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

Exp Hematol. 1999 May;27(5):904-15. doi: 10.1016/s0301-472x(99)00012-0.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Culture Techniques / methods*
  • Cell Division / drug effects
  • Culture Media, Serum-Free
  • Hematopoietic Stem Cells / cytology*
  • Hematopoietic Stem Cells / immunology
  • Hematopoietic Stem Cells / ultrastructure
  • Humans
  • Immunophenotyping
  • Mice
  • Microscopy, Electron, Scanning
  • Proto-Oncogene Proteins / pharmacology
  • Receptor Protein-Tyrosine Kinases / pharmacology
  • Stromal Cells / cytology
  • Thrombopoietin / pharmacology
  • Tumor Cells, Cultured
  • Umbilical Cord / cytology*
  • fms-Like Tyrosine Kinase 3


  • Culture Media, Serum-Free
  • Proto-Oncogene Proteins
  • Thrombopoietin
  • FLT3 protein, human
  • Flt3 protein, mouse
  • Receptor Protein-Tyrosine Kinases
  • fms-Like Tyrosine Kinase 3