Study On Sorting And Ex-Vivo Expansion Of CD133～+ Cells Derived From Umbilical Cord Blood

Objective: Human umbilical cord blood (UCB) is rich in primitive hematopoietic l progenitor and transplantable stem cells. It is an attractive alternative source of hematopoietic stem/progenitor cell (HSPC) to bone marrow or mobilized peripheral blood. However, there is a limitation of the number of HSPC in single UCB sample, which is limits the widespread use of UCB in clinical setting. Ex-vivo expansion of HSPC is expected to solve this limitation, but it is inevitable that the differentiation of HSPC is usually occurred during expansion , which will decrease the primitive HSPC compartment and engraftment of expanded products. Therefore, selection for appropriate target cells and regulation of differentiation of HSPC in ex-vivo expansion is. a major topic in expansion of HSPC. CD133+ cells may be more primitive progenitors than CD34+ cells, and have an advantage in ex-vivo expansion. TGF-βl , which is an important negative haematopoietic regulator, can maintain primitive human HSPC in quiescence and decrease over-differentiation of HSPC, thus increase the content of HSPC in expanded products. To investigate the optimal means of expansion of HSPC, study on ex-vivo expansion and regulation for differentiation of CD133+ cells from human umbilical cord blood were performed. Methods: Harvested CD133+ cells from umbilical cord blood by Magnetic Cell Sorting(MACS),and at regular intervals of ex-vivo expansion, the number of nuclear cells (NC), colonogenic assay, immunophenotyping, and other biological properties of the cells from CD 133+ cells expansion group were examined. Results: 1. The content of CD 13 3+ cells in fresh UCB were about 0.62%, with MACS can get (1.05±0.98)xl06 CD133+ cells from single UCB,and the purity was 75.7±0.2%. 2. At initial, the expansion folds of NC of Test B was low than that in Test A and control ,were 9.90 times, 13.21 times and 12.63 times respectively. 3. The expansion systems which were added TGF-βl showed had more CD133+ cells than control at each interval. 4. At each interval, the plate efficiencies and expansion folds of CFU-GM,BFU-E,CFU-Mix of TGF-βl group more than that of control.Conclusion: 1.Magnetic Cell Sorting(MACS) is k kind of efficient and practical sorting method ,after single sorting the purity of CD133+ cells was about 75.7%. 2. By NC counU colonogenic assay immunophenotype examination,attested that our systems had notable expansion effection on NC> CD133+HSPCandCFU-GM> BFU^ CFU-Mix colony forming ratio. 3.In our systems,IL-3 had not notable effect on proliferation of UCB CD133+ cells,removing IL-3 from culture systems maybe favorable to delay over-differentiation of HSPC. 4. The low concentration of TGF-βl can delay over-differentiation of HSPC,is a kind of indispensable haematopoietic regulators.