- Email: [email protected]
O-13 Hematopoietic stem cells in normaland preleukemic disease states
Oral Presentations
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not always take advantage to decide when to start clinical intervention such as chemotherapy. Thus, we investigated whether Bmi-1 protein in CD34+ cells could be correlated with disease progression of MDS or prognosis of patients. Bone marrow cells were stained with anti-CD34-antibodyPE, fixed in PFA followed by staining with anti-Bmi-1antibody-FITC, and subjected to flow cytometry. Bmi-1 expression in CD34+ cells was preferentially seen in RAEB, RAEB-T and MDS-AML compared with RA and RARS; 14.09% in RA (n 3), 38.29% in RARS (n 2), 63.05% in RAEB (n 6), 56.78% in RAEB-T (n 3), 91.20% in MDS-AML (n 3). Patients were dead with >70% at Bmi-1 expression level. Moreover, patients with RA and RARS at >10% of Bmi-1 made disease progression to RAEB in a while. In contrast, one patient with RAEB-T at <10% of it is still alive. Alternatively, IPSS was closely correlated with Bmi-1 expression. These results suggest that the expression of Bmi-1 can distinguish the proliferating leukemic stem cells from resting cells by using flow cytometry. Taken together, Bmi-1 expression could predict the disease progression and prognosis of MDS.
Stem cell biology I
I
•
E
X
VIVO EXPANSION OF HUMAN HEMATOPOIETIC STEM CELLS
T. Nakahata*. Department of Pediatrics, Graduate School of Medicine, Kyoto University, Japan *E-maih [email protected] o-u.ac .jp The recent development of assays measuring the ability to reconstitute human hematopoiesis in NOD/SCID mice enabled investigators to evaluate the stem cell activity of expanded human HSC. This mouse is a useful tool for evaluating human HSC, however, the engraflability of human HSC is not satisfactory and T lymphogenesis is not observed even if a large number of human HSC are transplanted into the mice. More recently, we developed a new strain of immunodeficient NOD/SCID/y~ ~ (NOG) mice in which successful engraftment was achieved even if less than 103 cord blood CD34 ÷ cells were transplanted into the mice. We observed human myeloid, erythroid, B, T, NK, DC, mast cells and megakaryocytes in various hematopoietic tissues of NOG mice. NOG mice may be useful for analysis of human hematopoiesis and the evaluation of human HSC expanded in ex eivo. We demonstrated the significant expansion of human HSC by a combination of SCF, FL, TPO, and an IL-6/sIL-6R complex, using xenoplantation into NOD/SCID and NOG mice. We have succeeded the discovery of a serum-free closed culture system of human HSC and plan to start a clinical study using expanded HSC. I will present our recent studies on molecular cloning of self-renewal factors and plasticity of human HSC. (Presented in the co-operate session with Chugai.)
[O~
PRIMING OF TRANSCRIPTION FACTORS AND HEMATOPOIETIC CELL FATE DECISION
K. Akashi*. Graduate School of Medical Sciences, Kyushu University, Japan *E-maih akashi@canc er.med.kyushu-u.ac.j p Hematopoiesis is a paradigmatic system for progressive restriction of cell fate potentials from hematopoietic stem cells (HSCs) to lineage-restricted progenitors that produce all lineages of mature blood cells. A fi.mdamental question is how the progeny of multipotent cells adopts one fate from a choice of several. Lineage commitment and subsequent differentiation of multipotent cells likely involves the selective activation and silencing of particular gene expression programs. We systematically profiled gene expression in rigorously purified self-renewing HSCs and lineage-restricted progenitors including common lymphoid progenitors (CLPs) and common myeloid progenitors (CMPs). CLPs and CMPs co-expressed T/B lymphoid and myelo-erythroid transcription factors, respectively, indicating that the transcription of lineage-related genes can occur prior to lineage commitment. This lineage "priming", which might allow flexibility for cell fate decisions by quantitative competition of more than two transcription factors, disappears progressively as cells lose their multipotentiality to become lineage-restricted. We have also identified eosinophil progenitors (EoPs) and basophil progenitors (BaPs) downstream of GMPs. Although both EoPs and BaPs co-express C/EBP¢~ and GATA-2, the decision of commitment to the eosinophil or the basophil lineage was achieved by the order of expression of these factors during the early stage of development. Thus, the appropriate control of the expression level and timing of transcription factors might be critical to maintain the hierarchical hematopoietic lineage decision. (Presented in the co-operate session with Chugai.)
[O~
HEMATOPOIETIC STEM CELLS IN NORMAL AND PRELEUKEMIC DISEASE STATES
C. Eaves*, X. Jiang, A. Eaves. Terry Fox Laboratory, British Columbia Cancer Agency arid University of British Columbia, Canada *E-mail: [email protected] A variety of in vitro and in vivo (xenotransplant) assays can now be used to distinguish different early stages of differentiation of normal human hematopoietic cells and allow these compartments to be objectively and reproducibly quantified. These include cells with both short and longterm repopulating activity and cells with varying degrees of lineage restriction that can also be phenotypically distinguished consistent with a hierarchical model of normal
Stem cell biology H hematopoiesis. Accumulating evidence suggests that many diseases that are first diagnosed in a "preleukemic" stage are clonal disorders of hematopoiesis that arise in the stem cell compartment and display growth control abnormalities before additional genetic or epigenetic alterations cause major perturbations in their ability to differentiate into normal blood cells. Accordingly, assays developed for primitive normal human cells can often be usefully applied to analyze the composition of preleukemic clones and investigate mechanisms underlying their deregulated amplification. We are currently exploiting this strategy in concert with genomic studies to gain insights into the pathogenesis of chronic myeloid leukemia and the myelodysplastic diseases. In this presentation, a review of our recent findings and new approaches to therapy will be reviewed. (Presented in the co-operate session with Chugai.)
~ A
REVERSIBLE TWO-STEP MODEL OF MDS/PRELEUKEMIA: TRANSGENIC MICE EXPRESSING INDUCIBLE BCL2 AND MUTANT NRAS
R.A. Padua 1 *, N. Omidvar2, S. Kogan3, T.-H. Phan 1, R. West 2, D. Felsher 4, I. Weissmann4, S. Thomas 5, J. Whittaker2, A. janinl, M. Pla 1, G. Mufti5, J.M. Bishop3, C. Chomienne 1. lInstitut Universitaire d'Hematologie,
Inserm U71& Paris, France; :School of Medicine, Cardiff University, UK, 3University of California, San Francisco, USA; 4Stanford University, Stanford, CA, USA; 5King~ College London, UK *E-maih [email protected] Myelodysplastic syndromes(MDS) are clonal stem cell hematological disorders characterized by ineffective hematopoiesis that frequently evolve to acute myeloid leukemia(AML) and are thus a model of multistep leukemogenesis. We present a reversible two-step model of MDS/preleukemia. The model is based on candidate genes, NRAS and BCL-2, frequently altered in MDS patients at risk of acute transformation. Expression of both genes was directed to committed myeloid progenitors via the myeloid MRP8 promoter. MRP8-NRASD12 and MRP8-BCL-2 mice have normal survival though demonstrate increased dysplastic myeloid cells detected by immunnophenotyping. MRP8BCL-2 mice present with expansion of bone marrow primitive Scat+ blast cells. In contrast, NRASD12/BCL-2 mice have a significantlyreduced life span presenting with disease characteristic of MDS/preleukemia, together with increased bone marrow blasts and infiltrated tissues and a penetrance of 50% with a latency period of 3 6 months, which is transplantable. Increased RAS activity was observed in the NRASD12 transgenic mice and both the Mact+ and Scat+ compartments had an increase in RAS/BCL-2 complexes in the double transgenic mice. By creating a tetracyclineinducible model, TBCL-2/MMTVtTA/NRASDt2, we found
$5
that the mechanism of transformation to leukemia can be rescued or re-established by switching BCL-2 expression off or on respectively. These results suggest that the inhibition of one step may reverse the evolution of the disease to leukemia, identifying potential targets for therapy. (Presented in the co-operate session with Chugai.)
Stem ceg biology II
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CD45-NEGATIVE CLONAL CELLS WITH VERY IMMATURE PHENOTYPE (CD45-CD34-CD38-Lin-) IN PATIENTS WITH MYELODYSPLASTIC SYNDROMES
K. Ogata 1 *, C. Satoh 2, M. Tachibana 1, H. Hyodo 1, H. Tamura 1, K. Dan 1, T. Kimura 3, Y. Sonoda 4, T. Tsuji5.
1Division of Hematology, Department of Medicine, Nippon Medical School, eDepartment of Bioregulation, Department of Medicine, Nippon Medical School, 3Department of Molecular-Targeting Cancer Prevention, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, 4Department of Hygiene, Kansai Medical University, 5Department of Industrial Science arid Technology, Tokyo University of Science, Japan *E-mail: [email protected] CD45 is a hematopoietic lineage-restricted antigen that is expressed on all hematopoietic cells except for some mature cell types. CD45+CD34+CD38 L i n cells are well-documented hematopoietic stem cells (HSCs), and CD45+CD34 CD38 L i p cells are probably less mature HSCs. In this study, we detected CD45 CD34 CD38 L i p cells in the peripheral blood and bone marrow of patients with myelodysplastic syndromes (MDS). Fluorescence in situ hybridization showed that CD45 CD34 CD38 L i p cells had the same chromosomal aberration as the myeloblasts. Generally, MDS cells have extremely reduced hematopoietic potential compared with normal cells, but we documented the following in some patients. Freshlyisolated CD45 CD34 CD38 L i p cells did not form any hematopoietic colonies but had long-term culture-initiating cell activity. When co-cultured with stroma cells, the cell proliferation kinetics of CD45 CD34 CD38 L i p cells differed from those of CD34+ myeloblasts. The number of cells in CD45 CD34 CD38 L i n cell cultures decreased during the first 7 t0 days, while clusters consisting of 2 4 cells began to appear on days 7 t0. These cells showed only weak potential for proliferation/differentiation, yet differentiated into CD34+ cells and then mature myeloid cells. In contrast, in the CD34+ myeloblast culture, cell clusters began to appear on day 3 and a marked increase in cells was observed on day 7. The CD45 CD34 CD38 L i p cell population represents the most immature immunophenotype so far identified in the hematopoietic lineage and is involved in the malignant clone in MDS.
$4
not always take advantage to decide when to start clinical intervention such as chemotherapy. Thus, we investigated whether Bmi-1 protein in CD34+ cells could be correlated with disease progression of MDS or prognosis of patients. Bone marrow cells were stained with anti-CD34-antibodyPE, fixed in PFA followed by staining with anti-Bmi-1antibody-FITC, and subjected to flow cytometry. Bmi-1 expression in CD34+ cells was preferentially seen in RAEB, RAEB-T and MDS-AML compared with RA and RARS; 14.09% in RA (n 3), 38.29% in RARS (n 2), 63.05% in RAEB (n 6), 56.78% in RAEB-T (n 3), 91.20% in MDS-AML (n 3). Patients were dead with >70% at Bmi-1 expression level. Moreover, patients with RA and RARS at >10% of Bmi-1 made disease progression to RAEB in a while. In contrast, one patient with RAEB-T at <10% of it is still alive. Alternatively, IPSS was closely correlated with Bmi-1 expression. These results suggest that the expression of Bmi-1 can distinguish the proliferating leukemic stem cells from resting cells by using flow cytometry. Taken together, Bmi-1 expression could predict the disease progression and prognosis of MDS.
Stem cell biology I
I
•
E
X
VIVO EXPANSION OF HUMAN HEMATOPOIETIC STEM CELLS
T. Nakahata*. Department of Pediatrics, Graduate School of Medicine, Kyoto University, Japan *E-maih [email protected] o-u.ac .jp The recent development of assays measuring the ability to reconstitute human hematopoiesis in NOD/SCID mice enabled investigators to evaluate the stem cell activity of expanded human HSC. This mouse is a useful tool for evaluating human HSC, however, the engraflability of human HSC is not satisfactory and T lymphogenesis is not observed even if a large number of human HSC are transplanted into the mice. More recently, we developed a new strain of immunodeficient NOD/SCID/y~ ~ (NOG) mice in which successful engraftment was achieved even if less than 103 cord blood CD34 ÷ cells were transplanted into the mice. We observed human myeloid, erythroid, B, T, NK, DC, mast cells and megakaryocytes in various hematopoietic tissues of NOG mice. NOG mice may be useful for analysis of human hematopoiesis and the evaluation of human HSC expanded in ex eivo. We demonstrated the significant expansion of human HSC by a combination of SCF, FL, TPO, and an IL-6/sIL-6R complex, using xenoplantation into NOD/SCID and NOG mice. We have succeeded the discovery of a serum-free closed culture system of human HSC and plan to start a clinical study using expanded HSC. I will present our recent studies on molecular cloning of self-renewal factors and plasticity of human HSC. (Presented in the co-operate session with Chugai.)
[O~
PRIMING OF TRANSCRIPTION FACTORS AND HEMATOPOIETIC CELL FATE DECISION
K. Akashi*. Graduate School of Medical Sciences, Kyushu University, Japan *E-maih akashi@canc er.med.kyushu-u.ac.j p Hematopoiesis is a paradigmatic system for progressive restriction of cell fate potentials from hematopoietic stem cells (HSCs) to lineage-restricted progenitors that produce all lineages of mature blood cells. A fi.mdamental question is how the progeny of multipotent cells adopts one fate from a choice of several. Lineage commitment and subsequent differentiation of multipotent cells likely involves the selective activation and silencing of particular gene expression programs. We systematically profiled gene expression in rigorously purified self-renewing HSCs and lineage-restricted progenitors including common lymphoid progenitors (CLPs) and common myeloid progenitors (CMPs). CLPs and CMPs co-expressed T/B lymphoid and myelo-erythroid transcription factors, respectively, indicating that the transcription of lineage-related genes can occur prior to lineage commitment. This lineage "priming", which might allow flexibility for cell fate decisions by quantitative competition of more than two transcription factors, disappears progressively as cells lose their multipotentiality to become lineage-restricted. We have also identified eosinophil progenitors (EoPs) and basophil progenitors (BaPs) downstream of GMPs. Although both EoPs and BaPs co-express C/EBP¢~ and GATA-2, the decision of commitment to the eosinophil or the basophil lineage was achieved by the order of expression of these factors during the early stage of development. Thus, the appropriate control of the expression level and timing of transcription factors might be critical to maintain the hierarchical hematopoietic lineage decision. (Presented in the co-operate session with Chugai.)
[O~
HEMATOPOIETIC STEM CELLS IN NORMAL AND PRELEUKEMIC DISEASE STATES
C. Eaves*, X. Jiang, A. Eaves. Terry Fox Laboratory, British Columbia Cancer Agency arid University of British Columbia, Canada *E-mail: [email protected] A variety of in vitro and in vivo (xenotransplant) assays can now be used to distinguish different early stages of differentiation of normal human hematopoietic cells and allow these compartments to be objectively and reproducibly quantified. These include cells with both short and longterm repopulating activity and cells with varying degrees of lineage restriction that can also be phenotypically distinguished consistent with a hierarchical model of normal
Stem cell biology H hematopoiesis. Accumulating evidence suggests that many diseases that are first diagnosed in a "preleukemic" stage are clonal disorders of hematopoiesis that arise in the stem cell compartment and display growth control abnormalities before additional genetic or epigenetic alterations cause major perturbations in their ability to differentiate into normal blood cells. Accordingly, assays developed for primitive normal human cells can often be usefully applied to analyze the composition of preleukemic clones and investigate mechanisms underlying their deregulated amplification. We are currently exploiting this strategy in concert with genomic studies to gain insights into the pathogenesis of chronic myeloid leukemia and the myelodysplastic diseases. In this presentation, a review of our recent findings and new approaches to therapy will be reviewed. (Presented in the co-operate session with Chugai.)
~ A
REVERSIBLE TWO-STEP MODEL OF MDS/PRELEUKEMIA: TRANSGENIC MICE EXPRESSING INDUCIBLE BCL2 AND MUTANT NRAS
R.A. Padua 1 *, N. Omidvar2, S. Kogan3, T.-H. Phan 1, R. West 2, D. Felsher 4, I. Weissmann4, S. Thomas 5, J. Whittaker2, A. janinl, M. Pla 1, G. Mufti5, J.M. Bishop3, C. Chomienne 1. lInstitut Universitaire d'Hematologie,
Inserm U71& Paris, France; :School of Medicine, Cardiff University, UK, 3University of California, San Francisco, USA; 4Stanford University, Stanford, CA, USA; 5King~ College London, UK *E-maih [email protected] Myelodysplastic syndromes(MDS) are clonal stem cell hematological disorders characterized by ineffective hematopoiesis that frequently evolve to acute myeloid leukemia(AML) and are thus a model of multistep leukemogenesis. We present a reversible two-step model of MDS/preleukemia. The model is based on candidate genes, NRAS and BCL-2, frequently altered in MDS patients at risk of acute transformation. Expression of both genes was directed to committed myeloid progenitors via the myeloid MRP8 promoter. MRP8-NRASD12 and MRP8-BCL-2 mice have normal survival though demonstrate increased dysplastic myeloid cells detected by immunnophenotyping. MRP8BCL-2 mice present with expansion of bone marrow primitive Scat+ blast cells. In contrast, NRASD12/BCL-2 mice have a significantlyreduced life span presenting with disease characteristic of MDS/preleukemia, together with increased bone marrow blasts and infiltrated tissues and a penetrance of 50% with a latency period of 3 6 months, which is transplantable. Increased RAS activity was observed in the NRASD12 transgenic mice and both the Mact+ and Scat+ compartments had an increase in RAS/BCL-2 complexes in the double transgenic mice. By creating a tetracyclineinducible model, TBCL-2/MMTVtTA/NRASDt2, we found
$5
that the mechanism of transformation to leukemia can be rescued or re-established by switching BCL-2 expression off or on respectively. These results suggest that the inhibition of one step may reverse the evolution of the disease to leukemia, identifying potential targets for therapy. (Presented in the co-operate session with Chugai.)
Stem ceg biology II
•
CD45-NEGATIVE CLONAL CELLS WITH VERY IMMATURE PHENOTYPE (CD45-CD34-CD38-Lin-) IN PATIENTS WITH MYELODYSPLASTIC SYNDROMES
K. Ogata 1 *, C. Satoh 2, M. Tachibana 1, H. Hyodo 1, H. Tamura 1, K. Dan 1, T. Kimura 3, Y. Sonoda 4, T. Tsuji5.
1Division of Hematology, Department of Medicine, Nippon Medical School, eDepartment of Bioregulation, Department of Medicine, Nippon Medical School, 3Department of Molecular-Targeting Cancer Prevention, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, 4Department of Hygiene, Kansai Medical University, 5Department of Industrial Science arid Technology, Tokyo University of Science, Japan *E-mail: [email protected] CD45 is a hematopoietic lineage-restricted antigen that is expressed on all hematopoietic cells except for some mature cell types. CD45+CD34+CD38 L i n cells are well-documented hematopoietic stem cells (HSCs), and CD45+CD34 CD38 L i p cells are probably less mature HSCs. In this study, we detected CD45 CD34 CD38 L i p cells in the peripheral blood and bone marrow of patients with myelodysplastic syndromes (MDS). Fluorescence in situ hybridization showed that CD45 CD34 CD38 L i p cells had the same chromosomal aberration as the myeloblasts. Generally, MDS cells have extremely reduced hematopoietic potential compared with normal cells, but we documented the following in some patients. Freshlyisolated CD45 CD34 CD38 L i p cells did not form any hematopoietic colonies but had long-term culture-initiating cell activity. When co-cultured with stroma cells, the cell proliferation kinetics of CD45 CD34 CD38 L i p cells differed from those of CD34+ myeloblasts. The number of cells in CD45 CD34 CD38 L i n cell cultures decreased during the first 7 t0 days, while clusters consisting of 2 4 cells began to appear on days 7 t0. These cells showed only weak potential for proliferation/differentiation, yet differentiated into CD34+ cells and then mature myeloid cells. In contrast, in the CD34+ myeloblast culture, cell clusters began to appear on day 3 and a marked increase in cells was observed on day 7. The CD45 CD34 CD38 L i p cell population represents the most immature immunophenotype so far identified in the hematopoietic lineage and is involved in the malignant clone in MDS.