The Studies On The Committed Differntiation Of Human Umbilical Cord Blood CD34～+ Cells Into T Lymphocytes In Vitro

Hematopoietic stem cells (HSCs) are the principal cell formation in humans and also it has been studied for longer times of all kinds of stem cells. The production of mature blood cells is a continual process that is the result of proliferation and differentiation of HSCs and committed progenitor cells, and differentiated cells.Hematopoietic cells have the potential for providing benefit in a variety of clinical settings. A number of malignancies can be successfully treated by high does therapy /irradiation and haematopoietic progenitor cell transplantation (HPCT).Umbilical cord blood has recently received attention as a rich, readily available source of stem cells and has been successfully exploited in partially mismatched transplantation. Umbilical cord blood has been demonstrated has more hematopoietic stem cells than peripherial blood and it has been used as an alternative source of bone marrow hematopoietic stem cells for transplanation in children.Umbilical cord blood is sufficient in our country.progenitor cells from umbilical cord blood,fetal, postnatal, and adult tissues have been shown to undergo differentiation along myeloid erythroid megakaryocyte B lymphoid lineages, NK cells, and dentritic cells In vitro. However, T lymphopoiesis have proved more difficult to study.CD34+ cells represent 0. 5 to 1% of bone marrow and umbilical cord blood mononuclear cells;they consist predominantly of lingeage-committed progenitors that coexpress CD38 and myeloid- or lymphoid-specific antigens. In the present study, we used thymic stromal monolayer system to demonstrate that lineage-depleted CD34+ cells from human umbilical cord blood mononuclear cells can differentiate into mature, functional T cells. The lymphoid-associated cell surface markers CD3,CD4, and CD8 were expressed in a temporal sequence that recapitulated in vivo thymopoiesis.Objective: To establish the technique for T lymphocytes inducted and differentiated by using human hematopoietic stem/progenitor cells ex vivo and to provide a technological platform for studing T cells biological characteristics and cellular immune.Methods: Human CD34- positive cells were isolated from umbilical cord blood by using a high-gradient magnetic activated cell sorting(MACS) system and CD34+ cells were seeded on human fetal thymic stromal monolayer system. Liquid culture system contains hematopoietic cytokines (FL+IL-12+IL-7+IL-2)and 20% human AB serium. Non-adhension cells were obtained after 7, 14, 28, 35, 42 days of culture respectively to analysis T cells phenotype by FACS using T cells specific Monoclonal Antibody. T cells morphology and the killing activity with LDH assay were also studied.Results: After 2 weeks of culture CD4+CD8+immature T lymphocytes were about 0. 3%~ 13. 3% and the peak value is 16. 6%~26. 5% at 4 - 5 weeks of culture. CD4XD8+ and CD4+CD8~ T. cells that coexpressed CD3 cells increased at 26. 5%~64. 9% and 11. 6% - 38. 9% after 6 weeks of culture. Wright staining shows that the mature T cells harvested after mitogenic stimulation have the presence of large cells withlymphoblast morphology. After 5 weeks of culture T cells had killing tumor cells ability.Conclusion: Human umbilical cord blood CD34+ cells could be differentiated into T lymphocytes by the culture conditions of human fetal thymic stromal monolayer system and the cytokine combination of FL+IL-12+IL-7+IL-2, and the T cells can be stimulated by IL-2+PHA. T cells maturing in the thymic monolayer system had killing tumor cells ability. In summary, therefore, this study emphasizes the potential of cord blood as an alternative source of multipotent hematopoietic progenitor cells with conserved T lymphoid capacity.