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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.
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 mesenchymal stem cells (tfhMSCs) as feeder. METHODS: UCB CD34 ~ + cells were isolated and cultured using four culture systems in serum-containing or serum-free medium. Xuitable aliquots of cultured cells wereused to monitor cell production, clonogenic activity, and long-term culture-initiating culture (LTC-IC) output. , 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 thenmber of total nucleated cells among different culturesystems in serum Now, on d 14, the outputs of CD34 ~ + cells, CFU-C and CFU-GEMM in ffh MSCs coculture system weresignificantly enhanced. LTC-IC assay that that tfh MSCs cocul ture system had the most powerfulactivity. severe-combined immunodeficient (SCID) mouse repopulating cell (SRC) assay confirmed extensive ability of the expanded cells to reconstitute long-term hematopoiesis. Further ed. PCR analysis demonstrated the presence of human hematopoietic cells in the bone marrow and peripheral blood cells of NOD / SCID mice. CONCLUSION: The TPO / FL-transduced hMSCs, incombination with additive cytokines, can effectively express and hematopoietic progenitors from UCB in vitro and the tfhMSCs coculture system may be a suitable system for ex vivo manipulation of primitive progenitor cells under contact culture conditions.