In vitro frequency analysis of spleen colony-forming and marrow-repopulating hemopoietic stem cells in the mouse

Summary An assay is described for Day-12 spleen colony-forming cells (CFU-S-12) and hemopoietic stem cells with marrow-repopulating ability (MRA) in the mouse using a miniaturized stroma-dependent bone marrow culture assay in vitro. Bone marrow cells are grown in liquid culture in microtiter wells, and the resulting adherent stromal layers are depleted of all hemopoietic activity by 20 Gy gamma irradiation. Subsequently, single cell suspensions containing stem cells are overlaid in a range of concentrations, and the presence of one or more emerging phase nonrefractive cell clones (cobblestone areas) in a single well scored as positive. The frequencies of cobblestone area-forming cells (CAFC) are then calculated by employing Poisson statistics. It is shown that the CAFC Day-10 and CAFC Day-28 frequencies closely correlate with those of CFU-S-12 and MRA cells, respectively.