- Email: [email protected]
Cycling status of primitive malignant progenitors from patients with acute myelogenous leukemia (Aml)
80
Abstracts/Experimental Hematology 28 (2000) 31–131
fection was obtained in CBA mice by intraperitoneal injection of 2 ⫻ 102 tachyzoites. Changes in the number of granulocytemacrophage (CFU-GM) and erythroid (BFU-E and CFU-E) progenitor cells, were evaluated in the liver and spleen. Specimens of the spleen and the liver were histopathologically examined by light and electron microscopy. The results obtained demonstrated significant changes in the number of different splenic hematopoietic cells from the 3rd hour of toxoplasmic infection onward, together with histologically confirmed myeloid, erythroid and megakaryocytic hyperplasia in the spleen. Analysis of the liver specimens revealed significant extramedullary hematopoiesis within hepatic lobules. The changes observed during acute toxoplasmosis confirmed the presence of greatly stimulated hematopoiesis, which is not confined to the spleen, an active hematopoietic organ in mice, but also present in the liver which is normally postnatally devoid of hematopoiesis. The liver hematopoietic involvement suggests that the host defense system during a massive severe infection such as toxoplasmosis activates all hematopoietic mechanisms available, including those that are normally operative only during fetal life. 156
Sunday, July 9, 2000 (18:30–19:30) Poster Session I: Chronic Myelogenous Leukemia
HUMAN INTERFERON-ALPHA GENE TRANSFER IMPROVE ENGRAFTMENT OF CD34⫹ CELLS IN SCID MICE Hatem E. Sabaawy, Karen Seiter, Shuo Quan, Tauseef Ahmed and Nader G. Abraham Departments of Pharmacology and Medicine, New York Medical College, Valhalla, NY Systemic administration of Interferon-alpha (IFN-␣) enhances survival of CML patients, however, it is associated with deleterious side effects. Gene transfer of IFN-␣ into CD34⫹ stem cells represents a novel strategy to achieve long-term expression of IFN-␣. IFN-␣ gene transfer using adenoviral and retroviral vectors was associated with normal proliferation of CD34⫹ progenitors as measured by CFU-GM and BFU-E assays. Flow Cytometric Analysis revealed no significant difference in cell viability and analysis of mRNA from CD34⫹ harvested CFU-GM progenitors by RT/ PCR demonstrated the expression of human IFN-␣ mRNA. RIA revealed that IFN-␣ infected CD34⫹ cells produced 72.2 ⫾ 15.4 U/ml/106 cells/24 hs compared to 8.3 ⫾ 2.1 and 4.3 ⫾ 1.2 U/ml/ 106 cells/24 hs in control vector infected and non-infected cells respectively. We evaluated the In Vivo long-term expression of IFN-␣ gene by transplanting the infected CD34⫹ cells into severe combined immune deficient (SCID) mice. Engraftment of CD34⫹ cells infected with IFN-␣ gene in NOD/SCID mice was successful for 30 days. Next, we studied the effects of local IFN-␣ expression on the cellular adhesion molecules, VLA-4, Mac-1, ICAM-1, and L-Selectin in K562 cells and human umbilical endothelial vein cells. K562 cells infected with IFN-␣ gene showed significantly higher levels of VLA-4, Mac-1 and ICAM-1. We conclude that viral expression of IFN-␣ gene may provide feasible approach to enhance stem cells engraftment in bone marrow transplantation.
157
Sunday, July 9, 2000 (18:30–19:30) Poster Session I: Gene Expression-Transcription Factors
SEVERE VALVULAR AND AORTIC ARCH CALCIFICATION IN A PATIENT WITH GAUCHER’S VARIANT HOMOZYGOUS FOR D409H MUTATION George Roshini*, Bruce Lytle*, Alan Lichtin Taussig Cancer Center, Cleveland Clinic, Ohio Gaucher’s disease is an autosomal recessive inherited defect of the lysosomal enzyme glucocerebrosidase which leads to glucocerebroside accumulation in the reticuloendothelial system. The most common mutations are N370S and 84 gg which lead to bone fractures, organomegaly and cytopenias. We present a case of a 17 year old Palestinian patient who presented in 7/97 with severe aortic and mitral valvular calcification, as well as calcification of the ascending aorta, the aortic arch and the ostia of his coronary arteries. The patient was confirmed to be homozygous for the D409H mutation of the glucocerebrosidase gene. The patient’s enzyme assay for glucocerebrosidase activity was 5 nm/hr/mg protein (normal 13–22 nm/hr/mg). The patient presented with symptoms of dyspnea and chest pain. He had a history of documented aortic valve calcification that was diagnosed in 1991 by echocardiogram after two of his older brothers died of congestive heart failure and severe valvular calcification. Cardiac catheterization on 7/30/99 showed a severely calcified aorta with almost no motion of the aortic valve leaflets and severe calcification of the mitral valve and the mitral valvular apparatus. On 8/5/97 patient underwent extensive cardiac surgery with aortic and mitral valve replacements and intraoperative findings confirmed calcification of the entire aortic root. Electron microscopy of the valves confirmed the presence of Gaucher’s bodies. Enzyme therapy with imiglucerase was initiated in the perioperative period and patient was in stable condition at last follow-up in 12/99. Previous reports of patients with the D409H mutation note this unusual phenotype with severe calcification of the aortic and mitral valves. 158
Sunday, July 9, 2000 (10:30–12:30) Session I-1: Acute Leukemia: Basic Research
CYCLING STATUS OF PRIMITIVE MALIGNANT PROGENITORS FROM PATIENTS WITH ACUTE MYELOGENOUS LEUKEMIA (AML) Y. Guan* and D. Hogge Terry Fox Laboratory, B.C. Cancer Agency, Vancouver, Canada Hematopoietic progenitors in normal steady state peripheral blood (PB) are quiescent. In contrast, using an overnight 3H-Thymidine (3H-Tdr) assay, we previously found that many AML colony forming cells (AML-CFCs) and long term culture-initiating cells (AML LTC-ICs) were actively cycling. The same 3H-Tdr suicide assay is now used to compare the proliferative status of AML progenitors detected in NOD/SCID mice (NOD/SCID leukemiaIC or NOD/SL-IC) to that of AML-CFC and LTC-IC detected in 6 newly-diagnosed AML patient samples. Culture of AML cells for 16 h without 3H-Tdr in serum-free medium with 50 ng/ml Steel Factor, 20 ng/ml IL-3 and 20 ng/ml G-CSF maintained AML-CFC and LTC-IC numbers at near input levels (mean % input ⫾ SD, 123 ⫾ 72 and 117 ⫾ 47, respectively) but reduced the % CD45⫹ AML cells detected in NOD/SCID mouse bone marrow 8 wks after i.v. injection as compared to uncultured cells (mean % input ⫾
Abstracts/Experimental Hematology 28 (2000) 31–131
SD, 58 ⫾ 41). The addition of 20 Ci/ml high specific activity 3HTdr to such cultures reduced the numbers of both AML CFC and LTC-IC significantly (mean % kill ⫾ SD 57 ⫾ 42 and 68 ⫾ 30, respectively). However, ⬍50% kill of AML-CFC and/or LTC-IC was seen for 3 of 6 samples. NOD/SL-ICs from these 3 samples were also largely quiescent (% kill 16-43% at 8 wks). In one sample, AML CFC, LTC-IC and NOD/SL-IC were all actively cycling (⬎80% kill). In the remaining 2 cases, although ⬎90% kill of AML LTC-IC and 61 and 99% kill of AML-CFC were detected, NOD/SL-IC showed 26 and 0% kill at 8 wks. Thus, although a larger proportion actively cycling CFC and LTC-IC are detected in AML blood than in normal steady state PB, a significant proportion of primitive leukemic progenitors from most patient samples are quiescent. These cells may be relatively resistant to standard therapeutic regimens for AML. 159
Monday, July 10, 2000 (9:45–11:15) Session III-1: Acute Leukemia: Clinical and Basic Research
SELECTIVE CYTOTOXICITY OF DIPHTHERIA TOXININTERLEUKIN 3 FUSION PROTEIN FOR ACUTE MYELOID LEUKEMIA (AML) STEM CELLS M. Feuring-Buske*1, A. E. Frankel2, B. Gerhard*1, D. E. Hogge1 The Terry Fox Laboratory, B. C. Cancer Agency, Vancouver, Canada. 2Department of Cancer Biology, Wake Forest University School of Medicine, Winston-Salem, NC, USA Recently we showed that a diphtheria toxin (DT) GM-CSF fusion protein produced up to 1.6 log kill of primitive AML progenitors measured in 5 wks stromal co-cultures (AML longterm culture-initiating cells or LTC-IC), suspension culture (AML SC-IC) and in vivo in NOD/SCID mice. However, DT-GMCSF failed to kill AML progenitors from some patient samples. 80% of leukemic blasts from AML patients proliferate in response to interleukin 3 (IL3) while in some reports primitive normal progenitors appear not to express IL-3 receptors. Thus, to engineer a toxin which could potentially target primitive leukemic but not normal cells human IL3 was linked to a truncated form of DT with a (G4S)2 linker (DTLIL3). 24 h exposure of AML cells to 50 ng/ml DTLIL3 produced a mean (range) log kill of AML CFC, LTC-IC and SCIC of 0.78 (0.28–1.19) (n ⫽ 6); 0.6 (0.31 and 0.77) (n ⫽ 2) and 0.56 (0.39–0.76) (n ⫽ 3), respectively. The mean reduction in AML cell engraftment in NOD/SCID mice by the toxin-treated cells was 1.74 log at week 4 (range 1.14–2.62) and 0.6 log (range 0–1.9) at week 8 (n ⫽ 4). Normal bone marrow (n ⫽ 3) incubated with 50 ng/ml DTLIL3 for 24 h showed a mean log kill of 0.3 in the CFC assay. Interestingly, no cell kill was observed in the LTCIC and SC-IC assay of all 3 normal bone marrow samples tested even when the concentration of DT-IL3 was increased to 250 ng/ ml. Thus, DTLIL3 shows selective toxicity for leukemic but not normal progenitors and may prove to be useful therapeutic agent for some patients with AML.
160
81
Tuesday, July 11, 2000 (10:15–12:15) Session V-5: Stem and Progenitor Cell Transplantation: Experimental II
GETTING CLOSER TO THE HAEMOPOIETIC STEM CELL UTILISING INTERNAL CD34 EXPRESSION D. Pearce1/2*, R. Pettengell2*, E. C. Gordon-Smith2 & C. P. McGuckin1 1 Kingston University, Sch. Life Sciences; 2St George’s Hospital Medical School, Dept., of Haematology & 1/2The King-George Lab, London CD34 surface molecules has been exhaustively used as surrogate markers for primitive haemopoietic cells, but, increasing immunophenotyping combined with SCID mice / sheep repopulation evidence since 1997 suggests extremely primitive haemopoietic cells exist that do not express external (e)CD34. CD38 is expressed in increasing amounts on (e)CD34⫹ cells as they mature. Most primitive (⬵10%) (e)CD34⫹ cells, are also characterized by lack of CD38. AC133 (a novel 5-transmembrane glycoprotein), is expressed on early (e)CD34⫹ subsets. CD34 can be quickly upregulated (1 min) independently of transcription / translation, probably from preformed intracellular stores. Our study investigated internal CD34, determining its significance in haemopoietic hierarchy, via triple flow cytometry examining (i)CD34 & (e)CD34, with one other haemopoietic cell marker CD38 or AC133).
(i)CD34⫹ Subset
% of CBMNC
% CD38low/⫺
%AC133⫹
(i)CD34⫹/(e)CD34neg (i)CD34⫹/(e)CD34⫹ (i)CD34low/(e)CD34⫹ (i)CD34neg/(e)CD34lo Mean⫾SEM
0.06%⫾0.02% 0.38%⫾0.08% 0.30%⫾0.05% 0.15%⫾0.03% From CD38 data
40.86%⫾9.24% 11.94%⫾2.09% 7.08%⫾1.31% 5.30%⫾1.55% N ⫽ 10
4.17%⫾1.58% 88.68%⫾1.55% 81.91%⫾3.14% 30.91%⫾8.60% N⫽6
Such statistically different staining patterns suggests the CD34 molecule translocates from internal stores to surface during haemopoietic cell development. (i)CD34⫹/(e)CD34neg cell expression profiles are markedly different to (e)CD34⫹ cells indicating they are a separate distinct subset. Although (i)CD34 is not a feasible “positive” marked for harvest/selection, it’s analysis allows a new haemopoietic developmental stage to be determined. Further immunophenotyping of these (i)CD34⫹ cells is being performed to elucidate the significance of internal CD34 expression, and its relationship to reversible CD34 expression. 161
Monday, July 10, 2000 (16:00–17:00) Poster Session II: Stem Cell Biology
IS AC133 INVOLVED IN DIRECT CELL TO CELL COMMUNICATION? C. P. McGuckin1, R. Pettengell2*, K. Jones1*, F. Martin1*, E. C. Gordon-Smith2, & D. Pearce1/2* 1 Kingston University, School of Life Sciences, Surrey, UK; 2 St George’s Hospital Medical School, Dept. Haematology & 1/2 King-George Lab. London, UK AC133 has shown significant sequence homology to the murine molecule prominin, and a Caenorhabtidus elegans protein. Mouse prominin is detectable at early stages of murine embryonic
Abstracts/Experimental Hematology 28 (2000) 31–131
fection was obtained in CBA mice by intraperitoneal injection of 2 ⫻ 102 tachyzoites. Changes in the number of granulocytemacrophage (CFU-GM) and erythroid (BFU-E and CFU-E) progenitor cells, were evaluated in the liver and spleen. Specimens of the spleen and the liver were histopathologically examined by light and electron microscopy. The results obtained demonstrated significant changes in the number of different splenic hematopoietic cells from the 3rd hour of toxoplasmic infection onward, together with histologically confirmed myeloid, erythroid and megakaryocytic hyperplasia in the spleen. Analysis of the liver specimens revealed significant extramedullary hematopoiesis within hepatic lobules. The changes observed during acute toxoplasmosis confirmed the presence of greatly stimulated hematopoiesis, which is not confined to the spleen, an active hematopoietic organ in mice, but also present in the liver which is normally postnatally devoid of hematopoiesis. The liver hematopoietic involvement suggests that the host defense system during a massive severe infection such as toxoplasmosis activates all hematopoietic mechanisms available, including those that are normally operative only during fetal life. 156
Sunday, July 9, 2000 (18:30–19:30) Poster Session I: Chronic Myelogenous Leukemia
HUMAN INTERFERON-ALPHA GENE TRANSFER IMPROVE ENGRAFTMENT OF CD34⫹ CELLS IN SCID MICE Hatem E. Sabaawy, Karen Seiter, Shuo Quan, Tauseef Ahmed and Nader G. Abraham Departments of Pharmacology and Medicine, New York Medical College, Valhalla, NY Systemic administration of Interferon-alpha (IFN-␣) enhances survival of CML patients, however, it is associated with deleterious side effects. Gene transfer of IFN-␣ into CD34⫹ stem cells represents a novel strategy to achieve long-term expression of IFN-␣. IFN-␣ gene transfer using adenoviral and retroviral vectors was associated with normal proliferation of CD34⫹ progenitors as measured by CFU-GM and BFU-E assays. Flow Cytometric Analysis revealed no significant difference in cell viability and analysis of mRNA from CD34⫹ harvested CFU-GM progenitors by RT/ PCR demonstrated the expression of human IFN-␣ mRNA. RIA revealed that IFN-␣ infected CD34⫹ cells produced 72.2 ⫾ 15.4 U/ml/106 cells/24 hs compared to 8.3 ⫾ 2.1 and 4.3 ⫾ 1.2 U/ml/ 106 cells/24 hs in control vector infected and non-infected cells respectively. We evaluated the In Vivo long-term expression of IFN-␣ gene by transplanting the infected CD34⫹ cells into severe combined immune deficient (SCID) mice. Engraftment of CD34⫹ cells infected with IFN-␣ gene in NOD/SCID mice was successful for 30 days. Next, we studied the effects of local IFN-␣ expression on the cellular adhesion molecules, VLA-4, Mac-1, ICAM-1, and L-Selectin in K562 cells and human umbilical endothelial vein cells. K562 cells infected with IFN-␣ gene showed significantly higher levels of VLA-4, Mac-1 and ICAM-1. We conclude that viral expression of IFN-␣ gene may provide feasible approach to enhance stem cells engraftment in bone marrow transplantation.
157
Sunday, July 9, 2000 (18:30–19:30) Poster Session I: Gene Expression-Transcription Factors
SEVERE VALVULAR AND AORTIC ARCH CALCIFICATION IN A PATIENT WITH GAUCHER’S VARIANT HOMOZYGOUS FOR D409H MUTATION George Roshini*, Bruce Lytle*, Alan Lichtin Taussig Cancer Center, Cleveland Clinic, Ohio Gaucher’s disease is an autosomal recessive inherited defect of the lysosomal enzyme glucocerebrosidase which leads to glucocerebroside accumulation in the reticuloendothelial system. The most common mutations are N370S and 84 gg which lead to bone fractures, organomegaly and cytopenias. We present a case of a 17 year old Palestinian patient who presented in 7/97 with severe aortic and mitral valvular calcification, as well as calcification of the ascending aorta, the aortic arch and the ostia of his coronary arteries. The patient was confirmed to be homozygous for the D409H mutation of the glucocerebrosidase gene. The patient’s enzyme assay for glucocerebrosidase activity was 5 nm/hr/mg protein (normal 13–22 nm/hr/mg). The patient presented with symptoms of dyspnea and chest pain. He had a history of documented aortic valve calcification that was diagnosed in 1991 by echocardiogram after two of his older brothers died of congestive heart failure and severe valvular calcification. Cardiac catheterization on 7/30/99 showed a severely calcified aorta with almost no motion of the aortic valve leaflets and severe calcification of the mitral valve and the mitral valvular apparatus. On 8/5/97 patient underwent extensive cardiac surgery with aortic and mitral valve replacements and intraoperative findings confirmed calcification of the entire aortic root. Electron microscopy of the valves confirmed the presence of Gaucher’s bodies. Enzyme therapy with imiglucerase was initiated in the perioperative period and patient was in stable condition at last follow-up in 12/99. Previous reports of patients with the D409H mutation note this unusual phenotype with severe calcification of the aortic and mitral valves. 158
Sunday, July 9, 2000 (10:30–12:30) Session I-1: Acute Leukemia: Basic Research
CYCLING STATUS OF PRIMITIVE MALIGNANT PROGENITORS FROM PATIENTS WITH ACUTE MYELOGENOUS LEUKEMIA (AML) Y. Guan* and D. Hogge Terry Fox Laboratory, B.C. Cancer Agency, Vancouver, Canada Hematopoietic progenitors in normal steady state peripheral blood (PB) are quiescent. In contrast, using an overnight 3H-Thymidine (3H-Tdr) assay, we previously found that many AML colony forming cells (AML-CFCs) and long term culture-initiating cells (AML LTC-ICs) were actively cycling. The same 3H-Tdr suicide assay is now used to compare the proliferative status of AML progenitors detected in NOD/SCID mice (NOD/SCID leukemiaIC or NOD/SL-IC) to that of AML-CFC and LTC-IC detected in 6 newly-diagnosed AML patient samples. Culture of AML cells for 16 h without 3H-Tdr in serum-free medium with 50 ng/ml Steel Factor, 20 ng/ml IL-3 and 20 ng/ml G-CSF maintained AML-CFC and LTC-IC numbers at near input levels (mean % input ⫾ SD, 123 ⫾ 72 and 117 ⫾ 47, respectively) but reduced the % CD45⫹ AML cells detected in NOD/SCID mouse bone marrow 8 wks after i.v. injection as compared to uncultured cells (mean % input ⫾
Abstracts/Experimental Hematology 28 (2000) 31–131
SD, 58 ⫾ 41). The addition of 20 Ci/ml high specific activity 3HTdr to such cultures reduced the numbers of both AML CFC and LTC-IC significantly (mean % kill ⫾ SD 57 ⫾ 42 and 68 ⫾ 30, respectively). However, ⬍50% kill of AML-CFC and/or LTC-IC was seen for 3 of 6 samples. NOD/SL-ICs from these 3 samples were also largely quiescent (% kill 16-43% at 8 wks). In one sample, AML CFC, LTC-IC and NOD/SL-IC were all actively cycling (⬎80% kill). In the remaining 2 cases, although ⬎90% kill of AML LTC-IC and 61 and 99% kill of AML-CFC were detected, NOD/SL-IC showed 26 and 0% kill at 8 wks. Thus, although a larger proportion actively cycling CFC and LTC-IC are detected in AML blood than in normal steady state PB, a significant proportion of primitive leukemic progenitors from most patient samples are quiescent. These cells may be relatively resistant to standard therapeutic regimens for AML. 159
Monday, July 10, 2000 (9:45–11:15) Session III-1: Acute Leukemia: Clinical and Basic Research
SELECTIVE CYTOTOXICITY OF DIPHTHERIA TOXININTERLEUKIN 3 FUSION PROTEIN FOR ACUTE MYELOID LEUKEMIA (AML) STEM CELLS M. Feuring-Buske*1, A. E. Frankel2, B. Gerhard*1, D. E. Hogge1 The Terry Fox Laboratory, B. C. Cancer Agency, Vancouver, Canada. 2Department of Cancer Biology, Wake Forest University School of Medicine, Winston-Salem, NC, USA Recently we showed that a diphtheria toxin (DT) GM-CSF fusion protein produced up to 1.6 log kill of primitive AML progenitors measured in 5 wks stromal co-cultures (AML longterm culture-initiating cells or LTC-IC), suspension culture (AML SC-IC) and in vivo in NOD/SCID mice. However, DT-GMCSF failed to kill AML progenitors from some patient samples. 80% of leukemic blasts from AML patients proliferate in response to interleukin 3 (IL3) while in some reports primitive normal progenitors appear not to express IL-3 receptors. Thus, to engineer a toxin which could potentially target primitive leukemic but not normal cells human IL3 was linked to a truncated form of DT with a (G4S)2 linker (DTLIL3). 24 h exposure of AML cells to 50 ng/ml DTLIL3 produced a mean (range) log kill of AML CFC, LTC-IC and SCIC of 0.78 (0.28–1.19) (n ⫽ 6); 0.6 (0.31 and 0.77) (n ⫽ 2) and 0.56 (0.39–0.76) (n ⫽ 3), respectively. The mean reduction in AML cell engraftment in NOD/SCID mice by the toxin-treated cells was 1.74 log at week 4 (range 1.14–2.62) and 0.6 log (range 0–1.9) at week 8 (n ⫽ 4). Normal bone marrow (n ⫽ 3) incubated with 50 ng/ml DTLIL3 for 24 h showed a mean log kill of 0.3 in the CFC assay. Interestingly, no cell kill was observed in the LTCIC and SC-IC assay of all 3 normal bone marrow samples tested even when the concentration of DT-IL3 was increased to 250 ng/ ml. Thus, DTLIL3 shows selective toxicity for leukemic but not normal progenitors and may prove to be useful therapeutic agent for some patients with AML.
160
81
Tuesday, July 11, 2000 (10:15–12:15) Session V-5: Stem and Progenitor Cell Transplantation: Experimental II
GETTING CLOSER TO THE HAEMOPOIETIC STEM CELL UTILISING INTERNAL CD34 EXPRESSION D. Pearce1/2*, R. Pettengell2*, E. C. Gordon-Smith2 & C. P. McGuckin1 1 Kingston University, Sch. Life Sciences; 2St George’s Hospital Medical School, Dept., of Haematology & 1/2The King-George Lab, London CD34 surface molecules has been exhaustively used as surrogate markers for primitive haemopoietic cells, but, increasing immunophenotyping combined with SCID mice / sheep repopulation evidence since 1997 suggests extremely primitive haemopoietic cells exist that do not express external (e)CD34. CD38 is expressed in increasing amounts on (e)CD34⫹ cells as they mature. Most primitive (⬵10%) (e)CD34⫹ cells, are also characterized by lack of CD38. AC133 (a novel 5-transmembrane glycoprotein), is expressed on early (e)CD34⫹ subsets. CD34 can be quickly upregulated (1 min) independently of transcription / translation, probably from preformed intracellular stores. Our study investigated internal CD34, determining its significance in haemopoietic hierarchy, via triple flow cytometry examining (i)CD34 & (e)CD34, with one other haemopoietic cell marker CD38 or AC133).
(i)CD34⫹ Subset
% of CBMNC
% CD38low/⫺
%AC133⫹
(i)CD34⫹/(e)CD34neg (i)CD34⫹/(e)CD34⫹ (i)CD34low/(e)CD34⫹ (i)CD34neg/(e)CD34lo Mean⫾SEM
0.06%⫾0.02% 0.38%⫾0.08% 0.30%⫾0.05% 0.15%⫾0.03% From CD38 data
40.86%⫾9.24% 11.94%⫾2.09% 7.08%⫾1.31% 5.30%⫾1.55% N ⫽ 10
4.17%⫾1.58% 88.68%⫾1.55% 81.91%⫾3.14% 30.91%⫾8.60% N⫽6
Such statistically different staining patterns suggests the CD34 molecule translocates from internal stores to surface during haemopoietic cell development. (i)CD34⫹/(e)CD34neg cell expression profiles are markedly different to (e)CD34⫹ cells indicating they are a separate distinct subset. Although (i)CD34 is not a feasible “positive” marked for harvest/selection, it’s analysis allows a new haemopoietic developmental stage to be determined. Further immunophenotyping of these (i)CD34⫹ cells is being performed to elucidate the significance of internal CD34 expression, and its relationship to reversible CD34 expression. 161
Monday, July 10, 2000 (16:00–17:00) Poster Session II: Stem Cell Biology
IS AC133 INVOLVED IN DIRECT CELL TO CELL COMMUNICATION? C. P. McGuckin1, R. Pettengell2*, K. Jones1*, F. Martin1*, E. C. Gordon-Smith2, & D. Pearce1/2* 1 Kingston University, School of Life Sciences, Surrey, UK; 2 St George’s Hospital Medical School, Dept. Haematology & 1/2 King-George Lab. London, UK AC133 has shown significant sequence homology to the murine molecule prominin, and a Caenorhabtidus elegans protein. Mouse prominin is detectable at early stages of murine embryonic