Isolation, Culture And Molecular Identification Of Human Cord Blood Hematopoietic Stem/Progenitor Cells

In addition to the adult bone marrow (BM) and the peripheral blood, cord blood is another potential source of hematopoietic stem/progenitor cells (HSPCs). It has been considered as prospective stem cell in tissue engineering, cell transplantation, gene therapy and clinic use. Researchers have studied a lot about HSPCs in the cell level and molecular level. The study in the cell level focuses on how to expand HSPCs and maintain the active self-renewing and multi-differentiate capacity of HSPCs. Now it has some results. The study in the molecular level mainly focuses on the gene therapy that has successfully been applied in animal models. But now we still know very little about HSPCs. By now we still don't know the mechanism of its active self-renewing and multi-differentiate capacity. We can also not identify the genes that are related with the self-renewing and multi-differentiate capacity and other capacities. In the cell level, different labs use different ways to expand HSPCs, so we can't make sure which way is the most effective way.In our study, the mononuclear cells (MNCS) and CD34 cells were isolated from the cord blood by Ficoll density centrifugation, and were cultured and expanded in vitro in IMDM containing FBS. Averagely, 5.84 0.98 10 MNCs and 4.67 0.79 106 CD34 cells were obtained from 80 15ml cord blood. The CD34 cells const itute 0.8 0.1%of the total MNCs. The culture of two types of cells in vitro with or without cell growth factors both showed the Loigistic curve. In our study, the IMDM medium is the most proper medium for expansion of HSPCs in vitro compared to 1640 medium. The cocktail of several cell growth factors: SCF (50ng/ml) IL-3 (10ng/ml) FL (50ng/ml) TPO (20ng/ml) GM-CSF (10ng/ml) was used to expand CD34 cells in vitro. A week later, the cells expanded about 9.84fold; two weeks later, the cells expanded about 15. 35 fold. DD-RT PCR was used to identify the gene differential display between the CD34 cells and the CD34 cells, and between the non-expanded CD34 cells and the expanded CD34 cells. One different fragment was showed between the uncultured CD34 cells and the CD34 cells and analyzed the sequence of the fragment, the result is that the fragment is one part of human CD34 gene. No any difference of gene differential display between the non-expanded CD34 cells and the expanded CD34 cells. This result indicated that the way of using DD-RT PCR to identify HSPCs in molecular is feasible.