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

Abstract

We have developed an in vitro clonal assay of murine ematopoietic precursor cells that form spleen colonies FU-S day 12) or produce in vitro clonable progenitors in the arrow (MRA cells) of lethally irradiated mice. The assay is sentially 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 facilitating both the quantitative assessment of a series of subsets in the hematopoietic stem cell hierachy and the study of single long-term repopulating cells in vitro. © 1991 by The American Society of Hematology.