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Use of limiting-dilution type long-term marrow cultures in frequency
analysis of marrow-repopulating and spleen colony-forming hematopoietic
stem cells in the mouse
RE Ploemacher, JP van der Sluijs, CA van Beurden, MR Baert and PL Chan
Department of Cell Biology II, Erasmus University, Rotterdam, The
Netherlands.
We have developed an in vitro clonal assay of murine hematopoietic
precursor cells that form spleen colonies (CFU-S day 12) or produce in
vitro clonable progenitors in the marrow (MRA cells) of lethally irradiated
mice. The assay is essentially a long-term bone marrow culture in
microtiter wells containing marrow-derived stromal "feeders" depleted for
hematopoietic activity by irradiation. To test the validity of the assay as
a quantitative in vitro stem cell assay, a series of unsorted and
physically sorted bone marrow cells were simultaneously assayed in vivo and
overlaid on the feeders in a range of concentrations, while frequencies of
cells forming hematopoietic clones (cobblestone area forming cells, CAFC)
were calculated by means of Poisson statistics. Linear regression analysis
of the data showed high correlations between the frequency of CFU-S day 12
and CAFC day 10, and between MRA cells and CAFC day 28. A majority of MRA
activity and CAFC day 28 was separable from CFU-S day 12 and CAFC day 10.
This correlation study validates the CAFC system as a clonal assay
facilitation both the quantitative assessment of a series of subsets in the
hematopoietic stem cell hierarchy and the study of single long-term
repopulating cells in vitro.
Volume 78,
Issue 10,
pp. 2527-2533,
11/15/1991
Copyright © 1991 by The American Society of Hematology
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