- Email: [email protected]
Functional characterization of minimal residual disease in aml - simultaneous detection of p-glycoprotein function and aberrant phenotypes
Abstracts/Experimental Hematology 28 (2000) 31–131
These in vivo observations were complemented by in vitro analysis of donor BM. Colony-assays (HPP-CFC), thymidine suicide tests (percentage of cycling cells), and lineage analysis were performed. Significant asynchronous fluctuations of HPP-CFC numbers were seen while proliferation varied inversely with engraftment. These data provide statistically significant evidence of circadian variations of BM engraftability and form a base for further studies of stem cell chronobiology. 220
Tuesday, July 11, 2000 (10:15–12:15) Session V-4: Stem and Progenitor Cell Transplantation: Clinical
THE WORLDWIDE HEMATOPOIETIC CELL TRANSPLANTATION EXPERIENCE FOR CEREBRAL X-ADRENOLEUKODYSTROPHY C. Peters, S. Abel*, T. DeFor*, G. Korblick*, K. Dusenbery*, L. A. Lockman*, R. Ziegler*, E. G. Shapiro*, W. Krivit, and The International Storage Disease Collaborative Study Group Univ. of Minnesota, Minneapolis, MN. USA The International Storage Disease Collaborative Study Group has evaluated the effectiveness of hematopoietic cell transplant (HCT) to halt the progressive neurologic deterioration, dementia, and early death of cerebral X-adrenoleukodystrophy (CXALD) in 126 patients (? donor: 3) from July 1982 to January 1999.
Characteristics
Related (n⫽56)
Unrelated (n⫽67)
Median age at HCT (range) BM: 6/6, 5/6, 4/6, ? UCB: 6/6, 5/6, 4/6 Preparative therapy Chemo, Chemo⫹XRT XRT alone, ? Median VIQ (range) Median PIQ (range) Outcomes Survival at 5 y (95% CI) Deaths and causes CXALD, GVHD Median VIQ (range) Median PIQ (range)
9.2 y (1.9–15.6) 41, 5, 6, 3 1, 0, 0
8.0 y (4.9–16.1) 33, 15, 0, 7 4, 6, 2
38, 14 0, 4 93 (47–115) 85 (blind–115)
29, 36 1, 1 93 (59–124) 87 (blind–138)
68% (54–82%) 16 8, 3 83 (40–116) 82 (blind–112)
54% (41–67%) 27 8, 1 73 (46–123) 69 (blind–123)
Abbreviations: ?, unknown; y, years; BM, bone marrow; UCB, umbilical cord blood; chemo, chemotherapy; XRT, radiation therapy; VIQ, verbal IQ; PIQ, performance IQ.
HCT can achieve neurologic and neuropsychologic stabilization as well as preservation of life in boys with CXALD especially when performed early in the disease course. A centralized HCT CXALD database is needed so that clinical trials can be performed.
221
101
Sunday, July 9, 2000 (18:30–19:30) Poster Session I: Graft Versus Host Disease
KINETICS OF SERUM CYTOKINE LEVELS IN PEDIATRIC PATIENTS WITH ACUTE GRAFT-VERSUSHOST DISEASE (GVHD) AFTER TRANSPLANTATION K. W. Chik*, P. M. P. Yuen*, K. Li, H. Pong*, M. M. K. Shing*, C. K. Li* Department of Paediatrics, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, HKSAR, China We aimed to study the correlation of serum cytokine levels with GVHD after hematopoietic stem cell transplantation (HST). From 1997 to 1999, serum samples were serially collected (3 times per week) from 20 HST subjects. Serum levels of interferon (IFN) ␥, interleukin (IL)-10, IL-12 and IL-15 were assessed using commercial ELISA kits, (R&D Systems, Minneapolis, USA). The median age of transplant patients was 8.46 years. There were 1 subject with grade IV disease, 2 with grade III disease, 6 with grade II disease, 3 with grade I disease and 8 without any clinical evidence of GVHD. The mean peak serum levels of IL-10 and IL-15 were 43.2 ⫾ 37.5 pg/ml and 49.3 ⫾ 16.2 pg/ml in GVHD subjects respectively while they were 18.6 ⫾ 9.0 pg/ml and 19.9 ⫾ 5.9 pg/ml in subjects without GVHD (p ⬍ 0.05). The peak level occurred around one week from transplant. Serum IFN and IL-12 levels were not significantly different between the 2 groups. We suggested that the serum level of IL-10 and IL-15 may reflect the development of GVHD, possibly mediated by the disturbance of T cells and monocytes/macrophages. The response of the individual to the transplant conditioning could be a factor leading to the surge of IL-10 and IL-15 levels within 1 week following HST. 222
Monday, July 10, 2000 (9:45–11:15) Session III-1: Acute Leukemia: Clinical and Basic Research
FUNCTIONAL CHARACTERIZATION OF MINIMAL RESIDUAL DISEASE IN AML - SIMULTANEOUS DETECTION OF P-GLYCOPROTEIN FUNCTION AND ABERRANT PHENOTYPES M. A. van der Pol1*, J. M. Pater1*, N. Feller1*, A. van Stijn1*, A. H. Westra1*, G. J. Ossenkoppele1, H. J. Broxterman2, G. J. Schuurhuis1* Departments of 1Hematology and 2Medical Oncology, University Hospital Vrije Universiteit, Amsterdam, The Netherlands P-glycoprotein (Pgp) expression in de novo AML is associated with poor prognosis, with in part of the cases higher Pgp expression found in refractory and relapsed AML. The question whether such is due to either i) chemotherapy-induced selection of resistant subpopulations or ii) upregulation of Pgp function or iii) simply reflects, in cases with a Gaussian distribution of Pgp activity, survival of larger fractions of cells when the Pgp activity is higher, should be answered by functionally characterizing minimal residual disease (MRD). Pgp function was detected using Syto16/ PSC833, a fluorescence shift ⬎1.0 indicating Pgp activity. In de novo blasts from 44 patients (pts), a median shift of 1.5 (range: 1.0–31.7) was found. In 88% (39/44) of pts a leukemia associated phenotype (LAP) could be established for subsequent detection of MRD. A very sensitive and reproducible 4 colour FACS assay has been developed that can simultaneously detect Pgp function and LAP. Thus far the hypotheses have been tested in 30 samples from
102
Abstracts/Experimental Hematology 28 (2000) 31–131
ten pts at different stages of disease: de novo, after 1st-3th course of chemotherapy, in the stem cell graft, after PBSCT, at relapse. Homogenous Pgp function was found in these de novo blasts with a median shift of 1.4 (5 pts ⬎ 1.5; 5 pts ⬍ 1.5). No consequent change in Pgp function was found in the follow-up samples, showing a mean ⫾ SEM of 102 ⫾ 8% of shift of the corresponding de novo sample. Therefore, we conclude that the prognostic value of Pgp in AML is likely due to survival of larger fractions of cells, including AML stem cells, in cases of higher mean Pgp activity in cells with homogenous (Gaussian) distribution of Pgp activity. 223
Sunday, July 9, 2000 (18:30–19:30) Poster Session I: Acute Leukemia: Basic Research
CLONOGENIC ABILITY AND APOPTOSIS RESISTANCE ARE HIGHER FOR CD34⫹ THAN FOR CD34⫺ BLASTS IN AML A. van Stijn*, P. M. Bontje*, M. A. van der Pol*, N. Feller*, G. J. Ossenkoppele and G. J. Schuurhuis* Department of Hematology, University Hospital Vrije Universiteit, Amsterdam, The Netherlands Many cellular and functional characteristics of acute myeloid leukemia (AML) blasts have been correlated with clinical parameters; these include spontaneous apoptosis and response to chemotherapy (Smith et al., Br J Haematol, 1998, 102:1042; Wuchter et al., Leukemia, 1999, 13:1943). Since there is accumulating evidence that the leukemogenic cell in AML resides in the CD34⫹ cell population, CD34⫹ and CD34⫺ AML cells were separated using the MiniMACS system and evaluated for a) clonogenic ability in the CFU-L assay and b) apoptosis resistance in a serum-free culture system. Initial percentage of CD34⫹ ranged between 4 and 88%. After correction for impurities in both fractions, the clonogenic ability of CD34⫺ blasts in 10/17 samples was 0, while CD34⫹ blasts had a median of 547 CFU-L per 106 cells (range 71–3673). In 6/17 cases, both fractions had clonogenic ability, of which the CD34⫹ fraction had the highest in 4 cases; in 1/17 cases both fractions had no clonogenic ability. Spontaneous apoptosis was measured in 14 cases during 6 hours using Annexin-V/7-AAD and Syto16/PSC833/7-AAD, a sensitive method to detect early apoptosis (Schuurhuis et. al., Blood, 1998, 92:suppl 1:121a). Compared to the CD34⫹, the CD34⫺ fraction displayed a significantly higher median value of spontaneous (early and late) apoptosis (mean % CD34⫺/ mean % CD34⫹ at t ⫽ 0 h: 32/19, p ⫽ 0.003; at t ⫽ 2 h: 36/22, p ⫽ 0.005; at t ⫽ 6 h: 42/27, p ⫽ 0.021). These results show that in a CD34⫹ AML, the CD34⫹ compartment differs from the CD34⫺ compartment concerning at least spontaneous apoptosis and clonogenic ability. This should be considered when correlating any blast cell property with clinical outcome. 224
Sunday, July 9, 2000 (18:30–19:30) Poster Session I: Cytokines, Growth Factors and Receptors
THYMOSIN 4, A NEGATIVE REGULATOR OF HEMATOPOIETIC PROGENITORS PRODUCED BY BONE MARROW ENDOTHELIAL CELLS Q. R. Wang*, W. Q. Huang*, L. M. Cheng*, B. H. Wang*. (Intro by C. T. Wu) Experimental Hematology Laboratory, Human Medical University Changsha, China For further study the hematopoietic inhibitors which elaborated by bone marrow endothelial cells (a cell line, published previ-
ously), the serum-free bone marrow endothelial cell conditioned medium (BMEC-cm) was collected. The components of different molecular weight (MW) ⬎ 10 KD, ⬍ 10 KD, ⬍ 0.5 KD were separated from BMEC-cm by means of serial ultrafiltration. In the component of MW ⬎ 10 KD, the solutes of MW ⬍ 10 KD were already removed by repeated filtrations. The effects of BMEC-cm as a whole and its ultrafiltration-prepared components on hematopoietic progenitors were investigated in vitro by culturing the cells in a liquid culture system contained growth factors. The results showed that BMEC-cm significantly increased the number of HPP-CFC but exerted much less influence on CFU-GM. Meanwhile the component of MW ⬎ 10 KD significantly increased the number of CFU-GM but decreased the number of HPP-CFC. Both the components of MW ⬍ 10 KD (or T4 as control) and the component of MW ⬍ 0.5 KD (or AcSDKP as control) significantly decreased the number of HPP-CFC and CFU-GM. The results of HPLC analysis showed that the thymosin 4-like material and AcSDKP-like material were present in ⬍ 10 KD and ⬍ 0.5 KD component respectively. The detection of expression of mRNA demonstrated that thymosin 4 mRNA was strongly expressed in BMEC. ELISA assays of BMEC-cm showed that BMEC elaborated high levels of T4 and AcSDKP. The above results suggested that T4 (MW ⫽ 4982) may be one of the inhibitors existed in ⬍ 10 KD component and AcSDKP (MW ⫽ 487) which can be generated from T4 by a one-step enzymatic cleavage may be one of the inhibitors existed in ⬍ 0.5 KD component. Since both T4 (or ⬍ 10 KD BMEC-cm) and AcSDKP (or ⬍ 0.5 KD BMEC-cm) inhibite hematopoietic progenitors entering into cell cycle. Hence, they may be useful in protecting hematopoietic progenitors from the damaging effect of cytotoxic agents and may be useful also in enhancing the expansion of early hematopoietic progenitors meanwhile decreasing their differentiation when they were employed together with growth factors in liquid cultures. 225
Monday, July 10, 2000 (16:00–17:00) Poster Session II: Stem Cell Biology
FLOW CYTOMETRY ANALYSIS OF MURINE HEMATOPOIETIC PROGENITORS RECOVERING FROM CYCLOPHOSPHAMIDE DAMAGE L. Sˇ efc, E. Necˇ as Institute of Pathophysiology, 1st Faculty of Medicine, Charles University, Prague, Czech Republic Along with damage to hematopoietic cells, cyclophosphamide damages also cells of hematopoietic microenvironment. Their uneven regeneration leads to a prolonged dysregulation of supporting functions of the microenvironment. The bone marrow progenitors follow a unique regeneration curve characterized by an early rapid recovery followed by secondary depression. We have analyzed the cellular composition of the bone marrow in mice recovering from single dose of cyclophosphamide by means of flow cytometry and microscopy. Sca-1⫹ progenitors have been magnetically separated and analyzed for c-kit, flk2/flt3, CD71 and lin⫹ markers. Their proliferation activity and apoptotic rate was also determined. The number and percentage of Sca-1⫹ cells in the bone marrow increased rapidly 2 to 3 days after cyclophosphamide. Their proliferation rate was high and they showed an increase in CD71 expression. The number and percentage of Sca-1⫹ progenitors 3 to 7 days after cyclophosphamide dropped considerably. Their proliferation rate was low along with lower CD71 and flt-3 expression.
These in vivo observations were complemented by in vitro analysis of donor BM. Colony-assays (HPP-CFC), thymidine suicide tests (percentage of cycling cells), and lineage analysis were performed. Significant asynchronous fluctuations of HPP-CFC numbers were seen while proliferation varied inversely with engraftment. These data provide statistically significant evidence of circadian variations of BM engraftability and form a base for further studies of stem cell chronobiology. 220
Tuesday, July 11, 2000 (10:15–12:15) Session V-4: Stem and Progenitor Cell Transplantation: Clinical
THE WORLDWIDE HEMATOPOIETIC CELL TRANSPLANTATION EXPERIENCE FOR CEREBRAL X-ADRENOLEUKODYSTROPHY C. Peters, S. Abel*, T. DeFor*, G. Korblick*, K. Dusenbery*, L. A. Lockman*, R. Ziegler*, E. G. Shapiro*, W. Krivit, and The International Storage Disease Collaborative Study Group Univ. of Minnesota, Minneapolis, MN. USA The International Storage Disease Collaborative Study Group has evaluated the effectiveness of hematopoietic cell transplant (HCT) to halt the progressive neurologic deterioration, dementia, and early death of cerebral X-adrenoleukodystrophy (CXALD) in 126 patients (? donor: 3) from July 1982 to January 1999.
Characteristics
Related (n⫽56)
Unrelated (n⫽67)
Median age at HCT (range) BM: 6/6, 5/6, 4/6, ? UCB: 6/6, 5/6, 4/6 Preparative therapy Chemo, Chemo⫹XRT XRT alone, ? Median VIQ (range) Median PIQ (range) Outcomes Survival at 5 y (95% CI) Deaths and causes CXALD, GVHD Median VIQ (range) Median PIQ (range)
9.2 y (1.9–15.6) 41, 5, 6, 3 1, 0, 0
8.0 y (4.9–16.1) 33, 15, 0, 7 4, 6, 2
38, 14 0, 4 93 (47–115) 85 (blind–115)
29, 36 1, 1 93 (59–124) 87 (blind–138)
68% (54–82%) 16 8, 3 83 (40–116) 82 (blind–112)
54% (41–67%) 27 8, 1 73 (46–123) 69 (blind–123)
Abbreviations: ?, unknown; y, years; BM, bone marrow; UCB, umbilical cord blood; chemo, chemotherapy; XRT, radiation therapy; VIQ, verbal IQ; PIQ, performance IQ.
HCT can achieve neurologic and neuropsychologic stabilization as well as preservation of life in boys with CXALD especially when performed early in the disease course. A centralized HCT CXALD database is needed so that clinical trials can be performed.
221
101
Sunday, July 9, 2000 (18:30–19:30) Poster Session I: Graft Versus Host Disease
KINETICS OF SERUM CYTOKINE LEVELS IN PEDIATRIC PATIENTS WITH ACUTE GRAFT-VERSUSHOST DISEASE (GVHD) AFTER TRANSPLANTATION K. W. Chik*, P. M. P. Yuen*, K. Li, H. Pong*, M. M. K. Shing*, C. K. Li* Department of Paediatrics, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, HKSAR, China We aimed to study the correlation of serum cytokine levels with GVHD after hematopoietic stem cell transplantation (HST). From 1997 to 1999, serum samples were serially collected (3 times per week) from 20 HST subjects. Serum levels of interferon (IFN) ␥, interleukin (IL)-10, IL-12 and IL-15 were assessed using commercial ELISA kits, (R&D Systems, Minneapolis, USA). The median age of transplant patients was 8.46 years. There were 1 subject with grade IV disease, 2 with grade III disease, 6 with grade II disease, 3 with grade I disease and 8 without any clinical evidence of GVHD. The mean peak serum levels of IL-10 and IL-15 were 43.2 ⫾ 37.5 pg/ml and 49.3 ⫾ 16.2 pg/ml in GVHD subjects respectively while they were 18.6 ⫾ 9.0 pg/ml and 19.9 ⫾ 5.9 pg/ml in subjects without GVHD (p ⬍ 0.05). The peak level occurred around one week from transplant. Serum IFN and IL-12 levels were not significantly different between the 2 groups. We suggested that the serum level of IL-10 and IL-15 may reflect the development of GVHD, possibly mediated by the disturbance of T cells and monocytes/macrophages. The response of the individual to the transplant conditioning could be a factor leading to the surge of IL-10 and IL-15 levels within 1 week following HST. 222
Monday, July 10, 2000 (9:45–11:15) Session III-1: Acute Leukemia: Clinical and Basic Research
FUNCTIONAL CHARACTERIZATION OF MINIMAL RESIDUAL DISEASE IN AML - SIMULTANEOUS DETECTION OF P-GLYCOPROTEIN FUNCTION AND ABERRANT PHENOTYPES M. A. van der Pol1*, J. M. Pater1*, N. Feller1*, A. van Stijn1*, A. H. Westra1*, G. J. Ossenkoppele1, H. J. Broxterman2, G. J. Schuurhuis1* Departments of 1Hematology and 2Medical Oncology, University Hospital Vrije Universiteit, Amsterdam, The Netherlands P-glycoprotein (Pgp) expression in de novo AML is associated with poor prognosis, with in part of the cases higher Pgp expression found in refractory and relapsed AML. The question whether such is due to either i) chemotherapy-induced selection of resistant subpopulations or ii) upregulation of Pgp function or iii) simply reflects, in cases with a Gaussian distribution of Pgp activity, survival of larger fractions of cells when the Pgp activity is higher, should be answered by functionally characterizing minimal residual disease (MRD). Pgp function was detected using Syto16/ PSC833, a fluorescence shift ⬎1.0 indicating Pgp activity. In de novo blasts from 44 patients (pts), a median shift of 1.5 (range: 1.0–31.7) was found. In 88% (39/44) of pts a leukemia associated phenotype (LAP) could be established for subsequent detection of MRD. A very sensitive and reproducible 4 colour FACS assay has been developed that can simultaneously detect Pgp function and LAP. Thus far the hypotheses have been tested in 30 samples from
102
Abstracts/Experimental Hematology 28 (2000) 31–131
ten pts at different stages of disease: de novo, after 1st-3th course of chemotherapy, in the stem cell graft, after PBSCT, at relapse. Homogenous Pgp function was found in these de novo blasts with a median shift of 1.4 (5 pts ⬎ 1.5; 5 pts ⬍ 1.5). No consequent change in Pgp function was found in the follow-up samples, showing a mean ⫾ SEM of 102 ⫾ 8% of shift of the corresponding de novo sample. Therefore, we conclude that the prognostic value of Pgp in AML is likely due to survival of larger fractions of cells, including AML stem cells, in cases of higher mean Pgp activity in cells with homogenous (Gaussian) distribution of Pgp activity. 223
Sunday, July 9, 2000 (18:30–19:30) Poster Session I: Acute Leukemia: Basic Research
CLONOGENIC ABILITY AND APOPTOSIS RESISTANCE ARE HIGHER FOR CD34⫹ THAN FOR CD34⫺ BLASTS IN AML A. van Stijn*, P. M. Bontje*, M. A. van der Pol*, N. Feller*, G. J. Ossenkoppele and G. J. Schuurhuis* Department of Hematology, University Hospital Vrije Universiteit, Amsterdam, The Netherlands Many cellular and functional characteristics of acute myeloid leukemia (AML) blasts have been correlated with clinical parameters; these include spontaneous apoptosis and response to chemotherapy (Smith et al., Br J Haematol, 1998, 102:1042; Wuchter et al., Leukemia, 1999, 13:1943). Since there is accumulating evidence that the leukemogenic cell in AML resides in the CD34⫹ cell population, CD34⫹ and CD34⫺ AML cells were separated using the MiniMACS system and evaluated for a) clonogenic ability in the CFU-L assay and b) apoptosis resistance in a serum-free culture system. Initial percentage of CD34⫹ ranged between 4 and 88%. After correction for impurities in both fractions, the clonogenic ability of CD34⫺ blasts in 10/17 samples was 0, while CD34⫹ blasts had a median of 547 CFU-L per 106 cells (range 71–3673). In 6/17 cases, both fractions had clonogenic ability, of which the CD34⫹ fraction had the highest in 4 cases; in 1/17 cases both fractions had no clonogenic ability. Spontaneous apoptosis was measured in 14 cases during 6 hours using Annexin-V/7-AAD and Syto16/PSC833/7-AAD, a sensitive method to detect early apoptosis (Schuurhuis et. al., Blood, 1998, 92:suppl 1:121a). Compared to the CD34⫹, the CD34⫺ fraction displayed a significantly higher median value of spontaneous (early and late) apoptosis (mean % CD34⫺/ mean % CD34⫹ at t ⫽ 0 h: 32/19, p ⫽ 0.003; at t ⫽ 2 h: 36/22, p ⫽ 0.005; at t ⫽ 6 h: 42/27, p ⫽ 0.021). These results show that in a CD34⫹ AML, the CD34⫹ compartment differs from the CD34⫺ compartment concerning at least spontaneous apoptosis and clonogenic ability. This should be considered when correlating any blast cell property with clinical outcome. 224
Sunday, July 9, 2000 (18:30–19:30) Poster Session I: Cytokines, Growth Factors and Receptors
THYMOSIN 4, A NEGATIVE REGULATOR OF HEMATOPOIETIC PROGENITORS PRODUCED BY BONE MARROW ENDOTHELIAL CELLS Q. R. Wang*, W. Q. Huang*, L. M. Cheng*, B. H. Wang*. (Intro by C. T. Wu) Experimental Hematology Laboratory, Human Medical University Changsha, China For further study the hematopoietic inhibitors which elaborated by bone marrow endothelial cells (a cell line, published previ-
ously), the serum-free bone marrow endothelial cell conditioned medium (BMEC-cm) was collected. The components of different molecular weight (MW) ⬎ 10 KD, ⬍ 10 KD, ⬍ 0.5 KD were separated from BMEC-cm by means of serial ultrafiltration. In the component of MW ⬎ 10 KD, the solutes of MW ⬍ 10 KD were already removed by repeated filtrations. The effects of BMEC-cm as a whole and its ultrafiltration-prepared components on hematopoietic progenitors were investigated in vitro by culturing the cells in a liquid culture system contained growth factors. The results showed that BMEC-cm significantly increased the number of HPP-CFC but exerted much less influence on CFU-GM. Meanwhile the component of MW ⬎ 10 KD significantly increased the number of CFU-GM but decreased the number of HPP-CFC. Both the components of MW ⬍ 10 KD (or T4 as control) and the component of MW ⬍ 0.5 KD (or AcSDKP as control) significantly decreased the number of HPP-CFC and CFU-GM. The results of HPLC analysis showed that the thymosin 4-like material and AcSDKP-like material were present in ⬍ 10 KD and ⬍ 0.5 KD component respectively. The detection of expression of mRNA demonstrated that thymosin 4 mRNA was strongly expressed in BMEC. ELISA assays of BMEC-cm showed that BMEC elaborated high levels of T4 and AcSDKP. The above results suggested that T4 (MW ⫽ 4982) may be one of the inhibitors existed in ⬍ 10 KD component and AcSDKP (MW ⫽ 487) which can be generated from T4 by a one-step enzymatic cleavage may be one of the inhibitors existed in ⬍ 0.5 KD component. Since both T4 (or ⬍ 10 KD BMEC-cm) and AcSDKP (or ⬍ 0.5 KD BMEC-cm) inhibite hematopoietic progenitors entering into cell cycle. Hence, they may be useful in protecting hematopoietic progenitors from the damaging effect of cytotoxic agents and may be useful also in enhancing the expansion of early hematopoietic progenitors meanwhile decreasing their differentiation when they were employed together with growth factors in liquid cultures. 225
Monday, July 10, 2000 (16:00–17:00) Poster Session II: Stem Cell Biology
FLOW CYTOMETRY ANALYSIS OF MURINE HEMATOPOIETIC PROGENITORS RECOVERING FROM CYCLOPHOSPHAMIDE DAMAGE L. Sˇ efc, E. Necˇ as Institute of Pathophysiology, 1st Faculty of Medicine, Charles University, Prague, Czech Republic Along with damage to hematopoietic cells, cyclophosphamide damages also cells of hematopoietic microenvironment. Their uneven regeneration leads to a prolonged dysregulation of supporting functions of the microenvironment. The bone marrow progenitors follow a unique regeneration curve characterized by an early rapid recovery followed by secondary depression. We have analyzed the cellular composition of the bone marrow in mice recovering from single dose of cyclophosphamide by means of flow cytometry and microscopy. Sca-1⫹ progenitors have been magnetically separated and analyzed for c-kit, flk2/flt3, CD71 and lin⫹ markers. Their proliferation activity and apoptotic rate was also determined. The number and percentage of Sca-1⫹ cells in the bone marrow increased rapidly 2 to 3 days after cyclophosphamide. Their proliferation rate was high and they showed an increase in CD71 expression. The number and percentage of Sca-1⫹ progenitors 3 to 7 days after cyclophosphamide dropped considerably. Their proliferation rate was low along with lower CD71 and flt-3 expression.