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1.
Full-term cord blood (TCB) hematopoietic stem/progenitor cells (HSC/HPCs) are used for stem cell transplantation and are well characterized. However, the properties of preterm cord blood (PCB) HSC/HPCs remain unclear. In the present study, we compared HSC/HPCs from TCB and PCB with respect to their expression of surface markers, homing capacity and ability to repopulate HSCs in the NOD/Shi-scid mice bone marrow. The proportion of CD34+CD38− cells was significantly higher in PCB. On the other hand, the engraftment rate of TCB CD34+ cells into NOD/Shi-scid mice was significantly higher than PCB CD34+ cells. The expression of VLA4 was stronger among TCB CD34+ cells than PCB CD34+ cells. Moreover, there was a positive correlation between the proportion of CD34+CXCR4+ cells and gestational age. These data suggest that the homing ability of HSCs increases during gestation, so that TCB may be a better source of HSCs for transplantation than PCB.  相似文献   

2.
Ex vivo expansion of residual autologous hematopoietic stem and progenitor cells collected from victims soon after accidental irradiation (autologous cell therapy) may represent an additional or alternative approach to cytokine therapy or allogeneic transplantation. Peripheral blood CD34+ cells could be a useful source of cells for this process provided that collection and ex vivo expansion of hematopoietic stem and progenitor cells could be optimized. Here we investigated whether mesenchymal stem cells could sustain culture of irradiated peripheral blood CD34+ cells. In vitro irradiated (4 Gy 60Co gamma rays) or nonirradiated mobilized peripheral blood CD34+ cells from baboons were cultured for 7 days in a serum-free medium supplemented with stem cell factor+thrombopoietin+interleukin 3+FLT3 ligand (50 ng/ml each) in the presence or absence of mesenchymal stem cells. In contrast to cultures without mesenchymal stem cells, irradiated CD34+ cells cultured with mesenchymal stem cells displayed cell amplification, i.e. CD34+ (4.9-fold), CD34++ (3.8-fold), CD34++/Thy-1+ (8.1-fold), CD41+ (12.4-fold) and MPO+ (50.6-fold), although at lower levels than in nonirradiated CD34+ cells. Fourteen times more clonogenic cells, especially BFU-E, were preserved when irradiated cells were cultured on mesenchymal stem cells. Moreover, we showed that the effect of mesenchymal stem cells is related mainly to the reduction of apoptosis and involves cell-cell contact rather than production of soluble factor(s). This experimental model suggests that mesenchymal stem cells could provide a crucial tool for autologous cell therapy applied to accidentally irradiated victims.  相似文献   

3.
The good outcome of hematopoietic stem cell (HSC) transplantation is hampered by low doses of CD34+ cell infusion. Transplanted HSCs undergo a replicative stress that causes accelerated senescence due to rapid telomere shortening. The expansion of human cord blood HSCs is instrumental in obtaining a large number of "good quality" cells, in terms of telomere length and telomerase activity compared to adult HSCs.  相似文献   

4.
Hematopoietic stem cells (HSCs) are used in transplantation therapy to reconstitute the hematopoietic system. Human cord blood (hCB) transplantation has emerged as an attractive alternative treatment option when traditional HSC sources are unavailable; however, the absolute number of hCB HSCs transplanted is significantly lower than bone marrow or mobilized peripheral blood stem cells (MPBSCs). We previously demonstrated that dimethyl-prostaglandin E2 (dmPGE2) increased HSCs in vertebrate models. Here, we describe preclinical analyses of the therapeutic potential of dmPGE2 treatment by using human and nonhuman primate HSCs. dmPGE2 significantly increased total human hematopoietic colony formation in?vitro and enhanced engraftment of unfractionated and CD34(+) hCB after xenotransplantation. In nonhuman primate autologous transplantation, dmPGE2-treated CD34(+) MPBSCs showed stable multilineage engraftment over 1 year postinfusion. Together, our analyses indicated that dmPGE2 mediates conserved responses in HSCs from human and nonhuman primates and provided sufficient preclinical information to support proceeding to an FDA-approved phase 1 clinical trial.  相似文献   

5.
Umbilical cord blood (UCB) transplantation has emerged as a promising therapy, but it is challenged by scarcity of stem cells. Eltrombopag is a non-peptide, thrombopoietin (TPO) receptor agonist, which selectively activates c-Mpl in humans and chimpanzees. We investigated eltrombopag's effects on human UCB hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) expansion, and its effects on hematopoiesis in vivo. Eltrombopag selectively augmented the expansion of human CD45+, CD34+, and CD41+ cells in bone marrow compartment without effects on mouse bone marrow cells in the NOD/SCID mice xenotransplant model. Consequently, eltrombopag increased peripheral human platelets and white blood cells. We further examined effects in the STAT and AKT signaling pathways in serum-free cultures. Eltrombopag expanded human CD34+ CD38-, CD34+, and CD41+ cells. Both eltrombopag and recombinant human TPO (rhTPO) induced phosphorylation of STAT5 of CD34+ CD41-, CD34- CD41+, and CD34- CD41- cells. rhTPO preferentially induced pSTAT3, pAKT, and more pSTAT5 in CD34- C41+ cells, while eltrombopag had no effects on pSTAT3. In conclusion, eltrombopag enhanced expansion of HSCs/HPCs of human UCB in vivo and in vitro, and promoted multi-lineage hematopoiesis through the expansion of bone marrow HSCs/HPCs of human UCB in vivo. Eltrombopag differed somewhat from rhTPO in the signal transduction pathways by favoring earlier HSC/HPC populations.  相似文献   

6.
Mouse hematopoietic stem cells (HSCs) are the best-studied stem cells because functional assays for mouse HSCs were established earliest and purification techniques for mouse HSCs have progressed furthest. Here we describe our current protocols for the purification of CD34-/lowc-Kit+Sca-1+lineage marker- (CD34-KSL) cells, the HSC population making up approximately 0.005% of bone marrow cells in adult C557BL/6 mice. Purified HSCs have been characterized at cellular and molecular levels. Since clonal analysis is essential for the study of self-renewal and lineage commitment in HSCs, here we present our single-cell colony assay and single-cell transplantation procedures. We also introduce our immunostaining procedures for small numbers of HSCs, which are useful for signal transduction analysis. The purification of CD34-KSL cells requires approximately 6 h. Initialization of single-cell culture requires approximately 1 h. Single-cell transplantation requires approximately 6 h. Single-cell immunostaining requires approximately 2 d.  相似文献   

7.
Keyser KA  Morris JC  Kiem HP 《Cytotherapy》2005,7(4):345-352
BACKGROUND: There is ongoing controversy about the transdifferentiation of hematopoietic stem cells (HSC) into different tissues such as mesenchymal cells. This transdifferentiation or 'plasticity' would be an appealing concept for many therapeutic strategies. While studies in the murine model show encouraging results, reports from clinical allogeneic stem cell transplantations do not support the concept of HSC plasticity. Our aim was to determine whether transplantation of transduced autologous marrow CD34+ cells leads to long-term engraftment of gene-marked cells with mesenchymal characteristics in the baboon. METHODS: We analyzed marrow of two baboons that had received green fluorescence protein (GFP)-marked CD34+ autologous marrow cells after myeloablative conditioning. Marrow was obtained 1 and 2.5 years after transplantation and adherent CD11a- (pan-leukocyte Ab) cells were cultured for 3 weeks. Cultures were then analyzed by flow cytometry and fluorescence microscopy for the presence of GFP+ cells. For further analysis fresh and cultured cells were also labeled with multiple Ab and functional analysis was performed. RESULTS: Both animals showed persistent and stable GFP marking by flow cytometry in peripheral blood leukocytes as well as in CD34+ marrow cells at 1 and 2.5 years after transplantation. There was no evidence of GFP+ mesenchymal cells by either flow cytometry or fluorescence microscopy, while functional and phenotypical analysis identified mesenchymal stem cells in these cultures. DISCUSSION: We conclude that genetically modified CD34+ cells do not contribute to the adherent marrow-derived mesenchymal cell population after autologous transplantation.  相似文献   

8.
Cord blood is a source of hematopoietic stem cells used in transplantation in which hematopoietic reconstitution is necessary. This transplant modality requires the cryopreservation of hematopoietic stem cells (HSCs). Dimethyl sulfoxide has been used as a cryoprotectant (CPA) in the cryopreservation of HSCs; however, it has been demonstrated that Me2SO exhibits toxic side effects to the human body. Due to its stability upon freezing, disaccharides such as trehalose have been investigated as a cryoprotectant. This study investigated the hypothesis that a cryopreservation solution containing intracellular and extracellular trehalose improves the recovery of stem cells after cryopreservation. After thawing, the cells were tested for their viability using the 7AAD stain, CD45+/CD34+ cells were assessed using flow cytometry and the MTT viability assay, and the proportion of hematopoietic progenitor cells was measured using the CFU assay. Our results showed the effectiveness of the solution containing intracellular and extracellular trehalose in the cryopreservation of cord blood cells, demonstrating that trehalose may be an optimal cryoprotectant when present both inside and outside of cells.  相似文献   

9.
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.  相似文献   

10.
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.  相似文献   

11.
Over the years, various biological parameters have been proposed for predicting rapidity and long term maintenance of hematopoietic engraftment after peripheral blood stem cell transplantation (PBSCT). Determination of the graft content in CFU-GM was the only one available until the end of the eighties. But, for technical reasons, and also because it does not actually evaluate the self-renewal potential of the cell products reinfused, it has now been commonly replaced by the determination of CD34+ cell amounts, which are known to contain the pluripotent hematopoietic stem cells. However, a frequent discrepancy still exists between the number of CD34+ cells reinfused and the engraftment efficiency. We have recently demonstrated a higher accuracy of the numbers of CD34+38- cells contained in graft products to predict rapidity of trilineage engraftment, which has further been confirmed by other investigators. Furthermore, we and others, have proposed a threshold dose of 5 x 10(4) CD34+38- cells/kg b.w. below which the trilineage engraftment kinetics are significantly slower and unpredictible. This "cut-off" value also appears to be a realistic clinical tool to decide if hematopoietic growth factor(s) must be administered or not after PBSCT. Indeed, when for example, rh-G-CSF administration after transplant of CD34+38- amounts < 5 x 10(4) kg has indisputable positive effects on the rapidity of neutrophil engraftment, length of hospitalization and posttransplant costs, enough to make it fully justified in this situation, it is absolutely not the case when it is administered after reinfusion of CD34+38- cell amounts > 5 x 10(4) /kg. In this case, posttransplant rh-G-CSF administration could even result in a decrease in stem cells with self-renewal potential of the graft, which should still raise more concerns for its indiscriminate and costly use.  相似文献   

12.

Background

The green fluorescent protein (GFP) has proven a useful marker in retroviral gene transfer studies targeting hematopoietic stem cells (HSCs) in mice. However, several investigators have reported very low in vivo peripheral blood marking levels in nonhuman primates after transplantation of HSCs transduced with the GFP gene. We retrovirally marked cynomolgus monkey HSCs with the GFP gene, and tracked in vivo marking levels within both bone marrow progenitor cells and mature peripheral blood cells following autologous transplantation after myeloablative conditioning.

Methods

Bone marrow cells were harvested from three cynomolgus macaques and enriched for the primitive fraction by CD34 selection. CD34+ cells were transduced with one of three retroviral vectors all expressing the GFP gene and were infused after myeloablative total body irradiation (500 cGy × 2). Following transplantation, proviral levels and fluorescence were monitored among clonogenic bone marrow progenitors and mature peripheral blood cells.

Results

Although 13–37% of transduced cells contained the GFP provirus and 11–13% fluoresced ex vivo, both provirus and fluorescence became almost undetectable in the peripheral blood within several months after transplantation regardless of the vectors used. However, on sampling of bone marrow at multiple time points, significant fractions (5–10%) of clonogenic progenitors contained the provirus and fluoresced ex vivo reflecting a significant discrepancy between GFP gene marking levels within bone marrow cells and their mature peripheral blood progeny. The discrepancy (at least one log) persisted for more than 1 year after transplantation. Since no cytotoxic T lymphocytes against GFP were detected in the animals, an immune response against GFP is an unlikely explanation for the low levels of transduced peripheral blood cells. Administration of granulocyte colony stimulating factor and stem cell factor resulted in mobilization of transduced bone marrow cells detectable as mature granulocyte progeny which expressed the GFP gene, suggesting that transduced progenitor cells in bone marrow could be mobilized into the peripheral blood and differentiated into granulocytes.

Conclusions

Low levels of GFP‐transduced mature cells in the peripheral blood of nonhuman primates may reflect a block to differentiation associated with GFP; this block can be overcome in part by nonphysiological cytokine treatment ex vivo and in vivo. Copyright © 2002 John Wiley & Sons, Ltd.
  相似文献   

13.
Background:Parathyroid hormone (PTH) is a calcium homeostasis regulator and can affect bone marrow niche. PTH leads to the bone marrow stem cell niche expansion as well as the induction of stem cell mobilization from the bone marrow into peripheral blood. In this study, we evaluated the association between pre- transplantation serum PTH levels and the number of circulating CD34+ cells along with the platelets/white blood cells (Plt/WBC) engraftment in patients who underwent autologous Hematopoietic Stem Cell Transplantation.Methods:Subjects for the study were 100 patients who received autologous hematopoietic stem cell transplantation (auto-HSCT), retrospectively. Serum levels of PTH, calcium, phosphorus, and alkaline phosphatase were measured before mobilization. Their impacts were measured on the number of mobilized CD34+ hematopoietic stem cells, and Plt/WBC engraftment.Results:High levels of serum PTH (> 63.10 pg/mL) was significantly associated with higher number of CD34+ cells in peripheral blood after granulocyte- colony stimulating factor (G-CSF)-induced mobilization (p= 0.079*). Serum calcium at low levels were associated with higher number of circulating CD34+ cells post mobilization. Pre- transplantation serum levels of phosphorus and alkaline phosphatase on CD34+ numbers were not statistically significant. Serum Plt/WBC engraftment was not improved in presence of high levels of serum PTH.Conclusion:We suggested that serum PTH levels before transplantation could be influential in raising the number of circulating CD34+ hematopoietic stem cell after mobilization.Key Words: Auto-HSCT, CD34+ Cell, Pre- transplant PTH  相似文献   

14.
15.
The adult bone marrow retains two populations of stem cells with emerging importance for the treatment of diverse liver diseases: hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). However, the mechanisms that control liver regeneration after bone marrow cell transplantation are still controversial. Liver regeneration after partial hepatectomy is a complex process that requires the proliferation of all hepatic cells. Growth factors, cytokines and extracellular matrix molecules are key elements in this process. Laminins are a family of extracellular matrix proteins with adhesive and chemotactic functions, expressed in the portal and centrolobular veins of the normal liver. The aim of this study was to investigate laminin expression during liver regeneration induced by partial hepatectomy followed by bone marrow mononuclear cell (BMMNC) transplantation. Rat BMMNCs were isolated by Ficoll-gradient centrifugation, stained with DAPI and injected into recently hepatectomyzed rats via the portal vein. Liver sections obtained 15 min, 1 day and 3 days after the surgery were immunolabeled with anti-rat CD34 and/or laminin primary antibodies and observed under a laser scanning confocal microscope. Results showed that 15 min after partial hepatectomy, a transplanted CD34+ HSC was found in contact with laminin, which was localized in the portal and centrolobular veins of rat livers. Furthermore, 1 and 3 days after hepatectomy, transplanted BMMNCs were found in the hepatic sinusoids expressing laminin. These results strongly suggest that laminin might be an important extracellular matrix component for bone marrow cell attachment and migration in the injured liver.  相似文献   

16.
A major problem hampering effective stem cell-based therapies is the absence of a clear understanding of the human hematopoietic stem cell (HSC) pool composition. The severe combined immunodeficiency (SCID) repopulating cell (SRC) xenotransplant assay system provides a powerful tool for characterizing the frequency, cell surface markers, cell cycle status, homing and response to cytokine stimulation of human HSCs. Clonal tracking of retrovirally transduced SRCs and transplantation of specific subpopulations revealed SRC classes with distinct repopulation potentials. However, all HSC repopulation assays are based on intravenous injection, a complex process that requires circulation through blood, recognition and extravasation through bone marrow vasculature, and migration to a supportive microenvironment. Thus, some classes of HSCs may remain undetected. By direct intrafemoral injection, we identified rapid SRCs (R-SRCs) within the Lin-CD34+CD38loCD36- subpopulation. R-SRCs rapidly generate high levels of human myeloid and erythroid cells within the injected femur, migrate to the blood and colonize individual bones of non-obese diabetic (NOD)-SCID mice within 2 weeks after transplantation. Lentivector-mediated clonal analysis of individual R-SRCs revealed heterogeneity in their proliferative and migratory properties. The identification of a new HSC class and an effective intrafemoral assay provide the tools required to develop more effective stem cell-based therapies that rely on rapid reconstitution.  相似文献   

17.
Flow cytometric enumeration of CD34+ hematopoietic stem and progenitor cells (HSCs) is widely used for evaluation of graft adequacy of peripheral blood and bone marrow stem cell grafts. In the present study, we review and compare the major counting techniques of stem and progenitor cells. The methods are: the Milan/Mullhouse protocol, two-platform ISHAGE (International Society of Hematotherapy and Graft Engineering) and single-platform ISHAGE analysis system. According to the Milan/Mulhouse protocol, HSCs are identified by CD34 antibody staining and easy gating strategy. The ISHAGE guidelines for detection of CD34+ cells are based on a four-parameter flow cytometry method (CD34PE/CD45PerCP staining, side and forward angle light scatter) thus employing multiparameter gating strategy. With two-platform ISHAGE protocol, an absolute CD34+ count is generated by incorporating the leukocyte count from an automated hematology analyser. The single-platform ISHAGE method to determine the absolute CD34+ count directly from a flow cytometer includes the use of Trucount tubes (Becton Dickinson) with a known number of fluorescent beads. CD34+ cells were quantified in mobilized peripheral blood, collected by leukapheresis, and bone marrow from 42 samples from patients with hematological malignancies. The differences against the means display low disagreement between the Milan/Mulhouse and ISHAGE protocols, with discrepancies of up to 2.5% (two-platform ISHAGE)--2.6% (single-platform ISHAGE) in enumeration of CD34+ cells in leukapheresis product and 4.8% (two-platform ISHAGE)--4.9% (single-platform ISHAGE) in bone marrow. Our results show high correlation among all three methods. Since the three protocols are compatible, choosing the most convenient in terms of costs, simplicity and compliance with clinical results appears to be a logical consequence.  相似文献   

18.
Hematopoietic stem cells (HSCs) are widely used in transplantation therapy to treat a variety of blood diseases. The success of hematopoietic recovery is of high importance and closely related to the patient’s morbidity and mortality after Hematopoietic stem cell transplantation (HSCT). We have previously shown that SALL4 is a potent stimulator for the expansion of human hematopoietic stem/progenitor cells in vitro. In these studies, we demonstrated that systemic administration with TAT-SALL4B resulted in expediting auto-reconstitution and inducing a 30-fold expansion of endogenous HSCs/HPCs in mice exposed to a high dose of irradiation. Most importantly, TAT-SALL4B treatment markedly prevented death in mice receiving lethal irradiation. Our studies also showed that TAT-SALL4B treatment was able to enhance both the short-term and long-term engraftment of human cord blood (CB) cells in NOD/SCID mice and the mechanism was likely related to the in vivo expansion of donor cells in a recipient. This robust expansion was required for the association of SALL4B with DNA methyltransferase complex, an epigenetic regulator critical in maintaining HSC pools and in normal lineage progression. Our results may provide a useful strategy to enhance hematopoietic recovery and reconstitution in cord blood transplantation with a recombinant TAT-SALL4B fusion protein.  相似文献   

19.
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.  相似文献   

20.
Hepatocyte growth factor (HGF), which was originally isolated as a liver generating factor, enhances hematopoiesis. To study the effect of HGF on hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs), we generated severe combined immunodeficiency (SCID) mice producing human (h) HGF and/or stem cell factor (SCF) by transferring the relevant genes to fertilized eggs, and then transplanted hematopoietic progenitors from human cord blood into the transgenic (Tg) SCID mice. Six months after transplantation, a significantly larger number of human cells were found in the Tg SCID mice than in non-Tg controls. Characteristically, the recipient SCID mice producing h HGF (HGF-SCID) had a significantly increased number of h CD41+ cells, whereas the SCF-SCID recipients had more CD11b+ cells. Significantly large numbers of CD34+ progenitors were found in the SCID mice transferred with both h HGF and h SCF genes (HGF/SCF-SCID) when compared with HGF-SCID or SCF-SCID mice. These results imply that HGF supports the differentiation of progenitors in megakaryocyte lineage, whereas SCF supports that in myeloid lineage. The results also imply that HGF acts on HSCs/HPCs as a synergistic proliferative factor combined with SCF. We have demonstrated the advantage of the human cytokine-producing animal in the maintenance of human HSCs.  相似文献   

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