Neonatal gene transfer leads to widespread correction of pathology in a murine model of lysosomal storage disease

For many inborn errors of metabolism, early treatment is critical to prevent long-term developmental sequelae. We have used a gene-therapy approach to demonstrate this concept in a murine model of mucopolysaccharidosis type VII (MPS VII). Newborn MPS VII mice received a single intravenous injection with 5.4 x 10(6) infectious units of recombinant adeno-associated virus encoding the human beta-glucuronidase (GUSB) cDNA. Therapeutic levels of GUSB expression were achieved by 1 week of age in liver, heart, lung, spleen, kidney, brain, and retina. GUSB expression persisted in most organs for the 16-week duration of the study at levels sufficient to either reduce or prevent completely lysosomal storage. Of particular significance, neurons, microglia, and meninges of the central nervous system were virtually cleared of disease. In addition, neonatal treatment of MPS VII mice provided access to the central nervous system via an intravenous route, avoiding a more invasive procedure later in life. These data suggest that gene transfer mediated by adeno-associated virus can achieve therapeutically relevant levels of enzyme very early in life and that the rapid growth and differentiation of tissues does not limit long-term expression.     

细胞因子体外扩增脐血CD+34细胞移植SCID鼠人类免疫功能的建立

目的：探讨扩增的脐血ＣＤ３４细胞体内造血功能。方法：ＦＬＴ３ 配体联合血小板生成素体外培养脐血ＣＤ＋３４细胞２～３ 周后移植ＳＣＩＤ鼠,采用ＦＣＭ 检测ＳＣＩＤ鼠骨髓、外周血、脾脏中的人类ＣＤ４５、ＣＤ３、ＣＤ１９抗原。结果：脐血ＣＤ＋３４细胞扩增１０～３７ 倍,移植６．６×１０４～６．９×１０５ 脐血ＣＤ＋３４细胞于ＳＣＩＤ鼠,６ 周后１０ 只ＳＣＩＤ鼠骨髓中均检测到人类ＣＤ４５抗原（０．２％ ～４．０％ ）,６ 只ＳＣＩＤ鼠外周血、脾脏中检测到人类ＣＤ３ 抗原（０．１％ ～２．３％ ）,３ 只ＳＣＩＤ鼠中外周血、脾脏中检测到人类ＣＤ１９抗原（０．３％ ～１．３％ ）。结论：ＦＬＴ３ 配体联合血小板生成素体外扩增的脐血ＣＤ＋３４细胞能重建ＳＣＩＤ鼠人类免疫功能。     

Murine mucopolysaccharidosis type VII: The impact of therapies on the clinical course and pathology in a murine model of lysosomal storage disease

Murine mucopolysaccharidosis type VII (MPS VII) is a lysosomal storage disease caused by a recessively inherited deficiency of the lysosomal enzyme -glucuronidase. Affected mice have clinical, biochemical and pathological findings similar to those seen in humans with MPS VII (Sly syndrome), including growth retardation, facial dysmorphism, deafness, behavioural deficits and widespread glycosaminoglycan storage in lysosomes in the viscera, skeleton and brain. This mouse model is a useful tool for the evaluation of the effectiveness and experimental therapies for the MPS disorders. Syngeneic bone marrow transplantation performed in newborn MPS VII animals – before clinical evidence of disease is pronounced – prolongs life, improves hearing and bone growth, and prevents lysosomal storage in many sites, but does not correct the central nervous system disease. Enzyme therapy with -glucuronidase from the first days of life does reduce lysosomal storage in the brain in murine MPS VII. The enzyme-replaced mice also have reduced visceral lysosomal storage, impressive normalization of their phenotype and an improved life span. The effectiveness of gene therapy for the treatment of lysosomal storage disease has also been tested using the MPS VII model. When transplanted into MPS VII mice, syngeneic haematopoietic stem cells or mouse skin fibroblasts infected with retrovirus expressing -glucuronidase decreased storage, but only in the liver and spleen. Injection of an adenovirus vector expressing -glucuronidase into the vitreous of the MPS VII mice reduced storage in the retinal pigment epithelium and corneal endothelium. Intravenous administration of the adenovirus vector transduced with the -glucuronidase gene reduced liver and spleen storage and, when instilled into the cerebral ventricles, this viral vector caused -glucuronidase production in epithelial cells lining the ventricles. Recently, retroviral vector-corrected MPS VII fibroblasts secreting high levels of -glucuronidase were engrafted directly into the brains of adult MPS VII mice with resultant reduction in storage in neurons and glia adjacent to the grafts. Future efforts aimed at prolonging expression of the -glucuronidase gene by viral vectors and more precisely directing the therapeutic effect to the skeleton and brain will be important in optimizing treatments for murine MPS VII and extending the results of such therapies to humans with MPS.     

Circulating human CD34+ progenitor cells modulate neovascularization and inflammation in a nude mouse model

CD34+ progenitor cells hold promise for therapeutic neovascularization in various settings. In this study, the role of human peripheral blood CD34+ cells in neovascularization and inflammatory cell recruitment was longitudinally studied in vivo. Human CD34+ cells were incorporated in Matrigel, implanted subcutaneously in nude mice, and explanted after 2, 4, 7, or 14 days. Cell-free Matrigels served as controls. Histochemical analyses demonstrated that neovascularization occurred almost exclusively in CD34+ implants. Cellular and capillary density were increased in cell-loaded Matrigels after 2 days and further increased at 14 days. Human CD34+ cells did not incorporate in neovessels, but formed vWF+/CD31+/VEGF+ cell clusters that were present up to day 14. However, CD34+ cells induced host neovascularization, as demonstrated by increased presence of murine CD31+ and vWF+ vasculature from day 7 to 14. Moreover, recruitment of murine monocytes/macrophages was significantly enhanced in CD34+ implants at all time points. Gene expression of chemotactic cytokines MCP-1 and IL-8 was detected on CD34+ cells in vitro and confirmed immunohistochemically in cell-loaded explants at all time points. Our data indicate that human CD34+ cells, implanted in a hypoxic environment, generate an angiogenic niche by secreting chemotactic and angiogenic factors, enabling rapid neovascularization, possibly via recruitment of monocytes/macrophages.     

Hyperbaric oxygen improves engraftment of ex-vivo expanded and gene transduced human CD34+ cells in a murine model of umbilical cord blood transplantation

Delayed engraftment and graft failure represent major obstacles to successful umbilical cord blood (UCB) transplantation. Herein, we evaluated the use of hyperbaric oxygen (HBO) therapy as an intervention to improve human UCB stem/progenitor cell engraftment in an immune deficient mouse model. Six- to eight-week old NSG mice were sublethally irradiated 24 hours prior to CD34⁺ UCB cell transplant. Irradiated mice were separated into a non-HBO group (where mice remained under normoxic conditions) and the HBO group (where mice received 2 hours of HBO therapy; 100% oxygen at 2.5 atmospheres absolute). Four hours after completing HBO therapy, both groups intravenously received CD34⁺ UCB cells that were transduced with a lentivirus carrying luciferase gene and expanded for in vivo imaging. Mice were imaged and then sacrificed at one of 10 times up to 4.5 months post-transplant. HBO treated mice demonstrated significantly improved bone marrow, peripheral blood, and spleen retention and subsequent engraftment. In addition, HBO significantly improved peripheral, spleen and bone marrow engraftment of human myeloid and B-cell subsets. In vivo imaging demonstrated that HBO mice had significantly higher ventral and dorsal bioluminescence values. These studies suggest that HBO treatment of NSG mice prior to UCB CD34⁺ cell infusion significantly improves engraftment.     

Engraftment kinetics of human CD34+ cells from cord blood and mobilized peripheral blood co-transplanted into NOD/SCID mice

We have reported short periods of post transplant neutropenia in human patients co-transplanted with cord blood (CB) and low numbers of haploidentical mobilized peripheral blood (MPB) CD34+ cells. To investigate the effect that the proportion of MPB to CB cells may have on engraftment kinetics, we have co-transplanted fixed numbers of human CB CD34+ cells mixed with different numbers of MPB CD34+ cells into NOD/SCID mice. We periodically quantified the proportion of human cells and the relative contribution of MPB and CB cells to the human engraftment on marrow aspirates. At the lowest MPB/CB ratios (5 : 1, 10 : 1), the contribution of CB cells predominated at all time points analyzed, and in three out of four experiments MPB cell contributions progressively decreased from day +15. At higher MPB/CB ratios, MPB cells had a more important contribution to both early and late engraftment, with the highest cell ratio resulting in only marginal CB cell engraftment. Therefore, our results showed greater potential, on a per cell basis, of human CB vs MPB cells for competitive sustained engraftment in the xenogeneic model used, which was only abrogated by the co-infusion of very high numbers of MPB cells.     

Engraftment after infusion of CD34+ marrow cells in patients with breast cancer or neuroblastoma.

The CD34 antigen is expressed by 1% to 4% of human and baboon marrow cells, including virtually all hematopoietic progenitors detectable by in vitro assays. Previous work from our laboratory has shown that CD34+ marrow cells can engraft lethally irradiated baboons. Because the CD34 antigen has not been detected on most solid tumors, positive selection of CD34+ cells may be used to provide marrow cells capable of engraftment, but depleted of tumor cells. In seven patients with stage IV breast cancer and two patients with stage IV neuroblastoma, 2.5 to 17.5 x 10(9) marrow cells were separated by immunoadsorption with the anti-CD34 antibody 12-8 and 50 to 260 x 10(6) positively selected cells were recovered that were 64 +/- 16% (range 35% to 92%) CD34+. The patients received 1.0 to 5.2 x 10(6) CD34-enriched cells/kg after marrow ablative therapy. Six patients engrafted, achieving granulocyte counts of greater than 500/mm3 at 34 +/- 10 (range 21 to 47) days and platelets counts of greater than 20,000/mm3 at 46 +/- 14 (range 28 to 66) days posttransplant. Five of these patients showed durable engraftment until the time of death 82 to 386 days posttransplant. One patient failed to sustain engraftment associated with metastatic marrow disease. Three patients died at days 14, 14, and 17 posttransplant, two of whom had evidence of early engraftment. These studies suggest that CD34+ marrow cells are capable of reconstituting hematopoiesis in humans.     

Cocultivation of umbilical cord blood cells with endothelial cells leads to extensive amplification of competent CD34+CD38- cells

Objective: In this report, methods to expand the number of human cord blood hematopoietic stem cells were explored. MATERIALS AND METHODS: CD34+ cord blood cells were expanded in the presence of various cytokine combinations in either a stroma-free cell culture system or a preformed porcine microvascular endothelial cell layer. After 7 to 21 days, stem cell number and function were monitored. In addition, the replicative history of stem cells was tracked using the fluorescent dye, PKH26. RESULTS: With the addition of various cytokine combinations, total cellular expansion was equivalent for both culture systems, although the endothelial cell-based system contained statistically greater numbers of CD34+ cells. By day 21, the endothelial-based system receiving the FLT3L, SCF, IL-6, and GM-CSF cytokine combination contained five-fold greater numbers of CD34+ than the stroma cell-free culture cell system. Endothelial-based cultures receiving these four cytokines plus megakaryocyte growth and development factor produced a 640-fold expansion of CD34+CD38- cells as compared to a four-fold expansion in the stroma-free system. The number of progenitor cells generated was similar with both systems, yet the greatest degree of expansion of cobblestone area-forming cells was observed in the endothelial based cultures (11-fold vs four-fold). Virtually all CD34+ and CD34+CD38+ cells expanded in the presence of endothelial cells had undergone self replication by day 10, yet stromal cell-free cultures contained a significant number (4.8%) of quiescent cells. Identical numbers of re-isolated cord blood CD34+ cells expanded in both systems exhibited a similar ability to engraft and generate cells belonging to multiple hematopoietic lineages in human fetal bones implanted in immunodeficient mice. CONCLUSIONS: These results suggest that the use of preformed endothelial cell monolayers might permit the ex vivo generation of sufficient numbers of cord stem cells to serve as successful grafts for adult transplant recipients.     

Engraftment potential into NOD/SCID mice of CD34+ cells derived from human fetal liver as compared to fetal bone marrow

BACKGROUND AND OBJECTIVES: We hypothesized that qualitative or quantitative differences in hematopoietic stem cells from fetal liver (FL) and fetal bone marrow (FBM) may be the cause of their organ specificity. DESIGN AND METHODS: To analyze possible differences in vivo, we compared the engraftment potential of equal numbers of CD34+ cells isolated from human FL or FBM into immunodeficient NOD/SCID mice. RESULTS: Mice showing engraftment following transplantation of CD34+ cells from FL demonstrated 14% (range 2-76%) CD45+ cells of human origin in the bone marrow compared to significantly lower levels of engraftment (4%, range 2-20%, p < 0.04) of FBM CD34+ cells. Likewise, the percentage of CD34+ CD38- cells in FBM was 4 times lower than the percentage in FL (1.4+/-0.9% and 5.6+/-0.7%, respectively). Similar organ distribution of engrafted human cells was found. Subset analysis of human cells in bone marrow of engrafted mice revealed identical distribution of the lymphoid, myeloid and erythroid lineages after transplantation of CD34+ cells from FL or FBM. INTERPRETATION AND CONCLUSIONS: The FL CD34+ cells showed a four-fold higher content of the CD34+ CD38- subset coinciding with a four-fold higher engraftment of CD34+ cells into NOD/SCID mice. Since the organ distribution and differentiation potential of the cells engrafted were similar, we concluded that CD34+ hematopoietic cells derived from FL and FBM have quantitatively different, but qualitatively the same potential for engraftment into NOD/SCID mice.     

Engraftment of human central memory-derived effector CD8+ T cells in immunodeficient mice

In clinical trials of adoptive T-cell therapy, the persistence of transferred cells correlates with therapeutic efficacy. However, properties of human T cells that enable their persistence in vivo are poorly understood, and model systems that enable investigation of the fate of human effector T cells (T(E)) have not been described. Here, we analyzed the engraftment of adoptively transferred human cytomegalovirus pp65-specific CD8(+) T(E) cells derived from purified CD45RO(+)CD62L(+) central memory (T(CM)) or CD45RO(+)CD62L(-) effector memory (T(EM)) precursors in an immunodeficient mouse model. The engraftment of T(CM)-derived effector cells (T(CM/E)) was dependent on human interleukin-15, and superior in magnitude and duration to T(EM)-derived effector cells (T(EM/E)). T-cell receptor Vβ analysis of persisting cells demonstrated that CD8(+) T(CM/E) engraftment was polyclonal, suggesting that the ability to engraft is a general feature of T(CM/E.) CD8(+) T(EM/E) proliferated extensively after transfer but underwent rapid apoptosis. In contrast, T(CM/E) were less prone to apoptosis and established a persistent reservoir of functional T cells in vivo characterized by higher CD28 expression. These studies predict that human CD8(+) effector T cells derived from T(CM) precursors may be preferred for adoptive therapy based on superior engraftment fitness.     

Simultaneous generation of CD34+ primitive hematopoietic cells and CD73+ mesenchymal stem cells from human embryonic stem cells cocultured with murine OP9 stromal cells

OBJECTIVE: Human embryonic stem cells (hESCs) have been shown to generate CD34(+) primitive hematopoietic cells after several days of coculturing with the OP9 murine stromal cell line. CD73(+) multipotent mesenchymal cells have also been isolated from hESC/OP9 cocultures after several weeks. We hypothesized that generation of CD34(+) hematopoietic cells and CD73(+) mesenchymal stem cells (MSCs) may follow similar kinetics, so we investigated the generation of CD73(+) cells in the first 2 weeks of hESC/OP9 cocultures, at a time when CD34(+) cells are generated. MATERIALS AND METHODS: We cocultured hESCs with OP9 cells and examined the time course of appearance of human CD34(+) and CD73(+) cells using flow cytometry. We tested the hematopoietic progenitor potentials of CD34(+) cells generated using hematopoietic colony-forming assays, and the multipotent mesenchymal properties of CD73(+) cells generated using in vitro differentiation assays. RESULTS: We observed that in the first 2 weeks of the hESC/OP9 coculture system CD34(+) hematopoietic and CD73(+) MSC generation follows a similar pattern. We sorted the CD34(+) cells and showed that they can generate hematopoietic progenitor colonies. Starting with cocultured cells on day 8, and through an enrichment procedure, we also could generate a pure population of MSCs. These hESC-derived MSCs had typical morphological and cell surface marker characteristics of adult bone marrow-derived MSCs, and could be differentiated toward osteogenic, adipogenic, and chondrogenic cells in vitro, a hallmark property of MSCs. CONCLUSIONS: OP9 cells when cocultured with hESCs support simultaneous generation of CD34(+) primitive hematopoietic cells and CD73(+) MSCs from hESCs.     

Identification of human T-lymphoid progenitor cells in CD34+ CD38low and CD34+ CD38+ subsets of human cord blood and bone marrow cells using NOD-SCID fetal thymus organ cultures

In contrast to myeloid and B-lymphoid differentiation, which take place in the marrow environment, development of T cells requires the presence of thymic stromal cells. We demonstrate in this study that human CD34+, CD34+ CD38+ and CD34+ CD38(low) cells from both cord blood and adult bone marrow reproducibly develop into CD4+ CD8+ T cells when introduced into NOD-SCID embryonic thymuses and further cultured in organotypic cultures. Such human/mouse FTOC fetal thymic organ culture) thus represents a reproducible and sensitive system to assess the T-cell potential of human primitive progenitor cells. The frequency of T-cell progenitors among cord-blood-derived CD34+ cells was estimated to be 1/500. Furthermore, the differentiation steps classically observed in human thymus were reproduced in NOD-SCID FTOC initiated with cord blood and human marrow CD34+ cells: immature human CD41(low) CD8- sCD3- TCR alphabeta- CD5+ CD1a+ T cells were mixed with CD4+ CD8+ cells and more mature CD4+ CD8- TCR alphabeta+ cells. However, in FTOC initiated with bone marrow T progenitors, <10% double-positive cells were observed, whereas this proportion increased to 50% when cord blood CD34+ cells were used, and most CD4+ cells were immature T cells. These differences may be explained by a lower frequency of T-cell progenitors in adult samples, but may also suggest differences in the thymic signals required by bone marrow versus cord blood T progenitors. Finally, since cytokine-stimulated CD34+ CD38(low) cells retained their ability to generate T cells, these FTOC assays will be of value to monitor, when combined with other biological assays, the influence of different expansion protocols on the potential of human stem cells.     

Granulomonocyte-associated lysosomal protein expression during in vitro expansion and differentiation of CD34+ hematopoietic progenitor cells

Using an in vitro expansion and differentiation system for human CD34+ cord blood (CB) progenitor cells, we analyzed the induction and expression kinetics of the granulomonocyte associated lysosomal proteins myeloperoxidase (MPO), lysozyme (LZ), lactoferrin (LF), and macrosialin (CD68). Freshly isolated CD34+ CB cells were negative for LZ and LF, and only small proportions expressed MPO (4% +/- 2%) or CD68 (3% +/- 1%). Culturing of CD34+ cells for 14 days with interleukin (IL)- 1, IL-3, IL-6, stem cell factor, granulocyte-macrophage colony- stimulating factor (GM-CSF), and G-CSF resulted in on average a 1,750- fold amplification of cell number, of which 83% +/- 7% were MPO+. Without addition of GM-CSF and G-CSF, lower increases in total cell numbers (mean, 211-fold) and lower proportions of MPO+ cells (54% +/- 11%) were observed. The proportion of MPO+ cells slightly exceeded but clearly correlated with the proportion of cells positive for the granulomonocyte associated surface molecules CD11b (Mac-1), CD15 (LeX), CD64 (Fc gamma RI) CD66, or CD89 (Fc alpha R). At day 14 MPO+ and LZ+ cells were virtually identical. However, at earlier time points during culture (days 4 and 7), single MPO+ or LZ+ cell populations were also observed, which only later acquired LZ and MPO, respectively. Maturation of cells into the neutrophilic pathway was indicated by the acquisition of MPO, followed by LZ. In contrast, maturation of cells into the monocytic pathway was indicated by the acquisition of LZ followed by MPO and CD14. CD68 was found to be expressed at day 4 by the majority of cells and was not restricted to the granulomonocytic cells, as cells with megakariocytic (CD41+) or erythroid (CD71hi) features were CD68+. LF expression was observed only in GM- plus G-CSF- supplemented cultures, in which only 26% +/- 5% of cells expressed LF by day 14.     

Differential regulation of the human and murine CD34 genes in hematopoietic stem cells

Human CD34 (hCD34)-positive cells are used currently as a source for hematopoietic transplantation in humans. However, in steady-state murine hematopoiesis, hematopoietic stem cells (HSCs) with long-term reconstitution activity are found almost exclusively in the murine CD34 (mCD34)-negative to low fraction. To evaluate the possible differences in hCD34 and mCD34 gene expression in hematopoiesis, we made transgenic mouse strains with human genomic P1 artificial chromosome clones spanning the entire hCD34 genomic locus. In all transgenic mouse strains, a vast majority of phenotypic and functional HSC populations including mCD34(-/lo) express the hCD34 transgene. These data strongly support the notion that hCD34(+) human bone marrow cells contain long-term HSCs that can maintain hematopoiesis throughout life.     

Reduced mobilization of CD34+ stem cells in advanced human immunodeficiency virus type 1 disease

Granulocyte colony-stimulating factor (r-met Hu G-CSF; filgrastim; 10 microgram/kg/day for 7 days) was used to mobilize CD34+stem cells into the peripheral blood of human immunodeficiency virus type 1 (HIV-1)-infected individuals and a group of HIV-1-uninfected donors as a measure of immunologic reserve in HIV-1-infected people. G-CSF mobilized CD34+ cells of HIV-1-infected individuals with cell counts >500 CD4+ cells/mm3, as well as in HIV-1-uninfected donors. In contrast, CD34 cell mobilization was significantly blunted in HIV-1-infected individuals with cell counts <500 CD4+ cells/mm3 (<200 cell days vs. >650 cell days, P<.0005, compared with the >500 CD4+ cell cohort). At least 1.75x10(7) CD34 cells were harvested by leukapheresis from patients in each study cohort. CD34+ cell viability and the ability to differentiate precursor cells into myeloid and erythroid progenitor cells were not affected by HIV-1 infection.     

Engraftment defect of cytokine-cultured adult human mobilized CD34(+) cells is related to reduced adhesion to bone marrow niche elements

In vitro exposure of haematopoietic stem and progenitor cells (HSPC) to cytokines in expansion or gene therapy protocols reduces homing and engraftment in vivo. We have previously reported that this is related in part to altered tissue specificity of short‐term homing, leading to loss of cells in non‐haematopoietic tissues. Here we demonstrate that defective engraftment persists when cultured HSPC are transplanted by intrabone injection. Changes in engraftment function occur within 24 h of cytokine exposure, and are evident when engraftment is analysed solely in the injected bone. A novel ex vivo model of the bone marrow was developed, in which the attachment of infused HSPC in rodent long bones is reduced following culture with cytokines. Finally, cultured HSPC demonstrated reduced adhesion to N‐cadherin, osteopontin and vascular cell‐adhesion molecule‐1, ligands present in bone marrow niches. These changes in adhesive function occur rapidly, and are not related to downregulation of the relevant receptors. Our findings suggest that cytokine exposure of adult human HSPC results in altered adhesion within bone marrow niches, further leading to reduced engraftment potential in vivo.     

Autologous CD34+ and CD133+ stem cells transplantation in patients with end stage liver disease

AIM:To assess the utility of an autologous CD34 + and CD133 + stem cells infusion as a possible therapeutic modality in patients with end-stage liver diseases.METHODS:One hundred and forty patients with endstage liver diseases were randomized into two groups.Group 1,comprising 90 patients,received granulocyte colony stimulating factor for five days followed by autologous CD34 + and CD133 + stem cell infusion in the portal vein.Group 2,comprising 50 patients,received regular liver treatment only and served a...     

Cocultivation of umbilical cord blood CD34^＋ cells with retro-transduced hMSCs leads to effective amplification of long-term culture-initiating cells

AIM:To establish a novel coculture system for ex vivoexpansion of umbilical cord blood(UCB)hematopoieticprogenitors using thrombopoietin(TPO)/Flt-3 ligand(FL)-transduced human marrow-derived mesenchymalstem cells(tfhMSCs)as feeder.METHODS:UCB CD34~ cells were isolated and culturedusing four culture systems in serum-containing or serum-free medium.Suitable aliquots of cultured cells wereused to monitor cell production,clonogenic activity,and long-term culture-initiating culture(LTC-IC)output.Finally,the severe-combined immunodeficient(SCID)mouse-repopulating cell(SRC)assay was performed toconfirm ability of the cultured cells to reconstitute long-term hematopoiesis.RESULTS:There were no significant differences in thenumber of total nucleated cells among different culturesystems in serum-containing medium during 21-dculture.However,on d 14,the outputs of CD34~ cells,CFU-C and CFU-GEMM in ffhMSCs coculture system weresignificantly enhanced.LTC-IC assay demonstrated thatthe tfhMSCs coculture system had the most powerfulactivity.The severe-combined immunodeficient(SCID)mouse repopulating cell(SRC)assay confirmed extensiveability of the expanded cells to reconstitute long-termhematopoiesis.Furthermore,PCR analysis demonstratedthe presence of human hematopoietic cells in the bonemarrow and peripheral blood cells of NOD/SCID mice. CONCLUSION:The TPO/FL-transduced hMSCs,incombination with additive cytokines,can effectivelyexpand hematopoietic progenitors from UCB in vitro andthe tfhMSCs coculture system may be a suitable systemfor ex vivo manipulation of primitive progenitor cellsunder contact culture conditions.