title: Rapid ex vivo expansion of human umbilical cord hematopoietic progenitors using a novel culture system.
abstract: 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 CD34bright/CD38dim 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 CD34bright/CD38dim cells was noted. Colony-forming units in culture (CFU-C) and mixed colonies (CFU-GEMM) were enhanced by 10- to 30-fold and 10- to 20-fold, respectively. Moreover, generation of long-term-culture-initiating cells (LTC-IC) from CD34bright/CD38dim 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.
authors.affiliation: Research Center for Genetic Engineering and Cell Transplantation, Department of Internal Medicine, Tokai University School of Medicine, Isehara, Kanagawa, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Kawada
authors.forename: K
authors.initials: K
authors.lastname: Ando
authors.forename: T
authors.initials: T
authors.lastname: Tsuji
authors.forename: Y
authors.initials: Y
authors.lastname: Shimakura
authors.forename: Y
authors.initials: Y
authors.lastname: Nakamura
authors.forename: J
authors.initials: J
authors.lastname: Chargui
authors.forename: M
authors.initials: M
authors.lastname: Hagihara
authors.forename: H
authors.initials: H
authors.lastname: Itagaki
authors.forename: T
authors.initials: T
authors.lastname: Shimizu
authors.forename: S
authors.initials: S
authors.lastname: Inokuchi
authors.forename: S
authors.initials: S
authors.lastname: Kato
authors.forename: T
authors.initials: T
authors.lastname: Hotta
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002455:Cell Division / Q000187:drug effects; D016895:Culture Media, Serum-Free; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000276:immunology / Q000648:ultrastructure; D006801:Humans; D016130:Immunophenotyping; D051379:Mice; D008855:Microscopy, Electron, Scanning; D011518:Proto-Oncogene Proteins / Q000494:pharmacology; D020794:Receptor Protein-Tyrosine Kinases / Q000494:pharmacology; D017154:Stromal Cells / Q000166:cytology; D013926:Thrombopoietin / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014470:Umbilical Cord / Q000166:cytology*; D051941:fms-Like Tyrosine Kinase 3
chemical_list: D016895:Culture Media, Serum-Free; D011518:Proto-Oncogene Proteins; D013926:Thrombopoietin; C497970:FLT3 protein, human; C497971:Flt3 protein, mouse; D020794:Receptor Protein-Tyrosine Kinases; D051941:fms-Like Tyrosine Kinase 3