Rapid ex vivo expansion of human umbilical cord hematopoietic progenitors using a novel culture system

Cell numbers limit the widespread clinical use of cord blood (CB) for gene therapy and marrow replacement in adults; a simple and effective method for ex vivo expansion of CB primitive progenitor cells (PPC) is required, Recently, the combination of thrombopoietin (TPO) and Flk-2/Flt-3 ligand (FL-2) was reported to support slow proliferation of CB-PPC in stroma-free liquid culture. We established a novel culture system in which the murine stromal cell line HESS-5 dramatically supports the rapid expansion of cryopreserved CB-PPC in synergy with TPO/FL-2, Furthermore, while HESS-5 cells directly adhered to human progenitors during culture, the cultured human cells could easily be harvested without contamination by HESS-5 cells. Within 7 days of culture, a 100-fold increase in CD34(bright)/CD38(dim) cells was obtained in serum-containing culture, When HESS-5 cells were physically separated from human progenitor cells in the presence of TPO/FL-2, synergy was blocked, suggesting that HESS-5 cells support proliferation of PPC by direct cell-to-cell interaction, The hematopoietic-supportive effects of this xenogeneic coculture system were then assessed in a very short-term (5 days) serum-free culture, Expansion was further enhanced by addition of stem cell factor (SCF) or interleukin-3 (IL-3), As a result, a 50- to 100-fold increase in CD34(bright)/CD38(dim) cells was noted, Colony-forming units in culture (CFU-C) and mixed colonies (CFU-GER IRI) were enhanced by 10- to 30-fold and 10- to 20-fold, respectively, Moreover, generation of long-term-culture-initiating cells (LTC-IC) from CD34(bright)/CD38(dim) cells was amplified by 25-fold, The severe-combined immunodeficient (SCID) mouse-repopulating cell (SRC) assay confirmed extensive ability of the expanded cells to reconstitute long-term hematopoiesis. These results indicate that this xenogeneic coculture system, in combination with human cytokines, can rapidly generate PPC from cryopreserved CB, (C) 1999 International Society for Experimental Hematology. Published by Elsevier Science Inc.