Accelerated recovery of peripheral blood cell counts in mice transplanted with in vitro cytokine-expanded hematopoietic progenitors.

Interleukin 1 (IL-1) and interleukin 3 (IL-3) act synergistically in stimulating the growth of primitive hematopoietic progenitors. Murine bone marrow (BM) harvested 24 h after 5-fluorouracil (5-FU) administration (d1 5-FU BM) was stimulated with IL-1 and IL-3 to expand its progenitor pool during 7 days of suspension culture (delta-culture), and this in vitro expanded BM was compared to fresh d1 5-FU BM in its ability to reconstitute lethally irradiated or high-dose 5-FU-treated hosts. Transplantation with expanded delta-culture BM was found to dramatically shorten the period of cytopenia following lethal irradiation as compared to animals receiving d1 5-FU BM. Recipients of delta-cultured BM demonstrated accelerated recoveries of peripheral blood leukocytes, neutrophils, platelets, and erythrocytes. Furthermore, expansion of BM in vitro reduced the number of BM cells required for engraftment following lethal irradiation. Treatment of lethally irradiated mice with IL-1 and granulocyte colony-stimulating factor (G-CSF) following transplantation with delta-cultured BM or d1 5-FU BM further improved the recovery of neutrophils in these hosts. In conjunction with G-CSF post-transplantation cytokine therapy, high-dose 5-FU-treated mice transplanted with delta-cultured BM also demonstrated improved recovery kinetics of neutrophils and erythrocytes. Five and 10 weeks after BM transplantation, a decrease in the proliferative capacity of the earliest hematopoietic progenitors, detected in assays of primary and delta-culture generated-secondary high proliferative potential colony-forming cells (HPP-CFC), was found in all transplanted mice following a chemotherapy challenge with 5-FU. However, this impairment in the early progenitor/stem cell pool was not noticeably worsened by the expansion of BM in delta-cultures. The decrease in host hematopoietic proliferative potential associated with transplantation of limiting numbers of BM cells was not reversed over the 10 weeks of this study. The expansion of BM progenitor cells without loss of long-term proliferative potential may be of clinical importance in the fields of BM transplantation and gene therapy.     

Ex vivo culture rescues hematopoietic stem cells with long-term repopulating capacity following harvest from lethally irradiated mice

OBJECTIVE: High-dose ionizing radiation can cause lethal myeloablation in exposed individuals. We examined whether ex vivo culture could rescue hematopoietic stem cells with repopulating capacity following harvest from lethally irradiated animals. METHODS: We exposed B6.SJL mice to 1050 cGy, harvested their irradiated bone marrow (BM), and examined whether ex vivo culture of the irradiated BM mononuclear cells (MNC) with porcine microvascular endothelial cells (PMVEC) or cytokines alone could rescue hematopoietic cells with in vitro colony-forming activity, in vivo radioprotective capacity, and long-term repopulating potential. RESULTS: PMVEC coculture supported the recovery of fourfold and 80-fold greater numbers of total cells and colony-forming cells (CFC) compared to cyokines alone following 1050 cGy irradiation. All control mice irradiated with 1050 cGy died by day 30, as did mice transplanted with 1050 cGy-irradiated BM MNC. In contrast, transplantation of 1050 cGy-irradiated/PMVEC-cultured BM was fully radioprotective in 12 of 16 recipient mice (75%) exposed to 1050 cGy. Six of the 12 CD45.2+ mice (50%) transplanted with 1050 cGy-irradiated/PMVEC-cultured cells showed long-term (>6 months) multilineage repopulation derived from irradiated donor CD45.1+ cells. Surprisingly, transplantation of identical doses of 1050 cGy-irradiated/cytokine-cultured BM was also radioprotective in 50% of irradiated recipient mice and 50% of these mice demonstrated donor-derived repopulation. CONCLUSIONS: Fully functional BM stem and progenitor cells can be rescued following harvest from lethally irradiated animals via ex vivo culture with PMVEC or cytokines alone. This method can serve as a model for the rapid ex vivo rescue and transplantation of autologous BM progenitors in the treatment of victims of radiation injury.     

Differential homing and engraftment properties of hematopoietic progenitor cells from murine bone marrow, mobilized peripheral blood, and fetal liver

The rate of reconstitution following hematopoietic stem cell (HSC) transplantation differs widely depending on the tissue source of the cells infused. To test the hypothesis that variability in engraftment kinetics is related to differences in the efficiency with which intravenously transplanted HSCs "home" to the bone marrow (BM), the homing properties of murine fetal liver (FL), adult BM, and mobilized peripheral blood (MPB) cells were compared. Lethally irradiated mice transplanted with 2 x 10(6) FL, BM, or MPB cells exhibited sequentially slower recovery of circulating leukocytes and platelets that correlates with the progressively lower frequency of colony-forming cells (CFCs) in these tissues. However, differences in the rate and degree of early and long-term reconstitution were maintained even after infusing equal numbers of CFCs derived from FL, BM, and MPB. To compare the homing of progenitors from these tissues, cells were labeled with fluorescent PKH26 dye and injected into lethally irradiated hosts. Three hours later, PKH26(+) cells were reisolated from the BM and spleen by fluorescence-activated cell sorting and assayed for in vitro CFCs. Despite the higher level of very late antigen (VLA)-2, VLA-4, and VLA-5 on Sca-1(+)c-kit(+) cells from FL compared to BM, 10-fold fewer FL CFCs homed to hematopoietic organs than those from BM. MPB cells homed slightly better, but still less efficiently than BM cells. Therefore, clonogenic cells from different tissues exhibit striking variations in homing efficiency that does not necessarily correlate with engraftment kinetics. Homing is likely counterbalanced by intrinsic differences in proliferative potential that ultimately determine the rate of hematopoietic reconstitution.     

Homing and engraftment defects in ex vivo expanded murine hematopoietic cells are associated with downregulation of beta1 integrin.

OBJECTIVE: Hematopoietic progenitors generated by ex vivo expansion "home" less efficiently to the bone marrow (BM) after intravenous transplantation than fresh cells. To explore the underlying cause of this transplantation defect, we examined the homing and engraftment properties in vivo of fresh and cultured marrow cells differing in beta1 integrin expression. MATERIALS AND METHODS: Fresh murine BM cells, or the expanded progeny of enriched Sca-1(+) c-kit(+)Lin(-) stem cells, were fractionated into beta1(-/lo) and beta1(+) subpopulations by cell sorting. These populations were assayed for their content of in vitro colony-forming cells (CFCs), cells able to provide radioprotection, and early and long-term multilineage hematopoietic reconstitution following transplantation into myeloablated recipients. These endpoints were correlated with the homing properties of beta1(-/lo) and beta1(+) cells that were labeled with 5- (and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE) and tracked to hematopoietic organs 24 hours after injection into lethally irradiated mice. RESULTS: Most normal stem and progenitor cells express high levels of beta1 integrin. In contrast, most clonogenic cells generated in vitro are beta1(-/lo). Consequently, expanded beta1(-/lo) progenitors failed to provide radioprotection or repopulate the hematopoietic system following intravenous transplantation. Defective engraftment of expanded cells was associated with reduced homing of beta1(-/lo) cells to the bone marrow. CONCLUSION: Downregulation of beta1 integrin on primitive hematopoietic cells during ex vivo expansion reduces their homing efficiency and negatively impacts hematopoietic reconstitution in vivo. Strategies directed at preserving beta1 integrin expression during culture may improve the clinical utility of expanded hematopoietic cells.     

In vivo trafficking,cell cycle activity,and engraftment potential of phenotypically defined primitive hematopoietic cells after transplantation into irradiated or nonirradiated recipients

Recent interest in bone marrow (BM) transplantation in nonconditioned or minimally conditioned recipients warrants investigation of homing patterns of transplanted hematopoietic progenitor cells (HPCs) in irradiated and nonirradiated recipients. To this end, phenotypically defined populations of BM cells were tracked in lethally irradiated or nonirradiated mice at 1, 3, 6, and 24 hours after transplantation. Recovery of transplanted cells at all time points was higher in BM of nonirradiated mice, similar to earlier suggestions. The percentage of lineage-negative Sca-1(+) cells and Sca-1(+) cells expressing CD43, CD49e, and CD49d steadily increased in BM of nonirradiated mice up to 24 hours, while fluctuating in irradiated mice. Cell cycle status and BrdU incorporation revealed that less than 20% of Sca-1(+) cells and fewer Sca-1(+)lin(-) cells had cycled by 24 hours after transplantation. To more directly examine trafficking of primitive HPCs, purified grafts of CD62L(-) or CD49e(+) subfractions of Sca-1(+)lin(-) cells, previously shown to be enriched for long-term repopulating cells, also were tracked in vivo. Recovery of purified cells was similarly increased in BM of nonirradiated mice. When 50 to 100 of these BM-homed cells were examined in serial transplantation studies, BM-homed cells from initially nonirradiated mice were enriched 5- to 30-fold for cells capable of long-term hematopoiesis in secondary recipients. Collectively, these data suggest that homing or survival of transplanted cells in irradiated recipients is less efficient than that in nonirradiated recipients, implicating an active role of radiation-sensitive microenvironmental cues in the homing process. These results may have important clinical implications in the design of BM transplantation protocols.     

Increased numbers of circulating ECs are associated with systemic GVHD

Introduction: Circulating endothelial cells (ECs) are known to reflect endothelial injury, and endothelial injury is associated with graft‐versus‐host disease (GVHD). We hypothesised that circulating ECs might be associated with systemic acute graft‐versus‐host disease (aGVHD). Methods: BALB/c (H‐2k^d) mice were treated with total body irradiation and then infused with C57B/6‐derived T‐cell‐depleted bone marrow (TCD‐BM) cells or TCD‐BM cells and splenocytes. Cyclosporine was used to prevent aGVHD. Circulating ECs and allogeneic lymphocytes were analysed by flow cytometry at multiple time points. The morphology and ultrastructure of the endothelium were examined by light microscopy or transmission electron microscopy. Results: The results indicated that the number of circulating ECs peaked at day 5 after lethal irradiation in all mice; allogenic transplanted mice (TCD‐BM cells and splenocytes) developed typical aGVHD beginning at day 7, exhibiting both histological and clinical symptoms of disease. Circulating ECs peaked a second time at day 9 with aGVHD progression. However, following the administration of CSA, an absence of or a reduction in the amount of subsequent endothelial injury was observed. Conclusion: Circulating ECs might be associated with systemic aGVHD.     

Bone marrow origin of hematopoietic progenitors and stem cells in murine muscle

It has been reported that mononuclear cells harvested from murine skeletal muscle are capable of hematopoietic reconstitution of lethally irradiated mice. First, the nature of the hematopoietic progenitors in the muscle of C57BL/6-Ly-5.1 mice was examined by means of methylcellulose culture. The types and incidences of colonies grown from muscle mononuclear cells were different from those cultured from bone marrow (BM) or peripheral blood mononuclear cells. The next step was to examine the origin of the hematopoietic progenitors and stem cells in the muscle with the use of Ly-5.2 mice that had been made chimeric by transplantation of Ly-5.1 BM cells. The percentages of Ly-5.1 cells cultured from the muscle of the chimeric mice correlated with those cultured from BM, indicating BM origin of hematopoietic progenitors in the muscle. Long-term hematopoietic engrafting cells in the muscle of the chimeric mice were also derived from BM. However, mobilization of progenitors into circulation by granulocyte colony-stimulating factor did not change the population of hematopoietic progenitors in the muscle. It is proposed that hematopoietic progenitors and stem cells in the muscle tissue are of BM origin but their transition from BM to muscle may be a slow process.     

Induction of donor-type chimerism and transplantation tolerance across major histocompatibility barriers in sublethally irradiated mice by Sca-1(+)Lin(-) bone marrow progenitor cells: synergism with non-alloreactive (host x donor)F(1) T cells.

Induction of transplantation tolerance by means of bone marrow (BM) transplantation could become a reality if it was possible to achieve engraftment of hematopoietic stem cells under nonlethal preparatory cytoreduction of the recipient. To that end, BM facilitating cells, veto cells, or other tolerance-inducing cells, have been extensively studied. In the present study, we show that BM cells within the Sca-1(+)Lin(-) cell fraction, previously shown to be enriched for early hematopoietic progenitors, are capable of reducing specifically antidonor CTL-p frequency in vitro and in vivo, and of inducing split chimerism in sublethally 7-Gy-irradiated recipient mice across major histocompatibility complex barriers. The immune tolerance induced by the Sca-1(+)Lin(-) cells was also associated with specific tolerance toward donor-type skin grafts. The minimal number of cells required to overcome the host immunity remaining after 7 Gy total body irradiation is very large and, therefore, it may be very difficult to harvest sufficient cells for patients. This challenge was further addressed in our study by demonstrating that non-alloreactive (host x donor)F(1) T cells, previously shown to enhance T-cell-depleted BM allografts in lethally irradiated mice, synergize with Sca-1(+)Lin(-) cells in their capacity to overcome the major transplantation barrier presented by the sublethal mouse model.     

Distinct functional properties of highly purified hematopoietic stem cells from mouse strains differing in stem cell numbers

We have previously demonstrated that young adult DBA/2 (DBA) mice have more stem cells than C57BL/6 (B6) mice, as measured in a cobblestone area-forming cell (CAFC) assay using unfractionated marrow. To study the nature of this difference, we have now compared the proliferative fate of single, highly enriched Sca-1(+)c-kit(+)Lin(-) stem cells from these strains. Although equal in frequency, functional comparison revealed that Sca-1(+)c-kit(+)Lin(-) cells from DBA mice contained twice as many cells with CAFC activity. DBA clones persisted much longer in vitro, and developed later in time. To assess whether these differences were of any functional relevance in vivo, we compared engraftment of lethally irradiated mice transplanted with 1000 B6 or DBA Sca-1(+)c-kit(+)Lin(-) cells. Recipients of enriched DBA cells recovered much faster than animals transplanted with B6 cells. We also studied endogenous hematopoietic recovery after 5-fluorouracil (5-FU) treatment in vivo. Progenitors and peripheral blood cells recovered twice as fast in DBA mice. Thus, DBA stem cells have superior proliferative potential compared with phenotypically identical stem cells obtained from B6 mice. Such genetically determined quantitative and qualitative differences in stem cell behavior likely contribute to the dramatically different hematopoietic recovery rates observed in human transplant patients. (Blood. 2000;96:1374-1379)     

Both alleles of PSF1 are required for maintenance of pool size of immature hematopoietic cells and acute bone marrow regeneration

Hematopoietic stem cells (HSCs) have a very low rate of cell division in the steady state; however, under conditions of hematopoietic stress, these cells can begin to proliferate at high rates, differentiate into mature hematopoietic cells, and rapidly reconstitute ablated bone marrow (BM). Previously, we isolated a novel evolutionarily conserved DNA replication factor, PSF1 (partner of SLD5-1), from an HSC-specific cDNA library. In the steady state, PSF1 is expressed predominantly in CD34(+)KSL (c-kit(+)/Sca-1(+)/Lineage(-)) cells and progenitors, whereas high levels of PSF1 expression are induced in KSL cells after BM ablation. In 1-year-old PSF1(+/-) mice, the pool size of stem cells and progenitors is decreased. Whereas young PSF1(+/-) mutant mice develop normally, are fertile, and have no obvious differences in hematopoiesis in the steady state compared with wild-type mice, intravenous injection of 5-fluorouracil (5-FU) is lethal in PSF1(+/-) mice, resulting from a delay in induction of HSC proliferation during ablated BM reconstitution. Overexpression studies revealed that PSF1 regulates molecular stability of other GINS components, including SLD5, PSF2, and PSF3. Our data indicate that PSF1 is required for acute proliferation of HSCs in the BM of mice.     

Anti-asialo GM1 antiserum treatment of lethally irradiated recipients before bone marrow transplantation: evidence that recipient natural killer depletion enhances survival, engraftment, and hematopoietic recovery

Natural killer (NK) cells are reported to have an important role in the resistance of lethally irradiated recipients to bone marrow transplantation (BMT). Therefore, we investigated the effects of recipient NK depletion on survival, chimerism, and hematopoietic reconstitution after lethal irradiation and the transplantation of limiting amounts of T-cell-deficient bone marrow (BM). When administered before BMT, anti-asialo GM1 (ASGM1) antiserum treatment, effective in depleting in vivo NK activity, was associated with a marked increase in survival in 3 of 3 allogeneic combinations (BALB/c into C3H/HeN, C57B1/6, or C3B6F1). This enhanced survival was independent of the susceptibility of each recipient strain to accept BALB/c BM. Moreover, recipient anti-ASGM1 treatment was also effective in increasing survival in recipients of syngeneic BM, suggesting that NK cells can adversely affect engraftment independent of genetically controlled polymorphic cell surface determinants. Analysis of chimerism in surviving animals 2 months post-BMT showed that recipient NK depletion significantly increased the level of donor engraftment when high doses of BM were transplanted. These studies also demonstrated that anti-ASGM1 pretreatment mainly resulted in an increase in extramedullary hematopoiesis in the second and third week after irradiation. Anti-ASGM1 treatment also dramatically accelerated the rate of appearance of donor-derived cells with a higher level of donor-cell engraftment apparent at a time when the differences in survival between NK-depleted and control BMT recipients became significant. Peripheral cell counts were also affected by NK depletion, with significantly enhanced platelet and red blood cell recovery and a moderate increase in granulocyte recovery. The overall favorable influence of anti-ASGM1 recipient treatment on hematopoietic events post-BMT suggests that, in humans, pretransplant regimens aimed toward NK depletion should be evaluated.     

Bone marrow transplantation with interleukin-1 plus kit-ligand ex vivo expanded bone marrow accelerates hematopoietic reconstitution in mice without the loss of stem cell lineage and proliferative potential

Cytokine combinations were tested for their ability to expand murine bone marrow (BM) progenitors in short-term suspension cultures (delta- cultures) with the aim of providing an enriched source of progenitors for BM transplantation (BMT). In a comparison of the efficacy of the combinations interleukin-1 (IL-1) + IL-3, IL-1 + kit-ligand (KL), and IL-1 + IL-6 + KL, BMT with IL-1 + KL expanded progenitors was found to be most effective in accelerating the recovery of peripheral blood leukocytes, platelets, and erythrocytes in lethally irradiated mice. The ex vivo expansion of BM in IL-1 + KL-stimulated delta-cultures also greatly reduced the number of transplanted cells needed to provide radioprotection. All mice survived at least 30 days when receiving 5 x 10(3) delta-cultured d1 5-fluorouracil (5-FU) BM cells (BM cells harvested 1 day after 5-FU administration), whereas complete survival of mice receiving fresh d1 5-FU BM required BMT with a 200-fold greater number of cells. BMT with expanded BM lead to predominantly donor- derived hematopoietic reconstitution for 280 days postprimary BMT and another 71 days after secondary BMT. The expansion of BM did not adversely effect the proliferative capacity and lineage potential of the stem cell compartment.     

Depletion of alloantigen-primed lymphocytes overcomes resistance to allogeneic bone marrow in mildly conditioned recipients

OBJECTIVE: Successful implantation of allogeneic bone marrow (BM) cells after nonmyeloablative conditioning would allow to compensate for the inadequate supply of compatible grafts and to reduce mortality of graft-vs.-host disease (GVHD). Recently, we proposed to facilitate engraftment of mismatched BM by conditioning for alloantigen-primed lymphocyte depletion (APLD) with cyclophosphamide (CY). Here we summarize the experimental results obtained by this approach. MATERIALS AND METHODS: Naive or mildly irradiated BALB/c mice were primed with C57BL/6 BM cells (day 0), treated with CY (day 1) to deplete alloantigen-primed lymphocytes, and given a second C57BL/6 BM transplant (day 2) for engraftment. Recipients were repeatedly tested for chimerism in the blood and followed for GVHD and survival. The protocol was also tested for inducing tolerance to donor tissue and organ allografts, and for treatment of leukemia, breast cancer, and autoimmune diabetes in NOD mice. RESULTS: APLD by 200 mg/kg CY provided engraftment of allogeneic BM from the same donor in 100% mildly irradiated recipients. Eighty percent chimeras remained GVHD-free more 200 days. All chimeras accepted permanently donor skin grafts and donor hematopoietic stromal progenitors. Allogeneic BM transplantation (BMT) after APLD had a strong therapeutic potential in BALB/c mice harboring malignant cells and in autoimmune NOD recipients. Tolerance-inducing CY dose could be reduced to 100 mg/kg. Conditioning for APLD resulted in engraftment of allogeneic BM after a significantly lower radiation dose than treatment with radiation and CY alone. CONCLUSION: Our results demonstrate that conditioning for APLD has a definite advantage over general immunosuppression with CY and radiation therapy.     

Smad5 is dispensable for adult murine hematopoiesis

Smad5 is known to transduce intracellular signals from bone morphogenetic proteins (BMPs), which belong to the transforming growth factor-beta (TGF-beta) superfamily and are involved in the regulation of hematopoiesis. Recent findings suggest that BMP4 stimulates proliferation of human primitive hematopoietic progenitors in vitro, while early progenitors from mice deficient in Smad5 display increased self-renewal capacity in murine embryonic hematopoiesis. Here, we evaluate the role of Smad5 in the regulation of hematopoietic stem cell (HSC) fate decisions in adult mice by using an inducible MxCre-mediated conditional knockout model. Surprisingly, analysis of induced animals revealed unperturbed cell numbers and lineage distribution in peripheral blood (PB), bone marrow (BM), and the spleen. Furthermore, phenotypic characterization of the stem cell compartment revealed normal numbers of primitive lin(-)Sca-1(+)c-Kit(+) (LSK) cells in Smad5(-)(/)(-) BM. When transplanted in a competitive fashion into lethally irradiated primary and secondary recipients, Smad5-deficient BM cells competed normally with wild-type (wt) cells, were able to provide long-term reconstitution for the hosts, and displayed normal lineage distribution. Taken together, Smad5-deficient HSCs from adult mice show unaltered differentiation, proliferation, and repopulating capacity. Therefore, in contrast to its role in embryonic hematopoiesis, Smad5 is dispensable for hematopoiesis in the adult mouse.     

Ex vivo expansion with stem cell factor and interleukin-11 augments both short-term recovery posttransplant and the ability to serially transplant marrow

The characterization of many cytokines involved in the control of hematopoiesis has led to intense investigation into their potential use in ex vivo culture to expand progenitor numbers. We have established the optimum ex vivo culture conditions that allow substantial amplification of transient engrafting murine stem cells and which, simultaneously, augment the ability to sustain serial bone marrow transplantation (BMT). Short-term incubation of unfractionated BM cells in liquid culture with stem cell factor (SCF) and interleukin-11 (IL- 11) produced a 50-fold amplification of clonogenic multipotential progenitors (CFU-A). Following such ex vivo expansion, substantially fewer cells were required to rescue lethally irradiated mice. When transplanted in cell doses above threshold for engraftment, BM cells expanded ex vivo resulted in significantly more rapid hematopoietic recovery. In a serial transplantation model, unmanipulated BM was only able to consistently sustain secondary BMT recipients, but BM expanded ex vivo has sustained quaternary BMT recipients that remain alive and well more than 140 days after 4th degree BMT. These results show augmentation of both short-term recovery posttransplant and the ability to serially transplant marrow by preincubation in culture with SCF and IL-11.     

Progenitor cell assays predict hematopoietic reconstitution after syngeneic transplantation in mice

Hematopoietic reconstitution following syngeneic bone marrow transplantation with graded doses of untreated and drug-treated bone marrow was studied in B6D2F1 mice. Granulocyte-macrophage colony- forming units (CFU-GM) and spleen colony-forming units (CFU-S) showed similar in vitro drug sensitivities. Both the speed of hematologic recovery and survival of mice transplanted with untreated or drug- treated bone marrow were directly related to the number of CFU-GM or CFU-S transplanted. Similar hematologic recovery was seen for untreated marrow transplants and treated transplants that had similar CFU-GM or CFU-S content. There is a minimum number of transplanted CFU-GM or CFU- S that allows survival of lethally irradiated mice. This number is present in a marrow transplant containing the equivalent of 5 X 10(3) untreated cells or producing one to two spleen colonies. There also exists a maximum value for the number of hematopoietic progenitors in a marrow graft, above which the rate of hematologic recovery following transplantation is rapid and no detectable increase in the rate is seen with increasing CFU-GM or CFU-S content. The presence of this maximum value for transplanted progenitors and variations in culture techniques are probably the reasons previous studies have not always shown a correlation between CFU-GM content and hematologic recovery after bone marrow transplantation.     

Expression of Fas and Fas-ligand in donor hematopoietic stem and progenitor cells is dissociated from the sensitivity to apoptosis

OBJECTIVE: The interaction between the Fas receptor and its cognate ligand (FasL) has been implicated in the mutual suppression of donor and host hematopoietic cells after transplantation. Following the observation of deficient early engraftment of Fas and FasL-defective donor cells and recipients, we determined the role of the Fas-FasL interaction. METHODS: Donor cells were recovered after syngeneic (CD45.1-->CD45.2) transplants from various organs and assessed for expression of Fas/FasL in reference to lineage markers, carboxyfluorescein succinimidyl ester dilution, Sca-1 and c-kit expression. Na?ve and bone marrow-homed cells were challenged for apoptosis ex vivo. RESULTS: The Fas receptor and ligand were markedly upregulated to 40% to 60% (p < 0.001 vs 5-10% in na?ve cells) within 2 days after syngeneic transplantation, while residual host cells displayed modest and delayed upregulation of these molecules ( approximately 10%). All lin(-)Sca(+)c-kit(+) cells were Fas(+)FasL(+), including 95% of Sca-1(+) and 30% of c-kit(+) cells. Fas and FasL expression varied in donor cells that homed to bone marrow, spleen, liver and lung, and was induced by interaction with the stroma, irradiation, cell cycling, and differentiation. Bone marrow-homed donor cells challenged with supralethal doses of FasL were insensitive to apoptosis (3.2% +/- 1% vs 38% +/- 5% in na?ve bone marrow cells), and engraftment was not affected by pretransplantation exposure of donor cells to an apoptotic challenge with FasL. CONCLUSION: There was no evidence of Fas-mediated suppression of donor and host cell activity after transplantation. Resistance to Fas-mediated apoptosis evolves as a functional characteristic of hematopoietic reconstituting stem and progenitor cells, providing them competitive engraftment advantage over committed progenitors.