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A murine stromal cell line allows the proliferation of very primitive human
CD34++/CD38- progenitor cells in long-term cultures and semisolid assays
C Issaad, L Croisille, A Katz, W Vainchenker and L Coulombel
INSERUM U362, Institut Gustave Roussy, Villejuif, France.
Analysis of molecular mechanisms associated with stem cell commitment and
differentiation requires an in vitro assay that identifies the most
primitive hematopoietic stem cells in human bone marrow. Such primitive
stem cells usually do not form colonies in short-term semisolid assays and
are best identified by their ability to initiate sustained hematopoiesis
when they are cocultured with competent stromal cells. In this study, we
investigated whether a murine marrow stromal cell line (MS-5) that supports
colony-forming unit-spleen (CFU-S) maintenance would permit, both in
short-term colony assays and long-term cultures, the development of
primitive human stem cells sorted on the basis of their high expression of
CD34 and lack of expression of CD38 antigen. In short-term colony assays,
this population included almost exclusively primitive progenitor cells.
MS-5 cells synergized with any combination of interleukin-3, Steel factor,
granulocyte colony- stimulating factor, agar-leukocyte conditioned medium,
and erythropoietin and increased at least twofold both the cloning
efficiency of CD34++/CD38- cells and the size of the colonies. Furthermore,
MS-5 cells triggered the development of multipotent blast cell progenitors
with a high proliferative potential, which in these conditions represented
1% to 2% of CD34++/CD38- cells. When MS-5 cells were substituted by human
stromal cells or when growth factor combinations were used in the absence
of stromal cells, much lower numbers of CFU-blast were detected. This
selective action of MS-5 on early progenitors was also observed when MS-5
cells were used as feeders in long-term cultures of CD34++/CD38- cells.
Murine cells promoted the expansion of high proliferative potential
primitive progenitor cells up to 3 months, although they did not support
their differentiation in mature clonogenic progenitors or terminally
differentiated cells. Sustained hematopoiesis in these longterm cultures
was accounted for by 2% to 5% of initial CD34++/CD38- cells as estimated by
limiting dilution experiments. Mechanisms by which murine stromal cells act
specifically on human primitive stem cells are unclear, but from our data
this effect is unlikely to be explained solely by known species
cross-reactive growth factors. Further manipulation of this long-term
coculture system should prove useful in identifying stromal molecules
regulating commitment and differentiation of early human progenitor cells.
Volume 81,
Issue 11,
pp. 2916-2924,
06/01/1993
Copyright © 1993 by The American Society of Hematology
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