Long-term engraftment of p18INK4C-deficient hematopoietic stem cells is enhanced in the sublethally-irradiated recipients

Non-myeloablative regimens for host conditioning have been widely used in clinical hematopoietic stem cell transplantation due to their reduced toxicity on the recipients. But a milder conditioning regimen may require a higher engrafting ability of donor stem cells in competing with endogenous stem cells. Thus, new strategies for enhancing the competitiveness of donor stem cells in non-myeloablative recipients would have important implications for current clinical stem cell transplantation. It is known that the absence of p18 ^INK4C (p18) gene can enhance the self-renewal potential of hematopoietic stem cells (HSCs). We applied the approach of competitive bone marrow transplantation to evaluate the impact of p18 gene deletion on long-term engraftment of HSCs in sublethally irradiated hosts. We found that p18 ^−/− HSCs had a significant advantage over wild-type HSCs during long-term engraftment in the mouse recipients that received a sub-lethal irradiation (5-Gy). The engraftment efficiency of p18 ^−/− HSCs in the sub-lethally irradiated recipients was similar to that in the lethally irradiated (10-Gy) recipients. Our current study demonstrates that enhanced engraftment of donor HSCs in the absence of p18 does not strictly depend on the dose of irradiation used for host conditioning. Therefore, p18 might serve as a potential drug target for increasing the efficacy of stem cell transplant in the patients that are preconditioned with either a myeloablative or non-myeloablative regimen.     

Long-term engraftment of p18 INK4C-deficient hematopoietic stem cells is enhanced in the sublethally-irradiated recipients

Non-myeloablative regimens for host conditioning have been widely used in clinical hematopoietic stem cell transplantation due to their reduced toxicity on the recipients. But a milder conditioning regimen may require a higher engrafting ability of donor stem cells in competing with endogenous stem cells. Thus, new strategies for enhancing the competitiveness of donor stem cells in non-myeloablative recipients would have important implications for current clinical stem cell transplantation. It is known that the absence of p18 ^INK4C (p18) gene can enhance the self-renewal potential of hematopoietic stem cells (HSCs). We applied the approach of competitive bone marrow transplantation to evaluate the impact of p18 gene deletion on long-term engraftment of HSCs in sublethally irradiated hosts. We found that p18 ^−/− HSCs had a significant advantage over wild-type HSCs during long-term engraftment in the mouse recipients that received a sub-lethal irradiation (5-Gy). The engraftment efficiency of p18 ^−/− HSCs in the sub-lethally irradiated recipients was similar to that in the lethally irradiated (10-Gy) recipients. Our current study demonstrates that enhanced engraftment of donor HSCs in the absence of p18 does not strictly depend on the dose of irradiation used for host conditioning. Therefore, p18 might serve as a potential drug target for increasing the efficacy of stem cell transplant in the patients that are preconditioned with either a myeloablative or non-myeloablative regimen.     

HoxB4 confers definitive lymphoid-myeloid engraftment potential on embryonic stem cell and yolk sac hematopoietic progenitors

The extent to which primitive embryonic blood progenitors contribute to definitive lymphoid-myeloid hematopoiesis in the adult remains uncertain. In an effort to characterize factors that distinguish the definitive adult hematopoietic stem cell (HSC) and primitive progenitors derived from yolk sac or embryonic stem (ES) cells, we examined the effect of ectopic expression of HoxB4, a homeotic selector gene implicated in self-renewal of definitive HSCs. Expression of HoxB4 in primitive progenitors combined with culture on hematopoietic stroma induces a switch to the definitive HSC phenotype. These progenitors engraft lethally irradiated adults and contribute to long-term, multilineage hematopoiesis in primary and secondary recipients. Our results suggest that primitive HSCs are poised to become definitive HSCs and that this transition can be promoted by HoxB4 expression. This strategy for blood engraftment enables modeling of hematopoietic transplantation from ES cells.     

Simultaneous knockdown of p18INK4C, p27Kip1 and MAD1 via RNA interference results in the expansion of long-term culture-initiating cells of murine bone marrow cells in vitro

A combination of extrinsic hematopoietic growth regulators, such as stem cell factor (SCF), interleukin (IL)-3 and IL-6, can induce division of quiescent hematopoietic stem cells (HSCs), but it usually impairs HSCs' self-renewal ability. However, intrinsic negative cell cycle regulators, such as p18^INK4c (p18) p27^Kip1 (p27) and MAD1, can regulate the self-renewal of HSCs. It is unknown whether the removal of some extrinsic regulators and the knockdown of intrinsic negative cell cycle regulators via RNA interference (RNAi) induce ex vivo expansion of the HSCs. To address this question, a lentiviral vector-based RNAi tool was developed to produce two copies of small RNA that target multiple genes to knockdown the intrinsic negative cell cycle regulators pl8, p27 and MAD1. Colony-forming cells, long-term culture-initiating cells (LTC-IC) and engraftment assays were used to evaluate the effects of extrinsic and intrinsic regulators. Results showed that the medium with only SCF, but without IL-3 and IL-6, could maintain the sca-1⁺c-kit⁺ bone marrow cells with high LTC-IC frequency and low cell division. However, when the sca-1⁺c-kit⁺ bone marrow cells were cultured in a medium with only SCF and simultaneously knocked down the expression of pl8, p27 and MAD1 via the lentiviral vector-based RNAi, the cells exhibited both high LTC-IC frequency and high cell division, though engraftment failed. Thus, the simultaneous knockdown of pl8, p27 and MAD1 with a medium of only SCF can induce LTC-IC expansion despite the loss of engraftment ability.     

Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells

Hematopoietic stem cells (HSCs) are characterized by their ability to differentiate into all hematopoietic cell lineages while retaining their capacity for self renewal. One of the predictions of this model is the existence of a heterogeneous pool of HSCs, some members of which are destined to become lineage restricted progenitor cells while others function to renew the stem cell pool. To test whether HSCs are heterogeneous with respect to cell cycle status, we determined the fraction of phenotypically defined murine HSCs (Thy1.1lo Lin-/lo Sca- 1+) that contain > 2n amount of DNA as measured by propidium iodide staining, Hoechst dye uptake and [3H]thymidine labeling; that fraction is 18-22%. In contrast, in the developing fetal liver, 40% of HSCs are in the S/G2/M phases of the cell cycle. Those HSCs which exhibit a low level of staining with rhodamine 123 are almost exclusively in G0/G1 (97%) whereas only 70% of HSCs which stain brightly for rhodamine 123 are in G0/G1. The injection of 100 G0/G1 HSCs rescued 90% of lethally irradiated mice in contrast to 100 S/G2/M HSCs, which protected only 25% of lethally irradiated recipients. Enhanced long-term donor-derived multilineage reconstitution of the peripheral blood was observed in recipients of 100 G0/G1 HSCs compared to recipients of 100 S/G2/M cells. These data indicate that a significant proportion of HSCs are actively proliferating during steady state hematopoiesis and that this subpopulation of cells exhibits reduced stem cell activity.     

Immunomodulatory effects induced by cytotoxic T lymphocyte antigen 4 immunoglobulin with donor peripheral blood mononuclear cell infusion in canine major histocompatibility complex-haplo-identical non-myeloablative hematopoietic cell transplantation

BACKGROUND AIMS. Previously, cytotoxic T lymphocyte antigen 4 (CTLA4) immunoglobulin (Ig) has been shown to allow sustained engraftment in dog leukocyte antigen (DLA)-identical hematopoietic cell transplant (HCT) after non-myeloablative conditioning with 100 cGy total body irradiation (TBI). In the current study, we investigated the efficacy of pre-transplant CTLA4-Ig in promoting engraftment across a DLA-mismatched barrier after non-myeloablative conditioning. METHODS. Eight dogs were treated with CTLA4-Ig and donor peripheral blood mononuclear cells (PBMC) prior to receiving 200 cGy TBI followed by transplantation of granulocyte-colony-stimulating factor (G-CSF) mobilized peripheral blood stem cells from DLA haplo-identical littermates with post-grafting immunosuppression. A control group of six dogs was conditioned with 200 cGy only and transplanted with grafts from DLA haplo-identical littermates followed by post-grafting immunosuppression. RESULTS. In vitro and in vivo donor-specific hyporesponsiveness was demonstrated on day 0 before TBI in eight dogs that received CTLA4-Ig combined with donor PBMC infusions. Four of five dogs treated with increased doses of CTLA4-Ig achieved initial engraftment but eventually rejected, with a duration of mixed chimerism ranging from 12 to 22 weeks. CTLA4-Ig did not show any effect on host natural killer (NK) cell function in vitro or in vivo. No graft-versus-host disease (GvHD) was observed in dogs receiving CTLA4-Ig treatment. CONCLUSIONS. Non-myeloablative conditioning with 200 cGy TBI and CTLA4-Ig combined with donor PBMC infusion was able to overcome the T-cell barrier to achieve initial engraftment without GvHD in dogs receiving DLA haplo-identical grafts. However, rejection eventually occurred; we hypothesize because of the inability of CTLA4-Ig to abate natural killer cell function.     

Mobilization‐based transplantation of young‐donor hematopoietic stem cells extends lifespan in mice

Mammalian aging is associated with reduced tissue regeneration and loss of physiological integrity. With age, stem cells diminish in their ability to regenerate adult tissues, likely contributing to age‐related morbidity. Thus, we replaced aged hematopoietic stem cells (HSCs) with young‐donor HSCs using a novel mobilization‐enabled hematopoietic stem cell transplantation (HSCT) technology as an alternative to the highly toxic conditioning regimens used in conventional HSCT. Using this approach, we are the first to report an increase in median lifespan (12%) and a decrease in overall mortality hazard (HR: 0.42, CI: 0.273–0.638) in aged mice following transplantation of young‐donor HSCs. The increase in longevity was accompanied by reductions of frailty measures and increases in food intake and body weight of aged recipients. Young‐donor HSCs not only preserved youthful function within the aged bone marrow stroma, but also at least partially ameliorated dysfunctional hematopoietic phenotypes of aged recipients. This compelling evidence that mammalian health and lifespan can be extended through stem cell therapy adds a new category to the very limited list of successful anti‐aging/life‐extending interventions. Our findings have implications for further development of stem cell therapies for increasing health and lifespan.     

Signal One and Two Blockade Are Both Critical for Non-Myeloablative Murine HSCT across a Major Histocompatibility Complex Barrier

Non-myeloablative allogeneic haematopoietic stem cell transplantation (HSCT) is rarely achievable clinically, except where donor cells have selective advantages. Murine non-myeloablative conditioning regimens have limited clinical success, partly through use of clinically unachievable cell doses or strain combinations permitting allograft acceptance using immunosuppression alone. We found that reducing busulfan conditioning in murine syngeneic HSCT, increases bone marrow (BM):blood SDF-1 ratio and total donor cells homing to BM, but reduces the proportion of donor cells engrafting. Despite this, syngeneic engraftment is achievable with non-myeloablative busulfan (25 mg/kg) and higher cell doses induce increased chimerism. Therefore we investigated regimens promoting initial donor cell engraftment in the major histocompatibility complex barrier mismatched CBA to C57BL/6 allo-transplant model. This requires full myeloablation and immunosuppression with non-depleting anti-CD4/CD8 blocking antibodies to achieve engraftment of low cell doses, and rejects with reduced intensity conditioning (≤75 mg/kg busulfan). We compared increased antibody treatment, G-CSF, niche disruption and high cell dose, using reduced intensity busulfan and CD4/8 blockade in this model. Most treatments increased initial donor engraftment, but only addition of co-stimulatory blockade permitted long-term engraftment with reduced intensity or non-myeloablative conditioning, suggesting that signal 1 and 2 T-cell blockade is more important than early BM niche engraftment for transplant success.     

Homing and long-term engraftment of long- and short-term renewal hematopoietic stem cells

Long-term hematopoietic stem cells (LT-HSC) and short-term hematopoietic stem cells (ST-HSC) have been characterized as having markedly different in vivo repopulation, but similar in vitro growth in liquid culture. These differences could be due to differences in marrow homing. We evaluated this by comparing results when purified ST-HSC and LT-HSC were administered to irradiated mice by three different routes: intravenous, intraperitoneal, and directly into the femur. Purified stem cells derived from B6.SJL mice were competed with marrow cells from C57BL/6J mice into lethally irradiated C57BL/6J mice. Serial transplants into secondary recipients were also carried out. We found no advantage for ST-HSC engraftment when the cells were administered intraperitoneally or directly into femur. However, to our surprise, we found that the purified ST-HSC were not short-term in nature but rather gave long-term multilineage engraftment out to 387 days, albeit at a lower level than the LT-HSC. The ST-HSC also gave secondary engraftment. These observations challenge current models of the stem cell hierarchy and suggest that stem cells are in a continuum of change.     

Adoptive transfer of T-cell precursors enhances T-cell reconstitution after allogeneic hematopoietic stem cell transplantation

Immunoincompetence after allogeneic hematopoietic stem cell transplantation (HSCT) affects in particular the T-cell lineage and is associated with an increased risk for infections, graft failure and malignant relapse. To generate large numbers of T-cell precursors for adoptive therapy, we cultured mouse hematopoietic stem cells (HSCs) in vitro on OP9 mouse stromal cells expressing the Notch-1 ligand Delta-like-1 (OP9-DL1). We infused these cells, together with T-cell-depleted mouse bone marrow or purified HSCs, into lethally irradiated allogeneic recipients and determined their effect on T-cell reconstitution after transplantation. Recipients of OP9-DL1-derived T-cell precursors showed increased thymic cellularity and substantially improved donor T-cell chimerism (versus recipients of bone marrow or HSCs only). OP9-DL1-derived T-cell precursors gave rise to host-tolerant CD4+ and CD8+ populations with normal T-cell antigen receptor repertoires, cytokine secretion and proliferative responses to antigen. Administration of OP9-DL1-derived T-cell precursors increased resistance to infection with Listeria monocytogenes and mediated significant graft-versus-tumor (GVT) activity but not graft-versus-host disease (GVHD). We conclude that the adoptive transfer of OP9-DL1-derived T-cell precursors markedly enhances T-cell reconstitution after transplantation, resulting in GVT activity without GVHD.     

Intra-bone marrow transplantation of human CD34+ cells into NOD/LtSz-scid IL-2rγnull mice permits multilineage engraftment without previous irradiation

Background aimsNon-irradiated immunodeficient recipients provide the best physiologic setting for revealing hematopoietic stem cell (HSC) functions after xenotransplantion. An approach that efficiently permits the detection of human hematopoietic repopulating cells in non-irradiated recipients is therefore highly desired.MethodsWe compared side-by-side the ability to reconstitute hematopoiesis via intra-bone marrow transplantation (IBMT) in three commonly used mouse strains avoiding previous irradiation.ResultsNon-irradiated NOD/SCID and NOD/SCID (β2m?/? mouse strains prevent engraftment even after IBMT. In contrast, combining the robustness of the NOD/SCID IL-2Rγ?/? recipient with the sensitivity of IBMT facilitates the detection, without previous host irradiation, of human SCID-repopulating cells 10 weeks after transplantation. The level of chimerism averaged 14% and multilineage engraftment (lymphoid dominant) was observed consistently in all mice. Analysis of injected and non-injected bones, spleen and peripheral blood demonstrated that engrafting cells were capable of in vivo migration and expansion.ConclusionsCombining the robustness of the NOD/SCID IL-2Rγ?/? mouse strain with the sensitivity of IBMT strongly facilitates long-term multilineage engraftment and migration for human CD34⁺ cells without the need for previous irradiation.     

Hematopoietic chimerism in sheep and nonhuman primates by in utero transplantation of fetal hematopoietic stem cells.

Bone marrow transplantation to reconstitute defective hematopoietic cell lines in children with congenital defects is limited by donor availability, graft rejection, and graft-versus-host disease (GVHD). These problems can be eliminated by transplanting normal preimmune fetal hematopoietic stem cells (HSC) into an unrelated preimmune fetal recipient. We show here that injections of allogeneic fetal stem cells into preimmune fetal lambs and monkeys result in long-term stable hematopoietic chimerism. HSCs harvested from the livers of preimmune fetal sheep and monkeys when injected into the peritoneal cavity of young unrelated fetal sheep and monkey recipients results in stable, long-term postnatal hematopoietic chimerism involving lymphoid, erythroid, and myeloid cells of donor origin. Donor cell engraftment was achieved without the use of cytoablative procedures and without the development of GVHD.     

Donor-type activated natural killer cells promote marrow engraftment and B cell development during allogeneic bone marrow transplantation.

Purified NK cells were obtained from mice with severe combined immune deficiency and were activated with human IL-2 (hrIL-2) in vitro to determine if, once activated, these cells could be transferred with compatible bone marrow cells (BMC) and promote marrow engraftment in irradiated allogeneic recipients. After culture with hrIL-2, these cells maintained a phenotypic and lytic spectrum consistent with a pure population of activated NK cells. These activated NK cells were then adoptively transferred with the donor BMC and rhIL-2 into lethally irradiated allogeneic hosts. The addition of NK cells with the BMC allowed for more rapid hematopoietic engraftment as determined through short term studies, and greater donor-derived chimerism with accelerated reconstitution of the B cell population as determined with long term analysis. No evidence of graft-vs-host disease was detected in the recipients receiving the activated NK cells with allogeneic T cell replete BMC and hrIL-2. The mechanism by which the transferred NK cells improved BMC engraftment was at least partly through the abrogation of the host effector cell's ability to mediate resistance to the marrow graft. Thus, the administration of donor-type activated NK cells with BMC and hrIL-2 may significantly augment hematopoietic engraftment and immune reconstitution in the clinical setting of allogeneic BMT without giving rise to graft-vs-host disease.     

Prevention of GVHD by modulation of rat bone marrow with UV-B irradiation: II. Kinetics of migration of UV-B-irradiated bone marrow cells in naive and lethally irradiated rats

UV-B irradiation (700 J/m2) of bone marrow (BM) cells prior to transplantation into lethally gamma-irradiated (1050 rad) allogeneic rats prevents the development of GVHD and results in a stable mixed lymphohematopoietic chimerism. To better understand the underlying mechanisms of the development of stable radiation chimeras in this model, this study was designed to examine whether the dose (700 J/m2) of UV-B irradiation used for the modulation of the BM inoculum would affect the homing pattern of radiolabeled BM cells compared to that of thoracic duct lymphocytes (TDL) in the naive and lethally irradiated recipients. The results showed that intravenously administered, 111Indium-oxine-labeled, unmodified TDL home specifically to the spleen, lymph nodes, and BM compartments with a subsequent recirculation of a large number of cells from the spleen to the lymph nodes. In contrast, radiolabeled, unmodified BM cells migrate specifically to the spleen, liver, and BM with the lymph nodes, thymus, and nonlymphoid organs containing very little amounts of radioactivity. The stable concentrations of radioactivity in the lymphoid and nonlymphoid compartments between 3 and 72 hr after injection suggest that BM cells, unlike TDL, do not recirculate. The migration pattern of BM cells in the naive recipient was not significantly different from that seen in lethally irradiated animals except for the higher concentration of radioactivity in the spleen and BM of irradiated animals compared to that seen in naive recipients. The similarity of tissue localization of BM cells in naive or in irradiated syngeneic recipients to that of allogeneic recipients suggests that the homing of BM cells is not MHC restricted. Our findings of similarity between tissue localization of UV-B-irradiated labeled BM cells and unmodified BM cells in naive and lethally irradiated recipients suggest that a dose of 700 J/m2 of UV-B irradiation is not capable of impairing BM cell migration although it is sufficient to abolish the homing of TDL to the HEV-bearing organs. Thus, our results show that BM cells are less susceptible to cell damage by UV-B irradiation than lymphocytes thereby providing the rationale for ex vivo modulation (rather than elimination) of mature T-lymphocytes in the donor BM inoculum with UV-B irradiation. This relatively simple and effective approach to modulation of T-cells in donor BM inoculum may be potentially useful in preventing GVHD without endangering successful engraftment in larger animals and in man.     

Promotive effect of macrophage colony-stimulating factor on long-term engraftment of murine hematopoietic stem cells

Large ex vivo expansion of hematopoietic stem cells (HSCs) sufficient for use in clinical applications has not been achieved, although the influence of some cytokines including SCF, IL-11, Flt3-L, and TPO for this purpose has been reported. We present evidence for an indirect effect of macrophage colony-stimulating factor (M-CSF) on expansion of murine HSCs. Fresh Lin(-/low) cells were isolated from Ly5.1 mouse bone marrow and cultured with or without M-CSF in the presence of SCF + IL-11 + Flt3-L or SCF + IL-11 + TPO for 6 days. The expanded cells were harvested and transplanted into lethally irradiated Ly5.2 recipients with competitor cells. Culture of Lin(-/low) cells with M-CSF significantly enhanced long-term engraftment. When the more enriched HSC populations of Lin(-/low) c-Kit(+) Sca-1(+) cells were used as a source of HSCs, such a promotive effect was not observed, in agreement with negative expression of the M-CSF receptor (c-Fms). However, co-culture with Lin(-/low) c-Fms(+) resulted in a significant increase of long-term engraftment. These results suggested that M-CSF is an indirect stimulator for ex vivo expansion of HSCs in the presence of SCF, IL-11, Flt3-L, and TPO. These observations provide new directions for ex vivo expansion and insight into new engraftment regulation through M-CSF signaling.     

Role of N-cadherin in the regulation of hematopoietic stem cells in the bone marrow niche

Cell-cell and cell-extracellular matrix interactions between hematopoietic stem cells (HSCs) and their niches are critical for the maintenance of stem cell properties. Here, it is demonstrated that a cell adhesion molecule, N-cadherin, is expressed in hematopoietic stem/progenitor cells (HSPCs) and plays a critical role in the regulation of HSPC engraftment. Furthermore, overexpression of N-cadherin in HSCs promoted quiescence and preserved HSC activity during serial bone marrow (BM) transplantation (BMT). Inhibition of N-cadherin by the transduction of N-cadherin short hairpin (sh) RNA (shN-cad) reduced the lodgment of donor HSCs to the endosteal surface, resulting in a significant reduction in long-term engraftment. shN-cad-transduced cells were maintained in the spleen for six months after BMT, indicating that N-cadherin expression in HSCs is specifically required in the BM. These findings suggest that N-cadherin-mediated cell adhesion is functionally essential for the regulation of HSPC activities in the BM niche.     

Factors affecting engraftment of allogeneic hematopoietic stem cells after reduced-intensity conditioning

Several factors influence the engraftment of allogeneic hematopoietic stem cells (HSC). Recently, there has been increased utilization of transplant-conditioning regimens that use reduced doses of chemotherapy and radiation that are considered to be non-myeloablative. These non-myeloablative (or reduced-intensity) allogeneic HSC transplants (RIST) decrease early post-transplant complications, but they are associated with higher incidences of mixed chimerism and graft rejection compared with transplantation after myeloablative condition-ing. RIST provides a unique opportunity to study allogeneic HSC engraftment. In particular, host immune status and stem cell graft composition have emerged as important factors affecting engraftment after RIST Based on these observations, it has been hypothesized that conditioning regimens and allograft composition can be tailored to an individual patients immune and disease status prior to transplant.     

Improved short-term engraftment of lentivirally versus gammaretrovirally transduced allogeneic canine repopulating cells

Gammaretroviral vectors require cell division for efficient transduction. Thus, extended cell culture times are necessary for efficient transduction with gammaretroviral vectors, which in turn can lead to stem cell loss and impaired engraftment. Lentiviral vectors transduce nondividing cells and are therefore able to transduce stem cells in short transduction protocols. Here, we compared the short-term engraftment of lentivirally and gammaretrovirally transduced canine allogeneic DLA-matched littermate cells. A reduced conditioning regimen of 400 cGy total body irradiation was used in preparation for clinical studies. Two dogs received a graft of gammaretrovirally transduced CD34-selected cells. CD34(+) cells were prestimulated for 30 h and then exposed twice to concentrated RD114 pseudotype vector. Three dogs received lentivirally transduced CD34-selected cells. Cells were transduced overnight with concentrated VSV-G pseudotype lentiviral vector. The animals in the lentiviral group showed a significantly faster granulocyte recovery. VNTR analysis 40-50 days after transplantation revealed higher donor chimerism for the lentiviral group compared to the retroviral group. These data suggest that short lentiviral transduction protocols may be superior to extended gammaretroviral transduction protocols with respect to engraftment potential of transduced CD34(+) hematopoietic repopulating cells.     

Human CD34+ CD133+ hematopoietic stem cells cultured with growth factors including Angptl5 efficiently engraft adult NOD-SCID Il2rγ-/- (NSG) mice

Increasing demand for human hematopoietic stem cells (HSCs) in clinical and research applications necessitates expansion of HSCs in vitro. Before these cells can be used they must be carefully evaluated to assess their stem cell activity. Here, we expanded cord blood CD34(+) CD133(+) cells in a defined medium containing angiopoietin like 5 and insulin-like growth factor binding protein 2 and evaluated the cells for stem cell activity in NOD-SCID Il2rg(-/-) (NSG) mice by multi-lineage engraftment, long term reconstitution, limiting dilution and serial reconstitution. The phenotype of expanded cells was characterized by flow cytometry during the course of expansion and following engraftment in mice. We show that the SCID repopulating activity resides in the CD34(+) CD133(+) fraction of expanded cells and that CD34(+) CD133(+) cell number correlates with SCID repopulating activity before and after culture. The expanded cells mediate long-term hematopoiesis and serial reconstitution in NSG mice. Furthermore, they efficiently reconstitute not only neonate but also adult NSG recipients, generating human blood cell populations similar to those reported in mice reconstituted with uncultured human HSCs. These findings suggest an expansion of long term HSCs in our culture and show that expression of CD34 and CD133 serves as a marker for HSC activity in human cord blood cell cultures. The ability to expand human HSCs in vitro should facilitate clinical use of HSCs and large-scale construction of humanized mice from the same donor for research applications.     

Flagellin, a TLR5 agonist, reduces graft-versus-host disease in allogeneic hematopoietic stem cell transplantation recipients while enhancing antiviral immunity

Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality in patients treated with allogeneic hematopoietic stem cell transplantation (HSCT). Posttransplant immunosuppressive drugs incompletely control GVHD and increase susceptibility to opportunistic infections. In this study, we used flagellin, a TLR5 agonist protein (～50 kDa) extracted from bacterial flagella, as a novel experimental treatment strategy to reduce both acute and chronic GVHD in allogeneic HSCT recipients. On the basis of the radioprotective effects of flagellin, we hypothesized that flagellin could ameliorate GVHD in lethally irradiated murine models of allogeneic HSCT. Two doses of highly purified flagellin (administered 3 h before irradiation and 24 h after HSCT) reduced GVHD and led to better survival in both H-2(b) → CB6F1 and H-2(K) → B6 allogeneic HSCT models while preserving >99% donor T cell chimerism. Flagellin treatment preserved long-term posttransplant immune reconstitution characterized by more donor thymic-derived CD4(+)CD25(+)Foxp3(+) regulatory T cells and significantly enhanced antiviral immunity after murine CMV infection. The proliferation index and activation status of donor spleen-derived T cells and serum concentration of proinflammatory cytokines in flagellin-treated recipients were reduced significantly within 4 d posttransplant compared with those of the PBS-treated control recipients. Allogeneic transplantation of radiation chimeras previously engrafted with TLR5 knockout hematopoietic cells showed that interactions between flagellin and TLR5 expressed on both donor hematopoietic and host nonhematopoietic cells were required to reduce GVHD. Thus, the peritransplant administration of flagellin is a novel therapeutic approach to control GVHD while preserving posttransplant donor immunity.