Ex Vivo Expansion of Human Cord Blood Stem Cells and Genetic Manipulation

Abstract

To introduce extrinsic genes into hematopoietic stem cells (HSC) by using retrovirus vectors, an efficient system for expanding HSC is required. We have established a novel culture system in which the murine stromal cell line HESS-5 dramatically supports the rapid expansion of cryopreserved cord blood primitive progenitor cells (CB-PPC) in synergy with TPO/FL. Within 5 days of serum-free culture in this system, a 50- to 100-fold increase in CD34+/CD38− cells was obtained; colony-forming units in culture (CFU-C) and mixed colonies (CFU-GEMM) were amplified by 10- to 30 fold and 10- to 20 fold, respectively. To further assess the long-term repopulating ability of those expanded cells, we performed a long-term culture-initiating cells (LTC-IC) assay and SCID-repopulating cells (SRC) assay using CD34+ cells cultured in this system. Within 5 days of culture, 5.1-fold amplification of the LTC-IC was obtained. SRC and their multilineage differentiation were detected in NOD/SCID mice 7 weeks after injection of these cultured cells. This system is further applicable to retrovirus mediated gene transfer to CB-PPC. The transduction efficiency of CD34+ cells was more than 40% when they were infected on HESS-5 monolayer cells. The engraftment of transduced cells in NOD/SCID mice 10 weeks after transplantation were confirmed by the presence of the gene introduced. These results indicate that this xenogeneic coculture system, in combination with human cytokines, can rapidly expand CB stem cells and is applicable to the efficient retrovirus-mediated gene transfer to them.