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Maintenance of primitive hematopoietic progenitor cell activity through successive in vitro divisions correlates with sustained long-term repopulating potential
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Abstracts/Experimental Hematology 28 (2000) 31–131
alone; IL1 alone; or no drug (control). Four biweekly doses were given and mice were sacrificed 4–6 weeks later. Their marrow (BM) cells were mixed with equal numbers (106 each) of competitor (B6HbbdGpi1a) cells; equal aliquots of this mixture were injected into lethally-irradiated B6 recipients. The number of mice in each group ranged from 8 to 21, with experiments repeated three times. At 150 days, the percentages of B6 BM were: control 49 ⫾ 3; IL1 only, 66 ⫾ 4; CTX, 32 ⫾ 2; IL1/CTX, 22 ⫾ 3. Differences were significant between control and CTX or IL1/CTX groups (p ⬍ 0.001; and between groups receiving IL1/CTX and CTX (p ⬍ 0.05). Absolute RU numbers and concentration respectively were: control, 596/11; IL1, 1239/ 23; CTX, 353/5; IL1-CTX, 195/3. These results indicate that IL1 use with CTX does not lead to PHSC sparing, but rather causes significant further quantitative and qualitative defect in the stem cell population. The previously reported animal sparing may result therefore from effects on organ systems other than the hematopoietic. 206
Monday, July 10, 2000 (16:00–17:00) Poster Session II: Signal Transduction
REGULATION OF SELF-RENEWAL DIVISIONS OF PRIMITIVE HUMAN HEMATOPOIETIC PROGENITOR CELLS X. Tong*, R. Abonour, E. F. Srour Indiana University School of Medicine, Indianapolis, IN, USA Whether self-renewal divisions of primitive hematopoietic progenitor cells is subject to extrinsic control mechanisms remains unclear. Using single cell assays, we examined whether the proliferative potential and self-renewal divisions of daughter cells of individual long-term culture-initiating cells (LTC-IC) can be modulated by exogenous cytokine stimulation. Single G0CD34⫹CD38⫺/ lo cells were deposited in individual wells of microtiter plates containing complete medium and the three cytokine combination SCF, Flt-3 ligand and MGDF (SFM). All wells were observed daily and after the first division of every primary cell (when two cells were evident in the well between days 1 and 7), one of the two daughter cells was transferred to a new plate prepared with a cytokine combination containing SFM plus IL3, IL6, GM-CSF and erythropoietin (SFM36GE). The destination of each daughter cell was recorded so that clonal pairs could be tracked. If cell growth in one well of a pair was not detected, the pair was discarded. Cells of each clone were harvested on day 7 and examined individually for their HPC content in a 6 week LTC-IC assay. Both daughter cells of 1259 clones split at the 2-cell stage supported continued cell proliferation. However, only 9% of clones in SFM contained ⱖ 8 cells on day 7 compared to 45% of their counterparts maintained with SFM36GE. Furthermore, while only 4.5% of clones remaining in SFM were scored at LTC-IC, 19.2% of the SFM36GE clones were determined to be LTC-IC, suggesting that this 7 cytokine combination recruited dormant LTC-IC into cell cycle. Approximately 1/3 of SFM LTC-IC clones were also scored as LTC-IC in SFM36GE suggesting that one of every three divisions of primitive HPC is a self-renewal division. However, of these pairs only 24% divided up to three times by day 7 in SFM compared to 65% in SFM36GE indicating that proliferation of progeny cells following self-renewal divisions may be extrinsically modulated. These results suggest that since the percentage of daughter cell-derived clones scored as LTC-IC and the proliferative potential of every LTC-IC were different under different cytokine conditions, cell cycle activation and fate may be extrinsically controlled.
207
Sunday, July 9, 2000 (10:30–12:30) Session I-5: Stem Cell Biology I
MAINTENANCE OF PRIMITIVE HEMATOPOIETIC PROGENITOR CELL ACTIVITY THROUGH SUCCESSIVE IN VITRO DIVISIONS CORRELATES WITH SUSTAINED LONG-TERM REPOPULATING POTENTIAL E. F. Srour, A. Jetmore*, P. A. Plett*, X. Tong*, D. Heilman*, R. Breese*, R. Abonour, C. M. Orschell-Traycoff Indiana University School of Medicine, Indianapolis, IN, USA Whether maintenance of primitive hematopoietic function through successive in vitro divisions of hematopoietic progenitor cells (HPC) can be modulated by cytokine stimulation was investigated using both in vitro and in vivo assays. Single human marrow CD34⫹CD38⫺/lo cells isolated in G0 phase of cell cycle (G0CD34⫹CD38⫺/lo cells) were cultured in either SCF, FL and MGDF (SFM) or SFM plus IL3, IL6, and GM-CSF (SFM36G) and the number of progeny cells in each clone was recorded on days 3, 5 and 7. Cells of each clone were harvested on day 7 and examined individually for their HPC content in a 6 seek LTC-IC assay. Being a single cell assay, presence of hematopoietic colonies at week 7 indicated that the primary cell was an LTC-IC with a known proliferation history. While the number of progeny cells per LTC-IC maintained in SFM for 7 days was 5.4 ⫾ 3.4 (mean ⫾ SD, n ⫽ 74), that for LTC-IC cultured in SFM36G was 27.1 ⫾ 42.1 (n ⫽ 78; p ⫽ 0.00002). Since the percentage of clones scored as LTC-IC was similar under both conditions, these data suggest that SFM36G may support additional in vitro cell divisions without loss of function compared to SFM. To test this hypothesis, BM CD34⫹ cells were expanded in SFM or SFM36G for 5 days then transplanted into conditioned NOD/SCID mice. Fresh CD34⫹ cells generated 39.0 ⫾ 13.8% chimerism in recipient mice 8 weeks post-transplantation. Progeny of equivalent cell numbers expanded with SFM supported 13.3 ⫾ 16.1% chimerism while progeny of cells maintained in SFM36G sustained 31.8 ⫾ 14.7% chimerism. Analysis of adhesion molecules expression did not reveal a phenotype common to fresh and SFM36G-expanded cells, suggesting that maintenance of repopulating potential of SFM36G-expanded cells may not be a function of enhanced homing. These data demonstrate that maintenance of primitive HPC function through successive in vitro cell divisions of single HPC may predict BM repopulating potential of these cells and suggest that SFM36G is superior to SFM in maintaining, and possibly expanding, the repopulating capacity of HPC. 208
Tuesday, July 11, 2000 (10:15–12:15) Session V-3: Signal Transduction II
HOMING EFFICIENCY AND EARLY CELL CYCLE KINETICS OF HUMAN CD34⫹ CELLS TRANSPLANTED INTO CONDITIONED NOD/SCID RECIPIENTS A. Jetmore*, P. A. Plett*, X. Tong*, F. M. Wolber*, R. Breese*, R. Abonour, C. M. Orschell-Traycoff, E. F. Srour Indiana University School of Medicine, Indianapolis, IN, USA Differences in the engraftment potential of hematopoietic progenitor cells (HPC) in distinct phases of cell cycle may result from the inability of cycling cells to home to the bone marrow (BM) microenvironment. Alternatively, the rate of entry of HPC homing to the BM into active phases of cell cycle may influence their lodge-
Abstracts/Experimental Hematology 28 (2000) 31–131
alone; IL1 alone; or no drug (control). Four biweekly doses were given and mice were sacrificed 4–6 weeks later. Their marrow (BM) cells were mixed with equal numbers (106 each) of competitor (B6HbbdGpi1a) cells; equal aliquots of this mixture were injected into lethally-irradiated B6 recipients. The number of mice in each group ranged from 8 to 21, with experiments repeated three times. At 150 days, the percentages of B6 BM were: control 49 ⫾ 3; IL1 only, 66 ⫾ 4; CTX, 32 ⫾ 2; IL1/CTX, 22 ⫾ 3. Differences were significant between control and CTX or IL1/CTX groups (p ⬍ 0.001; and between groups receiving IL1/CTX and CTX (p ⬍ 0.05). Absolute RU numbers and concentration respectively were: control, 596/11; IL1, 1239/ 23; CTX, 353/5; IL1-CTX, 195/3. These results indicate that IL1 use with CTX does not lead to PHSC sparing, but rather causes significant further quantitative and qualitative defect in the stem cell population. The previously reported animal sparing may result therefore from effects on organ systems other than the hematopoietic. 206
Monday, July 10, 2000 (16:00–17:00) Poster Session II: Signal Transduction
REGULATION OF SELF-RENEWAL DIVISIONS OF PRIMITIVE HUMAN HEMATOPOIETIC PROGENITOR CELLS X. Tong*, R. Abonour, E. F. Srour Indiana University School of Medicine, Indianapolis, IN, USA Whether self-renewal divisions of primitive hematopoietic progenitor cells is subject to extrinsic control mechanisms remains unclear. Using single cell assays, we examined whether the proliferative potential and self-renewal divisions of daughter cells of individual long-term culture-initiating cells (LTC-IC) can be modulated by exogenous cytokine stimulation. Single G0CD34⫹CD38⫺/ lo cells were deposited in individual wells of microtiter plates containing complete medium and the three cytokine combination SCF, Flt-3 ligand and MGDF (SFM). All wells were observed daily and after the first division of every primary cell (when two cells were evident in the well between days 1 and 7), one of the two daughter cells was transferred to a new plate prepared with a cytokine combination containing SFM plus IL3, IL6, GM-CSF and erythropoietin (SFM36GE). The destination of each daughter cell was recorded so that clonal pairs could be tracked. If cell growth in one well of a pair was not detected, the pair was discarded. Cells of each clone were harvested on day 7 and examined individually for their HPC content in a 6 week LTC-IC assay. Both daughter cells of 1259 clones split at the 2-cell stage supported continued cell proliferation. However, only 9% of clones in SFM contained ⱖ 8 cells on day 7 compared to 45% of their counterparts maintained with SFM36GE. Furthermore, while only 4.5% of clones remaining in SFM were scored at LTC-IC, 19.2% of the SFM36GE clones were determined to be LTC-IC, suggesting that this 7 cytokine combination recruited dormant LTC-IC into cell cycle. Approximately 1/3 of SFM LTC-IC clones were also scored as LTC-IC in SFM36GE suggesting that one of every three divisions of primitive HPC is a self-renewal division. However, of these pairs only 24% divided up to three times by day 7 in SFM compared to 65% in SFM36GE indicating that proliferation of progeny cells following self-renewal divisions may be extrinsically modulated. These results suggest that since the percentage of daughter cell-derived clones scored as LTC-IC and the proliferative potential of every LTC-IC were different under different cytokine conditions, cell cycle activation and fate may be extrinsically controlled.
207
Sunday, July 9, 2000 (10:30–12:30) Session I-5: Stem Cell Biology I
MAINTENANCE OF PRIMITIVE HEMATOPOIETIC PROGENITOR CELL ACTIVITY THROUGH SUCCESSIVE IN VITRO DIVISIONS CORRELATES WITH SUSTAINED LONG-TERM REPOPULATING POTENTIAL E. F. Srour, A. Jetmore*, P. A. Plett*, X. Tong*, D. Heilman*, R. Breese*, R. Abonour, C. M. Orschell-Traycoff Indiana University School of Medicine, Indianapolis, IN, USA Whether maintenance of primitive hematopoietic function through successive in vitro divisions of hematopoietic progenitor cells (HPC) can be modulated by cytokine stimulation was investigated using both in vitro and in vivo assays. Single human marrow CD34⫹CD38⫺/lo cells isolated in G0 phase of cell cycle (G0CD34⫹CD38⫺/lo cells) were cultured in either SCF, FL and MGDF (SFM) or SFM plus IL3, IL6, and GM-CSF (SFM36G) and the number of progeny cells in each clone was recorded on days 3, 5 and 7. Cells of each clone were harvested on day 7 and examined individually for their HPC content in a 6 seek LTC-IC assay. Being a single cell assay, presence of hematopoietic colonies at week 7 indicated that the primary cell was an LTC-IC with a known proliferation history. While the number of progeny cells per LTC-IC maintained in SFM for 7 days was 5.4 ⫾ 3.4 (mean ⫾ SD, n ⫽ 74), that for LTC-IC cultured in SFM36G was 27.1 ⫾ 42.1 (n ⫽ 78; p ⫽ 0.00002). Since the percentage of clones scored as LTC-IC was similar under both conditions, these data suggest that SFM36G may support additional in vitro cell divisions without loss of function compared to SFM. To test this hypothesis, BM CD34⫹ cells were expanded in SFM or SFM36G for 5 days then transplanted into conditioned NOD/SCID mice. Fresh CD34⫹ cells generated 39.0 ⫾ 13.8% chimerism in recipient mice 8 weeks post-transplantation. Progeny of equivalent cell numbers expanded with SFM supported 13.3 ⫾ 16.1% chimerism while progeny of cells maintained in SFM36G sustained 31.8 ⫾ 14.7% chimerism. Analysis of adhesion molecules expression did not reveal a phenotype common to fresh and SFM36G-expanded cells, suggesting that maintenance of repopulating potential of SFM36G-expanded cells may not be a function of enhanced homing. These data demonstrate that maintenance of primitive HPC function through successive in vitro cell divisions of single HPC may predict BM repopulating potential of these cells and suggest that SFM36G is superior to SFM in maintaining, and possibly expanding, the repopulating capacity of HPC. 208
Tuesday, July 11, 2000 (10:15–12:15) Session V-3: Signal Transduction II
HOMING EFFICIENCY AND EARLY CELL CYCLE KINETICS OF HUMAN CD34⫹ CELLS TRANSPLANTED INTO CONDITIONED NOD/SCID RECIPIENTS A. Jetmore*, P. A. Plett*, X. Tong*, F. M. Wolber*, R. Breese*, R. Abonour, C. M. Orschell-Traycoff, E. F. Srour Indiana University School of Medicine, Indianapolis, IN, USA Differences in the engraftment potential of hematopoietic progenitor cells (HPC) in distinct phases of cell cycle may result from the inability of cycling cells to home to the bone marrow (BM) microenvironment. Alternatively, the rate of entry of HPC homing to the BM into active phases of cell cycle may influence their lodge-