CD34high+CD38low/- cells generated in a xenogenic coculture system are capable of both long-term hematopoiesis and multiple differentiation

CD34+ cells isolated from human umbilical cord blood (HUCB) are thought to have potential in clinical applications such as transplantation and gene therapy. Recently, we developed a xenogenic coculture system involving HUCB-CD34+ cells and murine bone marrow stromal cells, HESS-5 cells, in combination with human interleukin-3 and stem cell factor. Under these xenogenic coculture conditions, the numbers of CD34high+ cells and primitive progenitor cells, such as CD34high+CD38low/− cells and high proliferative potential colony-forming cells (HPP-CFCs), increased dramatically by a factor of 102.1, 66.5 and 104.9, respectively. In the present study, we used a secondary culture of B progenitor cells and long-term culture (LTC)-initiating cells to characterize and compare the progenitor capability of re-isolated CD34high+CD38low/− cells, which have been identified as one of the most primitive progenitor cells, with that of freshly isolated CD34high+CD38low/− cells. Compared with freshly isolated CD34high+CD38low/− cells, the re-isolated CD34high+CD38low/− cells were equally as capable of proliferating and differentiating into myeloid and B progenitor cells. No significant differences were observed in the frequency of LTC-initiating cells in the re-isolated CD34high+CD38low/− cells compared with that in freshly isolated CD34high+CD38low/− cells. Furthermore, the re-isolated CD34high+CD38low/− cells were capable of long-term reconstitution and multiple differentiation in non-obese diabetic mice with severe combined immunodeficiency disease (NOD/SCID mice). The results demonstrate that this xenogenic coculture system can be used for successful in vitro expansion of HUCB-progenitor cells that possess the capability for both long-term hematopoiesis as well as multipotent differentiation into myeloid and lymphoid cells both in vivo and in vitro.