 2001). Here, we investigated whether loss of neurofibromin alters Ras effector pathways to enhance mast cell migration by utilizing Nf1 +/- and WT bone marrow derived mast cells (BMMCs). Using transwells and video microscopy, Nf1 +/- BMMCs show a two-fold increase in migration on fibronectin compared to WT cells in response to SCF. Since BMMCs adhere to the integrins VLA-4 and VLA-5 on fibronectin, we next tested whether increased migration of Nf1 +/- cells was mediated via increased adhesion to these two integrins. Nf1 +/- BMMCs show a two-fold increase in adhesion to both VLA-4 and VLA-5 compared to WT cells. Preincubation with blocking antibodies to VLA-4 or VLA-5 or use of fibronectin peptides mediating integrin-specific adhesion demonstrates that enhanced migration of Nf1 +/- BMMCs is mediated via VLA-4. Previous studies show that activation of PI-3 kinase and Rac2 are necessary for mast cell migration to SCF. Nf1 +/- mast cells show a two-fold increase in PI-3 kinase and Rac activity compared to WT cells with SCF stimulation. To genetically test whether hyperactivation of these pathways is responsible for the Nf1+/- phenotype, we intercrossed Rac2 -/- or p85a +/- mice (a regulatory subunit of PI-3 kinase) with Nf1 +/- mice. The increase in migration of Nf1 +/- mast cells was completely attentuated in Nf1 +/-; Rac2 -/- and Nf1 +/-; p85a-/- mast cells. Thus, these genetic and biochemical studies directly link hyperactivation of the Ras-PI-3K-Rac2 pathway and adhesion to VLA-4 to the enhanced migration of Nf1 +/- mast cells to fibronectin in response to SCF.

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Constitutive c-jun N-terminal kinase activity in acute 309 myeloid leukemia derives from activated Flt3: JNK inhibition unleashes catastrophic oncogene stress response

Clonal Adaptation and Evolution in Fanconi Anemia (FA) 311 A. Speckhart, M. Lensch, J. Yates, *G. Bagby,

A. Hartman*1, G. Burgess2, C. Phillips2, M. Su3, F. Salituro3, J. Alam3, V. Gelfanov2, H. Ramsey1, L. Cripe2, R. Hromas2, H. S. Boswell4,

Science Univ., U.S.A.

1 Walther Cancer Institute, U.S.A., 2Indiana University, U.S.A., 3Vertex Pharmaceuticals, U.S.A., 4Indiana Cancer Research Institute, U.S.A.

Oregon Health &

A functional relationship has been established between constitutive activity of c-jun N-terminal kinase, treatment failure in AML, and the existence of a multidrug resistance phenotype involving MRP1 , especially in response to the anthracycline drug class (Gripe, et al. In press). However, recently it has also become clear that oncogenesis, especially myeloid leukemogenesis, which occurs in the absence of mutations in key tumor suppressors p53 or p14 arf, imparts stress signaling pathways collectively referred to as “oncogene stress.” G-jun N-terminal kinase has been implicated in the homeostatic response to oncogene stress by virtue of a role for c-jun and junD in repression of p53 and p14arf, respectively. Therefore, a search was made for the upstream source of constitutive JNK signaling previously identified in virulent AML, and on the consequences of inhibition of JNK (measured by phospho-ser73 c-jun immunoblot) on cellular homeostasis and response/sensitivity to the anthracycline drug class. Herein, autoactivated Flt3 receptor, most commonly resulting from exon 11 internal tandem duplication (ITD) mutation (11/18 cases), was identified as the oncogenic origins of constitutive JNK activity in blast cells of 91% JNK+ve AML cases. In blast cells from 3 cases (of 18 total) without ITD by PGR, point mutation in exon 20 was further excluded by PGR, and yet maintenance of autoactivity of the overexpressed receptor protein was demonstrated in tissue culture without ligand. The components of a signaling pathway, from autoactive Flt3 via PI-3-kinase-to-JNK, were identified in blast cells. Selective inhibition of JNK within these blast cells, with a small molecule inhibitor, V-132, had dire consequences for cellular homeostasis, by proliferative inhibition, inducing apoptosis, and sensitizing cells to synergistic killing by the anthracycline, daunorubicin. These effects occurred without effects on the ERK or AKT pathways by V-132. Also, these effects were part of a coordinated unleashing of p53-dependent oncogene stress apoptotic response normally held in check by JNK operating downstream from Flt3.

Bone marrow failure is nearly universal in adults and children with FA and the relative risk of acute myeloid leukemia (AML) and myelodysplasia is 510,000. FANCC modulates apoptotic signals and inactivating mutations in FA patients result in a characteristic pro-apoptotic phenotype in hematopoietic cells. Seeking to determine linkage of bone marrow failure and the subsequent development of clonal neoplasia (myelodysplasia [MDS] and acute myeloid leukemia [AML]) we hypothesize that the apoptotic phenotype serves as a selective pressure for the emergence of stem cell clones that have undergone adaptive somatic mutations that lead to resistance to apoptotic cues. Interferon responsiveness of erythroid and myeloid progenitor cells from 9 normal volunteers, 7 FA patients without MDS/AML, and 3 FA patients with both FA and MDS/AML, was quantified in semisolid media containing Steel factor, IL-3, and erythropoietin. FA patients were hypersensitive to IFN-gamma but FA/MDS patients were resistant. Affymetrix GeneChip array analysis (U95A) was carried out using RNA prepared from freshly obtained low density bone marrow cells from a child with FA/MDS and his sister with uncomplicated FA. Reproducibility was high between chips for the same sample (r=0.945 [FA] and r=0.928 [FA/MDs]). We excluded 12,298 of 12,625 genes. Seventy of the 12,298 varied between replicates, 7,579 showed either no change or a less than twofold change, and 4,649 were absent in all samples. There were 327 differentially expressed genes of which approximately half were increased in the MDS sample. 7,906 genes were expressed in bone marrow cells from both siblings. There were 131 genes more highly expressed in the sibling with FA and MDS, 196 genes were expressed at a lower-level in this same individual. Of the differentially expressed genes at least 35 were candidates as causes of clonal evolution in FA stem cells. In preliminary experiments two epistatic genes, BAX (reduced in MDS marrow) and PSPHL (increased in MDS) have been identified. The transcriptomal approach holds the potential of defining pathways of clonal adaptation and early molecular events that account for clonal evolution.

Overexpression of EGR-1 and TAXREB107 in clonal cells 310 of PNH patients *J. Schubert, A. Lyakisheva, R. Schmidt, A. Ganser,

Highly efficient lentiviral transduction of NOD/SCID 312 repopulating cells but not long-term repopulating cells in the baboon in a direct comparison of the NOD/SCID assay versus

Medical School, Germany

autologous transplantation *P. Horn, B. Thomasson, J. Morris, H.-P. Kiem, Fred Hutchinson

Hannover

Cancer Research Center, U.S.A. Paroxysmal nocturnal hemoglobinuria is an acquired clonal defect of hematopoietic stem cells characterized by deficiency in GPI-anchored surface proteins. It is not yet known, how GPI-deficient stem cells are able to expand within the bone marrow and contribute considerably to the hematopoiesis. In PNH as well as in AA and MDS, genetic instability and increased mutation frequency have been detected. Therefore, a second event is very likely, such as additional mutations, leading to clonal expansion of GPI-deficient bone marrow stem cell in PNH. In order to elucidate the molecular basis of clonal expansion in PNH we identified several genes differentially expressed in normal and GPIdeficient cells of PNH patients by combination of RNA fingerprinting and cDNA array hybridization. Expression of two of these genes, EGR-1 and TAXREB107 has been further investigated. EGR-1 is upregulated in granulocytes of all PNH patients analyzed so far. In contrast, significant upregulation of TAXREB107 is present only in some of our PNH patients. Further analysis confirmed their overexpression in PNH and excluded a possible secondary event such as upregulated cytokines as the cause of the observed overexpression. Moreover, similar level of expression in cases of other clonal diseases such as MPS and MDS has been identified. Sequencing of promotor and coding regions of EGR-1 did not reveal any mutation. Our data suggest that additional genetic alterations apart from PIG-A mutations which might be located within upstream factors of EGR-1 would be present in PNH granulocytes. In addition, these genetic changes might contribute to clonal expansion of GPI-deficient cells in PNH.

The nonobese diabetic/severe combmed immune-deficient (NOD/SCID) mouse xenotransplant system is widely used as a surogate assay for hematopoietic stem cell engrafbnent and gene transfer. In this study we wished to directly compare oncoretroviral and lentiviral transduction efficiencies of repopulating cells m the NOD/SCID model with the autologous transplant setting in the baboon. We transplanted 3 baboons with CD34-enriched marrow cells transduced with GAL V pseudotype oncoretroviral vectors. Aliquots of the same cells that were reinfused into the baboon were also transplanted into irradiated NOD/SCID mice. At 6 weeks post transplant bone marrow was harvested from 32 mice and evaluated by flow cytometry for engraftment of baboon cells and transgene expression. Average engraftment of baboon cells was 4.5%. The average percentage of baboon cells expressing the transgene EYFP was 54-61% m the 3 groups of NOD/SCID mice. In the peripheral blood of the baboons 2 months lpost transplant 4.4-7. 7% of cells expressed EYFP. No immune response against Ithe transgene could be detected in the baboons by an intracellular cytokine stain. In 2 separate competitive repopulation assays in the baboon we compared lentiviral vectors at either different multiplicities of infection (MOl 10 vs. MOl 100) or with different growth factor combinations during the transduction (SCF only vs. IL-3, IL-6, SCF, MGDF, G-CSF, F1t3-L). Again, aliquots of the transduced cells were also transplanted into a total of 22 NOD/SCID mice. In both baboons marking with the lentiviral vectors decreased to levels below 0.1 % within 2 months for both experimental arms. However, up to 72% of engrafted baboon cells in the NOD/SCID mice expressed EGFP or EYFP at 6 weeks post transplant. Several of the mice were followed for up to 12 weeks. No decline in engraftment levels or the percentage of transgene expressing cells was observed. Analysis of engrafted cells revealed the presence ofCD34+, CD20+, and CDI3+ lcells, suggesting multilineage engraftment of baboon cells in NOD/SCID mice. In conclusion, there was a significant difference in transduction of NOD/SCID repopulating cells versus repopulating cells in the autologous transplant in the baboon. Marking was markedly higher m the NOD/SCID repopulating cells using both oncoretroviral and lentiviral vectors. This difference in gene transfer rates was much more pronounced with lentiviral vectors. Thus, it seems likely that a different, more easily transducible subset of repopulating cells is responsible for engraftment in the NOD/SCID mouse.

Abstracts / Experimental Hematology 30 (2002) 37-146

Extensive heterogeneity of AML stem cells revealed 313 by clonal tracking *K. Alexander, L. Jin, J. Dick,

Hospital for Sick Children, Canada

115

Interleukin-10 inhibits macrophage activation through 315 induction of SOCS3 P. Qasimi , A. Ghanipour , A. Yoshimura , J. Ihle , *A. Mui ,

1 1 2 3 1 1 Jack Bell Research Center, Vancouver Hospital & Health Sciences Centre, Canada, 2Kyushu University, Japan, 3St. Jude’s Hospital, U.S.A.

In AML, the leukemic clone is organized as a hierarchy which originates from a rare leukemic stem cell. Knowledge of the cellular and molecular program of these stem cells is central to elucidation of the leukemogenic process and for the development of effective therapies. Our recent finding that normal human stem cells have extensive variation in developmental potential raises the question of whether AML stem cells are also composed of distinct classes. Using the NOD/SCID leukemia model, the in vivo fates of individual SCID leukemia initiating cells (SL-IC) have been tracked following the detection of unique viral integration sites. Moloney-based retrovirus vectors are limited by the requirement for cell division for infection. Since AML stem cells do not cycle, lentivirus vectors, which have the capacity to transduce quiescent cells, have been used in our experiments to transduce SL-IC. A modified third generation lentivector encoding EGFP driven by the hPGK promoter, was used to transduce cells from patients with AML. High levels of GFP expression were obtained in leukemic blasts (31.1±21.5%) and clonogenic progenitors (40.0±29.5%). Similarly high levels of EGFP expressing cells were maintained in vitro up to 28 days indicating transduction of primitive progenitors. Transduced AML cells were injected into sub-lethally irradiated NOD/SCID mice. All transduced samples showed high levels of human engraftment (39.9±36.1% CD45+ cells) and EGFP expression (30.5±25.5%) in the bone marrow, showing efficient transduction of SL-IC. High resolution in vivo tracking of the clone emanating from each transduced SLIC was carried out by Southern integration site analysis on DNA isolated from sequential bone marrow aspirates. Some clones contribute to engraftment only at early time points while others contribute to long-term engraftment. Serial transplantation has shown that some clones appear dominant in secondary recipients but are undetectable in primary recipients while other clones are not observed in secondary mice. Our results are the first to identify SL-IC at the single cell level and show that heterogeneity in both proliferative and self-renewal potentials exists in the leukemic stem cell compartment. These findings will help guide new understanding of leukemic stem cell biology and new therapeutic strategies.

Interleukin-10 (IL-10) is an important immune regulator which inhibits macrophage production of proinflammatory mediators. The IL-10 receptor (IL-10R) belongs to the Class II cytokine family and like other members of this family, transduces signals through STAT transcription factors. We and others have shown that the anti-inflammatory action of IL-10 depends on STAT3 since macrophages deficient for STAT3 are resistant to the inhibitory action of IL-10. In order to understand the mechanism by which IL-10 inhibits TNFα production by lipopolysaccharide (LPS) activated macrophages, we searched for IL-10 regulated genes using DNA microarray methodology. The mRNA for SOCS3, a member of a newly described family of suppressors of cytokine signalling, was rapidly induced by IL-10. Using macrophages expressing IL-10Rs lacking the STAT3 activation motif or a dominant negative STAT3, we show SOCS3 is induced by IL-10 in a STAT3dependent manner. To determine the contribution of SOCS3 induction to the anti-inflammatory action of IL-10, we isolated SOCS3-/macrophages and examined the ability of IL-10 to antagonize LPSinduced TNFα expression. The ability of IL-10 to inhibit LPS activation of NFkB and TNFα production were both severely compromised in SOCS3-/- macrophages. Both of these IL-10 responses could be rescued by reconstituting the SOCS3-/- macrophages with the cDNA for SOCS3. These studies identify for the first time, a STAT3-regulated gene which mediates the anti-inflammatory action of IL-10 and futhermore describe a novel molecular target for SOCS3.

hTERT gene expression is increased by alternative 314 splicing of mRNA during the course of CML, and telomere loss at diagnosis is associated with early progression

A New Adult Mouse Model of Lethal b0-Thalassemia and 316 Its Rescue by Lentivirus-Mediated Globin Gene Transfer *S. Rivella , C. May , A. Chadburn , I. Riviere , M. Sadelain ,

*M. Drummond1, S. Hoare2, M. Alcorn3, S. Graham1, W. Keith2, T. Holyoake1, 1Glasgow University, U.K., 2Beatson Institute, U.K.,

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Memorial Sloan Kettering Cancer Center, U.S.A., 2Cornell University, U.S.A.

Glasgow Royal Infirmary, U.K.

Background: Chronic myeloid leukaemia (CML) is characterised by myeloid expansion, subsequent to acquisition of the Ph chromosome. Telomere shortening has been demonstrated in peripheral blood leucocytes (PBL) of CML patients, and a greater degree of loss may be associated with early blastic phase (BP). The regulation and role of hTERT during early disease remains poorly understood. We prospectively studied newly diagnosed CML patients with regard to degree of telomere loss and outcome, and examined levels of hTERT mRNA and its splicevariants in samples from all stages of CML. Methods: Flow-FISH telomere measurement of Ph- ex-vivo expanded T lymphocytes and Ph+ PBL was performed in 32 consecutive newly diagnosed patients, and delta-tel (Lymphocyte telomere signal –PBL telomere signal, ie relative degree of telomere shortening) correlated with Hasford score risk groups and time to progression. 51 samples from all stages of disease had hTERT mRNA and its splice variant transcripts (functional +α+β, and /or non-functional –α, -β, or –α-β) measured by Light Cycler and RT-PCR respectively. Telomerase activity was measured by TRAP assay. Results: Mean delta-tel was significantly higher in high-risk than low-risk score groups (3.4+2.8 kMESF vs 0.6+2.2 kMESF, mean+SD, p<0.05). Patients with a delta tel above the group mean had significantly reduced time to progression as compared to those below the mean (Log Rank test p<0.001). +α+β hTERT transcript levels increased significantly with disease progression, and were highest in blastic phase (BP). This was associated with a shift from a heterogeneous pattern of splice-variant hTERT mRNA at diagnosis to +α+β containing transcript patterns at BP. The presence of +α+β was required for telomerase activity, as demonstrated by TRAP assay. 53% of diagnostic samples did not express +α+β and there was a trend towards greater delta-tel values in this group. Conclusion: Telomere shortening in CML is greatest in high-risk score patients at diagnosis, and may predict for progression of disease. +α+β hTERT mRNA is upregulated by alternative splicing during disease progression, and although universally expressed in BP, is not in earlier phase disease. Lack of expression at diagnosis may be associated with greater telomere loss.

We have established a model of adult lethal ß°-thalassemia in order to evaluate the full therapeutic potential of lentiviral vectors encoding the human ß-globin gene. Mice engrafted with ß-globin-null (th3/th3) fetal liver cells died 7 to 10 weeks after engraftment with profound anemia and ineffective erythropoiesis (hemoglobin 3.6 ± 1.8g/dl, RBC 2.6 ± 1.2x10 6/mm3 with absent or very low reticulocyte counts). Pathology and FACS analyses showed in bone marrow (BM) and spleen a uniform erythroid hyperplasia but absence of mature erythroid cells. These mice can be rescued (n=12) by transplantation of unselected fetal liver cells transduced with TNS9, a vector encoding the human ß-globin gene. Molecular analyses were performed in long term studies (4 to 8 months) on 5 mice surviving with hemoglobin tetramers consisting mostly (>95%) of murine-α2:human-ß2. Hemoglobin was 5.4 ± 1.3g/dl and the copy number in bone marrow was 1.5 ± 0.6. We next analyzed expression of TNS9 in three genetic backgrounds (+/+, th 3/+ and th3/th3), comparing the ratio of human-ß/mouse-α mRNA in hematopoietic tissues to assess the effect of selective pressure on transgene function. While transcriptional activity of the human ß-globin gene, assessed in BM and spleen, is the same in three genetic backgrounds, we observed increasing amount of human ß-mRNA from BM to blood in mice engrafted with th3/th3 cells versus mice engrafted with wild-type cells (p<0.05). A selective model with its implications for gene therapy will be presented and discussed. In conclusion the level of human b-globin expression afforded by the TNS9 vector is sufficient to dramatically alter the course of severe ß°thalassemia.

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Role of mitogen-activated protein kinase family in serum317 induced leukemia inhibitory factor and interleukin-6 secretion by bone marrow stromal cells

In vivo selection of human clonogenic cells and 319 lymphomyeloid lineages derived from SCID-repopulating cells by expression of a mutant O6-methylguanine DNA

*T. Nakao, S. Kim, K. Ohta, H. Kawano, T. Asai, M. Hino, K. Miura, N. Tatsumi, H. Iwao, Osaka City University, Japan

methyltransferase (MGMTP140K) *K. Pollok1, J. Hartwell1, A. Braber1, M. Jansen2, E. Kreklau1, L. Erickson1, D. Williams2, 1Indiana University, U.S.A., 2Cincinnati

In the haematopoietic microenvironment, bone marrow stromal cells play an important role in regulating haematopoiesis by expressing various hematopoietic cytokines, including leukemia inhibitory factor (LIF) and interleukin-6 (IL-6). However, the intracellular signal that regulates cytokine secretion in bone marrow stromal cells has not been determined. The aim of this study was to evaluate the role of mitogen-activated protein kinase (MAPK) family in serum-induced secretion of LIF and IL-6 by bone marrow stromal cells. Transformed human bone marrow stromal cells (HS-5), which could sustain the proliferation of haematopoietic progenitor cells in vitro, were stimulated with fetal calf serum (FCS) to produce LIF and IL-6. We demonstrated that FCS also induced activation of extracellular signalregulated kinase (ERK), p38 MAPK and c-Jun NH2-terminal kinase (JNK) by Western blot analysis. To evaluate the role of these kinases in regulating the production of cytokines, we inhibited these pathways by using pharmacological inhibitors and expression of dominant-negative mutant. Both PD98059 (MAPK/ERK kinase inhibitor) and SB203580 (p38 MAPK inhibitor) attenuated FCS-induced LIF protein production and gene expression. SB203580 decreased IL-6 production and gene expression, but PD98059 had no effect on IL-6 production and gene expression. Expression of a dominant-negative mutant form of JNK1 that blocked FCS-induced JNK activity had no effect on protein production and gene expression of these cytokines. These findings demonstrate that both ERK and p38 MAPK are involved in FCS-induced LIF secretion, whereas only p38 MAPK is important for IL-6 secretion, and that FCS-induced activation of JNK has no effect on the production of LIF and IL-6. Although these cytokines both belong to the same cytokine family and share a number of biological activities, their secretion is regulated by different signal transduction pathways in bone marrow stromal cells. This distinct usage of MAPK pathways in the production of hematopoietic cytokines may reflect the tightly controlled regulation of cytokine productions and hematopoiesis within the marrow microenvironment.

Children’s Hospital, U.S.A. Expression of a chemoresistance gene in human hematopoietic stem and progenitor cells (SP) may allow for the enrichment of small numbers of transduced SP. In addition, chemoprotection of SP may allow for dose-intensified chemotherapy to treat a variety of drug-resistant cancers. We assessed whether SCID-repopulating cells could be protected from a combination therapy of O6benzylguanine (6BG) and 1,3-bis (2-chloroethyl)-1-nitrosurea (BCNU). CD34+ cells isolated from umbilical cord blood or G-CSF-mobilized peripheral blood were transduced with a GALV-pseudotyped bicistronic oncoretrovirus vector that coexpresses EGFP and a mutant form of O6-methylguanine DNA methyltransferase, MGMTP140K, that is resistant to 6BG and BCNU. Two different retroviral backbones, SF1 (kindly provided by Dr. Christopher Baum) and MSCV, were compared with regards to MGMTP140K expression. SF1- and MSCV-transduced CD34+ cells were sorted for EGFP+ cells and MGMTP140K activity was determined. MGMTP140K activity was 50% higher in SF1transduced CD34+ cells versus those transduced with MSCV. In addition, SF1- or MSCV-transduced SP were transplanted into NOD/SCID mice and subsequently mice were either not treated (n = 21) or treated once weekly (n = 43) for 2-4 weeks with 20-30 mg/kg 6BG and 5-10 mg BCNU. At 6-8 weeks post-injection, the bone marrow was analyzed for the presence of transduced human cells (EGFP+CD45+). Significant protection of human cells in the bone marrow was observed in both myeloid and B-cell lineages (nontreated vs. treated, p < 0.05). Up to 90% of the human cells in the bone marrow, expressed MGMTP140K, following in vivo treatment with 6BG/BCNU regardless of the retroviral backbone utilized. Colony forming unit assays indicated a 4-9-fold increase in the total number of EGFP+ colonies in the bone marrow of treated versus nontreated mice. Furthermore, MGMTP140K bioactivity was significantly increased in human colonies derived from treated mice. These data show that selection of human hematopoietic cells can be effectively accomplished in vivo using the combination of pharmacologic and genetic approaches.

Complete chimerism with persistent multilineage 318 transgene expression after immunoselection of retrovirally engineered hematopoietic stem cells

Retroviral-mediated gene transfer in human primary 320 T lymphocytes: impact of the type of stimulation on TCRB repertoire of gene-modified cells.

*B. Fehse1, Z. Li2, B. Schiedlmeier3, J. Düllmann4, O. Frank3, A. Zander4, W. Ostertag3, C. Baum2, 1University Hospital Hamburg-

*S. Coito1, D. Sauce1, A. Duperrier1, J.-M. Certoux2, F. Kuttler1, A. Collette3, E. Robinet1, P. Tiberghien1, C. Ferrand1, 1EFS-B/FC,

Eppendorf, Germany, 2Medical School Hannover, Germany, 3HeinrichPette-Institute, Germany, 4University Hospital Eppendorf, Germany

France, 2EFS-B/FC, Finland, 3Institut pasteur, France

Unpredictable interindividual variability and silencing of transgene expression over time are severe impediments to successful hematopoietic gene therapy. We demonstrate that homogenous transgene expression can be achieved in multiple hematopoietic lineages in vivo, using rather simple experimental procedures. Bone marrow of C57Bl/6 mice was transduced with retroviral vectors containing cis-active sequences improved for gene expression in primitive hematopoietic cells, but no additional element known to modify chromatin regulation. To monitor and allow enrichment of transduced cells, two variants of the human CD34 (hCD34) antigen were expressed, the full-length protein (flCD34), or a splice-variant lacking most of the cytoplasmic signal transduction domain (tCD34). Up to six months after transplantation into irradiated (10 Gy) recipients (n=6 per vector), transgene expression frequencies were rather constant, in the range of 15% of white blood cells, but with significant interanimal variability. Bone marrow cells of these mice were pooled and transplanted into a second cohort of irradiated (10 Gy) recipients (n=6 per vector), one group receiving unselected cells, the other flCD34+ or tCD34+ cells enriched to high purity (>95%) by immunoaffinity selection. Recipients of unselected cells showed a decline of hCD34+ cells over time, arguing against a selective advantage of hCD34+ cells. In contrast, recipients of immunoaffinity-enriched transgenic cells had a surprisingly homogenous pattern of transgene expression in multiple hematopoietic lineages (up to 100% hCD34+), without evidence for profound gene silencing or interanimal variability (SD <5%). Expression even persisted in tertiary CFU-S. Southern blot analysis revealed monoclonal or oligoclonal hematopoiesis, as opposed to evidence for polyclonal hematopoiesis in the unselected group. Thus, a simple retroviral vector in combination with a single enrichment procedure allows long-term, multilineage gene expression in serially transplanted hematopoietic cells, but this occurs at the expense of a reduction in the clonal repertoire.

Administration of donor T cells expressing the herpes simplex-thymidine kinase with a hematopoietic stem cell transplantation could allow, if graftversus-host disease was to occur, a selective in vivo depletion of these T-cells by the use of ganciclovir. Previous studies have shown that 12 days after PBMC activation with CD3 monoclonal antibody (CD3 mAb), TCRB repertoires of gene-modified cells (GMC) were skewed. The aim of this study was to determine the effect of the initial T-cell activation and of the transduction / selection step on subsequent TCRB repertoire alterations. The preparation of GMC required an initial stimulation with either CD3 mAb, CD3/CD28 beads (CD3/28), irradiated allogeneic PBMC (PBMC) or allogenic EBV-transformed cell lines (EBV), followed by a retrovirusmediated transduction from day 3 to day 4, and a final 7-day selection step based on the resistance of GMC to G418. TCRB repertoires of non transduced, non selected control cells (C0) and GMC, and their CD4+ and CD8+ subsets, were compared at day 12 to PBMC repertoires, and their CD4+ and CD8+ subsets, by the CDR3 spectratyping method which analyses the length of the CDR3 region of the TCRB chain. TCRB alterations of C0 and GMC were respectively 22.3 and 30.8% (CD3 mAb, n=8, p<0.05), 23.3 and 26.0% (CD3/CD28, n=3), 28.2 and 34.4% (PBMC, n=6) and 35.1 and 34.3% (EBV, n=3). TCRB alterations after CD3 mAb and CD3/CD28 activation were respectively 32.6 and 16.2% in CD4+ GMC (n=7, p<0.05) and 40.1 and 20.8% in CD8+ GMC (n=6, p<0.05). Thus, among the four treatments, CD3/CD28 was the one that best preserved the TCRB repertoire diversity of C0 cells or GMC, including when comparing with the CD3 mAb activation. Moreover, the transduction / selection increased significantly TCRB repertoires alterations after CD3 mAb activation, since GMC repertoires were more skewed than their respective C0 cells. In conclusion, we propose to use CD3/CD28 beads instead of CD3 mAb stimulation in order to maintain the most diverse TCRB repertoire, thus ensuring the broadest possible immune reactivity of GMC.

Abstracts / Experimental Hematology 30 (2002) 37-146

Tight regulation of plasmid based gene expression in vivo 321 following intramuscular injection *M. Margalith, A. Vilalta, P. Hobart, Vical Inc., U.S.A.

In vivo delivery of potent therapeutic proteins using plasmid DNA vectors will likely require tight regulation. The bacterial tetracycline repressor fused to a eukaryotic transcription activator (tTA) is a well-characterized system for regulating eukaryotic gene expression in vivo using the orally active, small molecule drug, doxycycline. We have designed a series of plasmids with two eukaryotic expression cassettes to deliver both the transactivator and the therapeutic gene on a single plasmid. Typically, tTA is expressed using a constitutive promoter while expression of the gene of interest (e.g. Epo, Factor IX, etc.) is under the control of a TetO7/minimal CMV promoter and dependent upon tTA. Thus, therapeutic protein expression can be modulated by by doxycycline. We found that strong enhancer elements in the constitutive promoter used in such single plasmid constructs result in low level, (“leaky”) expression of coding sequences downstream of the TetO7/minimal CMV promoter. Leakiness is unacceptable when regulating expression of potent cytokines, such as Epo. Testing a series of alternative promoters, we found that using RSV for constitutive expression of tetR/VP-16, eliminates leaky expression of the therapeutic gene of interest, such as Epo. Moreover, oral administration (drinking water) of doxycycline results in tightly controlled expression for a period of at least six months. These results indicate the importance of the design of the tTA based transcriptional control system for tight, longterm control of heterologous gene expression.

117

A genetic and genomic analysis identifies a cluster of genes 323 associated with hematopoietic cell turnover *L. Bystrykh , E. Weersing , B. Dontje , H. Geiger , N. Ivanova , 1

1

1

2

3

I. Lemischka3, E. Vellenga4, G. Van Zant2, G. de Haan1, 1State University of Groningen, The Netherlands, 2University of Kentucky, U.S.A., 3Princeton University, U.S.A., 4Academic Hospital Groningen, The Netherlands Hematopoietic stem cells from different strains of mice vary widely with respect to their cell cycle activity. In the present study we used complementary genetic and genomic approaches to identify molecular pathways affecting this complex trait. We identified a major quantitative trait locus (QTL) associated with variation in cell proliferation in C57BL/6 and DBA/2 mice to a 10 cM region on chromosome 11. A congenic mouse model confirmed that a genomic interval on chromosome 11 in isolation confers the proliferation phenotype. To detect candidate genes we performed subtractive hybridizations and gene arrays using cDNA from highly enriched stem cells from parental strains. Intriguingly, a disproportionate number of differentially expressed genes mapped to chromosome 11 and, more specifically, these transcripts occurred in three distinct clusters. The largest cluster colocalized exactly with the cell cycling QTL. Such clustering suggested the involvement of genetic variation that affects higher order chromosomal organization. This hypothesis was reinforced by the fact that differentially expressed genes mapped to recombination “coldspots”, as a consequence of which clustered genes are collectively inherited. These findings suggest the functional interdependence of these closely linked genes. Our data are consistent with the hypothesis that this isolated cell cycle QTL does not result from a mutation in a single gene, but rather is a consequence of variable expression of a collection of highly linked genes.

Enhanced Repopulating Ability and Protection 322 from Aberrant Proliferation in Fancc -/- Hematopoietic Stem Cells Transduced with Recombinant Fancc

Comparison of Gene Expression Patterns of Erythroid 324 and Myeloid Differentiation from Human Multipotential Progenitor Cells

*L. Haneline1, X. Li2, P. Hong2, A. Orazi2, E. Srour2, D. Clapp2,

*L. Chen1, J. Zhang2, D. Tang2, E. Fibach3, Y. Shi2, G. Rodgers2,

1

Indiana University, Cancer Research Institute, U.S.A., 2Indiana University, U.S.A.

1

Fanconi anemia (FA) is a chromosomal instability disorder characterized by progressive bone marrow (BM) failure and increased incidence of myeloid leukemias. Previously, we used a murine model containing a disruption in the Fanconi anemia group C (Fancc) gene to show that Fancc -/- hematopoietic stem cells (HSC) have a profound defect in repopulating ability (Haneline et al, BLOOD 1999). The current aim was to determine whether recombinant Fancc would restore normal repopulating ability in Fancc -/- HSC. Two retroviral vectors were evaluated. One utilized the MFG backbone containing the FANCC cDNA (MFG-FAC) while the second utilized the MSCV backbone containing the Fancc cDNA (MSCV-Fancc). Three independent competitive repopulation studies determined that repopulating ability of Fancc -/- HSC transduced with either MFG-FAC or MSCV-Fancc was 3.5 fold higher than uncorrected Fancc -/- HSC, demonstrated by repopulating units/10^6 cells (4.2±1.3 vs.1.2±0.2, p<0.05), but not statistically different from Fancc +/+ HSC (5.4 ±1.3). Surprisingly, transplantation of uncorrected Fancc -/- cells resulted in ~30% of recipients (7 of 22) developing aberrant proliferation suggestive of clonal hematopoiesis demonstrated by increased chimerism and single Sca1+lin- cell proliferation assays. Furthermore, ~one-third of recipients in this group developed histologic evidence of myelodysplasia or BM failure. These observations were never detected in the 24 mice transplanted with Fancc -/- cells transduced with either MSCV-Fancc or MFG-FAC. These data have important implications for understanding the potential of gene transfer to offer genome protection to FA patients as well as potential untoward effects if ex vivo manipulated HSC are not transduced.

The mechanisms underlying lineage-specific differentiation of erythroid and myeloid cells at molecular level remains unclear. To gain insight into this process, differentiation of human bone marrow AC133+ cells seeded in methylcellolse or liquid medium was induced with EPO or GCSF for 14-day in primary culture, and continued with the alternate cytokine for another 14-day in secondary cultures. Analysis of primitive hematopoietic markers CD34 and Notch1 or lineage-specific markers EPO-R and CD13 by single-cell RT-PCR showed individual colonies of 2 to 16 cells co-expressed EPO-R and CD13 on either CD34-positive or CD34-negative cells induced by EPO or G-CSF during primary and secondary semisolid cultures. In situ hybridization with the same cell surface markers in population of cells confirmed the single cell data. Rapid analysis of gene expression (RAGE) technique was employed to examine the changes of the differentiation-dependent gene expression during erythroid and myeloid development in primary and secondary liquid cultures. Using 40 unique gene amplification primer sets we found 6 genes of interest, including nuclear differentiation antigen mRNA, transcription factor genes and growth differentiation factors, and 1 previously unidentified gene. Analysis using 200 unique primer sets is now underway. Co-expression of EPO-R and CD13 strongly suggests that different lineage-associated genes co-exist within the same cell prior to exclusive commitment to either erythroid or myeloid lineage, indicating that these two lineages originate from the same precursors. Discrete gene changes between erythroid and myeloid development in this culture system may reveal a mechanistic basis for lineage-specific differentiation..

National Institutes of Health(NIH),NIDDK, U.S.A., 2National Institutes of Health, U.S.A., 3Hadassah University Hospital, Israel

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Abstracts / Experimental Hematology 30 (2002) 37-146

Purified Hematopoietic Stem Cells Show a Major Shift 325 of Gene Expression Through Cell Cycle that Coincides with an Engraftment Nadir

Microarray Analysis Reveals that Upregulated 327 Anti-apoptositic andProliferative Molecules, and Downregulated Pro-apoptotic Gene Expression are Present

*J.-F. Lambert1, M. Liu2, N. V. Baskaran3, G. Colvin1, C. I. McAuliffe1, B. G. Forget3, S. M. Weissman3, P. J. Quesenberry1,

in Human Leukemic Cells Stimulated by TNF-alpha *R. Liu1, F. Fan2, X. Li2, B. Whitehouse2, K. Zuckerman2, 1Moffitt

1

Roger Williams Medical Center, U.S.A., 2Harvard University, U.S.A., 3Yale University, U.S.A. We have shown major fluctuations of engraftability during the first cell cycle of cytokine stimulated whole bone marrow cells in in vitro culture. Here we examined ~urified lineage negative, Rhodamine low Hoechst 33342 low (Lin-Rh lowHOlow) cultured in IL-3, IL-6, IL-11 , and SCF for 0 to 48 hours. A volume corresponding to 500 male cells at time 0 was transplanted in lethally irradiated BALB/c females competed with 250,000 female bone marrow cells. We observed two nadirs of engraftment at 36 and 48 hours, representing 27 and 19% of time 0 engraftment, respectively. Two peaks of engraftment were observed at 38 and 42 hours, representing 90% and 90% of time 0 control respectively. At time 0 and 48-hours, cDNA was prepared from the Lin- RhlowHolow cell cultures and lineage+ cells (control) using a betaine-DMSO PCR. A modified 3’-end gel based differential display of cDNA was used to evaluate the pattern of gene expression in a panel of 251 “stem cell” targets selected by comparison of Lin-RhlowHolow (time 0) with lineage+ cells. Individual PCR bands were quantified using a 0 to 9 scale and results were visually compared using color-coded matrices. Gene expression analyzed at time 0 and 48 hours showed a major shift from “stem cell genes” being highly expressed at time 0 and turned off at 48 hours, while “cell division” genes were turned on at 48 hours. These observations suggest gene expression shifts through cell cycle in relation to cell cycle related alteration of chromatin coverage. The engraftment defect is related to a major phenotypic change of the stem cell.

Cancer Center, USF, U.S.A., 2Moffitt Cancer Center, U.S.A.

Identification of leukemia inhibitory factor (LIF)326 responsive genes in embryonic stem (ES) cells bearing a Shp-2 deletion mutation using microarray analysis

Long-term chimerism from T-cell depleted allogeneic 328 one marrow transplantation with low-dose busulfan and CD40-CD154 blockade

*R. Chan, W. Shelley, J. McClintick, H. Edenberg, M. Yoder,

*J. Li, J. Gorechald, C. Larsen, E. Waller, Emory University, U.S.A.

We found that many human leukemia cell lines are TNF-alpha resistant or stimulated by TNF-alpha, but U937 cells are sensitive to TNF-alpha-induced apoptosis. To search possible survival factors and understand mechanisms for TNF-alpha-induced cell proliferation, we used cDNA microarrays to analyze differences between Mo7e or U937 cells undergoing stimulatory and inhibitory treatments. Six groups for each cell line were used: (1) controls; (2) TNF-alpha; (3) anti-RI agonistic antibody; (4) anti-RII agonistic antibody; (5) TNF-alpha plus NF-kappaB inhibitor; and (6) TNF-alpha plus NF-kappaB and P38 MAPK inhibitors. Affymetrix U95A chips, including 12,000 known human genes, are used. By setting up cutoff thresholds of fold-changes at two, we build up a huge database for TNF-alpha-related proliferative and apoptotic cell lines undergoing activation or inactivation of TNF receptors. Our results showed that: (A) TNFalpha or activation of RI induced 18-30 fold increases of p50 and p52 NF-kappaB transcripts, but no change of p65 NF-kappaB in both cell lines. (B) TNF-alpha affected expression of several hundred of genes with limited similarity between U937 and Mo7e cells. Among TNF-alpha-activated kappaB-dependent genes in Mo7e cells, only 10% (8/78) were detected in U937. However, 59% (25/42) of TNF-alpha-induced kappaB- and p38-dependent genes expressed in Mo7e cells were detected in U937 cells. (C) TNF-alpha induced some highly expressed-genes (versus controls) in Mo7e cells, but not in U937 cells. They include: GM-CSF (28fold), CR7-5 (32-fold), Diubiquitin (19-fold), Ig-rearranged gamma chain (42fold), actin bundling protein (24-fold), IL-1 receptor (19-fold), MAP-2 (15-fold), OX-40 surface antigen (20-fold). (D) Interestingly, TNF-alpha induced 3.2-fold increase of BCL-xl transcripts over controls in Mo7e, but reduce 2.7-fold in U937 cells. Furthermore, TNF-alpha induced 31-fold decrease of pro-apoptotic BAX transcripts in Mo7e cells, but no change in U937 cells. (E) After subtracting the number of genes expressed differentially in TNF-alpha-treated Mo7e versus U937 cells, there are still 28 genes with decreased expression and 33 with increased expression in Mo7e cells exposed to TNF-alpha as compared with those with activation of RI and RII. In summary, data indicate that downregulated proapoptotic molecules and upregulated anti-apoptotic or proliferative molecules most likely play important roles, in TNF-alpha-induced leukemia cell proliferation.

Indiana University, U.S.A. LIF supports proliferation and maintains the pluripotency of undifferentiated ES cells, suggesting that the signaling pathways stimulated by LIF are critical for self-renewal. LIF binds the heterodimeric LIF receptor-gp130 complex causing activation of the Jak kinases with recruitment and phosphorylation of Shp-2 and STAT3. Homozygous mutant (Shp-2-/-) ES cells bearing a targeted exon 3 deletion exhibit increased sensitivity to LIF and increased frequency of secondary embryoid body formation upon secondary plating, implicating increased self-renewal potential. Consistent with ES cells bearing a mutation at the Shp-2-binding tyrosine of gp130, we found that the enhanced self-renewal phenotype of the Shp-2-/- ES cells correlates with increased LIF-stimulated STAT3 phosphorylation. However, the molecules regulated by LIF and phosphoSTAT3 mediating self-renewal are unknown. To address this question, Shp2-/- ES cells were treated with PBS or LIF at 1000 u/mL for 45 minutes (the time required to maximally induce expression of c-fos, a known LIFresponsive gene in ES cells). Performed in triplicate, total cellular RNA was prepared, labeled, and hybridized to Affymetrix Murine Genome U74A GeneChips® and analyzed with MAS4 software. We identified 62 genes or expressed sequence tags (ESTs) whose expression is significantly modified by LIF stimulation (p<0.01, as determined by t test on the average difference value). Among the most significantly induced genes were suppressor of cytokine signaling-3 (SOCS-3), tristetraprolin, serine/threonine kinase PIM3, and gut-enriched Kruppel-like factor (GKLF). The induction of SOCS-3 and GKLF has been verified by Northern blot analysis. By comparing the 62 sequences against the hematopoietic stem cell database (SCDb) compiled by Lemischka and colleagues (http://stemcell.princeton.edu/, Science 288, 2000), we found 6 genes and 1 EST also represented in the SCDb. These data provide novel insight into the gene products that mediate ES cell and HSC self-renewal.

Background: Previous studies of allogeneic transplantation using low-dose total body irradiation conditioning and co-stimulatory blockade with anti-CD154 monoclonal antibody resulted in chimerism levels of 10%, 24%, and 48% after exposure to 50, 100, or 200 cGy, respectively. Donor T-cells were essential for allo-engraftment (Blood 2001 98:467). We hypothesized that prior exposure to donor alloantigen in the presence of MR1 would enhance host tolerance to the graft and permit transplantation with T-cell depleted allogeneic bone marrow (TCD-BM) without a risk of graft-versus-host disease (GVHD). Methods: B10BR mice were conditioned with low-dose busulfan (20mg/kg, ip) one day before transplant (d-1), and given MR1 (500 microg) iv on days -7, -6, -4, -2, 0, and +7. On day -6 mice received viable allogeneic donor splenocytes or splenocytes treated with graded doses of ionizing radiation (7.5 to 30 Gy). Mice received 2 X 10E7 TCD-BM on day 0 and were monitored for survival, chimerism, and GVHD post-transplant. Results: all transplant recipients survived and gained body-weight at rates comparable to non-transplanted littermates without clinical sign of GVHD. Recipients given MR1 and TCD-BM alone had 39% and 50% donor cells in the blood on day +30 and +120, respectively. Recipients given MR1 and transplanted with viable allogeneic splenocytes on day –6 in addition to TCD-BM on day 0 had 64% donor hematopoietic chimerism at day +30, and 83% chimerism on day +120 (p<0.01, vs TCD-BM alone). Donor spleen-derived cells did not contribute to chimerism, and recipients transplanted without MR1 treatment did not engraft.Exposure of the day -6 splenocytes to gamma irradiation resulted in a loss of donor chimerism proportional to the radiation dose, such that recipients of donor splenocytes irradiated to 7.5, 15, and 30 Gy had 31%, 16%, and 6% donor cells at day +120, respectively. Conclusions: MR1 as a single agent was sufficient to permit establishment of stable mixed chimerism after transplantation with TCDBM from fully mismatched donors. Mixed chimerism was enhanced by prior exposure to viable allogeneic splenocytes on day -6 and diminished by exposure to apoptotic donor splenocytes, suggesting activation of specific host-versus-graft mechanisms by apoptotic donor cells.

Abstracts / Experimental Hematology 30 (2002) 37-146

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Mixed Hematopoietic Chimerism -A New Strategy 329 for Preventing GVHD in Nonmyeloablative Allogeneic Stem Cell Transplantation

Specific elimination of alloreactive T lymphocytes 331 by TH9402 based photodynamic therapy *R. Terra , G. Krosl , A. Balassy , M. Barrette , J. Rooney , D. Roy ,

*H. Ai1, R. C. Zhao2, M. Guo1, C. Yu1, D. Wang1, J. Qiao1, Q. Sun1, W. Sun1, S. Zhang1, 1North Tai Ping Road Hospital, China,

1

2

1

1

2

1

1

1

Hôpital Maisonneuve-Rosemont, Canada, 2Celmed BioSciences Inc., Canada

PUMC&CAMS, China

Nonmyeloablative allogeneic stem cell transplantation (NST) promises broad clinical use of stem cells in hematological disorders and nonmalignant diseases. We conducted a human clinical trial to explore the role of donor-recipient hematopoietic mixed chimerism (MC) in the prevention of graft-versus-host disease (GVHD) after NST. Nonmyeloablative pretreatment was used for NST in 33 patients. All patients recovered from the hematopoietic suppression and achieved engraftment. There were 11 cases of FDC (specify FDC) and 22 cases of MC. Of the 33 cases, 7 (21.2%) developed acute GVHD (aGVHD). The incidence of aGVHD in MC (2/22, 9.1%) was significantly lower than that in FDC (5/11, 45.5%, P< 0.05). In addition, 7 cases (21.2%) developed chronic GVHD (cGVHD). The incidence of cGVHD was also significantly lower in MC (3/22, 13.6%)- than that in FDC (4/11, 36.4%, P < 0.05). All patients had evidence of engraftment of neutrophils and platelets. The median survival time of 9 patients in the FDC group (9/11, 81.8%) is 6 months, compared with 9 months in 16 patients in the MC group (16/22,72.7%). However, there were no significant differences between FDC and MC groups in terms of survival rate and recovery time of neutrophils and platelets (P> 0.05). Mixed chimerism formed initially after transplantation, and lasted 3-6 months after NST. This early stage mixed chimerism significantly reduced the incidences of both acute and chronic GVHD without compromising hematopoietic reconstitution and GVL effect.

Stem cell transplantation (SCT) is currently used for the treatment of a variety of neoplastic diseases. However, the primary obstacle limiting the efficacy of allogeneic SCT is the occurrence of graft-versus-host-disease (GVHD). The purpose of this study was to determine whether selective depletion of donor alloantigen-specific T lymphocytes using photodynamic cell therapy (PDCT) would prevent GVHD in the context of MHC-mismatched stem cell transplantation. This question was addressed in a MHC-incompatible mouse model of GVHD. The donor (C57BL/6; H-2b) derived spleen cells were first activated against C3H/HeJ (H-2k) host spleen cells in a one-way mixed lymphocyte culture and then exposed to photodynamic treatment, using dibromorhodamine methyl ester (TH9402) as a photosensitizer. Activated T cells showed preferential retention of this photosensitizer compared to resting lymphocytes. In addition, in vitro experiments revealed that PDCT eradicates significantly higher proportion of activated than resting T cells. When lethally irradiated C3H/HeJ mice were transplanted with C57BL/6 derived T cell-depleted bone marrow cells supplemented with C57BL/6 derived spleen cells activated with C3H/HeJ targets, they rapidly succumbed to acute GVHD (within 10-20 days). In contrast, mice that received histoincompatible T cells previously exposed to photodynamique treatment survival until the end of observation period (>100 days). Additionally, transplantation of treated T cells induced GVHD in several different C57BL/6 histoincompatible strains of mice (third party), suggesting PDT specifically eradicated activated T cells while sparing most resting T lymphocytes. Analysis of immune recovery indicated that T and B cell reconstitution in animals transplanted with treated primed cells was similar to that of mice transplanted with syngenic bone marrow. Moreover, treated T cells could induce GVHD against MHC-mismatched mice indicating that immune reactivity toward third party cells was preserved. These results demonstrate that PDCT can selectively eliminate alloreactive T cells and prevent the development of GVHD, while sparing T cells reactive against non-host antigens, thus offering protection against infection and disease relapse.

Characterization of alloreactive T-cell clones by CDR3330 size spectratyping in a murine GVHD model after transplantation over minor Histocompatibility antigen mismatches

Tacrolimus and Mycophenolate Mofetil for GVHD 332 prevention in Adult Hematopoietic Allograft Recipients *P. McSweeney , S. Abhyankar , B. Blunk , A. Baron , J. Foran ,

*K. Schilbach1, J. Pippir2, H. Fluhr2, K. Marquordt2, B. Schütt2, D. Niethammer2, M. Eyrich2, 1Children´s Hospital University of

J. McGuirk2, P. Hardiman3, S. Picken2, S. Pavletic3, 1University of

Tuebingen, Germany, 2University of Tuebingen, Germany Minor histocompatibility antigens (miHags) play prominent roles in allorecognition and are recognized by CTLs that mediate this process. Our study focuses on tissue specific T-cell responses to miHag-encoded peptides between MHC matched mice in GvHD-target organs over the first 30 days post-transplant. After lethal irradiation (2x500cGy) BALB/c (H2d) mice were transplanted with 1x107 B10/D2 (H2d) bone marrow cells and 1x108 spleen cells to induce clinically significant GvHD. Cohorts of 3 animals were sacrificed on day 3, 9, 14, 21 and 30 post-transplant. RNA was isolated from skin, liver, ileum, colon, spleen and heart as a control. Complementarity-determining region 3 size spectratyping was used to study the diversities of Vß- and Jß-usage of CTLs infiltrating recipient tissues. Two distict patterns of T-cell repertoire diversities could be identified: In skin a restricted Vß-usage in combination with all Jß-segments contrasted a complete Vß-repertoire in intestinal organs combined with a restricted Jß-usage. Interestingly, T-cell repertoire in the heart showed almost complete identity with intestinal CDR3-size pattern. Sequencing of CDR3-regions expressing Vß8.1 revealed persisting clones in skin from day 9 to 30. In intestinal organs and heart, identical sequences could be identified at the respective time points, however, no persistence of sequences over several time points could be observed. Additionally, identical CDR3 loops were identified for 5 different Vß8.1/Jß combinations in intestinum and heart but not in skin. In summary, in skin a limited number of alloreactive T cell clones mediate and maintain GVHD continiously, while in the intestinum a vast temporary expansion of different clones dominates the GVHD process at the respective time points. Together with the observed unequal Jß-usage these results suggests that differences in the number of expressed miHags or modes of antigen presentation may contribute to the distinct patterns of T-cell recruitment during the process of GvHD in skin and intestinum. The striking correspondance of CDR3-size patterns and sequences in heart and intestinum with a simultaneously different clinical relevance point to the importance of organ specific environment (cytokine production, expression of MHC and adhesion molecules) in the regulation of GvHD.

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Colorado, U.S.A., 2St Lukes Hospital, Kansas, U.S.A., 3University of Nebraska, U.S.A. Purpose. To evaluate the safety and potential efficacy of Tacrolimus and mycophenolate mofetil (MMF) for prevention of acute GVHD. Methods: Twenty-four patients with hematologic malignancies underwent conventional allografting for malignancy with TBI-based or busulfan-based conditioning regimens. Tacrolimus 0.03 mg/kg i.v. was given by continuous infusion and converted at a 4:1 ratio for oral therapy with tapering between 2 and 6 months. MMF 15 mg/kg b.i.d. was given intravenously, then orally, until day +28 for related donors and until day +40 with tapering to day +56 for unrelated donor transplants. Transplants using PBSC (n=22) or bone marrow (n=2) were from HLA-matched siblings (n=19) and from HLAmatched (n=3) or one-antigen mismatched (n=2) unrelated donors. The median age was 38 (range 19-57). Diagnoses included ALL (n=4), AML (n=7), CML (n=2), CLL (n=3), NHL (n=5) and myelodysplasia (n=3). Results. Follow-up is at 131 (range 12-270) days. With follow-up at >40 days in 17 patients and >100 days in 14 patients acute grades II-IV GVHD occurred in 4/17 (23.5%) and grades III-IV GVHD occurred in 1 (5.9%, CI 0.1 –27.3%) patient. Engraftment of neutrophils (ANC > 500) occurred at 10 (range 8-18) days. Day 100 transplant mortality was 4.3 % (CI:0.1-22%). Chronic GVHD occurred in 6/13 (46%) evaluable patients with > 100 days follow-up. Nineteen (79%) patients are surviving with three deaths from relapse one from regimen-related toxicity, and one from infection associated with GVHD. Conclusions. Preliminary data indicates a good safety profile using Tacrolimus-MMF for acute GVHD prevention and suggests rapid engraftment, limited severe organ toxicities, and a low incidence of severe acute GVHD. Accrual and pharmacokinetic studies are ongoing to further define efficacy, safety, and optimization the combination.

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Abstracts / Experimental Hematology 30 (2002) 37-146

The Role of G-Protein Signaling in Hemopoietic 333 Stem/Progenitor Cell Mobilisation *T. Papayannopoulou, G. Priestley, B. Nakamoto,

The B-Cell Lymphoma Protein (BCL10) forms a large 335 proapoptotic complex in the cytoplasm *A. Ali-Saleh, C. Vijayasarathy,

Washington, U.S.A.

Arab Emirates

Chemokine signaling through their G-protein-coupled receptors (GPCRs) is important for adhesion and directed migration of leukocytes to infla,mmatory sites, and for lymphocyte trafficking in vivo. The mechanistic effects of chemokine signaling are dependent on integrin activation and are delivered through pertussis toxin (Ptx)-sensitive Gi-protein signaling. To directly test the influence of G-protein signaling in vivo on the mobilization or redistribution of stem/progenitor cells, we treated mice with Ptx and assessed the impact of this treatment on the numbers and distribution of progenitor cells between bone marrow and the periphery. We. found that a single injection of Ptx (100 µg/mouse) elicits a leukocytosis with a peak at about 3 days and lasting more than 10 days. Both lymphocytes (all subsets) and granulocytes are increased. Further, a progressive increase in circulating CFU-C(BFU-E, CFU-GM, CFU-Meg, or CFUMix) and CFU-S is seen with a peak at days 4-5 and lasting for ~2 weeks. To test whether the ribosyl transferase activity of Ptx is necessary for this effect, we evaluated the in vivo responses of B oligomer, which is responsible for receptor binding and mitogenic activity of Ptx, but lack any enzymatic activity. B oligomer treatment had no impact on leukocyte or progenitor levels in blood, suggesting that the ADP-ribosylase activity of Ptx is responsible for progenitor cell mobilization. Cholera toxin depressed all circulating cells (WBC and CFU-C) for several days, suggesting that activation of Gs protein signaling, directlyor indirectly, leads to opposite results than the inhibition of Gj signaling. Furthermore, when Ptx was given together with G-CSF, a dramatic synergy was seen. Phenotypic and functional studies on Ptx-mobilized cells indicate changes, likely attributable to inhibition of Gj signaling: a reduction in α4 expression of the α4/kit+ subset in the bone marrow, whereas in the blood only the subset with the downregulated α4 is mobilized; also, Ptx-mobilized cells respond less to SDF-I-dependent effects (i.e., migration and actin polymerization) in vitro. Whether bone marrow stromal cells, including endothelial cells, are also influenced by Ptx treatment will await further studies. The mobilizing effect of Ptx in vivo is antithetical to the in vitro Ptxtreated hemopoietic progenitor cells, which home normally to BM, whereas the in vitro Ptx-treated lymphocytes fail to home to lymphoid organs. The data provide a novel example of mobilization through in vivo pharmacologic modulation of molecular signaling.

The Bcl10 gene was recently isolated from the breakpoint region of t(1;14)(p22;q32) in Mucosa-Associated Lymphoid Tissue (MALT) lymphomas. Overexpression of BCL10 in mammalian cell lines promotes apoptosis and suppresses malignant transformation of rat embryonic fibroblasts. In addition, truncated mutations of the BCL10 that abolish its proapoptotic function, have been reported to occur at high frequencies in a number of malignancies, including hepatocellular carcinoma and colorectal cancer.These findings imply a critical role for BCL10 in regulating cellular growth and development. However, the mechanism through which BCL10 induces apoptosis and inhibits cellular transformation has not been determined. We have found that BCL10 promotes apoptosis by inducing the release of cytochrome c from the mitochondrial intermembrane space. In addition, purified recombinant BCL10 was also able to potentiate cytochrome c release from isolated rat mitochondria only in the presence of cytosolic (S100) extracts, suggesting that the proapoptotic function of BCL10 requires additional, unidentified, cytoplasmic factor. Our results suggest the existence of a novel cytoplasmic protein complex containing BCL10 which signals cell death by promoting the release of cytochrome c from mitochondria. Consistent with this model, gel-filtration of HeLa S100 extracts showed that 1020% of the cellular BCL10 was fractionated as a complex with an apparent molecular weight of 800 kDa. Affinity purification of this complex from S100 extracts identified at least nine additional proteins that associated specifically with BCL10. Western blotting analyses have identified the Death Associated Protein 3 (DAP3), inhibitor of metastatic progression (nm23), and Apoptosis-Inducing Factor (AIF) as members of this complex. The affinity-purified complex also contained JNK1, a member of the MAP kinase family. Purified JNK1 was able to directly phosphorylate recombinant BCL10. We have found that threonine163 is the target phosphorylation site by JNK1. Interestingly, mutation of amino acid 163 of BCL10 did not only abolish its phosphorylation by JNK1, but also enhanced its proapoptotic activity when overexpressed in mammalian cells. Thus, identification of all the components of the BCL10 complex and characterization the role of their association with BCL10 will provide critical information needed to understand the function of BCL10 and how mutations in this protein contribute to lymphomagenesis.

Potential role of Grb2 in sequestering P27Kip1 334 in the nuclear compartment of BCR/ABL-cells *M. Dao, C. Verfaillie,

Mechanism(s) of Matrix Metalloproteinase-2 Activation 336 in Acute Myelogenous Leukemia L. Marquez , L. Ross , N. Shrivaikar , L. Larratt , A. Turner ,

University of

University of Minnesota, U.S.A.

United Arab Emirates Univ., United

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*A. Janowska-Wieczorek2, 1Canadian Blood Services, Canada, 2

Purpose: By promoting the interaction between SOS and Grb2-SH3 domain, p210 bcr/abl indirectly activates Ras/MAPK signaling cascade. Interestingly, Grb2-SH3 domain can also interact with the proline rich region of a cyclin-dependent kinase inhibitor, p27Kip1, implicating an alternate route through which Grb2 might modulate cell cycle progression. Jiang et al. has previously reported an imbalance in the subcellular distribution of p27Kip1 in primary CML progenitors as well as an increase in the kinase activity of cyclinE/cdk2. Here, we investigate the possible interaction between p27Kip1 and Grb2 in MO7e/p210 cells and its impact on cell cycle control. Methods: MO7e cells were transduced with an amphotropic retroviral vector pMSCV-p210bcr/abl-IRES-eGFP and sorted by FACS. Prior to stimulation with serum or growth factor(s), cells were starved for 24 hours in serum-free medium with 300uM acyclovir or 2 ug/ml aphidicolin. Ki67/7AAD staining was done to compare the cell cycle kinetics of MO7e vs. MO7e/p210 cells. Cells were collected every 4 hour over a 24-hour period and subjected to subcellular fractionation to follow the subcellular localization kinetic of p27Kip1 protein. Immunoprecipitation experiments using antibodies against p27Kip1, Grb2, SOS, and cyclinE were done to determine their potential interactions in the presence of p210 bcr/abl. To identify the different complexes found between nuclear versus cytoplasmic compartment, cells were subfractionated prior to immunoprecipitation assays. Results: Differences in p27Kip1 subcellular localization correlated with differences in cell cycle kinetics between MO7e and MO7e/p210 cells. While there were no significant changes in protein levels of p27Kip1,SOS, and cyclinE, the level of tyrosine phosphorylated Grb2 was higher in MO7e/p210 cells. Cytoplasmic Grb2/SOS complexes were readily detectable in MO7e/p210 cells, in comparison to MO7e cells. Nuclear Grb2/p27 complexes were more prominent in MO7e/p210 cells than MO7e cells. Conclusions: Based on these observations, we conclude that Grb2 exists in both cytoplasmic and nuclear compartments of hematopoietic cells. In MO7e/p210 cells, the presence of Grb2/SOS in the cytoplasm corresponds with its role in enhancing Ras/MAPK pathway. The detection of Grb2/p27Kip1 in the nucleus suggests the possible role of Grb2 in sequestering p27Kip1 in a complex, thus preventing p27 from binding and inhibiting the kinase activity of cyclinE/cdk2.

University of Alberta, Canada

Matrix metalloproteinases (MMPs) belong to a family of zinc-dependent endopeptidases and are known to be involved in tumor progression, metastasis and angiogenesis. We have recently suggested that MMP-2 is implicated in leukemic dissemination (B J Haematol 1999, 2001). MMP-2 is secreted in latent form and mechanisms of its activation involve the formation of a ternary complex of pro-MMP-2, tissue inhibitor of metalloproteinase-2 (TIMP-2) and membrane-type MMPs (MT-MMPs). To elucidate the mechanism of MMP-2 activation in acute myelogenous leukemia (AML) we examined the expression of MMP-2 and the six MTMMPs identified to date, as well as TIMP-2, in mononuclear cells (MNC) obtained from 20 patients newly diagnosed with AML, four with myelodysplastic syndrome (MDS) and one AML patient in remission. MMP2 secretion was analyzed by zymography and MMP-2, TIMP-2 and MTMMP expression by RT-PCR. We found that 95% of AML samples (from 19/20 patients) expressed transcripts for MMP-2, 80% for MT1-MMP and MT2-MMP, 5% for MT3-MMP, 100% for MT4-MMP, 85% for MT5-MMP, 40% for MT6-MMP and 100% for TIMP-2, suggesting that MT-MMPs, especially MT1-, MT2- , MT4- and MT5-MMPs, have a role in MMP-2 activation in AML. Interestingly, in patients diagnosed with AML in remission and RA MDS, MMP-2 and MT1-MMP were not expressed. However, despite expression of MT-MMPs and TIMP-2, no active forms of MMP-2 were detected in serum-free media conditioned by AML blasts. Hence to mimic the bone marrow microenvironment we co-cultured AML blasts with stromal cells (fibroblastic, HUVEC) and found active MMP-2 in media from these co-cultures. We also found that bone marrow fibroblastic cells and HUVEC express transcripts for all six MT-MMPs. In conclusion, we suggest that stromal cell MT-MMPs may provide a necessary cell-surface anchor for a proteolytic cascade involving MMP-2 in AML, and could contribute to leukemic dissemination.

Abstracts / Experimental Hematology 30 (2002) 37-146

Cleavage of FLIPlong and Caspase-8 Are Associated 337 with CD44-Mediated Induction of Apoptosis in CD4 Memory Cells J. Panse, U. Platzbecker, T. Gooley, E. Santos, H. Deeg, *B. Sandmaier, Fred Hutchinson Cancer Research Center, U.S.A.

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The Role of flt3 and EPO Receptor Expressions on CD34+ 339 Hematopoietic Stem Cell Differentiation and Proliferation *J. Xu , H.-N. Hao ,

1 2 1 Shandong Provincial Hospital, China, 2Wayne State University, U.S.A.

Mab S5, which recognizes the adhesion molecule CD44, enhanced engraftment in canine myeloablative and nonmyeloablative MHCmismatched hematopoietic transplants. S5 induced apoptosis in human and canine PBMC and lymphocyte subsets. Other CD44 Mabs tested failed to enhance engraftment, and had comparatively modest effects on apoptosis. S5 also up-regulated Fas on T-cells, while other CD44 Mabs did not. CD4 cells showed the highest apoptosis rates after incubation with S5. CD4 memory cells were known to be relatively resistant to Fas-induced apoptosis due to expression of FLIP. We therefore studied the potential of S5 and other CD44 Mabs to induce apoptosis in CD4 memory cells (which express high levels of CD44) by AnnexinV/propidium iodide staining and analyzed the expression of FLIP and Caspase-8 by Western Blot. CD3+/CD4+/CD45RA- (memory) cells were isolated from human PBMC (n=7) by FACS sorting and incubated with 10 ug/ml of CD44 Mabs S5, Hermes-1, IM7, irrelevant control Mab or medium alone. Counterstaining of sorted cells confirmed high CD44 expression. All three CD44 Mabs induced apoptosis after 16 hrs, however, S5 induced significantly higher rates than other CD44 Mabs (p<0.04). Incubation of CD4 memory cells with S5 led to cleavage of FLIP(long) and Caspase-8 after 16 hrs and accumulation of FLIP(short). Relative resistance to apoptosis induced by Fas-activating Mab CH-11 in CD4 memory cells was characterized by consumption of FLIP. While CH-11 induced apoptosis of CD4 memory cells could be blocked by Caspase-8 inhibition, Caspase-8 inhibition did not prevent S5 induced apoptosis. These data suggested that CD44 mediated apoptosis in CD4 memory cells circumvented early cellular anti-apoptotic mechanisms. How CD4 memory cells contribute to rejection in MHC-mismatched marrow transplantation warrants further investigation.

The mechanism of that human fetal liver (FL) hematopoietic progenitor cells (HPCs) have higher yield erythropoiesis potential than cord blood (CB) HPCs have remains unclear. We hypothesize that flt3 receptor (flt3R) and erythropoietin receptor (EPOR) expressions may play important roles for HPC differentiation and proliferation. In the present study, we investigated the flt3R/EPOR expression on both CB- and FL-CD34+ cells using RT-PCR, immunoblot and flow cytometry with or without anti-flt3R or antiEPOR mRNA antisense treatment. We also estimated whether the colony formation potential of CB- and FL-CD34+ cells in methylcellulose culture influences by this antisense treatment. These results indicate that the EPOR expression on FL-CD34+ cells was 4 folds higher than that on CB-CD34+ cells, and both flt3R and EPOR expressions were response to their mRNA antisenses treatment. After 15 days in methycellulose culture, the numbers of CFU-GM were significant decreased in both CB and FL HPCs that were pretreated with antisense of flt3R. However, BFU-E formation in both CB and FL isolated cells was not influenced by flt3R mRNA antisense treatment. In contrast, EPOR antisense treated HPCs had less potential to generate BFU-E than non-treated HPCs had. In addition, the similar results were also shown in the flt3 or EPO ligand free methycellulose cultures. These observations suggest that flt3R and EPOR expressions are necessary in the augmentation of CFU-GM or BFU-E formations from both CB and FL HPCs.

GATA-transcription in a small Rho123 low, CD34+ 338 subpopulation of peripheral blood derived CD34- CD105+ adult stem cell lines

Isolation of a Mast Cell Progenitor Dependent Upon SCF 340 and IL-3: A Model of Stem Cell Signalling *I. McNiece, J. Brunetti, Z. Dai,

*C. Conrad1, B. Gottgens2, S. Kinston2, R. Huss1, 1University of

Sciences Center, U.S.A.

University of Colorado, Health

Munich, Germany, 2University of Cambridge, U.K. Purpose. We intended to isolate CD34- stem cell lines from human peripheral blood cells and obtain evidence for their multi-potency and plasticity. Materials and Methods. Adherent growing cells were isolated from PBMC and different cell clones were established after immortalization. The immunophenotype of the cell lines was investigated by flowcytometry. Cell clons were stained with Rhodamine 123, and the Rh123 low and the Rh123 high subpopulation was sorted for a RT-PCR survey of different transcription factors and distinct differences in morphology. Results. The peripheral blood-derived and fibroblast-like cell line V54/2 expressed high levels of CD10 and CD105 and showed only a very low level expression of CD34 (< 1.0 %) and CD117 (c-kit). Among the entire CD34-, CD105+ cell population that transcribed factors such as Myb, Tie-1 and VEGF, there was a small Rhodamine 123 low and CD34+ subpopulation, which transcribed significant levels of the GATA family. The morphology of the Rh123 low, CD34+ (also expressing the p-gp) was different as compared to the Rh123 high , CD34- population. Conclusion. The findings provide evidence that it is possible to isolate CD34- , CD105+ mesenchymal stem cell lines from the human peripheral blood cells which contain a small subpopulation of CD34+ and GATA-transcribing cells. Those cells are potential hematopoietic progenitors and can be recruited from the CD34- stem cell pool. The plasticity of stem cells seem to require essential molecular tools such as a panel of transcription factors to respond to the environmental demand within a biological system.

Hematopoietic stem cells (HSC) require two or more growth factors (CFs)for survival and proliferation. However, the mechanism of synergy is unknown. Recently we have isolated a cell population from mouse BM that has been grown continuously for more than 9 months and requires the combination of SCF plus IL-3 for survival and proliferation. These cells (MCS3) represent a mast cell progenitor with many granules staining intensely with toluidine blue and positive for both alcian blue and safranin. IL-4 alone has minimal stimulatory effect on MCS3 cells but IL-4 synergizes with either SCF or IL-3. Other cytokines, including GM-CSF, G-CSF, IL7, IL-2, IL-6, IL-l 1 , Flt-3L and LIF had no stimulatory effect alone or in combination with either SCF or IL-3. Sequential addition experiments demonstrate the requirement for both SCF and IL-3 at initiation of culture suggesting that the synergistic interaction of these factors is not through receptor upregulation. We are presently preparing molecular array chips to compare the signalling molecules in MCS3 cells compared to mast cells stimulated by SCF or IL-3 alone. Signalling intermediaries identified in these experiments will be evaluated in highly purified HSC. In summary, the MCS3 cells provide a model cell population to study the signalling mechanism of synergy between growth factors and may provide insights into growth factor signalling in HSC.

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One third of mice transplanted with single rhodamine123341 negative SP cells isolated from adult mouse bone marrow produce multilineage populations of blood cells for >6 months

Rhodamine123 exclusion identifies immature 343 hematopoietic progenitors after short-term culture M. von Planta , D. Suva , M. Duchosal , B. Chapuis , *V. Kindler ,

*N. Uchida, F. Leung, C. Eaves, BC Cancer Agency, Canada

1

Geneva University Hospital, Switzerland, 2Lausanne University Hospital, Switzerland

Hematopoietic stem cells (HSC) exhibit a Side Population (SP) phenotype based on their common verapamil-sensitive ability to efflux Hoechst 33342. However, this phenotype is also shared by a number of mature hematopoietic cell types including erythroid precursors and NK cells. In initial studies we found 0.2% (n=5) of adult mouse bone marrow (BM) cells to be SP cells, of which only 50% were lin- and only 2±1% were both lin- and also able to efflux Rhodamine123 (Rh). When 10 lin- Rh- SP cells were injected into each of 10 sublethally irradiated W41/W41 mice, 9 of the recipients showed multilineage blood cell regeneration from the injected cells for >6 months (5 - 66% of all the WBC derived from the 10 cells injected). Another 39 W41/W41 mice were transplanted with visually confirmed single cells and 13 of these also showed multilineage donor-derived WBC for >6 months (at levels ranging from 2% to 68%, with levels in 6 of the 13 mice at >40%). Limiting dilution assays performed on Rh± and Rh+ lin- SP cells showed that 89% of the longterm multilineage repopulating cells isolated were in the Rh- subset of the lin- SP cells with the remainder in the Rh± subset. These findings suggest that regulation of ABC transporter expression is tightly regulated during the earliest stages of hematopoietic stem cell differentiation with down-regulation of MDR preceding the down-regulation of ABCG2. They also suggest an important generic approach to obtaining highly purified stem cell populations from other tissues and species.

Freshly isolated immature hematopoietic progenitor cells (HPC) can be identified by their ability to exclude dyes such as rhodamine123 (rho) via the multidrug resistance (MDR) gene product. It is however not clear whether, after culture, this characteristics remains. To address this issue, we evaluated the frequency of late (CFU-C) and early (LTC-IC) HPC recovered from human CD34+ cells cultured for 5-7 days with thrombopoietin, in presence or absence of serum. These data were compared to those obtained with the rho exclusion assay, using the MDR-blocking drug verapamil to identify rholow and rho50 cells, that are enriched, ex vivo, in late and immature HPC respectively. CFU-C and rholow cell frequencies were marginally altered by the culture whereas LTC-IC frequencies decreased by 1.7- and 17-fold, and rho50 cell frequencies by 5- and 20-fold with and without serum respectively. In addition, when very immature CD34+CD38- and more mature CD34+CD38+ HPC were cultured in similar conditions, CFU-C and rholow cells were identical in both fractions, while preCFU were 40 ± 21-fold, and rho50 cells were 9 ± 7 fold higher in the CD34+CD38cultures (mean ± SD of 2 experiments). Altogether these data show that the modulation of rho50 cells was close to that of intermediate (preCFU) or immature (LTC-IC) HPC. This suggests that cultured rho50 cells are still enriched in immature HPC and that the rho assay may be regarded as an alternative mean to identify immature HPC in culture.

Phenotypic and genetic analysis of stem cell number and 342 mobilization potential in genetically distinct mouse strains E. J. Noach , A. Ausema , I. Akkerman , S. Koopal , E. Weersing ,

CD34 is a Marker of Mature Murine Mast Cells 344 *K. McNagny, E. Drew, H. Merkens, S. Chelliah, R. Doyonnas,

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E. Dontje2, *P. Wierenga2, E. Vellenga3, G. de Haan2, 1University of Groningen, Faculty of Medicine, The Netherlands, 2University of Groningen, The Netherlands, 3University Hospital Groningen, The Netherlands Genetically distinct mouse strains vary considerably in hematopoietic stem cell number. Specifically, AKR/J (AKR) mice have more stem cells that are mobilized more easily to peripheral blood than C57BL/6 (B6) mice. We aim to identify underlying mechanisms associated with these traits and started detailed phenotypic and genetic analyses of AKR and B6 stem cells. The frequency of primitive Lin-Sca-1+c-kit+ cells was identical in both strains. However, CAFC activity of single Lin-Sca-1+c-kit+ cells was 2-fold higher in AKR than in B6. Kinetics of peripheral blood reconstitution after transplantation of 1500 Lin-Sca-1+c-kit+ into lethally irradiated recipients was twice as fast in AKR mice compared to B6. In addition, two-fold more progenitors mobilized to peripheral blood after G-CSF challenge in AKR mice. Thus, AKR-Lin-Sca-1+c-kit+ cells have superior stem cell characteristics compared to B6 cells. We performed genetic linkage analysis to identify loci regulating mobilization potential using 57 polymorphic DNA markers. To this end, F2 AKRxB6 intercross mice were generated and treated with G-CSF. Mobilization potential varied widely and continuously, with values highly exceeding those of both parental strains, suggesting that multiple loci contributed to this trait. We found significant linkage for a locus on chromosome 3 and a putative locus on chromosome 15. B6-alleles at D3Mit19 and AKR-alleles at D15Mit241 correlated with increased mobilization response. Mice homozygous for B6-alleles at D3Mit19 and AKR-alleles at D15Mit241 had ~2-fold more circulating progenitors compared to animals carrying the opposite alleles at both loci. Currently, we are narrowing the regions in which potential candidate genes are located

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University of British Columbia, Canada

Objective: CD34 is a 90-120 kDa cell surface sialomucin that is widely used for the enrichment of human and murine hematopoietic stem cells (HSCs) because of its selective expression on progenitor cells and absence on mature hematopoietic cells. Recently we have found that CD34 is the prototypic member of a family of three proteins with similar structure and gene organisation. In light of this observation, we are further examining the distribution of CD34 family members in the mouse. Methods: Hematopoietic cell lines and primary tissues were evaluated for CD34 mRNA expression by Northern blot and protein expression by cell surface immunofluorescence. To confirm specific reactivity of the CD34 antibody, cells from CD34-deficient mice were used as controls. Results: Although CD34 mRNA was undetectable in all murine progenitor cell lines tested, high level expression was detected for bone marrow-derived mast cells (BMMCs). Likewise, cell surface immunofluorescence confirmed that CD34 is expressed by BMMCs and by in vivo peritoneal mast cells. No protein expression was observed for CD34-deficient mast cells. In addition, our data show that mast cells highly express the stem cell antigen, Sca-1, and the well-known stem cell and mast cell antigen, c-kit. Conclusion: Our results demonstrate that, contrary to current dogma, CD34 is expressed by one mature hematopoietic lineage: mast cells. Our data also demonstrate that antigenically, murine mast cells, and their precursors, closely resemble HSCs and suggest extreme care should be used in the phenotypic characterisation of HSC’’s to prevent mast cell contamination of stem cell preparations.

Abstracts / Experimental Hematology 30 (2002) 37-146

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Mobilized Peripheral Blood (MPB) as well as Bone 345 Marrow (BM) can be used to generate neuron-like cells in vitro

Thioredoxin overexpression mouse is less sensitive 347 to benzene hematotoxicity and leukemogenicity *Y. Hirabayashi , B. Yoon , Y. Kawasaki , J. Yodoi , T. Inoue ,

T. Tondreau, L. Lagneaux, A. Delforge, *D. Bron, Institut J. Bordet,

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Belgium

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National Institute of Health Sciences, Japan, 2Kyoto University, Japan

Introduction : MPB and BM contain both mesenchymal stem cells (MSC) that are able to self-renew, proliferate and differentiate into different cell lineages of the tissue in which they reside. We can also find MSC in many other tissues such as skin, intestine, muscle and recently in the brain. Materials and methods: In this study, we have evaluated different enrichment methods to enrich MSC from BM: RosetteSep, MACS methods using CD45 and GlyA coated beads and plastic adhesion. Their plasticity have been determined by their ability to differentiate in vitro into adipocytes, osteocytes, chondrocytes and neuron-like cells. Cultures BM derived MSC were performed in complete alpha-MEM medium while MPB derived MSC required bFGF for their growth. MSC were evaluated after direct/indirect immunofluorescence labelling using a mixture of antibodies: CD14, CD33, CD45, CD105, SH3 and Stro-1. Lipid vacuoles of adipocytes, mineral deposits of osteocytes and chondrocytes were determined after Oil Red O, Von Kossa and toluidin blue staining respectively. Neuronelike cells were detected after indirect immunofluorescence with beta III tubulin and TUJ-1 antibodies. Results: 0,6±0,2% and 2,9±0,9% of cells were obtained after RosetteSep and MACS methods from BM respectively . After four passages, we obtained respectively more than 84,5% and 74,7% of cells expressing the SH3 mesenchymal marker for subcultured BM and MPB. Concomitantly, these cells were differentiated into adipocytes, osteocytes, chondrocytes and neuronelike cells using adequate induction media. Conclusion: BM and MPB represent two easily accessible sources of MSC which could have therapeutic potential in various medical disorders.

Thioredoxin/adult-T cell leukemia derived factor (Trx/ADF) has been known to involve in the cellular defense mechanism for oxidative damage via regulation of intracellular redox status. Recently, we reported that hemopoietic stem cells carrying Trx/ADF transgene were resistant for cell damages from oxidative stress inducers, not only exposure to ultraviolet light, but also to paraquat or TCDD. In the present study, in association with a role of oxidative stress, we speculated a protective function of Trx/ADF against benzene-induced hematotoxicity using ADF-wild type and over-expressed transgenic (Tg-) mice. The mice were inhaled with 300 ppm of benzene 6 hrs/day, 5 days/wk for 2 weeks and then the blood counts and the marrow cellularity were measured; an in vitro colony for CFU-GMs were assayed as well. The expression of human-Trx transgene in bone marrow (BM) cells was analyzed by western blotting in ADF-wild and Tg-mice. After benzene exposure, significant decreases were observed in the peripheral blood counts, the BM cellularity, and number of CFUGMs/femurs in both ADF-wild type and the hetero-KO mice. On the contrary, benzene-induced hematotoxicity was considerably attenuated in Tg mice carrying Trx/ADF transgene. After 26 weeks inhalation of benzene, 30% of ADF-wild type mice died from lymphoma/leukemia by 42 week after starting benzene inhalation, whereas none of Tg-mice died by the time. Our results strongly suggest that the production of oxidativestress that results in disruption of redox regulation is an important mechanism in hematotoxicity triggered by benzene.

Activation of telomerase in bone marrow cells after 346 thymectomy of adult mice *T. Todria , A. Zander ,

Aryl hydrocarbon receptor (AhR) mediates benzene348 induced hematotoxicity *B.-i. Yoon , Y. Hirabayashi , J. Kanno , Y. Fujii-Kuriyama ,

1 2 1 Universitats Krankenhaus Eppen, Russian Federation, 2Universitats Krankenhaus Eppen, Germany

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T. Inoue1, 1National Institute of Health Sciences, Japan, 2Tohoku University, Japan

PURPOSE: BM from neonatally and A.Tx mice has an impaired radioprotective effect upon inoculation into lethally irradiated recipients due to decrease of HSC proliferative potential. The replicative cell senescence (telomere length) could be the cause of such decrease. As telomere length is difficult to measure in mice model we set out to check telomerase activity (TA). OBJECTIVES: Telomerase activity (TA) and cell cycle status of bone marrow (BM) cells of normal (N) and adult thymectomized (A.Tx) mice were studied during life-time in either steady state haematopoiesis or after transplantation of BM. METHODS: Female mice thymectomized at 4 weeks age were used as A.Tx. Lethally irradiated mice were reconstituted with BM of either N or A.Tx mice (12 months after surgery). Normal mice were of the same age as A.Tx mice. TA was determined by PCR-ELISA method. Cell cycle status was analysed by flow cytometry on FACScan. RESULTS: Low level of TA (2-5%) was detected in BM of normal mice in steady state haematopoiesis and in chimeras, reconstituted with BM of normal mice. Agedependent correlation of TA was not observed. However telomerase is expressed much more in mice after thymectomy. TA was 3 times higher (719%) in BM cells from either A.Tx donors or A.Tx BM reconstituted chimeras than in non-thymectomized mice. The number of cycling BM cells were the same in all four models. CONCLUSION: These data suggest that under steady-state conditions (natural ageing) mouse haemopoietic cells express telomerase over their life and the level of TA is not associated with thymus involution. However the alteration of telomerase dynamics, namely its activation, are detected in BM cells with a nearly complete block in T cell development: in both A.Tx mice and A.Tx reconstituted chimeras. These new findings suggest unexpected influence of T-cell depletion on the telomerase expression in mouse haemopoietic cells. The immunological status of animals became one of the factors of regulation of telomerase activity.

Benzene is a ubiquitous environmental contaminant that can induce hemopoietic toxicity and leukemia in human and mice. The phenolic metabolism of benzene by CYP2E1 enzyme in the liver is believed to be prerequisite for its cyto- and genotoxicity, yet the toxic mechanism of benzene is not fully understood. In the present study, we investigated the involvement of the aryl hydrocarbon receptor (AhR), a ligand-activated basic helix-loop-helix (bHLH) transcription factor, in the hematotoxicity using AhR wild-type (AhR+/+), heterozygous (AhR+/-) and homozygous (AhR-/-) male mice. Following a 2-week inhalation of benzene at 300 ppm, we have evaluated to changes in cellularity of the peripheral blood and the bone marrow (BM) and the levels of granulocyte-macrophage colonyforming units in the BM. The expressions of cyclin-dependent kinase inhibitor, p21, in the BM cells and CYP2E1 in the hepatic tissues respectively were evaluated by western blot analysis after benzene exposure. Our results clearly showed that AhR(-/-) mice were much more resistant to benzeneinduced hematotoxicity than AhR(+/+) wild-type mice. The finding that no change in p21 expression by BM cells could be detected in AhR(-/-) mice; whereas marked up regulation of p21 expression by BM cells was documented in AhR(+/+) mice, is a further indication of the resistance of AhR(-/-) mice to benzene hematotoxicity. The benzene resistance of AhR(-/) mice was abrogated by exposure to a combination of two major metabolites, phenol and hydroquinone, strongly supporting the concept that the AhR participates the metabolic pathway of benzene. Responsible metabolic CYPspecies are under investigation.

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Abstracts / Experimental Hematology 30 (2002) 37-146

Ex vivo amplification of human hematopoietic stem cells 349 by using passive transduction of HOXB4 homeoprotein *S. Amsellem , F. Pflumio , D. Bardinet , P. Charneau , A. Dubart-

Differential expression of alpha2 integrin separates 351 long-term and short-term reconstituting Lin(-)Thy1.1(lo)ckit(+)Sca-1(+) hematopoietic stem cells

Kupperschmitt2, S. Fichelson2, 1Institut Cochin - INSERM, France,

*A. Wagers1, I. Weissman2, 1Stanford University School of Medicine, U.S.A., 2Stanford University, U.S.A.

1

2

2

2

3

Institut Cochin, France, 3Institut Pasteur, France

Therapeutic protocols would be greatly improved by the availability of large numbers of hematopoietic stem cells (HSCs). The ordinary ex vivo expansion techniques for human HSCs generally involve the use of cytokine mixtures that commonly leads to cell commitment and differentiation. This is the reason why the development of alternative approaches to ex vivo expand undifferentiated and unmodified HSCs represents a crucial goal. HOXB4 homeoprotein is a particularly attractive candidate for HSC amplification since Sauvageau et al. reported that retrovirus-mediated over-expression of the HoxB4 gene in murine hematopoietic cells preferentially amplified the most primitive cell populations. Peripheral blood cell populations remained balanced and no in vivo leukemogenic process was observed, even in long-term experiments. We have developed a model of non-viral transient transduction of the human HOXB4 protein (hHOXB4) into HSCs, which relies (i) on the properties of homeodomaincontaining proteins to spontaneously pass through cell membranes and, (ii) on the capacity of stromal cells to support human hematopoiesis in long-term cultures. Murine stromal MS-5 cells have been transduced by the hHoxB4 cDNA preceded or not by a signal peptide coding sequence to favor externalization from producing cells. We show that (i) HOXB4 protein was found in the supernatant of MS-5 transduced by peptide signal-containing constructs and (ii) HOXB4 produced by MS-5 cells could be detected in both the nucleus and cytoplasm of human CD34+ cells after co-cultures with MS-5 cells containing HOXB4/signal peptide constructs. Human CD34+ or CD34+/CD38low cells were long-term co-cultured with hHOXB4producing MS-5 cells. We reproducibly observed a 2-6-fold increase in the number of clonogenic progenitors after 2-5 weeks of co-culture compared to co-cultures performed with MS-5 cells either non-transduced or transduced by an EGFP-coding control vector. Limiting dilution assays in extended long-term cultures showed that this increase was related to an amplification of more immature LTC-ICs in the presence of HOXB4. Finally, the functionality of HOXB4-amplified human HSCs is currently tested in xenogenic transplantations assays, using NOD-SCID immunodeficient mice. The present work provides a new approach to ensure ex vivo expansion of human immature HSCs without any genetic modification of these cells.

Self-renewing, multipotent hematopoietic stem cells are highly enriched within the Lin(-)Thy1.1(lo)c-kit(+)Sca-1(+) subset of normal murine bone marrow. However, heterogeneous expression within this population of certain cell surface markers raises the possibility that it may be further fractionated phenotypically, and perhaps functionally. We previously identified alpha2 integrin (CD49b) as a surface marker with heterogeneous expression on Lin(-)Thy1.1(lo)c-kit(+)Sca-1(+) stem cells. To determine if differences in alpha2 expression were indicative of differences in stem cell function, we purified alpha2(-) and alpha2(hi) stem cells by fluorescence activated cell sorting, and analyzed their function in long- and short-term hematopoietic reconstitution assays. Both alpha2(-) and alpha2(hi) cells could give rise to mature lymphoid and myeloid cells following transplantation into lethally irradiated, congenic recipients. However, alpha2(hi) cells supported hematopoiesis only for a short time (<4 weeks), while alpha2(-) cells reproducibly yielded robust, long-term (>20 weeks) reconstitution, suggesting that alpha2(-) cells represent a more primitive population than alpha2(hi) cells. Consistent with this idea, alpha2(-)Lin(-) Thy1.1(lo)c-kit(+)Sca-1(+) cells exhibited an ~6-fold decreased frequency of spleen colony forming units (day 12) versus alpha2(hi) cells. Furthermore, bone marrow cells isolated from animals transplanted >20 weeks previously with 20 alpha2(-)Lin(-)Thy1.1(lo)c-kit(+)Sca-1(+) cells included both alpha2(-) and alpha2(hi) stem cells of donor origin, indicating that alpha2(hi) cells are likely lineal descendents of alpha2(-) cells. Interestingly, alpha2 integrin expression is significantly reduced on lineage-restricted oligopotent progenitors in the marrow, suggesting that high level expression of alpha2 selectively marks a subset of primitive hematopoietic cells that retains multilineage reconstitution potential but exhibits reduced self-renewal capacity.

Ex Vivo Expansion of CD34+ Stem Cells in 350 HSCEM/Stemline(TM) Medium Leads to Increased Levels of Total Nucleated Cells and CD34+ Cells

Constitutive Circulation of HSC in Fetal Blood 352 *J. Christensen, D. Wright, I. Weissman,

Stanford University, U.S.A.

*F. Swartzwelder1, J. D. Tario, Jr.1, D. W. Allison2, S. L. Leugers2, H. N. Loke2, L. M. Donahue2, J. A. Harrington3, I. K. McNiece3, 1

Stemgenix, U.S.A., 2Sigma-Aldrich, U.S.A., 3Univ. of Colorado, U.S.A.

Purpose: In recent years, human hematopoietic stem cells (HSC) have become a valuable resource for the repopulation of the hematopoietic system following high-dose chemotherapy. HSC can be purified from several sources, most prominently bone marrow, peripheral blood and umbilical cord blood. In many cases, the cell number obtained after purification is not sufficient for transplant and must be increased utilizing ex vivo expansion. A successful expansion must provide enough fully functional material to combat neutropenia and thrombocytopenia in the early stages post-transplant and lead to long-term engraftment of the patient. To this end, we have co-developed a serum-free, animal protein-free medium (marketed as Hematopoietic Stem Cell Expansion Medium [HSCEM], Stemgenix, Amherst, NY and as Stemline, Sigma-Aldrich, St. Louis, MO) for the optimal expansion of HSC. Methods: Our product was compared with other commercially available serumfree expansion media for the ability to expand total nucleated cells (TNC) and CD34+ cells in a 24-well microplate culture system and in a 2-step, clinical-scale protocol using Teflon® culture bags. Clinical scale cultures were also assayed for presence of committed progenitors (GM-CFC) and primitive, high proliferative potential progenitors (HPP-CFC). Results: In the microplate culture system, use of HSCEM/Stemline, when compared to other serum-free media, provides a significantly increased expansion of TNC from cultures of CD34+ cord blood cells, bone marrow and mobilized peripheral blood. Flow cytometric data indicates an increased specific expansion of CD34+ cells and clinical scale data also supports the overall greater expansion of TNC and CD34+ cells in HSCEM/Stemline, as well as the expansion of both committed and primitive progenitor compartments. Conclusions: Results indicate that HSCEM/Stemline provides a significant benefit over other commercially available serum-free formulations, such as X-VIVO 15(TM) and StemSpan H2000(TM), for the expansion of TNC, committed progenitor and primitive progenitor compartments. This suggests that in the clinical stem cell transplant setting, HSCEM/Stemline may provide significant benefit toward reducing time-to-engraftment and may also result in the reduction in frequency and/or severity of neutropenia and thrombocytopenia.

The major site of hematopoiesis moves from the fetal liver to the fetal spleen and bone marrow late in fetal development. To date experiments have not been performed to evaluate the migration and seeding of hematopoietic stem cells (HSC) during this period in ontogeny. It has been proposed that developmentally timed waves of hematopoietic stem cells enter the bloodstream to seed the fetal spleen and bone marrow. Using competitive reconstitution assays to measure HSC activity, we determined the localization of HSC in the mid to late gestation embryo. We show that seeding of these fetal organs by fetal liver HSC occurs over a several days, possibly while stem cell niches form. Changes in the level of factors relevant to the homing and engraftment of HSC in these tissues was monitored by RT-PCR. We show that the seeding of fetal organs by fetal liver HSC does not require large fluxes of HSC entering the fetal bloodstream, and that HSC constitutively circulate at low levels from E12 to E17.F

Abstracts / Experimental Hematology 30 (2002) 37-146

Cytokine deprivation improves the homing 353 of long-term cultured human hematopoietic stem cells after transplantation int NOD/SCID mice

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Flk1+ CD34- Fetal BoneMarrow-derived Cells Having 355 Characteristics of Hemangioblast H. Guo, Z. Zhao, *J. Liu, H. Chen, R. Zhao,

PUMC&CAMS, China

*T. Kerre1, B. Vandekerckhove2, F. Offner1, J. Plum1, 1Ghent University Hospital, Belgium, 2Bloedtransfusiecentrum Oost-Vl, Belgium Long-term culture of human hematopoietic stem cells (HSC) is clinically important for gene therapy and for expansion of umbilical cord blood (UCB) HSC to enable transplantation of older children and adults. However, reports in literature on long-term engraftment of cultured HSC in NOD/SCID mice are mostly disappointing, suggesting a decrease in HSC activity after culture. We wanted to investigate the behavior of these cells early after transplantation, and therefore introduced UCB HSC that were cultured for 2 weeks in serum-free medium supplemented with SCF, TPO and Flt-3L, into our short-term in vivo trafficking assay (T. Kerre et al, J. Immunol. 2001, 167:3692-8). In this model, freshly isolated UCB CD34+ cells home specifically to the murine bone marrow and spleen, but only in the bone marrow proliferation exceeds apoptosis and cells expand, with kinetics depending on the source (UCB>mPB>BM) and the expression of CD38. In comparison, cultured CD34+ cells show an impaired homing capacity (prolonged presence in the circulation) and altered organ selectivity (homing to spleen and bone marrow, but also to liver and lung). Apoptosis on the other hand, is less pronounced. Markedly, the bone marrow homed cultured CD34+ cells show a reduction in early expansion (1 week / 4 weeks). All these data show that long-term culture of HSC affects all stages in the early transplantation phase. Study of the expression of homing molecules before and after culture showed that cultured cells downregulate CXCR4, a specific homing receptor for CD34+ cells to the bone marrow and upregulate the adhesion molecules VLA-4 and VLA-5, which may cause homing to inappropriate sites. Deprivation of cytokines for 1 or 2 days resulted in normalization in the expression of these homing molecules. When these cells were injected in NOD/SCID mice, the homing to the bone marrow increased while homing to the lungs decreased, in comparison with non-deprived cells. Preliminary results show that early expansion was not influenced by cytokine deprivation. In conclusion, our study showed that long-term culture decreases the homing ability of human hematopoietic stem cells, and that deprivation of cytokines can partially counter this defect.

Stem cells having bipotential of differentiating into endothelial and hematopoietic cells are defined as hemangioblast. To investigate whether human fetal bone marrow derived Flk1+ cells have characteristics of hemangioblasts, we isolated and cultured Flk1+CD34- cells which were vWF-, CD34-, CD45-, CD11b-, Glycophorin A-, and alpha-SMA- analyzed by flow cytometry. The Flk1+ CD34- cells seeded in ECM in the presence of VEGF and bFGF formed a three-dimensional spherical structure. The spherical structure sprouted, and turned into a microvascular-like structure. The distance between two adjoining cells, stained CD31+ vWF+, lining along vascular-like tubules was 109.09 +/- 2.43 um. We showed that the vascular structures were formed by endothelial cells identified morphologically by transmission electron microscope. The vascular structures were totally prevented after addition of anti-angiogenesis reagent suramin in the beginning of the culture. This further supported the role of Flk1+CD34- cells in vascularization. In addition, we identified a population of dendritic-like cells adhered around vascular structures. They were alphaSMA positive known as pericytes. Therefore, the Flk+CD34- cells behave likes angioblasts which differentiated into endothelial cells and pericytes. We also observed that there was a third population of cells with diameter of 15 um and round in shape emerged around vascular structures. They were CD34+ a character of hematopoietic phenotype. The in vitro observations were consistent with our in vivo studies. Flk1+CD34- cells isolated from rat bone marrow were transplanted into the syngeneic rat underwent partial hepatectomy. The marked engrafts were incorporated into the regenerating liver vascular systems. We are the first to show that Flk1+CD34- cells isolated from bone marrow have characteristics of hemangioblasts, which could differentiate into endothelial and hematopoietic cells.

Identification and Characterization of Multipotential 354 Stem Cell in the Human Dorsal Root Ganglion F. Zhang , Y. Hu , Y. Tan , G. Hong , F. Wang , L. Li , E. Fan ,

A Phase Space Model of Hemopoiesis 356 *M. Kirkland , K. Borokov ,

1

1

1

2

2

3

1

*R. C. H. Zhao4, 1PUMC&CAMS, China, 2Tongji Medical College ,

1

2 1

Barwon Health, Australia, 2University

of Melbourne, Australia

China, 3 Tianjin Medical University, China, 4Institute of Hematology, CAMS and PUMC, China We identified a new stem cell population in the human dorsal root ganglion (DRG) isolated from the aborted 16-26 week-old fetuses. The purified DRG cells clonally proliferated in vitro forming sphere colonies that composed of neural stem/precursor cells stained positive with antinestin antibodies. DRG stem cells were expanded 100 times in 6 passages when cultured with EGF and bFGF and remained stable in long-term cultures (> 2 months). To determine the multipotentiality of the human DRG stem cells, we plated the cells into a poly-L-lysine coated plate under differentiating conditions by removal of the mitotic factors, and demonstrated that they differentiated into DRG-specific cell types, including sensory neurons, satellite cells, and Schwann cells identified by immunocytochemistry. To further characterize the distribution and proliferation responses of DRG stem cells to injury, we analyzed DRG tissue sections prepared from animals that had an injury in the peripheral nerves. The proliferating cells were labeled in vivo by BrdU that was detected by immuno-staining of the DRG tissue sections with anti-BrdU antibodies. 8 hr after a transection of right sciatic nerves, there was almost 7-fold increase in BrdU-immunoreactive cells in the tissue sections comparing with the uninjured side of the DRG sections. Our results suggest that DRG stem cells undergo cell division in response to injuries of the peripheral nerves. This study is the first report in isolation of stem cells from DRG that can be induced to differentiate into multiple lineages of peripheral neuron system.

Standard models of stem cell growth and differentiation are based on the concepts of compartments and self renewal. Implicit in such modeling is the concept of a hierarchy of discontinuous compartments, with transitions between these compartments being described by discrete probabilities. However, if the stem cell population is viewed as a continuum rather than being composed of discrete states then a probability function must be used to describe the outcome of a cell division, and such functions must include the possibility that some daughter cells will have greater “stemness” than the parent cells in a renewing model, i.e. the potential for de-differentiation is required. We have developed a phase space model of hemopoiesis and stem cell proliferation and differentiation based on these concepts. The phase space can be used to describe the differentiative state of cells in two or more dimensions. Mathematically, the movement of cells through this space is described using a technique known as a Markov branching process. Each cell is described by the probability of movement in different directions within the space, either towards a more differentiated state (known as “terminal states”), or towards a less differentiated state. “Attractors” within the space, representing the effect of growth factors, can influence movement towards a differentiated state. Analysis of the phase space can be simplified by “regionalising” the space. At one extreme, such regionalisation becomes equivalent to the current model of stem cell renewal, i.e the current model is a subset of the phase space model. A number of observations arise from this model: 1. renewal is a function of the whole population rather than of individual cells, and 2. all cells that have not reached the terminal states have some probability of moving to a less differentiated state, and so contribute to the total “stemness” of the system. This has implications for the interpretation of the results of stem cell transplantation and expansion studies, and on models of aging of stem cell populations. This concept can also be extended to include non-hemopoietic cell populations, and so may serve as a model of stem cell plasticity.

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Functional differences between individual hematopoietic 357 stem cell candidates in vitro are correlated with telomere length measured by Flow-FISH

CD133: Internal & External Antigen Distribution 359 by Confocal Laser Scanning Microscope Analysis and 3-Dimensional Imaging

*K. Bartolovic, B. Berner, S. Balabanov, A. Wahl, A. Marxer, L. Kanz, T. Brummendorf, University of Tübingen , Germany

*K. Whiting1, C. McGuckin2, N. Forraz3, D. Wertheim2, R. Pettengell4, 1St George’s Hospital Medical School, U.K., 2Kingston

Early human hematopoietic stem/progenitor cells are enriched in the fraction of cells with the phenotype CD34+CD38-. However, cells with this phenotype are still heterogeneous and only a minor fraction is capable of long-term engraftment in irradiated immunodeficient mice. The proportion of cells with extensive expansion potential within this fraction decreases from fetal liver to cord blood to bone marrow. In previous studies we have shown that single sorted CD34+38stem cell candidates (HSC) from fetal liver display extensive functional heterogeneity in terms of growth kinetics when expanded in growth factor supplemented serum-free medium (J. Exp. Med. 188:1117-24). Interestingly, a positive correlation was found between cell cycle time and proliferative potential of the individual clone indicating that the slowest growing clones were characterized by the highest proliferative potential. Re-cloning experiments indicated that diversity in the proliferative properties of primitive CD34+CD38- cells is generated at a single cell level. In order to investigate the role of telomere biology for the expansion potential of individual SCC, we expanded 595 single sorted CD34+38- cells from three individual human cord blood specimen in serum-free medium containing Steel factor, Flt-3, Il-3, Il-6, G-CSF and TPO. After several weeks of culturing, 66 colonies consisting of more than 5 x104 cells could be devided into fast, intermediate and slowly growing clones based on the time period it took the individual clone to reach 5 x104 cells. 27 clones yielded enough cells to allow telomere length analysis by Flow FISH. Substantial differences in telomere length were observed between the different clones, ranging from 9 to 23 kMESF. A highly significant positive linear correlation between cell cycle time and telomere length of the individual clone was observed (R=0.7; p<0.0001). Significantly longer telomere length was found in slowly (n=5) and intermediate (n=4) growing clones compared to fast (n=18) growing clones (deltaTel = 6.0 kMESF; p<0.0001). In agreement with previous studies, these data provide further support for a functional hierarchy in the human HSC compartment and suggest that telomere length might be a useful marker for the identification of more primitive subsets among CD34+38- HSC.

University, U.K., 3King-George Laboratory, U.K., 4St George’s Hospital, U.K. Recently, evidence has suggested that hematopoietic and endothelial populations are derived from a common developmental precursor. Therefore, external membrane CD133 expression is a potential marker for early populations. We have investigated the possibility of internal stores of CD133 with the aim of identifying an earlier multipotential subset. CD133+ cells were positively selected from umbilical cord blood (n=3) by Miltenyi MACS Separation and AC133/1 antibody (anti-CD133). To determine external and internal CD133 antigen distribution, 200,000 CD133+ cells were seeded onto Poly L-Lysine slides for 1 hour prior to antibody labeling. Subsequent dual external and internal labeling of the CD133 antigen was carried out with Mouse AC133/2 and Molecular Probes fluorescentconjugated antibody, (Alexa fluor 488, for external labeling, and Alexa fluor 594, for internal labeling). Internal and external CD133 distribution was determined by confocal laser scanning microscope analysis (Zeiss LSM410) and 3-dimensional computer representation, by applying a system we developed in house, using Iris Explorer (NAG Ltd, Oxford, UK). External CD133 was found to be expressed by approximately 90% of adhered cells. 2.2% of external CD133+ cells co-expressed internal CD133. Purity of CD133+ cells from positive selection was determined by FACS analysis and was shown to be 92.70% +/-2.81SEM (range 84.51-97.15) (n=5). Although CD133+ populations represent an extremely rare population of cells, we have developed a working method for identification of these cells by confocal microscopy in low cell numbers at varying magnification levels and in 3dimensional space. We previously reported external CD133 distribution to be associated with pseudopodia formation during cell-to-cell interaction. In the current work, distribution variations were found between well-defined membrane pockets and more widely distributed crescents, which may correlate to the activation state of the cell membrane. Confocal 3-dimensional analysis further identified dense, localized internal stores of the CD133 antigen, possibly Golgi associated. Such accurate internal identification allows further membrane tracking analysis to be developed. The presence of internal CD133 may identify more closely, candidate hemangioblasts, capable of hematopoietic reconstitution and vasculogenesis.

Distinct characteristics of hematopoietic stem cells 358 in AGM and fetal liver *M. Takeuchi, T. Sekiguchi, A. Miyajima,

Highly sensitive ligation-mediated PCR technique 360 demonstrates that multiple clones reconstitute human hematopoiesis in the bone marrow of NOD/SCID mice

Japan

*S. Laufs1, B. Gentner1, K. Nagy1, K. Kühlcke2, J. Topaly3, B. Schiedlmeier4, W. Zeller1, A. Ho3, S. Fruehauf3, 1German Cancer

The University of Tokyo,

Definitive hematopoietic stem cells (HSCs) arise in the aorta-gonadmesonephros (AGM) region at embryonic day 10.5 (E10.5) in mice. HSCs generated in AGM migrate to fetal liver (FL) where HSCs proliferate actively along with production of mature blood cells from E11.5 to neonatal stage. To analyze these processes, we developed in vitro culture systems composed of E14.5 FL stromal cells and CD34+cKit+ HSCs derived either from E11.5 AGM or from E14.5 FL and demonstrated that AGM-derived HSCs expand and produce mature blood cells much more efficiently than E14.5 FL HSCs in FL microenvironment. To uncover the difference between AGM-derived HSCs and FL-derived HSCs, we compared the dynamic processes of production of blood cells. While the number of total hematopoietic cells from E11.5 AGM-HSCs expanded exponentially for 10 days, that from E11.5 FL-derived HSCs reached plateau within 4 days of co-culture. After 10 days of co-culture with FL stromal cells, AGM-HSCs generated fifty times more hematopoietic cells than FL-HSCs. It was shown previously that HSCs are derived from hemangioblasts that are present in the CD45- cells in AGM, In consistent with this, 80% of CD34+c-Kit+ AGM HSCs did not express CD45, while both CD45+ and CD45- AGMCD34+c-Kit+ cells gave rise to blood cells. In contrast, 70% of CD34+cKit+ HSCs in E11.5 FL and almost all CD34+c-Kit+ HSCs in E14.5 FL expressed CD45. These results clearly indicate that the characteristics of AGM-stem cells and FL-stem cells are quite different, even in the same stage of embryos and suggest that hemangioblasts generated in AGM become HSCs in FL.

Research Center, Germany, 2EUFETS GmbH, Germany, 3University of Heidelberg, Germany, 4Heinrich-Pette-Institut, Germany The hu-NOD/SCID mouse system allows to study human hematopoietic stem cells with marrow repopulation potential. Knowledge on the clonal diversity of human cells repopulating NOD/SCID mice is still limited. Human CD34-purified peripheral blood progenitor cells were transduced with retroviral supernatant and transplanted into immunodeficient NOD/SCID mice. After 8 weeks of engraftment, whole bone marrow DNA was analyzed for the number of different retroviral integration sites (clones). Highly sensitive solidphase ligation mediated PCR (LM-PCR) was developed that allowed us to simultaneously amplify multiple restriction fragments containing vector-flanking DNA junctions. In chimeric NOD/SCID bone marrows we detected between 6-19 integration sites per mouse (mean: 11; n=5). These integration sites were specific, in spite of the presence of mouse DNA and DNA from untransduced human cells. By repeated analyses of one bone marrow with different restriction enzymes we even found new additional integration sites, indicating that a single analysis with a single restriction enzyme underestimates the real number of contributing clones. After four analyses, we found up to 30 different clones per mouse bone marrow. Considering that 6.1% of the human cells were MDR1 transduced and analyzed, the total number of active clones would thus be estimated at close to 500 clones/mouse. This protocol allows highly sensitive and specific analysis of the development of individual vector-marked human hematopoietic stem cells and their progeny in the NOD/SCID mouse, even in a high mouse DNA background. Our data suggest that human hematopoiesis in the NOD/SCID mouse is polyclonal, as has been described for the repopulation of human/primate bone marrows, giving evidence that the NOD/SCID model is a suitable assay to study human hematopoiesis posttransplantation. Our method opens the door for a multitude of studies on the biology of stem cell transplantation and also on the biology of retroviral integration.

Abstracts / Experimental Hematology 30 (2002) 37-146

Ontogenic Fate of Mesenchymal Cell Associated Hepatic 361 and Hematopoietic stem cells in the Mouse Liver [or Niches Save the Queen]

127

Restricted transgene expression in human CD34+ cells 363 mediated by novel lentiviral vectors Z. Ye, *L. Cheng, Johns Hopkins University, U.S.A.

*I. Blazsek, J. Chagraoui, E. Oberlin, G. Uzan, B. Péault, INSERM U-506, France Molecular signaling between the cardiac/septum transversum mesoderm and primitive endoderm that specifies foregut patterning has been recently identified. We investigated the role of mesoderm in subsequent fate of hepatic (HepSC) and hematopoietic (HemSC) stem cells during liver organogenesis. Whole-mount stereomicroscopy and histology show that hemangiopoietic cell aggregates appear in the cardiac mesoderm that interdigitates with budding hepatic cords at E8.5-9.0. Real-time and time-lapse microcinematography performed in explant cultures show that this mesendodermal boundary autonomously generates primitive, erythroid cobblestone-area-forming cells (EryP-CAFCd7). Treatment of explants with mVEGF, HGF/SF and Epo revealed that primitive erythroid cells emerge together with functional cardiac myoblasts and angioblasts. However, definitive hematopoietic colony-forming cells (GM-CFU, BFU-E, CAFCd7-14) appeared in the liver from E9.5 and HemSC (LTC-IC/CAFCd35) from E12.5.Gain of hematopoietic competence required contact with mesenchymal cells (FGF2R/Bek+, desmin+) that bring together presumptive HepSCs (CK-19+, c-met+) and/or HemSC/CAFC in compact aggregates (niches), analogues to marrow hematons. Niches provide a local production of growth factors and functional morphogens (HGF/SF, SDF-1, BMP-4, retinol) as shown by immunocytochemistry, gold impregnation or by treatments with neutralizing antibodies and inhibitors of signaling pathways (geldanamycin, cyclopamine). In long-term culture these evolved into hepatic colony-forming units (Hep-CFU) with branching cholangiocytes, hepatoblasts/hepatocytes (CK-18+, AFP+/alb+) and sprouting vascular endothelium (EphB4+, EphrinB2+). At acrophase (E16), 30-40% of niches included HemSC (LTC-IC/CAFCd35). The deaggregated fraction developed many individual hepatocyte clusters, hematopoietic cobblestone areas and non-adherent spheroid epithelial follicles, but was unable to form organized Hep-CFU. Thus, in the liver primordium, the developmental fate of endodermal and hematopoietic stem cells is determined in compact morphogenetic units (niches), containing mesenchymal cells, that create an instructive microenvironment and continuous selective pressure during organogenesis.

Introduction of growth-promoting genes to hematopoietic stem and progenitor cells (HSPCs) has been proposed to enhance HSPC survival and proliferation. However, this approach was often associated with leukemia or myelo-proliferative diseases. This is likely due to the dysregulated transgene expression in differentiating cells by existing retroviral vectors that express constitutively. Therefore, the success of this gene transduction approach requires regulated transgene expression, in addition to efficient and stable gene transfer into engrafting HSPCs. Recently, we and others demonstrated that specific transgene expression controlled by a chosen promoter can be readily achieved using lentiviral vectors (LVs) with the self-inactivating (SIN) modification. Using in vitro assays and the NOD/SCID mouse model, we showed specific transgene expression after stable gene transfer into multi-potent HSPCs by LVs with a lineage-selective promoter (Blood, 99:399; 2002). In search of genes that are selectively expressed in human CD34+ HSPCs, we cloned recently a novel cytokine-like gene C17 (Genomics, 65:238; 2000). C17 gene is expressed in both CD34-Lin- and CD34+Lin- cells from human bone marrow (BM) and in CD34+ hematopoietic progenitor cell lines such as TF1, but not in mature hematopoietic cells from BM and blood cells that are CD34-. Having examined many human adult tissues and cell lines, we found C17 expression is highly restricted to cells within the hematopoietic and vascular systems. We have begun to elucidate the C17 gene regulation mechanisms and the promoter sequence in addition to its biological functions. Using our SIN LVs, we constructed a series of LVs containing variable sizes of the C17 promoter. The smallest one contains the 512 bp proximal genomic (plus 11 bp cDNA) sequence. The 523 bp C17 promoter is sufficient to direct transgene expression in TF1 progenitor cells, but inactive in HL-60 (CD34-/C17-) hematopoietic cells. Similar results were obtained with other CD34+/C17+ and CD34-/C17- cell lines. Therefore, the 523 bp C17 promoter sufficient to direct transgene expression specifically in cultured CD34+/C17+ progenitor cells. Once validated with primary human CD34+ HSPCs, this system of combined stable gene transfer and targeted transgene expression (selectively in HSPCs) should provide a powerful tool for HSPC biology and engineering.

Contributions of spleen, liver, and bone marrow 362 in maintaining hematopoietic homeostasis in the neonatal mouse

Functional properties of muscle-derived CD45-Sca-1+c364 kit- cells fractionated on the basis of CD34 and Thy-1.2 expression

*F. Wolber, E. Leonard, M. Yoder, E. Srour, Indiana University,

*J. Howell1, M. Yoder2, E. Srour2, 1Indiana University School of

U.S.A.

Medicine, U.S.A., 2Indiana University, U.S.A.

In the neonatal mouse, both spleen and bone marrow (BM) are organs of hematopoiesis. While migration of hematopoietic cells between organs occurs in the fetus, it has been speculated that organ-specific pools of cells might cooperatively or independently maintain hematopoietic homeostasis after birth. We measured hematopoietic progenitor cells (HPC) and hematopoietic stem cells (HSC) in the BM, spleen, blood and liver of fetal and neonatal mice by assessing clonogenic cells in vitro and long-term reconstitution following transplantation in vivo. HPC remained elevated in the blood from 16 days (d) post-coitus (pc) to 4d after birth. HSC were present in the blood at 2-3d post-birth and also detectable in the liver up to 8d after birth, suggesting that migration of both HPC and HSC from the fetal liver to the neonatal BM occurs over a prolonged period. Total HPC content in BM and spleen of 2d pups was similar, implying that these organs make equal contributions to hematopoiesis in the neonate. To examine whether BM and spleen hematopoiesis in the neonate are independent, we splenectomized 1-3d pups and examined them at 7d and 18d. Surprisingly, the liver displayed elevated hematopoiesis following splenectomy. The livers of splenectomized animals, as compared to control or sham-treated animals, contained 2-fold more total non-parenchymal cells, and 2 to 5-fold more HPC. HSC frequency was also significantly (p=0.008) increased: mice transplanted with 10(E5) liver cells from splenectomized or sham-treated pups displayed a mean blood chimerism of 56% (n=4) or 12% (n=3), respectively. In contrast, no differences in BM total cells, HPC or HSC were observed between splenectomized and control pups. Furthermore, blood, but not liver or BM, of 4d pups splenectomized one day earlier appeared to contain a higher frequency of HSC. Together, these data suggest that following neonatal splenectomy, HSC and HPC recirculate for more than 24 hours before returning to the liver, which then acts as a site of extramedullary hematopoiesis and compensates for the absence of the spleen. Thus it is likely that the spleen, liver and BM cooperatively maintain hematopoietic homeostasis in the neonate.

We previously demonstrated that modest hematopoietic engraftment in irradiated recipients can be sustained long-term following transplantation of 2.5x10e4 skeletal muscle CD45-Sca-1+c-kit- cells from 4-7 day-old mice. The hematopoietic potential of CD45-Sca-1+c-kit- cells assessed in competitive repopulation assays increased 16.9-fold in expansion cultures supplemented for 9 days with 5 ng/ml mSCF, mFlt3L, MGDF, BMP-4, and mVEGF (5 cytokines) relative to the engraftment capacity of freshly isolated cells. Furthermore, cells from these cultures, but not freshly isolated cells, expressed mRNA of the hematopoietic-specific genes PU.1, SCL, and c-myb suggesting that, under these culture conditions, maturation of CD45-Sca-1+c-kitcells was directed toward hematopoietic lineages. We hypothesized that addition of BMP-2 and BMP-7 to the cocktail of 5 cytokines (7 cytokines) would further augment the in vivo hematopoietic potential of CD45-Sca-1+c-kit- cells following 9 days of culture. Infusion of 4x10e4 freshly isolated cells displayed 4.1±2.4% donor chimerism at 2 months post-transplantation (PT), while 4x10e4 7 cytokine-stimulated cells expanded 11.2-fold ex vivo and led to 8.1±1.8% donor chimerism; a 22-fold increase in repopulating potential. Since CD45-Sca-1+c-kit- cells are a heterogeneous population of cells, we hypothesized that further fractionation of this population using anti-CD34 and anti-Thy-1.2 monoclonal antibodies could potentially enrich for HSC activity. Whereas both the CD34- and CD34+ fractions maintained in culture for 9 days appeared morphologically similar to the parent CD45-Sca-1+c-kit- population, Thy-1.2- cells remained round, non-adherent, and formed tight clusters; while Thy-1.2+ cells exhibited a completely adherent, stromallike appearance. Two of five mice transplanted with 3.0x10e2 CD34+ cells demonstrated donor chimerism (3.2% and 3.5%) at 4 mo. PT; however, 0/5 mice revealed detectable chimerism when similar numbers of CD34- cells were transplanted. At 1 mo. PT, 4x10e4 Thy-1.2- cells yielded higher donor cell chimerism in competitive repopulation assays than 4x10e4 Thy-1.2+ cells (5.7±2.1% vs. 2.8±3.4%). Taken together, these results suggest that BMP-2 and BMP-7 can further increase the hematopoietic repopulating potential of cultured muscle-derived CD45Sca-1+c-kit- cells. Furthermore, it appears that Thy-1.2 expression correlates with differences in CD45-Sca-1+c-kit- cell morphology in vitro, and that Thy-1.2- and CD34+ sub-fractions of these cells are enriched for hematopoietic repopulating ability in short-term analysis PT.

128

Abstracts / Experimental Hematology 30 (2002) 37-146

Non-peptide lipid mediators induce proliferation and 365 adhesion of CD34+ hematopoietic progenitor cells A. Boehmler , C. Denzlinger , L. Kanz , *R. Möhle ,

Efficient single hematopoietic cell transplantation 367 *S. Corbel, A. Lee, L. Yi, F. Rossi,

Cytokines and chemokines have been established as important regulators of hematopoietic progenitor cell (HPC) proliferation and migration. However, the role of non-peptide mediators in the hematopoietic microenvironment has remained elusive. We have recently shown that a receptor for cysteinyl leukotrienes, CysLT1, is highly expressed and functionally active in CD34+ HPC demonstrating that G-protein coupled seven-transmembrane receptors other than CXCR4 may be involved in progenitor cell trafficking and proliferation [Blood 97:3433-3440, 2001]. Production of lipid mediators, particularly leukotrienes, has been observed in the normal bone marrow, but their function in the hematopoietic microenvironment is still unknown. Therefore, we analyzed the effects of cysteinyl leukotrienes on proliferation and adhesion of mobilized human CD34+ HPC. In serum-free liquid cultures supplemented with SCF, IL-3, and FLT3L (each 100 ng/ml), leukotriene D4 (LTD4) augmented cytokine-induced expansion of HPC in a dose-dependent manner. After 7 days, the total cell number was 1.69 +/- 0.03-fold (mean +/- SEM) increased in cultures containing 100 nM LTD4 compared to cytokines alone. Interestingly, the maximum effect on proliferation was obsered at concentrations which also induced optimal progenitor cell chemotaxis. The proliferative effect of LTD4 was dependent on the presence of IL3, suggesting a specific effect of LTD4 on IL-3-mediated signaling. Preincubation with LTD also upregulated rapid adhesion of HPC to activated HUVEC monolayers in a time- and dose-dependent manner up to 1.8 +/- 0.1-fold with maximum adhesion being observed in cells treated with 1 uM LTD4 for 15 minutes. Similar effects were found in adhesion assays using primary bone marrow endothelial cells or VCAM-1 immobilized on a plastic surface. Both primitive CD34+/CD38- cells and lineage-committed clonogenic progenitors adhered after leukotriene preincubation. LTD4-triggered adhesion was blocked by VLA-4, but not LFA-1 antibodies, and by the specific CysLT1 receptor antagonist MK-571. Moreover, attachment of HPC to fibronectin was increased 1.5 +/- 0.2fold after LTD4 pretreatment, which was almost completely inhibited by VLA-5, but less effectively by VLA-4 antibodies. We conclude that non-peptide mediators, e.g. cysteinyl leukotrienes, can induce proliferation and beta1-integrin-mediated adhesion of HPC, and may therefore play a role in stem cell maintenance and homing to the bone marrow.

Hemopoietic stem cells (HSC) are able to replace any blood cell, and are widely used to cure hematological disorders. Recently, it has been shown that cells residing in the bone marrow can also participate in the regeneration of non-hemopoietic organs, such as brain, muscle or liver. However, the lineage to which these plastic cells belong is still elusive. In order to elucidate whether these different cell types can be generated from the same hemopoietic stem cell, or if distinct circulating progenitors exist for the different tissues, we reconstituted the hematopoietic system of irradiated mice with single HSC. We are using GFP transgenic mice as donor population and wild type C57Bl6 mice as recipient. We used flow cytometry to purify single HSCs from whole bone marrow. Before injection, the cell were visualized under microscope to ensure accuracy. Each cell was injected with GFP negative stem celldepleted helper bone marrow to ensure short term survival of the recipients. Preliminary results show that in average 1 out of 5 single HSC transplanted mice led to the appearance of GFP positive cells in the blood. We are currently investigating whether non-hemopoietic GFP expressing cells can be found in these animals.

Stem/Progenitor Cell Inversions:The Chiaroscuro Stem 366 Cell Model of Hematopoiesis *G. Colvin, J.-F. Lambert, C. McAuliffe, P. Quesenberry,

Green Fluorescent Protein (GFP) positive marrow 368 trafficking to injured murine liver *M. Abedi, G. Colvin, J. Lambert, C. McAuliffe, M. Dooner,

Williams Medical Center, U.S.A.

P. Quesenberry, Roger Williams Medical Center, U.S.A.

We have previously shown that cytokine stimulated primitive marrow hematopoietic stem cells progress through synchronized cell cycle in in vitro cultures and show an engraftment phenotype that fluctuates with cell cycle phase; engraftment is lost in late S/early G2 and recovered in the next G1 phase. Other, more preliminary studies suggest the sequence of events is a modulation of adhesion receptors followed by a defect of homing, which then determines engraftment. In the present studies, we have evaluated stem/progenitor expression through cytokine-stimulated cell cycle with thrombopoietin, FLT-3 ligand and steel factor with both an in vitro and in vivo assay. A 7-factor, 14 day soft-agar clonal assay for high proliferative colony forming cells (HPP-CFC) and colony forming unit culture (CFU-C) was compared with engraftable stem cells in a competitive 8 week transplant assay. Cultures were established in Teflon bottles or in microgravity rotating wall vessels (RWV). We observed marked increases in in vitro progenitor colony formation virtually always associated with marked decreases in engraftable stem cells (P<0.0001). These occurred during the first cell cycle transit, before any cell division and were reversible. There were clear stem/progenitor cell inversions. These data indicate that engraftable stem cells and progenitors are a continuum with reversible shifts in phenotype. We speculate that shifting chromatin coverage during cell cycle transit results in altered surface receptor expression and differential responses to microenvironment stimuli at different points in cell cycle. This is the chiaroscuro model of stem cell regulation.

Stem cell transdifferentiation to liver has been noted in many tissues including liver and Lagasse et al. (Nat Med 2000;11:1229) have shown restoration of liver function in FAH deficient mice from bone marrow stem cells. We have evaluated the impact of carbon tetrachloride (CCl4) and radiation on trafficking and transdifferentiation of bone marrow cells to liver cells. GFP+ transgenic mice were used as donor and C57BL/6 mice as recipient. Two models were investigated. In the first model male C57BL/6 mice were injected with 1000 µl/kg of CCl4 after 400, 500, and 900cGy of TBI. Four hours later they were transplanted with 25 million marrow mononuclear cells from GFP transgenic mice. Two months later mice were sacrificed and different organs, with a focus on liver, were evaluated. Frozen sections were evaluated with fluorescent microscopy for GFP expression and stained with CD45 and CD26 for cell phenotyping. Many GFP positive cells were seen in all samples, but hepatocytes defined by morphology and as CD45 neg., CD26 pos., were not seen in the CCl4+400cGy group, were rare in CCl4+500cGy group but were more frequent in 900cGy group. In the second model, marrow chimeric mice were established by transplantation of 25 million marrow cells from GFP transgenic mice after 400cGy of TBI. High levels of GFP+ cell chimerism was confirmed by FACS analysis of peripheral blood mononuclear cells. Two months after transplantation, one group received GCSF daily for 5 days and the two other group were injected with PBS. Three days later, mobilized animals and one of the control groups were injected with CCl4 intraperitoneally. The third group received corn oil. At 1 and 3 months after injury animals were sacrificed and organs were collected. In the injured group, despite the presence of rather significant number of GFP positive cells, no donor-derived hepatocytes were identified. Mobilization of bone marrow cells also did not improve transdifferentiation of GFP+ cells to hepatocytes. In both models GFP positive cells were seen in lung, myocardium, gut, and brain. These data indicate that liver injury is necessary to transdifferentiation and that irradiation is a critical component of such injury.

1 1 1 2 1 Dept. Med. 2, Univ. Tübingen, Germany, 2University of Tübingen, Germany

Roger

UBC, Canada

Abstracts / Experimental Hematology 30 (2002) 37-146

Computational and functional approaches for use 369 of bone marrow (BM) stem cells: Mesenchymal (MSC) and lymphohematopoietic (LHSC) for neuronal repair and/or replacement and identification of two molecular markers *P. Rameshwar1, J. Potian1, K. Sancilio1, P. Bandari1, L. Challenger2, E. Homsi2, J. McArdle1, 1UMDNJ-New Jersey Medical School, U.S.A., 2NJIT, U.S.A. Transdifferentiation of BM stem cells show promise for future use in tissue repair of distant organs. We propose two approaches can be used for neural repair by LHSC or MSC: I. Use of mathematical equations that will allow the direction of BM stem cells to injured areas at distant sites. The advantage of this approach is that it will allow for the use of syngeneic stem cells and applies to both types of stem cells. The equations can explain the feasibility of movement of stem cells in gradient changes of oxygen in the BM to the periphery. As the stem cells reach the sites of injuries, the equations could test different molecules with the microenvironment of the injury. It was determined that the mathematical equation provides insights in the manipulation of the microenvironment and/or the stem cells for efficient transdifferentiation to repair or replace a neuron. II. Transplanting fully differentiated neurons to the site of injury. This approach was addressed with MSC since they have been reported to elicit weak allogeneic responses. Human BM aspirates were cultured for MSC to ~75% confluence. Phenotypically, the cells were symmetrical and by immunofluorescence were SH2+/CD14-/CD45-/CD34-/CD29+/CD31-. MSC were transdifferentiated with retinoic acid to cells with neuronal phenotype (Nestin+/NeuN+). We next compared the expression of two genes in MSC and the transdifferentated cells: NK-1, a receptor for neurotransmitters that belong to the tachykinins, and HGFIN, a novel transmembrane protein that shares structural homology with NK-1. By northern analyses and immunoflurescence, we observed NK1 was expressed as the cells become neuronal and HGFIN was downregulated. These results are consistent with cells of BM and neuronal origin, suggesting that NK-1 and HGFIN might be used as molecular markers of transdifferentiation: from BM to neurons. Screening for NK-1 and HGFIN could be added to the indisputable power of the patch clamp technique for synaptic activity.

Reconstitution of B-lymphocite repertoire after allogeneic 370 hematopoietic stem cell transplantation in children analysis by HCDR3 fingerprinting *D. Di Martino1, A. Valetto2, M. Terranova1, P. Di Michele1, L. Scarso1, M. Iannacchino1, G. Morreale1, E. Lanino1, G. Dini1, 1

129

The marrow homing efficiency of murine hematopoietic 371 stem cells remains constant during ontogeny *S. Szilvassy , P. Ragland , C. Miller , C. Eaves ,

1 2 3 4 1 University of Kentucky, Lucille P. Markey Cancer Center, U.S.A., 2University of Kentucky, U.S.A., 3StemCell Technologies Inc., Canada, 4British Columbia Cancer Agency, Canada

Quantitative transplantation assays of hematopoietic stem cells and progenitors underestimate the true number of cells with repopulating potential by a factor (1) corresponding to the fraction of cells that reach sites in viva that are able to stimulate the generation of detectable clones of mature progeny. Although the f-factor for murine spleen colony-forming units (CFUS) was determined —40 years ago to be -10%, the seeding (or homing) efficiency ofHSC (competitive long- term repopulating units, CRU) is unknown. To measure the bone marrow (BM) seeding efficiency of HSCs from adult BM and day 14 fetal liver (FL), we determined the fraction of transplanted CRU that could be recovered from the 4 leg bones (representing 25% of the total BM) of lethally irradiated B6 mice 24 hr after their IV injection with Ly-5 congenic cells whose CRU content was determined by limiting- dilution transplantation assays in separate sets of primary and secondary mice. The seeding efficiency ofBM-CRU able to regenerate at least 50/(1 of the circulating lymphoc~es and myeloid cells 5 wk posttransplant was 12%. Analysis of the same mice after 10, 17 and 26 wk yielded similar values (7% in each case). The seeding efficiency determined for 5and 10-wk murine FL-CRU was 10%. The remarkable similarity of these values, both to each other and to those reported for human cord blood and FLCRU assayed in sublethally irradiated NOD/SCID mice, suggest common mechanisms regulating the BM homing of HSC in mouse and man throughout ontogeny. Interestingly, these values also predict maximally achievable CRU purities of 5-10% in enriched populations, whereas values as high as 300/(1 have been documented. This implies either that a significant proportion of transplanted CRU from adult BM or FL are not recoverable from the BM 24 hr later using conventional procedures or, more likely, that many HSCs home at least initially. and perhaps transiently. to other organ sites.

Detection of human cell factors in liver tissue of chimeric 372 goats engrafted with human hematopoietic stem cells *F. Zeng , H. Yam , C. Pang , S.-z. Huang ,

1 2 2 3 1 University of Pennsylvania, U.S.A., 2The Chinese University of Hong Kong, China, 3 Shanghai Institute of Medical Genetics, China

G.Gaslini Institute, Genoa, Italy, 2A.O. S. Chiara, Pisa, Italy

Immune reconstitution after allogeneic hematopoietic stem cell transplantation (alloHSCT) involves several components of the immune system (reappearance of functional B cells, T cells and NK cells). This process requires a variable period of time and this immunoincompetence along with occurrence of acute and chronic graft-versus-host-disease (GVHD) may cause significant morbidity and mortality. To better define the mechanisms and kinetic of B cell repertoire reconstitution after allo-HSCT in children, we have examined 12 patients referred to our Institute. The method used in this study is HCDR3 fingerprinting, that allows to analyze the different lengths of CDR3 of the emerging repertoire. HCDR3 is one of Ig heavy chain hypervariable segments that provides essential residues for the direct interaction with antigens. Reverse transcriptase polymerase chain reaction (RT-PCR)was performed on peripheral blood cells at various timepoints pre and post HSCT, two consecutive PCR were performed using specific primers for VH3 or VH6 gene familes and PCR products were separated on denaturing polyacrylamide gels and silver stained. HCDR3 fingerprinting showed in healthy donors 16-20 bands, each band corresponding to a particular CDR3 size and this can be considered the normal polyclonal situation. Patients analyzed before and after transplantation showed a strong oligoclonality (few bands detected) for VH3 family and almost monoclonality when VH6 family was studied. The return to a normal situation (more than 10 bands detected) is about 3-6 months after transplantation in all the patients. Fluorescent activated cell sorter (FACS) analysis with a large panel of monoclonal antibodies was also performed to analize B cell antigen expression and coexpression in the emerging population. Our preliminary data suggest that HCDR3 size distribution and diversification in the emerging repertoire is clonally restricted and displays an adult-type features.

Herein we describe whether human cell factors are present and functional in liver tissue of the human / goat xenogeneic transplant model. Human hematopoietic stem cells (hHSC) purified from umbilical cord blood were transplanted into fetal goats at 55-65 days gestation. After delivery, blood samples were assayed for the expression of human CD antigens and glycophorin A (GPA). Goat liver tissues were examined for the reactivity with monoclonal antibodies against human antigens, proliferating cell nuclear antigen (PCNA) and hepatocyte specific antigen (HSA). Expression of human HSA by the goat liver cells was also determined by FACS analysis. Tqtal RNA in liver was extracted for detection of human serum albumin (hAlE) and hepatocyte nuclear factor (hHNF)-3r3 by RT -PCR. The results showed that successful engraftment was confirmed by the detection of human blood cell chimerism in the circulation of transplanted goats. Human PCNA and HSA were found in liver specimens of transplanted goats but not of normal. FACS showed quantitative difference in the expression of human HSA by liver cells between normal and transplanted goats. RNA analysis showed that hAlE and hHNF-3r3 mRNAs were specifically expressed in liver tissues of chimeric goats. We conclude that goats en grafted with hHSC are capable of producing chimeric liver consisting of human cells. The presence of human hepatocytes in the liver tissue of such transplanted goats indicated the differentiation and reprogramming of hematopoeitic stem cells into hepatocytes. Furthermore, the chimeric goat livers were shown to be transcriptionally active for human hepatocyte specific genes.

130

Abstracts / Experimental Hematology 30 (2002) 37-146

Characterization of Chromatin-binding protein HMG-4 373 Function in hematopoietic stem cells *M. Nemeth , D. Curtis , A. Cline , S. Anderson , L. Garett , 1

2

3

1

1

D. Bodine1, 1National Human Genome Research Institute, U.S.A.,

Comparison Study of Human Fetal and Adult Bone 375 Marrow Derived Mesenchymal Stem Cells *Y. Hu1, L. Ma2, G. Ma2, X. Jiang2, R. C. Zhao2, 1

State Key Lab of

Hematology, China, 2PUMC&CAMAS, China

2 Royal Melbourne Hospital, Australia, 3National Human Genome Research Institute, Genetics , U.S.A.

To identify genes critical for hematopoietic stem cell (HSC) function we performed subtractive hybridization to detect genes preferentially expressed in the primitive lin- c-ki~ population of murine bone marrow. One gene we identified is the X-Iinked gene Hmg-4, a member of a family of chromatinbinding proteins that facilitate binding of transcription factors to DNA. To determine the role of Hmg-4 in HSC biology, we generated “knock-in” transgenic mice using homologous recombination to insert an IRES-GFP cassette into the 3’ UTR ofHmg-4 in male 129 mice. Lin- c-kit+ bone marrow cells from these mice were sorted into GFP +1- populations for use in different assays. In CFC assays, we found a 2.5-fold increase in the ability of GFP- cells to form CFU-C compared to GFP+ (p < 0.01). In competitive repopulation studies, 30,000 lin- c-ki~ GFP+ and 50,000 lin- c-ki~ GFP- cells respectively were mixed with 107 B6 bone marrow cells and injected into 10 lethally irradiated 129 x B6 F1 mice. Twelve weeks after transplan, hemoglobin electrophoresis demonstrated that mice receiving GFP- cells were 100% B6 phenotype whereas the recipients of GFP+ cells contained 28 :t 5% 129 phenotype. From these results, we hypothesized that GFP expression, and by extension Hmg-4 expression, co-localizes with long- term repopulating ability .To examine Hmg-4 function, we generated Hmg-4 null mice by homologous recombination (Hmg-4 KO). B6 marrow was mixed with equal amounts of control 129 or Hmg-4 KO marrow and 4 x 106 total cells were injected into irradiated 129 x B6 F1 mice. Twelve weeks after transplant, we observed that control 129 cells contributed equally to the repopulation of bone marrow (49 :t 6%), spleen (52 :t 5%), and thymus (39 :t 7%). Hmg-4 KO cells showed great variability in their ability to repopulate different hematopoietic lineages (66 :t 20%, 69 :t 19%, and 42 :t 30% of bone marrow, spleen, and thymus respectively), suggestive of reduced HSC numbers. We conclude that Hmg-4 plays a role in HSC repopulation and that its expression can serve as a marker for primitive hematopoietic cells.

To explore the differences of phenotype and biological characteristics between fetal and adult bone marrow derived mesenchymal stem cells. We cultured human fetal mesenchymal stem cells from 4-5 months old human aborted fetal and normal adult bone marrow low-density mononuclear cells. We performed studies on their growth curve, cell cycle, immunophenotype, ex vivo expansion potential, differential abilities, et al. We demonstrated that adherent fetal and adult bone bone marrow-derived cells cultured in the absence of differentiation stimuli give rise to a population of cells with phenotypical features of mesenchymal stem cells. Fetal and adult bone marrow-derived MSC are similar in cell morphology, antigenic phenotype, et al. Fetal bone marrow derived mesenchymal stem cells are and should be enough to sustain a steady supply of low differentiated cells upon proliferation. This cell may constitute an abundant and accessible cellular reservoir for stem cell bioengineering, otherwise adult bone marrow derived mesenchymal stem cell is more useful in hematopoietic reconstitute in the bone marrow transplant.

The hematopoietic system, DNA integrity and longevity: 374 a genetic connection *H. Geiger , J. True , G. van Zant ,

Modulation of the 1 alpha,25 dihydroxyvitamin D3 376 induced proliferation of a stem cell *D. Ben Alon , I. Nathan , G. Lugassy ,

2

2

1 1 2 1 University of Kentucky, U.S.A., University of Kentucky, Markey Cancer Center, U.S.A.

Previous experimental findings from our group have pointed towards a correlation between regulatory mechanIsms in hematopoietic stem/progenitor cells ;lnd murine longevity. We showed that a significant negative correlation exists between the percentage of hematopoietic progenitor oolls killed by hydroxy~rea (HU) and longevity. We then performed a genome-wide linked locus analysis for the trait longevity In the murIne BXD reCombinant inbred set. of strains and comp;lred the mapped loci to other loci affecting i) the number of hematopoietic stem and progenitor cells, ii) their change in number as animals age and iii) the fraction of progenitor cells killed by HU. Five out of seven loci linked to longevity .are: ;llso linked to a trai~ in the hematopoietic system. Animals reciprocally congenic for loci on chromosomes 7 and 11 show either a reduction or an increase in the number of progenitor cells killed by HU comp;lred to their background strain, thus confirming our linkage analysis (see table).

Congenic animals / Change of the frequency of progenitor cells killed by HU compared to background strain (%) Interestingly, progenitor cells from C57BU6.DBN2 (chr.11. 18-30 cM) animals are mQre sensitive to a radiation dose of 2 Gray (pc; 0.05), compared to C578U6 mIce. Progenitor cells bearing a huYAC encoding RAD50 showed a 12.5% increase in HUsusceptibility, pointing towards RADSO as a possible candidate gene in the interval on chromosome 11. We are currently investigating the molecular mechanisms 0f the phenotype by microarray analysis, comparing gene expression patterns of isolated stem cells of C57Bl/6.DBA/2 (chr.7. 0-37; chr. 11’ 0-30 cM) animals with C578BL/6 animals. Based on possible candidate genes in the mapped regions and in accordance with the reported phenotypes of the congenic animals, we proPQ~Q that DNA intesrity i~ :I rnsjor factor that regulates mammalian stem cells and longevity of mammals.

1

2

1 1

Barzilai Hospital, Israel,

Ben-Gurion University, Israel

The effect of 1 alpha,25 dihydroxyvitamin D3 (1,25(OH)2D3) on the proliferation of the TF-1 line of stem cells from erythroleukemia origin was investigated. In a medium supplemented with fetal calf serum (FCS) or with charcoal stripped FCS, 1,25(OH)2D3 enhanced TF-1 cells proliferation significantly. The cells responded to erythropoietin (Epo) and to 1,25(OH)2D3 by increased proliferation as measured by XTT method, labeled thymidine incorporation and counting. Their simultaneous addition potentiated cell proliferation in a synergistic manner. Calcium modifiers like Thapsigargin, an intracellular calcium releaser, and Ionomycin a Ca2+ ionophore enhanced the effect of 1,25(OH)2D3. BAPTA-AM, an intracellular Ca2+ chelator, caused a decrease in the proliferative effect of 1,25(OH)2D3. These results suggest a role for [Ca2+]i in mediating the proliferative effect of 1,25(OH)2D3 on the cells. However, there was no indication of a role for intracellular Ca2+ in the synergistic interaction between 1,25(OH)2D3 and Epo. The protein kinase C (PKC) inhibitor GF 109203X increased the 1,25(OH)2D3 activity suggesting an inhibitory effect of PKC on the 1,25(OH)2D3 induced proliferation. As for the interaction of 1,25(OH)2D3 and Epo, increasing intracellular Ca2+ or inhibiting PKC diminished the synergistic effect and the growth response to Epo. When working with serum free medium the proliferative effect of 1,25(OH)2D3 changed to an inhibitory effect but the synergistic effect of 1,25(OH)2D3 with Epo increased. Addition of vitamin D binding protein to the serum free medium did not change the inhibitory effect of 1,25(OH)2D3, pointing to the existence of a non steroid substance in the FCS capable of modulating the 1,25(OH)2D3 effect on the cells. The results shed light on the action of 1,25(OH)2D3 on early hematopoietic progenitors that involve Ca2+ ions.

Abstracts / Experimental Hematology 30 (2002) 37-146

131

Flt3 expression within the Lin-Sca1+c-kit+ stem cell pool 377 defines a novel progenitor population with lymphomyeloid but lineage-restricted reconstitution potential

The Long-Term Engraftment of Cord Blood CD34+ Cells 379 After Ex Vivo Culture *R. Brown , J. Zhang , G. Almeida-Porada , R. Herzig ,

*J. Adolfsson1, O. Borge2, D. Bryder2, K. Theilgaard-Mönch3, I. Åstrand-Grundström2, E. Sitnicka2, S. Jacobsen2, Y. Sasaki2,

E. D. Zanjani3, 1Quality Biological, Inc., U.S.A., 2James Graham

Laboratory Medicine, Sweden, 2Lund University, Sweden, 3 Copenhagen University, Denmark

1

2

3

2

Brown Cancer Cent, U.S.A., 3University of Nevada, Reno, U.S.A.

1

Flt3 and its ligand have been proposed to be involved in regulating the hematopoietic stem cell (HSC) pool. However, whereas the bone marrow Lin-Sca1+c-kit+flt3+ population is highly enriched in longterm repopulating HSC, Lin-Sca1+c-kit+flt3- cells completely lack such activity, but efficiently reconstitute B, T, and NK cells in vivo (Adolfsson et al Immunity 15:659, 2001). Although failing to in vivo long-term reconstitute myelopoiesis, in vitro single cell assays and short-term (1-2 weeks) in vivo reconstitution analysis revealed that virtually all Lin-Sca1+c-kit+flt3+ cells also have a granulocytemacrophage differentiation potential . In contrast, Lin-Sca1+ckit+flt3+ cells (unlike Lin-Sca1+c-kit+flt3- cells) have no ability to produce megakaryocytes in vitro. Furthermore, Lin-Sca1+ckit+flt3+ cells lack ability to reconstitute megakaryocyte or erythroid lineages 1-3 weeks following transplantation to lethally irradiated mice, although high levels of granylocyte-macrophage reconstitution could be observed. Thus, flt3 expression identifies a novel and primitive reconstituting progenitor cell within the LinSca1+c-kit+ HSC compartment, with a combined granulocyte, macrophage, B and T cell lineage potential, but with little or no megakaryocyte and erythroid differentiation potential.

Human Hematopoietic Stem Cells Can Be Induced 378 to Differentiate into Astroglial Cells In Vitro *H.-N. Hao , J. Zhao , R. L. Thomas , W. Lyman ,

1 2 2 2 1 Wayne State University, School of Medicine, U.S.A., 2Wayne State University, U.S.A.

Studies that used rodents have shown that neural cells can be generated from hematopoietic stem cells (HSC) in vivo. To test if HSC are able to give rise to neural cells, we incubated fetal human liver (FL) human CD133+/CD34+/CD3- HSC cells on the Poly-D-Lysine (PDL) coated dishes with either astrocytes in double-chamber tissue culture system or neural cell culture conditioned medium (astrocyte-enriched cell culture medium) for 14 days. Morphology characterizations of PDL adhering cells were studied using immunocytochemistry with neural specific marker antibodies. The immunostain results demonstrated that GFAP/S100 positive astroglial cells could be induced from fetal human liver derived HSC under these culture conditions. To facilitate the study of the multipotential precursor cell gene and cell phenotype specific protein expression, we estimated the bone morphogenetic protein 2 (BAM2), nestin, glial fibrillary acidic protein, O4 and beta-tubulin III mRNA and protein expression using RT-PCR and western blot assays. Both BAM and nestin mRNA expression were detected after HSC were cultured with neural-cell conditioned medium for 6 days. During differentiation, HSC only generated GFAP positive cells without showing oligodendroglial and neuronal makers under these culture conditions. These findings suggest that HSC are able to generate neural cells and the multipotential stem cell differentiation depends upon the growth environments.

Ex vivo expansion regimens for cord blood (CB) CD34+ cells that maintain their long-term engrafting ability holds great promise for adult transplantation. Data presented delineates clinically relevant ex vivo culture conditions that support long-term engrafting ability of CB CD34+ cells. CB CD34+ cells were cultured over 14 days in a clinical-grade serum-free medium, QBSF-60, supplemented with SCF, Flt-3 and Tpo. After 7 days, there was a median 55-fold expansion of nucleated cells, 26-fold expansion of CD34+ cells, and 47-fold expansion of CD34+CD38-Dr- cells. Expansion through day 14, yielded a median fold expansion of 545-fold for nucleated cells, 84-fold for CD34+ cells, and 47-fold for CD34+CD38-Dr- cells. The total number of LTC-IC cells increased during the expansion. Using the fetal sheep model for human hematopoiesis, cells expanded 7 days engraft and undergo multilineage differentiation in primary and secondary recipients equivalent to unexpanded cells. Interestingly, the 7 day expanded cells produced a higher level of differentiated cells in the primary animal than the unexpanded cell population, raising the possibility that expanded CB products may bring about an earlier reconstitution of the host. Day 14 expanded cells engrafted and produced multilineage blood cells in primary recipients, but not secondary recipients. CD34+ cells isolated from day 14 product transplanted into primary recipients resulted in significant engraftment and multilineage differentiation. Despite the presence of CD34+ cells in the bone marrow of these primary recipients, the cells failed to engraft secondary recipients. CD34+ cells isolated from the day 14 product that were transplanted into primary recipients resulted in significant engraftment and multilineage differentiation. The level of engraftment/blood formation was higher in these animals as compared to primary host transplanted with the un-selected product of the day 14 expansion. Of note, was the finding of a higher percent of CD34+ cells in this group of animals as compared to the un-selected expanded population. Despite the presence of CD34+ cells in the bone marrow of these primary recipients, the cells failed to engraft secondary recipients. These studies suggest it is possible to at least maintain longterm engrafting CB stem cells for 7-14 days under clinically relevant culture conditions.

A Novel Technique for Long-term Cryopreservation 380 of Hematopoietic Progenitor Cells using Intracellular Trehalose *S. Buchanan1, S. Gross1, J. Acker2, M. Toner2, J. Carpenter1, D. Pyatt1, 1University of Colorado, U.S.A., 2Harvard University, U.S.A. The purpose of this project was to establish an alternative methodology for the cryopreservation of hematopoietic progenitor and stem cells (HPC) using low concentrations of intracellular trehalose. We introduced this disaccharide into HPC using a genetically engineered mutant of the pore-forming protein, a-hemolysin from Staphylococcus aureus. By regulating extracellular zinc concentrations, this pore can be selectively opened and closed allowing for the diffusional transport of trehalose across the cell membrane. The human hematopoietic cell line TF-1 was used as a surrogate for primary HPC during these studies. Initial experiments were conducted to characterize the effects of poration on TF.1 cells and establish optimal conditions for trehalose loading and survival of the cells. After poration, cells were incubated in 200mM trehalose for 1 hour, pores were closed and the cells were frozen in a rate-controlled freezer and stored for one week at –80 C. Experimental cells were rapidly thawed and cultured either in liquid media or methylcellulose fortified with GM-CSF, IL-3, SCF and EPO. Endpoints measured after freezing included viability, differentiation ability and clonogenic output. Data obtained with this technique were compared to traditional freezing protocols using 10% DMSO. Freshly cultured, unfrozen TF.1 cells served as positive controls. Predictably, cells frozen with no cryoprotectant did not survive. Colonies generated from cells frozen in DMSO were 98% of control values while porated cells frozen with intracellular trehalose were 92% of control values. Additionally, no alterations were observed in the differentiation capabilities of frozen cells with either method. These data demonstrate that low concentrations of trehalose protect HPC during freezing and thawing. Additional studies are being conducted on primary HPC isolated from human cord blood.

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Biodistribution of Gene-marked Mesenchymal Stem Cells 381 in Immunocompetent Fisher Rats *R. Alur, J. Hendricks, S. Campbell, P. Vanguri,

Co-culture with Human Brain Endothelial Cells 383 Differentially Maintains Human Bone Marrow SCIDrepopulating cells in Vitro in the Absence of Exogenous Cytokines

Inc., U.S.A.

*J. Chute1, J. Park1, V. Tanavde2, D. Harlan2, C. Civin2,

Osiris Therapeutics,

1 NIDDK/Naval Medical Res Ctr, U.S.A., 2Johns Hopkins University, U.S.A.

Mesenchymal Stem Cells (MSCs) can be efficiently transduced with retroviruses to express high levels of transgene in vitro and in immunocompromised rats. In this study, we investigated the fate of gene marked rat MSCs (rMSCs) in immunocompetent rats in the absence of pharmacological Immunosuppression. Fisher rMSCs were transduced with a retroviral vector containing β-Galactosidase(β-Gal)-IRES-NeoR. Transduced MSCs pre-labeled with a nuclear marker DAPI (4’6-Diarnidino-2 phenylindole Dihydrochloride hydrate) were delivered to Fisher rats via intramuscular (IM) or intravenous (IV) injections. Animals were sacrificed on days 3,7, 14,21 and 28. In addition to skeletal muscles, various organs and bone marrow were collected and frozen for histochemistry and genomic DNA extraction. Donor MSCs were detected on frozen sections by fluorescence microscopy for DAPI. β-Gal expression was analyzed on frozen sections using X-gal as substrate. Muscles injected with r3-Gal-MSCs (about 60% β-Gal-positive) showed r3-Gal expression at all time points, but the number and intensity of r3-Gal positive cells decreased with time. However, transgene independent retention of DAPI- labeled cells was higher in the muscles at 28 days. This disparity suggests depletion of β-Gal-MSCs by non-specific and/or antigenspecific immune response against MSCs expressing the β-Gal protein, and/or potential silencing of retroviral transcription. Infiltration of macrophages to the site of injection was observed with acid-phosphatase staining. Following IV injection, β-Gal- positive cells were detected in bone marrow smears at all time points in several animals. β-Gal positive cells were not seen in any other organ with IV or IM injections. A FRET based Real Time PCR for the NeoR gene was developed, which allowed specific and quantitative measurement of β-Gal-IRESNeoR transduced rMSCs in culture. Using this assay to assess the engraftment in vivo, NeoR copies were detected in genomic DNA extracted from muscles, kidney and bone. In conclusion, rMSCs injected into muscle survived and expressed trans gene for up to 4 weeks. Thus, muscle can be used as a depot for MSC based gene delivery of proteins or for localized muscle specific expression. IV infusion of cells was followed by MSC detection in the bone marrow, in preference to other organs. The route of delivery of gene marked MSCs should be tailored to the therapeutic g~ne and the specific disease under study.

In vitro culture of hematopoietic stem cells with cytokine combinations designed to maximize cell expansion results in consistent losses of primitive repopulating cells. We recently reported that co-culture of human bone marrow (BM) CD34+ cells with a human brain endothelial cell line (HUBEC) in the presence of GMCSF+IL-3+IL-6+SCF+Flt-3 lig supported a 5.5 fold increase in SRC freqency, whereas exposure to the identical cytokines in the absence of HUBEC resulted in a complete loss of SRC. In this study, we investigated whether human BM SRC could be more optimally expanded via contact with HUBEC in the absence of exogenous cytokines. Using Ki67 and 7AAD intracellular staining, we determined that at day 7 of adhesion to HUBEC, 9.5% of the CD34+CD38- subset had exited G0 and entered G1, 2.8% had entered G2/S/M phase, and 87.5% remained in G0. Consistent with this low level of proliferation, HUBEC adhesion caused a 2.5 fold increase in total cells, but CD34+ cell numbers and total CFC did not increase as compared to input. In contrast, HUBEC-cultured cells (starting dose: 1x10e6 BM CD34+) engrafted in 12 of 19 NOD/SCID mice (63%)(mean 4.6% huCD45+ cells) with tri-lineage differentiation, whereas unmanipulated BM CD34+ cells injected into NOD/SCID mice at the same dose engrafted in only 2 of 10 recipients (mean 0.4% huCD45+ cells). These data indicate that human brain endothelial cells, in the absence of exogenous cytokines, preferentially activate the most primitive hematopoietic cell subsets. Culture conditions which favor low level cell division within primitive hematopoietic populations may represent a more effective strategy for the clinical expansion of adult source stem cells.

Marked regional stem/progenitor cell distribution 382 differences within different marrow compartments *J.-F. Lambert, G. Colvin, J. Carlson, P. Quesenberry,

Transplantation of Human hematopoietic stem cells 384 into fetal goats under B-Scan ultrasonographic guidance: An attractive approach to Prenatal therapy

Roger

Williams Medical Center, U.S.A.

*S.-Z. Huang, F. Zeng, Z. Ren, Y. Zeng, Shanghai Institute of Medical Genetics, China

Experimental murine studies utilize predominantly hind limb marrow as a source for transplantation and analysis. However, significant regional differences within and between marrow compartments have been reported. We addressed both in vitro and in vivo stem/progenitor cell potential of different mouse marrow compartments using 7-factor stimulated HPP-CFC progenitor assay with 14 days incubation in double agar dishes and competitive transplantation. Engraftment potential was assessed by a competitive transplant assay using whole marrow from HBSS flushed-6hind-bones (B6.SJL, CD45.1) competed with several (C57BL,CD45.2) skeletal marrow compartment groups. Assessed was flushed-6-hind-bones (control), crushed-6-hind bones, crushed-spine, crushed-femurs, and crushed-all-bones into lethally irradiated CD45.2 recipients. Alternatively, marrow from the different compartments (B6.SJL) was directly transplanted in 2 Gy irradiated hosts (C57BL). We measured peripheral blood engraftment by FACS from 3 to 6 weeks post-transplant using a FITC conjugated antiCD45.1 MoAb. We observed that bone marrow isolated from crushed spine (30% of total cellularity) had a 6.8 fold increase of HPP-CFC progenitor numbers compared to flushed-6-hind-bones control. Crushed-6-hind-bones and crushed-all bones were 3.8 and 3.7 fold higher than control respectively (all P<0.01). Peripheral blood engraftment in the crushed-spine group at 6 and 9 weeks was 160+/-20% and 167+/-40% of the flushed-6-hind-bones control group, while engraftment levels in crushed-6-bone, crushed-femur and crushed-all-bones were similar to controls. Bone marrow from different skeletal compartments show notable differences in progenitor or engraftable cells content and potential. These observations point to regional differences in progenitor or engraftable stem cell content of different bony areas.

In utero transplantation of hematopoeitic stem cells (hHSC) into fetuses for propagation, expansion and differentiation could become a methodology of choice for prenatal treatment. However, the routine surgery procedures are often traumatic, and result in a high risk of miscarriages; therefore it is not clinically applicable at this time. Recently, we developed a safe and reliable protocol for transplantation of human hHSC into fetuses to produce chimeric goats. We injected I x 105 Lin-CD34+CD38- or CD34+ cells obtained from human cord blood directly into each of 20 fetal goats’ peritoneal cavity between 45-55 gestation using B-scan ultrasonograph as guidance. The total procedure was completed within 5 minutes. After that we examined the fetal development with B-scan ultrasonographs at regular intervals. At present time, 8 normal goats were born. The other ten pregnancies appear to be normal, which assures normal deliveries. There were two miscarriages. The 8 live-born goats indicated positive for human CD34 and GPA in their blood as detected by FACS and molecular analysis. FISH experiment confirmed human cell chimerism. Furthermore, human-like liver and kidney cells were present in chimeric livers and kidneys by immunochemistry analyses. We also conducted transplantation on another 50 fetal goats using regular surgical method for comparison and 11 fetal goats were aborted. The percentage was twice as high. The fact that the new procedure makes it possible for transplantation at an early gestation period, reduces immunological responses, and without surgery, these decreased the possibility of miscarriages. We believe our new approach will be useful for prenatal treatment of severe and incurable congenital diseases in the future.

Abstracts / Experimental Hematology 30 (2002) 37-146

133

Selective depletion of donor-reactive cells as a 385 nonmyeloablative conditioning for allogeneic bone marrow transplantataion

Microchimerism and clinical evaluations in pigs submitted 387 to sequential kidney and bone marrow transplantation as experimental protocol to ameliorate the organ tolerance

*T. Prigozhina1, O. Gurevitch2, G. Elkin2, S. Morecki2, E. Yakovlev2, S. Slavin2, 1Hadassah University Hospital, Israel, 2Hadassah

*M. Bonfichi1, E. Ferretti2, C. Marseglia2, F. Abbiati3, M. Alessiani3, P. Bernasconi2, E. Alessandrino2, 1IRCCS, Institute of Hematology,

Hospital, Israel

Italy, 2I. Ematologia IRCCS S.Matteo, Italy, 3I. Pat. Chir I Univ of Pavia, Italy

Application of bone marrow transplantation (BMT) in clinical practice is limited by graft failure and graft-vs.-host disease (GVHD). Lowering the intensity of immunosuppressive conditioning for BMT improves the safety of the procedure but can be associated with graft rejection. We developed a new approach for BMT that provides sustained GVHD-free engraftment of allogeneic BM after mild irradiation (200cGy). The main innovation of our protocol consisted in replacement of the intensive irradiation by selective depletion of donor-reactive (DR) host cells before BMT. DR cells of recipient (BALB/c) were activated by inoculation with a large dose of donor (C57BL/6) BM and one day later eliminated by the injection with a nonmyeloablative dose of cyclophosphamide (200 mg/kg). A second transplantation with BM from C57BL/6 donors converted recipients to complete chimeras and induces robust tolerance to donor BM stromal precursors, heart muscle and skin grafts. Allografts from third party CBA donors were rejected by chimeras soon after transplantation. Less than 20% chimeras died from GVHD within the observation period (1 year). BMT to recipients inoculated with BCL1 leukemia cells or transplanted locally with 4T1 breast carcinoma prevented development of malignancy in 90% leukemia bearing mice and in 40% recipients harboring breast carcinoma cells. Immunization of donors of the second BM with 4T1 cells prevented development of breast carcinoma in 80% of 4T1 inoculated mice. Animals treated for malignancy died less from GVHD than mice transplanted with allogeneic BM after intensive irradiation. However, late GVHD-related mortality in mice treated for leukemia was higher than after nonmyeloablative BMT to naive recipients. Infusion of host-type anti-donor immune lymphocytes 8 days post BMT improved survival of recipients treated for leukemia without affecting engraftment and the GVL potential of donor BM. These results show that mild conditioning for selective depletion of DR host cells before mismatched BMT has a strong therapeutic potential both for organ transplantation and for treatment of cancer.

Human hematopoietic stem cell transplantation to treat 386 hereditary hepatic related disorders *G. Almeida-Porada , C. Porada , E. Zanjani ,

1 2 1 1 University of Nevada at Reno, VA Medical Center, U.S.A., 2University of Nevada at Reno, U.S.A.

Recent studies demonstrated that hematopoietic stem cells (HSC) are able to generate hepatocytes after transplantation, providing optimistic evidence that HSC could potentially be used clinically to generate hepatocytes for replacing damaged or deficient liver tissue. The fetal sheep model of in utero hematopoiesis has been pivotal in laying the ground work for new therapeutic approaches in utero. In the present studies, we utilized this unique system to study the feasibility of using in utero transplantation to ameliorate hereditary hepatic related diseases. To this end, we attempted to determine the optimal source of HSC for producing hepatocytes in vivo by examining the ability of human adult bone marrow (BM), cord blood (CB), and mobilized peripheral blood (mPB) HSC to produce significant numbers of functional human hepatocytes, endothelium and biliary cells in vivo. CD34+Lin- or CD34-Lincells were isolated from adult BM, mPB or CB, transplanted into fetal sheep recipients and at 60 days post-transplant analyzed for HSC engraftment as an indicator of a successful transplant. The highest level of HSC engraftment was achieved with mPB (3-6%) followed by CB (3-4%) and BM (1.9-2.3%). The potential to generate hepatocytes in vivo was evaluated at the same time point by immunohistochemistry with monoclonal antibodies against human albumin and hepatocytes and with in situ hybridization using a human Aluspecific probe. Human adult BM CD34+Lin- cell population generated hepatocytes with the most efficiency (0.6-1.2% human hepatocytes) followed by adult BM CD34-Lin- cells(0.4-0.6% human hepatocytes). CB CD34-Lingenerated roughly 0.5% human hepatocytes while their CD34+ counterpart generated only 0.2%. Very few human hepatocytes were produced by the CD34+Lin- fraction from mPB, while the CD34-Lin- cells from mPB generated 0.2-0.4% human hepatocytes. When other phenotypes of hematopoietic cells were tested under the same conditions, certain phenotypes were able to generate more hepatocytes/ HSC dose than others, suggesting that it may be possible find an HSC population that is enriched for hepatocytic potential to treat selected diseases in utero.

The solid organ acceptance seems to be increased by the appearance of microchimerism in recipient. This fact is reported to be increased by infusion of donor bone marrow (BM) cells. In 11 out 14 outbred non related piglets submitted to heterotopic kidney transplantation (KT) [group A= no immunosuppressive therapy (IS) (3 cases), group B= IS (0.6 mg/Kg/die oral FK 506+10 mg/Kg bid oral mycophenolate mofetil: from day 0 till +30)(5 cases), group C= IS (as group B)+ BM infusion at day +7 (1,25±0,73x108/kg mononuclear cells from donor iliac crest) (6 cases)] we evaluated the presence of microchimerism after kidney plus bone marrow transplantation or after kidney transplant alone. We previously reported that only in C group the majority of subjects, 4 over 6 animals considered, alived and well the end of the study (90 days). These animals maintained an acceptable BM viability with persistence of CFU-GM growth. Instead in group A 2 animals died for acute cellular reject (ACR) after 8 and 11 days and in group B only one animal survived well till day +90 while 4 subjects died at +17, +21, +56 and + 64, (respectively peritonitis, 2 pericarditis and pneumonia). To allow the evaluation of donor/recipient microchimerism, all the recipient were female while donors were male. In recipient mononuclear bone marrow cell samples at day: 0, +7, +15, +30, +45, +60, +90 from kidney transplantation a specific 163-bp region of donor’s Y chromosome (Sry, sex determining region Y) was amplified. After the kidney transplant the Y band was found in all the examined sequences, independently that they were from group B or C. This fact may be symptomatic of presence and persistence of donor cell after the solid organ transplantation. It is still dubious if the infusion of bone marrow cells increases the amount of donor hemopoietic cells in the recipient bone marrow to consent a correlation with better survival showed by C group.

Hepatocyte growth factor increases human albumin 388 expression in the livers of immune deficient mice transplanted with highly purified human hematopoietic stem cells *X. Wang, S. Ge, G. McNamara, Q. Hao, G. Crooks, J. Nolta, Children’s Hospital Los Angeles, U.S.A. Purpose of the Study: To determine whether highly purified human hematopoietic stem/progenitor cells have the capacity to form hepatocytelike cells in vivo, in an immune deficient mouse model of stem cell plasticity. Methods: CD34+ or highly purified CD34+CD38-CD7- human hematopoietic stem cells from umbilical cord blood and bone marrow were transplanted into immunodeficient mice, following sublethal irradiation. One month post-transplantation 0.4ml/kg carbon tetrachloride (CCl4) was administered into the mice to induce massive liver damage and hepatocyte proliferation. Mice were analyzed one month after liver damage, in comparison to CCl4 injured and non-injured controls transplanted with the same stem cell populations. Results: Human-specific albumin mRNA and protein was expressed in the mouse liver, and human albumin was detected in the murine serum, in mice that had received CCl4 injury. Human AFP was never expressed in the livers of any of the mice, but in some mice human cytokeratin 19 was expressed, which may indicate bile duct development in addition to the albumin secreting hepatocyte-like cells. Human albumin was not expressed in the starting stem cell populations, in injured, non-transplanted mice or in noninjured mice that had been transplanted with human stem cells. Human albumin expression was only detected in human stem cell transplanted, CCl4 – treated mice. Expression of human albumin was significantly increased by administration of human hepatocyte growth factor (HGF) 48 hours after the CCl4-mediated liver injury. Conclusion: Our studies provide evidence that human “hematopoietic” stem/progenitor cell populations have the capacity to respond to the injured liver microenvironment by inducing albumin expression

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Incidence and features of expanded large granular 389 lymphocytes (LGL) following allogeneic stem cell transplantation: a long term analysis

High incidence of fatty liver and low serum testosterone 391 level in long term surviving patients who underwent allogeneic and autologous bone marrow transplantation in

*M. Mohty, C. Faucher, N. Vey, C. Chabannon, D. Sainty, C. Arnoulet, B. Gaugler, D. Maraninchi, D. Olive, D. Blaise,

childhood *Y. Yasuda, T. Shimizu, Y. Inoue, H. Yabe, S. Kato, Tokai University,

Institut Paoli Calmettes, France

Japan

Large granular lymphocyte (LGL) proliferation follows a chronic course during which major features are cytopenia and immune abnormalities. Persistently elevated numbers of LGL have been shown to be increased in few cases after allogeneic stem cell transplantation (allo-SCT). In the present report, we undertook a retrospective analysis of all LGL cases occurring following allo-SCT in a large cohort of 201 patients. The aim of the study was to determine clinical and biological features associated with LGL expansion following allo-SCT. Six cases could be documented over a long follow-up period of seven years. We demonstrate that LGL expansion occurs more frequently following a reduced preparative regimen (4 cases), as compared to conventional myeloablative regimens (2 cases) (incidence 8.2% vs. 1.3%, P=0.04). Expansion of LGL was seen between 3 and 15 months following allo-SCT. Hematopoiesis, with mild to severe cytopenia, was a privileged target for LGL. Different autoimmune manifestations like polyarthritis and hypergammaglobulinemia were also observed. LGL proliferation was observed in the context of chronic antigenic stimulation associated with recurrent viral infections especially CMV. Moreover, 5 patients out of these 6 high risk patients, achieved a long term complete remission concomitant or following LGL expansion. These data suggest that LGL are a subset with the properties of effector lymphocytes which may represent an important component of the graft versus tumor effect.

Long-term surviving bone marrow transplant (BMT) recipients often show elevated liver enzyme levels, and fatty liver can be one of the causes of such liver abnormalities. We investigated the incidence, the severity and the course of fatty liver in 86 patients who had been given BMT and had been followed up at least once a year for 2 to 20 years (median 9 years) after BMT. There were 8 autologous and 78 allogeneic BMT recipients. 48 had malignancies and 38 non-malignancies. 63 patients received radiation-based conditioning, and 23 non-radiation. 29 patients (33.7%) had fatty liver on ultrasonography and/or abdominal CT scan at least once during the follow-up period. The age of diagnosis of fatty liver was 6 to 25 years old (median 14 years old), and the interval from BMT to diagnosis was 1 to 14 years (median 6 years). None of the following factors were associated with the occurrence of fatty liver; age at transplant, age at onset of fatty liver, sex, disease to which BMT was indicated, presence of radiation or any other specific drugs in conditioning, GVHD prophylaxis, severity of acute or chronic GVHD, use of steroids, serum cholesterol or triglyceride level at transplant. Serum AST, ALT and LDL cholesterol were higher in those with fatty liver than in those without. Body mass index tended to be lower in the patients with fatty liver than those without, while percentage of body fat did not differ between the two groups. Serum testosterone level in the boys with fatty liver was significantly lower than those without or normal controls (p<0.01, age matched analysis). Our study showed a very high incidence of fatty liver among BMT recipients and a strong relationship between the presence of fatty liver and low serum testosterone level in boys with with fatty liver. This decreased testosterone level seems to be the result of fatty liver or fat accumulation to the testicular glands.

Donor lymphocyte infusions for prevention of relapse 390 after stem cell transplantation in adults with high risk acute lymphoblastic leukemia

Consolidation following autologous stem cell transplant 392 of non-Hodgkins and Hodgkins Lymphomas *J.-F. Lambert, G. Colvin, R. Rathore, L. Lum, M. Falvey,

*R. Arnold1, G. Massenkeil2, O. Rosen2, B. Doerken2, D. Hoelzer3,

G. Elfenbein, Roger Williams Medical Center, U.S.A.

1

University Hospital Charite, Germany, 2Univ. Hospital Charite Berlin, Germany, 3Univ. Hospital Frankfurt, Germany Patients with high risk ALL (Ph+, high WBC and/or complete remission >4 weeks) are candidates for allogeneic stem cell transplantation (SCT) in first complete remission (CR). Despite SCT in CR1 relapse rates are high (2040%). Beyond CR1 the relapse rate after SCT increases. Prognosis of relapsed ALL after SCT is poor and could not be improved with donor lymphocyte infusions (DLI)(Collins et al). We therefore studied the effect of prophylactic DLI on the incidence of relapse after allogeneic SCT. 14 patients with median age of 29 years (18-35) underwent allogeneic SCT (related donors n=10, unrelated donors n=4). Stage of disease was CR1 in 6 patients (Ph+ n=3, präT/T n=2, secondary ALL n=1), CR2 in 3 patients (präB n=2, T n=1),relapse in 4 patients präB/cALL n=3, T n=1) and induction failure (cALL n=1). Conditioning regimen included 12 Gy TBI and chemotherapy (VP16 60 mg/kg n=2, Cyclophosphamide 120 mg/kg n=7, VP16 50 mg/kg + CY 100 mg/kg n=4 in 13 patients and 1 patient received reduced intensity conditioning because of fungal pneumonia. GvHD prohpylaxis was stopped after d +60 post SCT. When no evidence for acute GvHD was observed, DLI were given. In total, 3 doses per patient were planned (starting with 5 x 10^6 CD3+ cells/kg in unrelated transplantation and 1 x 10^7 CD3+ cells/kg in sibling transplantation). When GvHD occurred, DLI were stopped. With a follow up between 11 and 42 months, 10 patients are alive in CR after SCT. 4 patients relapsed and died 6, 19, 21 and 23 months after SCT. There is a correlation between GvHD and remission after SCT in these high risk ALL patients. 9 patients developed GvHD after SCT and 1 out of 9 patients relapsed. 5 patients had no GvHD, even after 3 doses of DLI. 3 out of 5 patients relapsed, 2 out of 5 patients are in remission (>11, >19 months). Conclusion: In adults with high risk ALL DLI might prevent relapse. Larger studies are warranted.

Purpose: To determine whether autologous stem-cell transplantation (ASCT) followed by consolidation with rituximab or irradiation is superior to ASCT alone in adults with advanced or relapsed lymphoma. Patients and methods: Fourteen consecutive lymphoma patients were entered onto this prospective single center study. Seventeen previously transplanted patients with continuous follow-up were used as historical controls. Patients with non-Hodgkins lymphoma (NHL, n=11) or Hodgkins disease (HD, n=3) received ASCT followed by consolidation with rituximab (375 mg/m2 q week x 4 given q 6-months x 5) or irradiation (20-30Gy), respectively. Results: Age, diagnosis (NHL vs. HD), B symptoms, risk factors, LDH, previous response, and histological type were well balanced between the two groups. With a median follow-up of 21.3 months, the 30-month relapse rate (RR) was 23% and 53% (P=0.045), disease-free survival (DFS) was 70% and 41% (P=0.03) and overall survival (OS) was 73% and 47% (P=0.07) for the consolidation group and historical controls, respectively. A multivariate analysis showed that age over 55 and abnormal pre-transplant LDH were predictors of poor outcome. When NHL patients (rituximab consolidation) were analyzed separately (n=24), 30-month RR was 27% and 63% (P=0.08), DFS was 73% and 47% (P=0.05) and OS was 70% and 40% (P=0.06) for rituximab and control arms, respectively. Conclusion: The use of ASCT followed by consolidation using rituximab (NHL) or irradiation (HD) in adults with advanced lymphoma showed markedly reduced relapse rate and improved disease free survival and overall survival compared with conventional ASCT. The NHL subgroup (rituximab) also showed a marked advantage for consolidation.

Abstracts / Experimental Hematology 30 (2002) 37-146

135

Recovery of lymphocyte and dendritic cell subsets 393 following reduced intensity allogeneic bone marrow transplantation

Glivec treatment for Ph-positive (Ph+) leukemias 395 who relapsed before or after allogeneic stem cell transplantation (ASCT) with intensive monitoring of minimal

*M. Mohty, B. Gaugler, C. Faucher, D. Sainty, M. Lafage-Pochitaloff, N. Vey, R. Bouabdallah, F. Viret, D. Blaise, D. Olive, Institut Paoli Calmettes, France

residual disease (MRD) *M. Yamada, K. Miyamura, O. Sasaki, J. Kameoka, K. Meguro, T. Sasaki, Tohoku University, Japan

Approaches using reduced conditioning regimens are being explored with good preliminary results concerning feasibility and engraftment. However many aspects remain under-evaluated and especially few data are available about immune and dendritic cell (DC) reconstitution after these highly immunosuppressive regimens. We present here our data in 20 patients receiving allogeneic bone marrow transplantation (allo-BMT) using a reduced preparative regimen (Fludarabine, busulfan and ATG). We evaluated in the first 3 months following allo-BMT, several immunological parameters including DC subsets, and compared them to historical results obtained in a group of myeloablative allo-BMT patients. We found an early recovery of leukocytes, CD8+ and NK lymphocytes. We also found a trend towards an improved B cell recovery. These results are somewhat in contrast to the altered immune recovery observed in the myeloablative setting. In addition, we found a significant early circulating DC recovery. Circulating blood DCs were also found to be of full donor origin as assessed by FISH in sexmismatched pairs. Nevertheless, naive CD4+CD45RA+ T cells were found to be profoundly reduced following such regimens. This may represent an unfavorable characteristic, predicting an increased rate of viral and other opportunistic infections. The latter has already been reported by our group in this category of patients (Mohty et al., Bone Marrow Transplant., 2000). The observation of early recovery of blood DCs of donor origin is of great importance, since the chimerism status of antigen presenting cells was shown to play a key role in the initiation of GVHD and graft versus tumor effect (GVT). Moreover, during acute GVHD which can correlate with the GVT effect, we could observe in some patients a complete disappearance of circulating DC subsets, thus further supporting this hypothesis. We are currently investigating the use of allogeneic peripheral blood stem cells in the reduced setting. We are also assessing the role of ATG dose, in conjunction with other GVHD prophylaxis regimens. Collectively, these data further enhance the overall benefits of reduced intensity regimens and the need for stringent biological monitoring for assessment of the potential advantages of reduced intensity allo-BMT in comparison with conventional allo-BMT.

The prognosis of patients with Ph+ALL who relapsed before or after ASCT is very poor. Also, that of the CML patients who relapse after SCT is not satisfying. Glivec, a specific inhibitor of BCR-ABL tyrosine kinase, is currently on clinical trials in CML with promising results, and is effective at least several months for Ph+ALL. However, the effects of Glivec in the setting of ASCT has not been established. Here we evaluated the safety and efficacy of the Glivec to the patients who relapsed molecularly or hematologically before or after ASCT by intensive monitoring of BCR-ABL mRNA using real-time quantitative PCR. Case 1. A 32-year-old female received renal transplantation from her farther in October 1998. She was diagnosed as CML in October 1999. She received SCT from her HLA-identical sibling in July 2000. Because of the prophylaxis for the rejection of the renal graft, she had to continue cyclosporine. She developed cytogenetic relapse at 8 months after the transplant. Despite the two times of donor lymphocyte infusions (DLI), eventually 16 of 20 metaphases had Ph. Three months after the treatment with Glivec, Ph chromosome disappeared and number of BCR-ABL decreased from 50000 copies/mg RNA to undetectable-level. Case 2. A 46-year-old male with Ph+ALL received ASCT in August 2000 at his 1st relapse. Though his bone marrow was CR at day28 after BMT, BCR-ABL was detected. After 1.5 months of administration of Glivec, BCR-ABL declined to undetectable level. MRD was not detected at day56 with concomitant occurring chronic GVHD. He remained molecularly CR thereafter. Case 3. A 32-year-old female with Ph+ALL who developed Aspergillus pneumonia just before ASCT at January 2002. We chose Glivec rather than chemotherapy and BCR-ABL mRNA declined to 150 copies before ASCT in March 2002. Case 4. A 34-year-old male with CML developed molecular relapse 12 months after ASCT. He received DLI twice, but eventually he developed cytogenetic relpase. We started Glivec administration in January 2002 and observe the course. The treatment with Glivec currently seems to be promising, and will be explored in this ongoing study.

Toward allogeneic stem cell transplantation (ASCY) 394 without transfusion V. Ivanov, C. Faucher, P. Ladaique, M. Mohty, K. Bilger, D. Sainty,

Unrelated hematopoietic stem cell transplantation for 396 acute myeloidleukemia *N. Basara, L. Kraut, S. Guenzelmann, M. Koldehoff, M. Kiehl,

C. Chabannon, N. Vey, *D. Blaise, Institut Paoli Calmettes, France

A. Fauser, Clinic for BMT and Hematology , Germany

Nonmyeloablative (NMA) ASCT seems to require less transfusion than conventional ASCT. In a retrospective study we addressed this question in the patients we treated in 2001 to determine if hemoglobin (HB) level prior to transplant may have an influence on RBC transfusion requirements after conditioning. In 2001, among 37 patients treated with ASCT, 27 were transfusion independent at time of transplant. Twenty patients were prepared with NMA (FBS: Fludarabine(30 mg/m2x6), Busulfan(1mg/kgx8) and ATG(Thymoglobuline:2.5 mg/kg/dx1) and 7 with conventional TBI-based regiments. The pts with HB level under 11 g/dl were considered as anemic. In the conventional group all 7 pts received RBC transfusion after transplant, independently from the preconditioning HB level(11,6g/dl(9,2–13,8)). They received a median of 4(2-6) RBC transfusions during the first month. First transfusion occurred on day +0,4(-3 – +8). Three patients (40%) required RBC support after day+30. In the NMA group (pre-conditioning HB=10,96 g/dl(9,2–13,5)(p>0,05), 11 pts had an HB level above11g/dl(11,9+0,6g/dl). Three pts in this group did not need any RBC support during allogeneic reconstitution. All others needed just one RBC transfusion during the first month after PBSCT on day +8(3-11). After what all of them became transfusion independent with stable allogeneic erythropoiesis reconstitution. Other nine FBS pts were transfusion independent, but anemic with HB levels of 9,7+0,9 g/dl. All patients were transfused, the first transfusion occurring on day +2,6(-2–+10). 3 pts required just one RBC transfusion, three pts two and three pts 3 transfusions. All of them became transfusion-free before day+22. We conclude that the pts treated with NMA need less RBC transfusions that patients treated with conventional regimens (p=0,003). The pts in NMA group start RBC support later than pts after conventional graft (p=0,02). Furthermore for patients with HB>11 g/dl requirements are minimal if not null. Based on these data we are presently investigating the use of erythropoietin during the weeks preceding the conditioning regimen in order to increase HB level and hopefully decrease RBC transfusions requirement post transplant.Preliminary data will be presented.

AML patients (pts) lacking a suitable sibling donor have a probability of 80% to find an HLA-compatible unrelated donor. Between 1994 and 2000, 68 pts with primary AML received an unrelated HSCT in our center. We analysed overall and disease-free survival in this group of patients. In addition, the outcome of this group of pts was compared to the results of 65 patients with AML treated with HSCT from related donors. Both groups were matched with regard to age, FAB classification, stage of the disease and HLAcompatibility of the donor. Median age of pts was 36 (range 16 to 55; unrelated) and 40 (range 22 to 61; related) years, respectively. The male to female ratio was 33/32 and 33/35, respectively. Conditioning for HSCT consisted either of a busulfan-based regimen in 65 pts (30 related, 35 unrelated) or total body irradiation in 68 pts (35 related, 33 unrelated). Graftversus-host disease prophylaxis consisted of cyclosporine and prednisolone without (related transplants, 72%) or with methotrexate (unrelated transplants, 84%). Seventy nine % of pts with unrelated HSCT and 77% of pts with related HSCT have received an HLA-A, -B, and -DRB1 loci matched transplants. Median and average post-transplant follow-up was 8 and 18 months, respectively, with the longest overall survival being 6 years. Disease free survival (DFS) in the unrelated group of pts at 5 years was 54% for transplants transplanted in first complete remission (CR1) (n=16), 47% in second CR (CR2) (n=16) and 13% for advancedstages of the disease (>CR2) (n=36). Overall survival at 5 years was 47% (CR1), 36% (CR2) and 9% (>CR2). The cumulative incidences for relapse in the unrelated group were 13%, 19% and 44% for the three groups, respectively. DFS in pts treated with related transplants at 5 years was 42% (CR1) (n=25), 31% (CR2) (n=9) and 25% (CR2), (n=31). The cumulative incidences for relapse in related group were 20%, 22% and 48% for the three groups, respectively. In conclusion, the results of this comparative study confirm that unrelated HSCT is an important treatment option for patients with standard and high-risk AML, if sibling donors are not available.

136

Abstracts / Experimental Hematology 30 (2002) 37-146

G-CSF administration after autologous peripheral blood 397 stem cell transplantation (PBSCT). Yes or no? *P. Henon , M. Ojeba-Uribe , Y. Arkam , D. Bourderont ,

Predictive Parameters for Mobilized Peripheral Blood 399 CD34+ Progenitor Ccell Collection in Pediatric Patients *C. J. Lyu, T. Ham, S. Won, C. Yang, S. Lee, B. Kim,

H. Sovalat2, 1I.R.H.T. Hopital du Hasenrain, France, 2CH Mulhouse,

University, Korea

1

2

2

2

Yonsei

France Patients with malignant diseases undergoing autologous PBSCT were enrolled in a prospective study in an attempts to better clarify if G-CSF administration after transplant (Tx) is actually useful. Decision for G-CSF administration was dependent on the number of CD34+38- cells reinfused. 35 patients who were reinfused >5xl04 CD34+38- cells/kg b.w. did not receive post- Tx G-CSF (Group la) when 31 patients transplanted with <5x 104 were administered s.c. G-CSF 5 ~g/kg/day from d+5 (Group IIa). These 2 groups were compared with two control groups: one group of 11 historical patients who received <5xl04 CD34+38cells/kg without post- Tx G-CSF (Group lb ), and one group of 28 patients who were administered post- Tx G-CSF although receiving >5x 104 CD34+38- cells as being enrolled in randomized trials (Group lIb). Considering short term post-Tx hematopoietic recovery kinetics, G-CSF had a significant corrective impact on ANC recovery, as rapid in Group lb as in Group la, when it was consistantly delayed in Group 1b; It was even a little more rapid in Group lIb than in Group lb (p<0.05), and ANC reached a maximum peak significantly higher (p<0.01) in the 2 G-CSF groups than in the 2 non-G-CSF ones. But there was no positive G-CSF impact on short-term platelet and reticulocyte recovery, which were only dependent on the number of cells reinfus~d. There was no more impact of G-CSF on occurrence and degree of mucositis or fever. Long term survey of blood cell counts from 1 to 18 months post-Tx was much more surprising: if long-term ANC kinetics did not differ whichever the group, platelet counts and Hb levels were significantly higher at almost all points in time in Group IIa than in the 3 other groups, these parameters being even much more lowered in Group lIb (p from <0.05 to <0.01). This curiously contrasts with rates of CFU-GM, total CD34+ and 38- cells in the bone marrow not different from those of the other groups at 3, 6 and 12 months post- Tx. Also, the median post- Tx overall survival at 4 years was significantly power in Group lIb than in Group IIa (20% vs 55% ) although the type of patients did not differ in each group. In conclusion, post- Tx G-CSF administration is certainly useful in case of transplantation of low amounts of CD34+38- progenitor cells by shortening the ANC recovery time; On the contrary , it seems to induce deleterious effects on long term hematopoiesis and maybe on overall survival in case of transplantation of high cell amounts.

Objectives: In children it is very important to search the optimum time to start peripheral blood stem cell(PBSC) collection. So we analyzed several predictive parameters for yield of PBSC mobilization. Method: We analyzed data from 19 patients that underwent 31 PBSC mobilization episodes and 44 leukapheresis procedures, retrospectively. In this study, CD34+ cell, total white blood cell(WBC), and monocyte counts in peripheral blood(PB) on the day of collection were used for analysis. Increment of WBC and monocyte count from nadir to harvest day, and [increment of WBC count from nadir to harvest day(dWBC)]/[duration from nadir to harvest day(dT)], [increment of monocyte count from nadir to harvest day(dMONO)]/[dT] were also analyzed as a predictive parameters for yield of progenitor cell mobilization. Results: Linear regression analysis revealed a strong correlation between CD34+ cell value in PB on the harvest day and the number of CD34+ cells collected (R=0.72, P<0.01). WBC counts in PB on the harvest day, and increment of WBC count did not correlate with collected CD34+ cells. The yield of PBSC mobilization showed significant correlation with monocyte counts on the harvest day(R=0.314, P<0.05), increment of monocyte counts(R=0.302, P<0.05), dWBC/dT(R=0.417, P<0.01) and dMONO/dT(R=0.342, P<0.05). Conclusions: Our results suggest that in pediatric patients the PBSC yield can be predicted from the measurement of circulating CD34+ cell concentration on the day of collection and that is correspond to the results previously published. In this study, we found other several predictive parameters for yield of peripheral blood stem cell mobilization. So, pre-harvest peripheral blood CD34+ cell, monocyte counts, the increment of monocyte counts, dWBC/dT and dMONO/dT may be particularly useful tools in determining the optimum time to start PBSC collection in pediatric patients.

Transplantation of Peripheral Blood Stem Cells 398 as Compared With Bone Marrow From HLA-identical Siblings in Patients With Acute Myeloid Leukemia and Acute

Higher freezing concentrations and its effect on ANC 400 and platelet recovery post infusion *M. Dozier, K. Dicke, J. Gandy, M. Patel,

Lymphoblastic Leukemia *O. Ringden1, M. Labopin2, A. Bacigalupo3, F. Mandelli4, U. Schaefer5, J. Vossen6, H. Koc7, E. Gluckman8, F. Frassoni3,

U.S.A.

1 Huddinge Hospital, Karolinska Institute, Sweden, 2Institut des Cordeliers, France, 3Ospedale San Martino, Italy, 4Univ. La Sapienza, Italy, 5University Hospital Essen, Germany, 6BMT Centre Leiden, The Netherlands, 7Ankara University, Turkey, 8Hopital St. Louis, France

Because there is inconsistency with regard to the outcome using allogeneic peripheral blood stem cells (PBSC), compared to bone marrow (BM), in patients with leukemia, we did a retrospective analysis in adult patients with AML (n=2294) and ALL (n=1171) reported to the EBMT and transplanted after 1994 using HLA-identical sibling BMT. The source was BM in 2563 patients and PBSC in 1102 patients. In the AML patients receiving BM, 70% were in CR1 vs. 61% receiving PBSC (p<0.0001). In the ALL patients, the corresponding figures were 62% and 55% in the two groups, respectively (p=0.04). Results: Engraftment occurred in 95% of the patients. Median time to ANC >0.5 x 109/l was 19 days in patients receiving BM vs. 14 for the PBSC group in patients with AML and ALL (p<0.0001). Time to reach platelets >50 x 109/l in AML patients receiving BM was a median of 27 days vs. 17 for PBSC (p<0.00001). For ALL patients, the corresponding days were 30 vs. 16 (p<0.00001). In multivariate analysis, PBSC was the strongest factor associated with engraftment by ANC and platelets. Acute GVHD did not differ in patients receiving BM or PBSC. However, chronic GVHD occurred in 32±2% of the AML patients receiving BM vs. 46±3% among those receiving PBSC (p<0.0001). For ALL patients, chronic GVHD appeared in 40±3% vs. 49±5% in the two groups, respectively (p<0.0001). TRM, relapse, LFS and survival did not differ in AML or ALL patients receiving BM or PBSC. In multivariate analysis, PBSC vs. BM was not significant in patients with AML or ALL. Factors of importance for these outcomes included remission status at transplant, FAB M3, female donor to male recipient, center and age for AML patients and remission status, immunosuppression including methotrexate for ALL patients. In conclusion, for patients with AML and ALL, those receiving PBSC compared to BM had a significantly faster engraftment of ANC and platelets and an increased risk of chronic GVHD. However, acute GVHD, TRM, relapse, LFS and survival did not differ in AML or ALL patients receiving BM or PBSC from HLA-identical sibling donors.

Arlington Cancer Center,

At Arlington Cancer Center, we analyzed reinfusions on 20 patients (14 breast cancer, 3 NHL, 1 Hodgkin’s lymphoma, 1 Multiple Myeloma and 1 AML) between 1999-2000 for final CD34 x 10*6 cells/kg, WBC concentration of the frozen apheresis product and the effect WBC concentration had on days to ANC recovery to 500 cells/mm3 and platelet recovery to 20,000 /mm3. Reinfusions were usually administered over two consecutive days with standard of care antibiotic and antifungal support. The median WBC concentration x 10*6/ml was 510 (183-890). There was no significant difference between days to ANC recovery or platelet recovery between reinfusions of products frozen at > 500 x10*6 WBC/ml (N=18 reinfusions) and products frozen at < 500 x10*6 WBC/ml (N=12 reinfusions), (14+4 days vs 13+3 days [p value=0.14] for ANC recovery and 16+11 days vs 14+9 days[p value=0.24] for platelet recovery, respectively). The concentration of CD34 cells/kg of body weight was higher in the patients receiving apheresis products at concentrations less than 500 WBC/ml (5 x 10*6/kg vs 3.5 x 10*6 kg, p value = 0.028). For those patients who mobilize at less than optimal CD34 percentage of apheresis product, freezing at higher WBC concentrations allows for a reduction in volume and therefore a reduction in DMSO usage, fewer bags and thus less freezer space used. Freezing at higher WBC concentrations does not appear to have an effect on days to ANC recovery to 500 cells/mm3 or platelet recovery to 20,000/mm3. There were no differences associated with a higher or lower concentrated product observed in the quality (clumping, or reinfusion side effects) of the reinfused product.

Abstracts / Experimental Hematology 30 (2002) 37-146

137

Thawed Umbilical Cord Blood (UCB) followed by ex vivo 401 engineering with anti-CD3, IL-2, IL-7 & IL-12 followed by recryopreservation significantly expands and activates

Reinjection of ex vivo expanded non-human primate bone 403 marrow mononucleated cells (BMMNC) strongly reduces radiation-induced aplasia

UCB cytotoxic lymphocytes (CTLs): Potential for UCB: donor lymphocyte infusion (DLI) post UCB transplantation *M. Cairo1, J. Ayello1, J. Kurtzberg2, C. Van de Ven1,

*J. M. Bertho, J. Frick, C. Demarquay, A. Chapel, N. Dudoignon, F. Trompier, J. Aigueperse, D. Thierry, Institut de Radioprotection et

1

Columbia University, U.S.A., 2Duke University, U.S.A.

We have previously demonstrated the ability to significantly expand and activate cryopreserved and thawed (CT) UCB with anti-CD3. IL-2. IL- 7 and IL-12. (Robinson et 81, Exp Hem, 2002, in press) We therefore compared ex vivo engineered CT UCB with cryopreserved-thawed-expanded-recryopreserved- thawed (crEcr) and cryopreserved-thawed-re-cryopreservedthawed- expanded (CTCfE) UCB. Cryopreserved UCB (COBL T) were thawed. resuspended in serum free (SF) AIM V and incubated overnight @ 5% C02, 37°C. Nonadherent cells were cultured in SF AIM V :t anti-CD3 (50 ng/mi), IL- 2 (5 ng/mi), IL-7 (10 ng/ml) and IL-12 (10ng/mi) (AB/CY) for 48 hrs @ 5% CO2, 37°C or re-cryopreserved and re-thawed (CTECT) or not expanded but re- cryopreserved. re-thawed and then engineered as described above (CTCTE) from the same UCB unit. There was significant fold expansion (p<0.001) compared to media alone with respect to CD3, CD4. CD8, CD4/25, CD812S and CD3/4SRO and significant enhancement (p<0.001) of CD3 proliferation, Th1 subset expansion (IFNy) and NK cytotoxicity.

The CTECT method was equivalent or significantly better than the CT method in all parameters with maintenance of >= 900% viability . Therefore, these results suggest that thawing of cryopreserved UCB and ex vivo engineering with a short incubation with this AB/CY cocktail for UCB DLI and re- cryopreserving and re-thawing provides similar or enhanced results as thawing UCB followed by ex vivo engineering (CT) and potentially could be utilized for UCB DLI post UCBT.

de Sureté Nucléaire, France Ex vivo expansion of hematopoietic cells is a promising approach for the treatment of pan-cytopenia. Numerous protocols have been studied, most of them starting from purified CD34+ hematopoietic cells. However, the ex vivo expansion of total BMMNC may present some advantages. BMMNC contain all differentiation stages present in the bone marrow, including precursor cells able to generate rapidly mature circulating cells, but also immature cells, able to ensure a long-term hematopoietic recovery. Moreover, BMMNC also contain accessory cells, including mesenchymal stem cells, which can sustain hematopoietic recovery. In order to assess the therapeutic efficacy of ex vivo expanded BMMNC, we have set up a non-human primate model. Two ex vivo expansion protocols for BMMNC were studied. The first consisted of a 7-day culture in the presence of SCF, Flt3ligand, TPO, IL-3 and IL-6, which induced preferentially the expansion of immature hematopoietic cells (3.1±1.4, 10.0±5.1, 2.2±1.9 and 1.0±0.3 fold expansion for MNC, CFU-GM, BFU-E and LTC-IC respectively). The second was with the same cytokine combination supplemented with G-CSF with an increased duration of culture up to 14 days and induced mainly the production of mature hematopoietic cells (17.2±11.7 fold expansion for MNC and no detectable BFU-E and LTC-IC) although expansion of CFU-GM (13.7±18.8 fold) and CD34+ cells (5.2±1.4 fold) was also observed. Results showed the presence of mesenchymal stem cells and cells from the lymphoid and the megakaryocytic lineages in 7 day expanded BMMNC. To test the ability of ex vivo expanded cells to sustain hematopoietic recovery after radiation-induced aplasia, 6 non-human primates were irradiated at a supra lethal dose of 8 Gy and received the product of either 7 day (24 hours after irradiation) or 14 day (8 days after irradiation) expanded BMMNC. Results showed that the 7 day ex vivo expanded BMMNC shortened the period and the severity of pan cytopenia and improved hematopoietic recovery, while the 14 day ex vivo expanded BMMNC mainly produced a transfusion-like effect during 8 days, followed by hematopoietic recovery. These results suggest that ex vivo expanded BMMNC during 7 days may be highly efficient in the treatment of radiation-induced aplasia.

Application of a human multidrug transporter (ABCG2) 402 variant as a potential in vivo selectable marker in gene transfer to haematopoietic progenitor cells

The role of different stromal support on the maintainance 404 and expansion of haemopoietic progenitor cells M. Berger , *F. Fagioli , L. Gastaldo , C. Niceforo , K. Mareschi ,

G. Varady1, O. Ujhelly1, J. Cervenak1, M. Grez2, A. Varadi3, B. Sarkadi1, *K. Nemet1, 1National Health Center, Hungary, 2George-

E. Biasin1, A. Palmero1, R. Pessolano1, E. Madon1, 1University of

1

2

1

1

1

Speyer-Haus, Germany, 3Hungarian Academy of Sciences, Hungary

Turin, Italy, 2Dept. of Pediatrics - Univ. of Torino, Ospedale Regina Margh, Italy

Stem cell based gene therapy is often unsatisfactory because of the low number of corrected cells. In order to provide a selective advantage for the modified cells we applied the human ABCG2 protein, a resident xenobiotic transporter in stem cells. The relatively small size of the cDNA coding for this protein is a potential advantage in gene therapy application. We used gp91phox as the therapeutic gene for X-linked chronic granulomatous disease (X-CGD), coding for subunit of the phagocyte superoxide-generating NADPH oxidase enzyme. Co-expression of gp91phox and ABCG2 proteins was achieved by constructing a bicistronic retroviral vector. Myelo-monocytic X-CGD (PLB985-CGD) and normal human cord blood CD34+ cells were transduced by the retrovirus. The expression of gp91phox was determined by a monoclonal antibody, while ABCG2 function was measured by mitoxantrone uptake. Transduced cells were selected by mitoxantrone (MX). Granulocyte maturation and the reconstitution of superoxide production were also determined. Five days after PLB985-CGD transduction 45 % gp91phox-positive cells were found, while this ratio increased to 98-100 % as a result of drug selection for 4 days. The selected cells displayed a specific inhibitor-sensitive (FTC) increase in MX extrusion. Full restoration of superoxide production was achieved in the selected cells. After retroviral transduction of CD34+ cells using the same vector as above, MX uptake indicated 36% of ABCG2 positive cells. This ratio increased to 62% after 3 days of MX selection, while non-transduced cells showed no MX extrusion. Granulocyte maturation induced by G-CSF was normal. Retroviral transfer of a chemoresistance gene together with gp91phox to myeloid progenitor cells revealed that these cells had a selective advantage over non-transduced cells, when exposed to drugs.

Long term cultures are models for the study of early progenitor cells (Dexter et al, J Cell Physiol, 1977) Aim of the study. Evaluate the progenitor cell committment over four different stromal supports in terms of cells, CD34+, CD34+/CD38-, CFU-GM and BFU-E. Material and methods. Mononuclear cells were obtained from 8 umbilical cord blood samples from full term deliveries. Cells were separated over Ficoll density centrifugation (Eurobio). Four different stromal supports were compared: 1) human bone marrow stroma, 2) M2-10B4, 3) Sl/Sl and 4) a 1/1 mixture of M210B4 and Sl/Sl (MIX). 6x105 umbilical mononuclear cells were seeded in four plates over stromal cells previously irradiated and seeded at 7x104 cells/cm2. Weekly cultures were demipopulated and aliquots were counted and analysed for CD34+, CD34+/CD38- and for clonogenic ability. For the flow cytometric analysis, cells were incubated with IgG to block aspecific binding (Pharma Biasini) for 20’’ at 4 C, washed and incubated with 5 microliters of antiCD34 and anti CD38 moAb (BD) for 20’’. For the clonogenic test cells were seeded in methylcellulose medium (Methocult) and the colonies were scored 14 days later. Results. After 5 weeks’’ culture the cell fold increase was: 1.5, 2.99, 0.75, and 2.055 for human bone marrow stroma, M2-10B4, Sl/Sl and MIX, respectively. For the CD34+ cells we had: 1.1, 0.285, 0.55 and 0.29 for human bone marrow stroma, M2-10B4, Sl/Sl and MIX, respectively. After five weeks we had no CD34+/CD38cells in all conditions. For CFU-GM we had 5.86, 3.73, 3.9, and 4.4 fold increase for human bone marrow stroma, M2-10B4, Sl/Sl and MIX. We had no BFU-E output after 5 weeks in all conditions. The comparison of human bone marrow stroma vs stromal murine cell lines gave no rise to statistical differences in term of cells, CD34+, CD34+/CD38-, CFU-GM and BFU-E. Conclusion. Data obtained have no demonstrated significant differences among stromal supports in terms of cells, CD34+, CD34+/CD38-, CFU-GM and BFU-E. This could mean that these stromal cells could be used indifferently to identify and monitor LTC-IC content. Experiments are ongoing to evaluate the LTC-IC and CFU-MK maintenance over these stromal supports.

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Telomerase Activity as Early detection of Preleukemia 405 *L. R. Marsaman , T. Achmad , A. Maskoen ,

1 2 2 1 Padjadjaran University, Hasan Sadikin General Hospital, Indonesia, 2 Padjadjaran University, Indonesia

The diagnosis of concealed hemoblastosis and minimal 407 residual disease by means of original culture methods *T. Saralidze , T. Shvelidze , L. Mochevishvili , C. Zakareishvili , 1

1

1

1

K. Rusishvili1, P. Gabunia2, A. Shengelaia1, 1Institute of Hematology and Transfusiology, Georgia, 2AIDS and Inf Diseases, Georgia

OBJECTIVES : Telomerase activity is important for carcinogenesis. High telomerase activity was detected in patients with cancer, including children with leukemia. Recognition of the preleukemic syndrome is difficult and often is done in retrospective. Somepatients have initial diagnosis as Aplastic Anaemia. The aim of this study was to find out whether patients with aplastic anaemia who become leukemia have had higher telomerase activity at the time they diagnosed as Aplastic Anaemia . METHODS: We analyzed telomerase activity in leukocytes taken from peripheral blood (PB) specimens of the subjects at the time they were diagnosed. Subjects were all pediatric patients who diagnosed as leukemia or aplastic anaemia. Telomerase activy was detected using Telomeric Repeat Amplifiction Protocol (TRAP) assay. Patients who diagnosed aplastic Anaemia were followed up clinically as long as 6 months for the possibility to develop Leukemia. The data was analyzed using chi square analysis RESULTS : The mean value of telomerase activity in healthy children was 0.31.u/2 million cells . Higher telomerase activity was detected in almost all 21 pediatric patients with Leukemia ( 11 ALL, 9 AML, 1 CML ) with mean value 0.88 u/2 million cells . However heterogenicity of telomerase activity was found amongst this group. Among 14 patients diagnosed as Aplastic Anaemia at the first time of diagnosis, 6 patients remained aplastic anaemia while 8 patients became leukemia ( Preleukemia). Surprisingly high telomerase activity was detected in these patients with means value was 1.69 ( range 0.46-6.24). Aplastic Anaemia patients who have had increased telomerase activity were considered to be at risk to develop Leukemia 2.3 fold ( p=0.017 ). There were significant association between high telomerase activity and leukocyte count > 100.000/mm3. CONCLUSION: Our study suggest that telomerase activity might be a useful diagnosis for early detection of leukemia in paediatric patients who initially diagnosed as aplastic anaemia without waiting for longer time, and might improve therapy and prognostic factor. This study open the possibility of further study about the use of telomerase inhibitors as an effective adjunct to the therapy of Leukemia and Preleukemia.

The exact diagnosis of hemoblastosis and its stage by means of morphocytochemical and immunophenotyping methods is often difficult because of a few amount of malignant cells in the investigative material (bone marrow, peripheral blood, cerebrospinal fluid) in cases of myelodysplastic syndrome, minimal residual disease, treatment with corticosteroids, approaching relapse of acute leukemia and leukemisation of lymphomas. By use of our metods of cultivation proliferation of blast cells is achieved (Shvelidze T., 1979,1983, Shvelidze T., Saralidze T. 2000,sakpatent). They maintain in vitro their morpho-cytochemical peculiarities that helps us to determine the exact diagnosis. In the cases of belinear and mixed forms of leukemia the cultural methods reveal the prolipherative clone and in the cases of the approached relapse the new prolipherative clone responsible for the severity of clinical onset of the disease, and helps a physician to choose the appropriate scheme of cytostatic therapy. Cultivating of cerebrospinal fluid is nesseceary especially in patients with leukemia when the disease begins with neuroleukemia. These methods can be successfully used also for the diagnosis of the malignant cell metastasis in bone marrow, peripheral blood, cerebrospinal fluid.

BCR/ABL-mediated increased expression of multiple 406 genes implicated in the pathogenesis of chronic myelogenous leukemia

Increased expression of Gibbon Ape Leukemia Viral 408 receptor on K562 cells and human CD34+ cells reverses inhibition of gene transfer seen in the presence of high

*S. Salesse, C. Verfaillie, University of Minnesota, U.S.A.

concentrations of GALV pseudotyped vector *T. Relander1, A. Brun1, L. Pedersen2, K. Olsson1, J. Richter1, 1Lund University, Sweden, 2Aarhus University, Gabon

The BCR-ABL chimeric protein plays a central role in the pathogenesis of Chronic Myelogenous Leukemia (CML). Intensive research has elucidated many of the signal transduction pathways activated by BCR-ABL. Activation of such pathways may affect the expression of genes that confer the malignant phenotype. However, few data that address BCR-ABL-dependent gene expression are available and only a few downsteam targets have been identified. In order to further define such downstream genes, we have performed a subtractive hybridization to isolate differentially expressed transcripts between human CD34+ cells that express or do not express p210BCR/ABL. Cord blood (CB) CD34+ cells were transduced with an MSCV-retrovirus vector containing either eGFP alone or p210 BCR/ABL-IRES-eGFP. GFP+ cells were FACS selected (>90% purity), and subtractive hybridization performed between the two populations using the Clontech PCR-Select System. 34 subtracted clones expressed in p210-eGFP but not eGFP-transduced CD34+ cells have been confirmed by Northern blot, and sequenced. 56% represent novel proteins and 44% are homologous to known genes. Quantitative Real time PCR analysis confirmed that 14 of 14 genes tested were also overexpressed in additional populations of p210 BCR/ABL-transduced CB CD34+ cells as well as in CD34+ cells from primary newly diagnosed CML patients versus GFP-transduced CB or samples from normal donors. Western blot analysis showed that the known sequences were also overexpressed at the protein level. Intriguingly, treatment of BCR-ABL-positive cells with Abl-specific tyrosine kinase inhibitor, STI571, induced a decrease expression at the mRNA as well as protein level of a part of these sequences while the other part remained unmodified, suggesting two different categories of effectors. Some of the overexpressed genes are implicated in cellular processes known to be disturbed in CML, including the MAPK or the ubiquitin pathway, while overexpression of other genes, including Ran and Nup98, may implicate new cellular pathways involved in CML. Thus, the identification of such downstream genes activated by BCR/ABL may lead to important new insights in the molecular mechanisms underlying CML and identify critical targets for novel therapies for this disease.

Previous results from our group show that oncoretroviral transduction of human cord blood CD34+ cells on Retronectin using amphotropic and GALVpseudotyped vectors is inhibited by high concentrations of vector containing medium (VCM) (J Gene Medicine 2001; 3:207-218). Hypothesis: this inhibition was caused by competition at the receptor level between infectious and noninfectious vector particles and this effect could be overcome by increasing the levels of GALV receptor Pit-1 on the target cells. To incr ease expression of Pit-1, cells were exposed to the phorbol ester PMA (10 ng/ml) at transduction. PMA increased Pit-1 expression on CD34+ CB cells 10-fold (reverse transcriptase quantitative PCR, RT-QPCR) and overall transduction was increased. The inhibi tion of transduction was completely reversed: TE 57.3±7.3 %(vector preloaded onto Retronectin), TE 22.4±4.2 % (preload+high titer VCM) vs TE 74.7±4.6 % (preload+VCM+PMA). To more specifically increase Pit-1 expression on target cells, we constructed an am photropic retroviral vector containing Pit-1, IRES and eGFP (MPIG). Mus Dunni tail fibroblasts were transduced and clones with different levels of GFP expression isolated. When these clones were transduced with a GALV pseudotyped vector containing the yel low fluorescent protein YFP (PG13-MYIN), transduction efficiency closely correlated to Pit-1 expression. To overexpress Pit-1 on CB CD34+ cells, these were cultured with SCF, TPO and flt3 ligand, all at 100 ng/ml. After MPIG transduction cells were 48 ho urs later transduced with PG13-MYIN under PL or PL+VCM conditions. FACS analysis showed that the inhibitory effect from VCM was completely abolished in the cells with overexpression of Pit-1(mean TE 51.4 % for PL alone vs 52 % for PL+VCM) but not in cells transduced with the control vector (38.1 % for PL vs 10.4 % for PL+ VCM). Similar results were seen when transducing K562 cells. RT-QPCR on CD34+ cells transduced with MPIG and sorted for GFP expression showed approx.10-fold increase of Pit-1 expression on MPIG+ cells. Conclusions: low levels of Pit-1 expression on CD34+ CB cells limits GALV vectors transduction, especially in the presence of high titer VCM. Inhibition of gene transfer from GALV VCM can be completely abolished by increasing Pit-1 express ion on the target cells.

Abstracts / Experimental Hematology 30 (2002) 37-146

Cord Blood Banking and Transplants: Experience 409 of a Single Center in China *Z. C. Han , L. Qiu , J. Han , Q. Li , C. Yang , M. Lu , H. Ying , M. 1

2

2

2

2

2

2

Wang2, M. Han2, 1Institute of Hematology and Blood Diseases

139

A novel factor capable of stimulating hematopoiesis 411 and angiogenesis *Z. C. Han , Y. Liu , Y. Cai , S. Lu ,

1 2 2 2 1 Institute of Hematology and Blood Diseases Hospital, China, 2Institute of Hematology, China

Hospital, China, 2Institute of Hematology and Blood Diseases Hospital, CAMS an, China We describe the experience with the cord blood (CB) banking and initial results of transplantation with unrelated CB in our institution. CB units were collected, processed and stored as described by Rubinstein et al. Reference samples were tested for CD34+ cell counting, progenitor assay, microbial detection, HLA-A, B and DRB1 low resolution SSO typing. 2568 qualified CB units were banked by Jan.31,2002. The average of volume, nuclear cell(NC), CD34+ cells, CFU-GM and CFU-GEMM prior to cryopreservation were 41.42±1.97ml, 10.8±3.03x108 , 2.69±1.56x106,1.59±0.90x106 and 0.79±0.21x106, respectively. The recovery of cell processing for NC, CD34+ cells and progenitors was 86.1±57.97%, 91.26±12.97% and 89.38 ±11.36%, respectively. The cell viability post-thaw was over 90%. The median recovery post-thawed for TNC, CD34+ cells and progenitors was 89%, 92% and 90%, respectively. The analysis of the HLA typing results indicated that the distribution of HLA antigen and gene frequency was agreed with the Northern Han population. The analysis of the unrelated donor matched probability indicated that for patients to find 5 or 6 loci of HLA-A, HLA-B and HLA-DR matched CB with the probability over 90%, the CB storage needs to be over 50,000 units. When the CB storage is over 500,000 units, 99% Northern Han patients would find all 6 loci of HLA-A, HLA-B and HLA-DR matched CB. Among the inquiries for CB from 62 patients, 6 loci matched CB units were found in 1 case, 22 cases with 5 loci matched CB units in our bank. Eight patients with high-risk acute leukemia received 5~6 loci matched unrelated CB transplantation in our transplant center. Six patients engrafted in 7 evaluated patients. The 6 patients were alive 60~320 days with 1 patient relapsed 90 days after transplant. In summary, we have established a CB stem cell bank with high quality. The results of our preliminary clinical studies indicate that the banking of unrelated CB would be a very important alternative resolution for crisis of HSC donation in China.

A novel factor, named hemangiopoietin (HAPO), was originally purified from the urinary extracts of patients with aplastic anemia and genetically cloned from human fetal liver cDNA library. The part of HAPO cDNA coding N-terminal region (262 amino acids) was amplified by PCR, cloned into the pET32c(+) expression vector. After transformation into Escherichia coli (BL21), the HAPO protein was expressed as soluble protein. Biological analysis found that HAPO supports significantly the proliferation and survival of LTC-IC and stromal/meshenchamyl stem cells in long-term murine and human bone marrow cultures. In the presence of HAPO, the number of CD34+, Scan-1+ and Flk-1+ cells was significantly increased. HAPO also promotes the growth of KG1a cells, a CD34+ leukemia cell line, and umbilical cord endothelial cells. In vivo, HAPO provided stimulation of hematopoiesis when injected for 7 days after total body irradiation of 4.5 Gy. Mice given HAPO had a significant increased bone marrow CD34+ cells, megakaryocytes and mononuclear cells compared with control mice. Although the full understand of biological function of HAPO requires further study, our data demonstrate for the first time the presence of a previously not described cytokine acting on both hematopoiesis and angiogenesis.

Abnormal expression of VEGF and its receptors in acute 410 myeloid leukemia Y. Wang , Z. Xiao , R. Yang , *Z. C. Han ,

Treatment of spinal injury by human umbilical cord blood 412 stem cell transplantation *Z. C. Han , Z. Zhao , H. Li , H. Liu , R. Yang ,

Angiogenesis is of prognostic importance not only in solid tumors but also in hematologic maliganancies. Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are important potential angiogenic factors. Cellular VEGF has been suggested to be a key prognostic factor in acute myeloid leukemia (AML) with high white blood cell (WBC). To elucidate the precise role of VEGF and its receptors in AML, we investigated the mRNA expression of VEGF and its receptors KDR, Flt1 by RT-PCR in 14 AML cell lines and 44 AML patients, and analyzed the plasma level of VEGF by ELISA. In 14 AML cell lines, the mRNA expression of VEGF and its receptors KDR, Flt1 were 14(100%), 7 (50.0%), 13 (92.9%) respectively. In 44 AML patients, the positive rates in their bone marrow mononuclear cells (BMMNCs) were 26 (59.1%), 1 (2.3%), 22 (50.0%) respectively. There was no detectable expression of VEGF and its receptors KDR, Flt1 in the BMMNCs and CD34-enriched cells from normal donors. The plasma VEGF level was statistical significantly increased in 39 AML patients who were newly diagnosed or relapsed (135.3±87.9 pg/ml) compared to that in 15 patients in complete remission (CR) (80.6 ±36.9 pg/ml) and 12 normal donors (80.6±33.1 pg/ml). The plasma VEGF level of 18 patients who achieved CR after one cycle of chemotherapy (97.3±26.9 pg/ml) was significantly lower than that of 17 non-CR patients (176.5±111.0 pg/ml, P=0.007). In addition, the mRNA expression of bFGF and its receptor FGFRs1-4 was also higher in AML patients and the 14 AML cell lines than in normal donors. However, the plasma level of bFGF was normal in AML patients. Our results demonstrate that the expression of VEGF and its receptors in AML blast cells and the patients’ plasma level of VEGF are abnormally increased, and suggest that such abnormality is associated with disease remission states of AML patients.

Human umbilical cord blood cells (HUCBC) contain a number of immature stem/progenitor cells with extensive proliferation capacity and therefore have been used as a source of transplantable stem and progenitor cells to reconstitute marrow in children with acute leukemia and severe Fanconi anemia. However, little is known about the survival and development of HUCBC transplantation in the central nervous system. In the present study, we tested whether local administration of HUCB CD34+ cells survive, differentiate in the spinal cord and repair neurological function after injury in rats. HUCB CD34+ cells were separated by MACS with a purity of 97%, and CD34+ cells were labeled with Bromodeoxyuridine (BrdU) (20mmol/L) 72 hours before administration. Adult Wistar rats (n=25) weighing 200-300g were employed in the experiments. Rats were anethetized with pentobarbitone. Animals were allowed to survive for 4 weeks after semispinal dissection. After surgery, 3X104 CD34+ cells were infused into the spinal cord. Behavioral tests were performed before surgery and 24 hours, 1 week, 2 weeks, 3 weeks and 4 weeks post surgery. Modified Tarlov score was used to evaluate neurological function recovery. Immunohistochemical staining was used to identify cells derived from HUCBC. BrdU positive cells and Glial fibrillary acidic protein (GFAP for astrocytes) and neuronal nuclear antigen (NeuN for neurons) positive cells were found in the local site of surgery. The Tarlov scores were significantly higher in the experimental group than in the control group (infused with PBS). These results indicate that HUCB CD34+ cells transplantation can significantly improve the neurological function of rats with semi-spinal dissection, and suggest that these might result from the transdifferentiation of HUCB CD34+ cells.

1 1 1 2 1 Institute of Hematology, China, 2Institute of Hematology and Blood Diseases Hospital, China

1 2 2 2 2 1 Institute of Hematology and Blood Diseases Hospital, China, 2Institute of Hematology, China

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Abstracts / Experimental Hematology 30 (2002) 37-146

Detection of Toxoplasma Gondii and Human Cytomegalo413 virus DNA in Blood from Transplant Recipients Using Multiplex Nested Polymerase Chain Reaction

Platelet- and megakaryocyte-derived microparticles 415 transfer CXCR4 receptor to CXCR4-null cells and make them susceptible to infection by X4-HIV

*N. Khodai Booran, V. Bakayev, Pasteur Institute of Iran, Iran

T. Rozmyslowicz1, M. Majka2, J. Kijowski2, G. Gaulton1, *M. Ratajczak2, 1University of Pennsylvania, U.S.A., 2University of Louisville, U.S.A.

Evidences from many studies suggested a polymerase chain reaction (PCR) as a valuable method for diagnosing infectious diseases in the transplants.We used this method for detection of Toxoplasma Gondii and human cytomegalovirus in blood speciemens from patients after bone marrow or kidney transplantation. DNA of both infectious agent were detected using two seperat sets of nested primers in the PCR. The condition for a multiplex nested PCR providing simultaneous identification of both pathogens in one tube were optimized.This assay provide an application in clinical research for diagnosis of infections in post-transplant recipients.

Under some circumstances the HIV virus may infect cells that do not express receptors essential to HIV-entry. We hypothesized that platelet- and megakaryocyte-derived microparticles (MP) could play a role in such infections. MP are circular membrane fragments shed from the surface of eukaryocytic cells. After adhesion to target cells, MP may transfer membrane-associated proteins to these cells. We found that peripheral blood platelet- (PMP) and megakaryocytederived microparticles (MegaMP) that highly express CXCR4 may transfer this receptor from the surface of platelets or megakaryocytes to the surface of CXCR4-null cells. Since this mechanism could potentially allow CD4+/CXCR4-null cells to become infected by Ttropic HIV, we incubated several human CD4+/CXCR4-null cells such as normal erythroblasts, glioblastomas U87, MAGI and hematopoietic cell lines UT-7, HEL and TF-1 with PMP or MegaMP, and found that these cells became CXCR4+. We next exposed these cells to X4-HIV (IIIB) and evaluated their susceptibility to infection by PCR, ELISA, and morphological analysis. We observed in all instances that after CD4+/CXCR4-null cell lines acquired CXCR4 from PMP or MegaMP, they could became infected by X4 HIV. We postulate that both PMP and MegaMP may play a novel and important role in spreading HIV-1 infection by transferring the CXCR4 co-receptor to CD4+/CXCR4-null cells.

Dynamics of the cytomegalovirus - dendritic cell 414 interaction:Dendritic cell maturation stage, major histocompatibility complex and viral gene functions control

Virus modulation effects on autoimmue idiopathic 416 thrombocytopenia andanemia models in the mouse *A. Musaji, J.-P. Coutelier,

T cell activation S. Mathys1, T. Schroeder2, M. Messerle1, U. Koszinowski1, *U. Just3,

Belgium

Max von Pettenkofer-Institut, Germany, 2GSF Clinical Molecular Biology, Germany, 3GSF, Institute of Clinical Molecular Biology, Germany Dendritic cells (DC) are potent antigen presenting cells and play a central role in inducing protective immunity to viral infection. Cytomegaloviruses (CMV) closely interact with hematopoietic cells and reside lifelong within the infected host. To analyse the impact of CMV infection on DC function, we infected defined differentiation stages of the multipotent mouse stem cell line FDCP-mix with a recombinant mouse CMV (MCMV) expressing green fluorescence protein as reporter gene. Both, immature and mature DC are targets for MCMV infection. Infection of immature DC results in virus production and DC activation, as indicated by the transient upregulation of MHC class I and II, CD80, CD86, and CD40 cell surface molecules. Immature DC migrate upon activation and may contribute to dissemination of CMV. The infection of mature DC is non-productive and restricted to immediate-early and early viral protein expression. During early stages of MCMV infection mature DC upregulate MHC and costimulatory molecules and activate autologous naive T cells. At later times of MCMV infection, DC prevent T cell activation in autologous and allogeneic mixed lymphocyte reactions by downregulating MHC and co-stimulatory molecules. Remarkably, infected DC fail to activate allogeneic T cells at any stage of virus infection. Thus, DC under influence of MCMV have a physiological dual role: to initiate and also to restrict T cell activation. The negative shift of this balance under allogeneic conditions may contribute to explain CMV risks following transplantation.

Université Catholique de Louvain,

1

In human pathology and mouse models viruses were shown to play a certain role in ethiology of autoimmune disease. Nevertheless, the modulation effects of virus infection on autoimmune pathology were studied with a lesser extent. The modulation effects of lactate dehydrogenase elevating virus (LDV) are studied on the mouse models of human Autoimmune Idiopathic Thrombocytopenia and Anemia (AITP and AIHA). AITP in mouse is caused in a newly established model of mouse i.p. immunization with rat platelets or is mimiced by a passive polyclonal antibody (from immunized rats and rabbits with mouse platelets) and IgM&IgG2a monoclonal antibody (from NZWxBXSB F1 mouse) transfer. The IgG1&IgG2a moAb transfer is also used to mimic AIHA in mouse. Only in mice with provoked or mimiced autoimmue disease, LDV causes the drastic platelet count drop in AITP models and Ht drop in AIHA model. LDV exaggeration of thrombocytopenia in poAb-mimiced AITP was clearly Fc-dependent. In AIHA model the icrease of autoantibody pathology against erythrocytes after LDV infection was shown to be in direct dependence of IFNgamma pathway since IFN-gamma receptor KO animals showed no modulatory effects of LDV. We hypothesize the uniform mechanism of virus action in at least AITP and AIHA were the virus causes the activation of macrophage-cell responsible for auto-antibody opsonized thrombocytes and erythrocytes elimination from the circulation. This activation is produced via cytokine loops, mainly IFN-gamma that allows macrophages to express more phagocytosis involved receptors such as Fc-gammaR and CR thus more effective trapping and phagocytosis of autoantibody-opsonzed target cells.

Abstracts / Experimental Hematology 30 (2002) 37-146

141

Qualitative and quantitative analysis of human 417 herpesviruses in chronic and acute B-cell lymphocytic leukemia and in multiple myeloma

Induction of Human Cytomegalovirus (CMV)-specific 419 Cytotoxic T-lymphocytes (CTLs) by Artificial Antigen Presenting Cells (AAPC)

*S. Hermouet1, C. Sutton2, T. Rose3, I. Corre4, J. Casey2, 1INSERM

*G. Papanicolaou1, J. Latouche2, M. Sadelain2, 1Memorial Sloan

U463, Institut de Biologie, France, 2Cornell University, U.S.A., 3 University of Washington, U.S.A., 4INSERM U463, Nantes, France

Kettering Cancer Center, U.S.A., 2Memorial Sloan-Kettering , U.S.A.

Real-time quantitative polymerase chain reaction (qPCR) was used to quantify viral loads of human herpesviruses (HHVs) at diagnosis in 61 samples of malignant B-cells: 21 chronic lymphocytic leukemia (BCLL), 29 acute lymphoblastic leukemia (B-ALL) and 11 multiple myeloma (MM); control samples were blasts from 16 acute myeloid leukemia (AML) and 24 blood or bone marrow samples from healthy donors. The majority of samples from healthy donors and patients were positive for EBV and contained < 1000 copies/µg DNA; EBV loads were occasionally high (> 5000 copies/µg DNA) in B-ALL (3/16) and B-CLL (2/21) tumor samples. The fraction of samples positive for HHV-8 and HHV-6A was high for adults with B-ALL (18.8% HHV-8+, 43.8% HHV6A+) or B-CLL (28.6% HHV-8+, 52.4% HHV-6A+) but was < 10% for MM patients. B-ALL, B-CLL and MM samples were rarely positive for HHV-6B and HHV-7. Lastly, 75% of B-ALL samples were positive for CMV, and CMV loads were significantly higher in B-ALL samples than in MM, B-CLL or AML samples. We also used PCR with consensusdegenerate hybrid oligonucleotide primers (CODEHOP) to look for novel HHVs in B-cell samples: no sequence indicative of a new HHV was detected. Altogether, the data indicate that the presence of several HHVs, including CMV and EBV at high loads, is not rare in adult BALL and B-CLL tumor cells. Future prospective studies should determine whether patients with high EBV/CMV loads at diagnosis in tumor samples face a higher risk of delayed hematological recovery, virus-related complications or relapse.

CMV disease accounts for high morbidity and mortality in bone marrow transplant patients. The transfer of CMV-specific CD8+ T lymphocytes derived from the donor is an effective way to reconstitute cellular immunity against CMV. The objective of this study is to generate and characterize CMV-specific CTLs using the AAPC system. AAPC were generated by stable transduction of mouse fibroblasts (NIH3T3) with 5 retroviral vectors encoding for HLAA0201, the costimulatory molecules B7, ICAM-1, LFA-3 and the full length CMVpp65 protein(AAPCpp65)or the immunodominant peptide p495 (NLVPMVATV) derived from pp65 and presented in the context of HLAA0201 (AAPC495). Results: T cells of HLAA0201, CMV-seropositive donors were cocultured with AAPCpp65 or AAPC495. Flow cytometry using p495 -HLA tetrameric complexes shows that CMV-specific CTLs which were detectable at 1-2% at the start of the coculture, represent 30-40% of the CD8+ T cells after 14 days of stimulation. The expansion of the CMV specific CD8+ T cells is 250-fold.CTLS express effector cell type phenotype: CD45RA-,CD62L- and CCR7-. These CTLs are able to kill specifically HLAA0201 T2 target cells loaded with p495 and EBV transformed Bcells (BLCLs) expressing pp65. Furthermore, the CTLs are able to kill CMVinfected fibroblasts (MRC5). Intracellular cytokine staining shows that 11.2% and 7% of CD8 cells secrete INF-g and TNF-a respectively. AAPCpp65 were equally potent to AAPC495 in inducing CTLs confirming that AAPCs can process pp65 and present p495 in the context of HLAA0201. Comparison of the AAPCs to established systems for CTL induction, showed the AAPC system to be largely superior to PBMCs and comparable to autologous BLCLs pulsed with p495 as stimulators. Conclusions: 1) AAPCs can be used to generate high numbers of CMV-specific cytotoxic CD8+ cells. 2) These CTLs are able to kill CMV-infected fibroblasts. 3) AAPCs can process pp65 and efficiently present the peptide p495 in the context of HLA0201. 4) Compared to other systems, the AAPCs offer the advantage of fast and efficient induction of CTLs from any HLAA0201 positive donor circumventing the need to generate autologous dendritic cells or BLCLs.

Implication of hepatitis C virus (HCV) and human 418 herpesvirus-8 (HHV-8) in the pathogenesis of a case of aggressive primary plasma cell leukemia (PCL)

Associated viral infections in patients with hematologic 420 malignancies after BMT K. Abdulkadirov, *V. Tchebotkevitch, S. Moiseev,

*S. Hermouet1, I. Corre2, M. Gassin3, E. Bigot-Corbel4, J. Casey5,

Inst.Hematol., Russian Federation

INSERM U463, Institut de Biologie, France, 2INSERM U463, Nantes, France, 3Virologie, CHU Nantes, France, 4Biochimie, CHU Nantes, France, 5Cornell University, U.S.A. We analysed the role played by HCV and HHV-8 in the pathogenesis of an unusual case of primary PCL diagnosed in a 32 year-old man. The patient, with a history of HCV infection but negative for human immunodeficiency virus, was admitted in intensive care for septick shock. An extensive infiltration of blood and bone marrow (BM) by plasmablasts (blood: 90%; BM: 47%), accompanied by a monoclonal kappa IgG in serum and urine, was discovered and the patient was diagnosed with PCL. The plasmablasts, CD56- and CD138/Syndecan1+++, were also CD19+ and CD5+ (weakly), a characteristic rarely found in multiple myeloma (MM). Conventional cytogenetics, followed by FISH, revealed an isolated t(9;14) (p13;q32) translocation involving PAX-5. This translocation has been described in lymphoplasmacytoid lymphoma, the type of lymphoma most frequently associated with HCV, and also in tumor cells of HCV+ patients with primary effusion lymphoma (PEL), a pathology associated with HHV-8. Indeed the patient had viremia for both HCV (1a genotype) and HHV-8 (C’ subtype). Analysis of the monoclonal IgGk showed that it was directed against HCV core protein. Quantitative PCR and immunofluorescence studies showed that 100% of plasmablasts were infected by HHV-8 (> 300 viral copies/blast); the majority of the blasts were co-infected by HCV. In conclusion, the data and chronology suggest that the following sequence of events may have led to primary PCL: 1) benign, HCV-driven oligoclonal expansion of B-lymphocytes, 2) blockade of plasmacytic differentiation consecutive to Pax-5 rearrangement, 3) acceleration of the PAX-5-rearranged, monoclonal plasmablast expansion after infection by HHV-8.

Russian

1

The aim of the study was to evaluate the frequency and clinical features of viral infections in patients (pts) with different forms of hematologic malignancies. Herpes viruses (HSV 1 and 2, CMV, Epstain-Barr virus) as well as respiratory viruses were controlled in our clinic. In this study 22 pts after allogeneic bone marrow transplantation (ALBMT) and 58 pts after autologous stem cells transplantation (ASCT) were observed. The most problematic group of viral infections was CMV. The probability of CMV infection was greater after ALBMT than after ASCT (10 from 22 - 45% versus 12 from 58 - 21%). The peculiarity of ALBMT was the high frequency (87,6%) of associated CMV and Respiratory Syncitial virus infections. In contrast such association was not demonstrated in ASCT group. The clinical signes of associated viral infections were differ from mono-CMV infection. The ptophylaxis of viral infections with IVIG “Cytotect” (Biotest, Germany) plus/or gancyclovir reduced the clinical manifestations of the disease. The results demonstrate the necessity of monitoring not only herpes viruses but also respiratory viruses in immunocompromised pts with hematologic malignancies.

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421 Withdrawn

Xenotransplant of Human Adipose-derived Mesenchymal 423 Stem Cells *T. Meyerrose , D. A. DeUgarte , G. McNamara , M. H. Hedrick , 1

2

3

2

J. A. Nolta1, 1Washington University, U.S.A., 2UCLA, U.S.A., 3

Children’s Hospital LA, U.S.A.

Purpose: This study was designed to investigate the reconstitution potential of human adipose-derived mesenchymal stem cells (AMSC) into various tissue compartments using a murine xenotransplant model. This unique population of cells was also characterized for proliferative ability, multilineage potential, and transgene retention, both in vitro and in vivo. Methods: Green fluorescent protein was introduced to purified AMSC via lentiviral or retroviral vectors to assist in identification of cells post-transplant as well as future clonal analysis. Transduced cells were administered to sub-lethally irradiated immune deficient mice through various routes including intravenous, intraperitoneal, intramuscular, and subcutaneous within a biodegradeable matrix. Up to 90 days post-transplant, tissues were harvested and DNA PCR was performed for specific vector sequences as well as human Alu repeat sequences. In situ hybridization and confocal microscopy were also utilized to examine the integration of donor cells into the murine tissue architecture. Explant culture through selection media further confirmed the continued expression of all transgene components. Current experiments are evaluating the long-term selfrenewal properties of these AMSC both in vitro and in vivo. Results: We have verified the presence of human AMSC in the lung, liver, spleen, intestine, heart, brain, skeletal muscle and associated connective tissue up to 90 days post-transplant. We have noted an absence of these cells in either the peripheral blood or bone marrow of recipient mice. A unique pattern of tissue specific localization was observed consistently across the various administration routes and independent of transduction parameters. Confocal microscopy has shown integration into the endogenous architecture of most tissues, and possible in vivo expansion under certain conditions. Conclusions: These AMSC represent a population of stem cells with a pattern of tissue distribution unique from traditional bone marrow mesenchymal stem cells. They are easily obtained and very amenable to current transduction protocols for either lentiviral or retroviral transduction, making them an excellent avenue for cell-based therapies involving a wide range of end tissue targets. We have shown multilineage potential in vitro, and are currently working on methods to create an increased selective advantage in vivo for future use in gene therapy applications.

High gene transfer efficiency and pancellular erythroid 422 expression resulting in correction of Anemia in ß-thalassemic mice with lentiviral-based human ß-globin vector *S. Imren1, R. Pawliuk2, B. Cavilla3, C. Eaves3, L. Wadsworth4, R. Nagel5, M. Fabry5, E. Bouhassira5, I. London6, P. Leboulch7, R. K. Humphries8, 1Terry Fox Laboratory, Canada, 2Genetix Pharmaceutical Ltd., U.S.A., 3Terry Fox Lab, BC Cancer Agency, Canada, 4Children’s and Women’s Health Center of BC, Canada, 5Albert Einstein College of Medicine, U.S.A., 6Massachusetts Institute of Technology, U.S.A., 7Harvard Medical School and Brigham & Women’s Hospital, U.S.A., 8British Columbia Cancer Research Center, Canada With oncoretroviruses carrying a human p-globin gene and GFP for F ACS preselection of transduced cells, we have previously reported complete reconstitution of mice by transduced hematopoietic stem cells. While significant levels of human p-globin gene expression were achieved, these were still suboptimal due to limited LCR sequences, low gene copy number and heterocellular expression. In an effort to overcome these problems, we have generated a lentiviral-based human p-globin gene vector consisting of more extensive LCR sequence (2.7 kb of HS2, HS3, HS4) an extended p-globin promoter and additional sequences ( e.g. central polypurine tract, RRE) to facilitate high viral titers. With this vector, we have achieved high concentration viral titers (> 109 /ml) that stably transmitted the provirus into target cells. Post 5- FU bone marrow from donor thalassemic mice, homozygous for deletion of the murine p-major gene (C57BV6 Hbbtli-l/Hbbth-I, hereafter termed THAL mice) were prestimulated with growth factors overnight, exposed to virus for 5 hrs. and then immediately injected into lethally irradiated THAL mice without selection. All recipients exposed to high titer viral preparations achieved complete and stable reconstitution with red blood cells (RBC) positive for human p-globin as assessed by F ACS in animals followed >8 months posttransplant and in secondary bone marrow recipients followed up to 6 months. Importantly, this high level of reconstitution was associated with a marked correction in all hematologic parameters including normalization of reticulocyte counts and RBC numbers (p<0.001), near normalization of hemoglobin levels and hematocrits, and complete elimination of free a-globin chains and splenomegaly. Consistent with these findings all mice had a high level of human p-globin (32 :t 4% of all p-like globin chains by HPLC) documented in RBCs. In contrast, mice transplanted with bone marrow exposed to lower titer virus (~3xI08 ml) achieved incomplete reconstitution (mean 35% human p-globin positive RBCs) associated with a lower proviral integration copy number (1 vs. >3). Together, these findings provide strong evidence that stem cell based genetic correction of p-thalassemia is achievable with vectors enabling high level expression of p-globin, multiple events of proviral integration and pancellular expression in generated erythroid progeny.

Differentiative Potential Of Human Metanephric 424 Mesenchymal Stem Cells *G. Almeida-Porada,

University of Nevada at Reno, VA Medical

Center, U.S.A.

In the present studies, we began to evaluate the ability of mesenchymal stem cells (MSC) derived from non-hematopoietic organs to form blood and other tissues in vitro and in vivo. Given its mesodermic derivation, we first used human fetal kidney as a source of MSC. Kidney MSC were Thy1+,CD51+,CD44+,CD45- and Vimentin+, a phenotype consistent with that of metanephric mesenchyme and were unable to form hematopoietic colonies in methylcellulose. Using microarray analysis and immunocytochemistry, we demonstrated that these kidney-derived MSC expressed numerous markers of early metanephric mesenchyme including vimentin, collagen type I, smooth muscle actin, FGF-7, and TIMP-3. We thus termed these cells metanephric MSC (MNMSC). Furthermore these cells also expressed c-met, the receptor for hepatocyte growth factor, which prompted us to examine whether these cells might possess the ability to generate hepatocytes under the appropriate conditions. Indeed, cells with hepatocyte-like morphology and phenotype were obtained from the MNMSC after in vitro culture in hepatocyte growth-inducing media. After transplantation into pre-immune sheep fetuses, MNMSC produced multi-lineage hematopoietic engraftment and gave rise to CD34+ cells. Successful hematopoietic engraftment in secondary recipients demonstrated the generation of long-term engrafting hematopoietic stem cells from these MNMSC. PCR analysis confirmed human hematopoietic engraftment and revealed that human cells were also present within other organs. Liver sections of transplanted animals contained human albumin-producing hepatocytes. In conclusion, a human MNMSC population simultaneously gave rise to human blood and liver suggesting that MSC may represent a broad population of putative stem cells present in multiple adult organs.