H. Kawada et al., Rapid ex vivo expansion of human umbilical cord hematopoietic progenitors using a novel culture system, EXP HEMATOL, 27(5), 1999, pp. 904-915
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, Rece
ntly, 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 cryopreserve
d CB-PPC in synergy with TPO/FL-2, Furthermore, while HESS-5 cells directly
adhered to human progenitors during culture, the cultured human cells coul
d 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 obtaine
d in serum-containing culture, When HESS-5 cells were physically separated
from human progenitor cells in the presence of TPO/FL-2, synergy was blocke
d, suggesting that HESS-5 cells support proliferation of PPC by direct cell
-to-cell interaction, The hematopoietic-supportive effects of this xenogene
ic coculture system were then assessed in a very short-term (5 days) serum-
free culture, Expansion was further enhanced by addition of stem cell facto
r (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 (C
FU-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-ini
tiating cells (LTC-IC) from CD34(bright)/CD38(dim) cells was amplified by 2
5-fold, The severe-combined immunodeficient (SCID) mouse-repopulating cell
(SRC) assay confirmed extensive ability of the expanded cells to reconstitu
te long-term hematopoiesis. These results indicate that this xenogeneic coc
ulture system, in combination with human cytokines, can rapidly generate PP
C from cryopreserved CB, (C) 1999 International Society for Experimental He
matology. Published by Elsevier Science Inc.