QUANTITATION OF MURINE HEMATOPOIETIC STEM-CELLS IN-VITRO BY LIMITING DILUTION ANALYSIS OF COBBLESTONE AREA FORMATION ON A CLONAL STROMAL CELL-LINE

Murine hematopoietic stem cells with varying proliferative capacity ca n be assayed by limiting dilution analysis of ''cobblestone area'' (CA ) formation on stromal layers in micro-long-term bone marrow cultures. Cobblestone area forming cell (CAFC) frequency determined at early ti me points (day 7) correlates with mature stem cells measured as day 8 CFU-S, whereas CAFC frequency determined at day 28 correlates more clo sely with long-term marrow repopulating ability. Here we report a modi fication of the CAFC assay in which a clonal bone marrow stromal cell Line, GBl/6, is substituted for fresh marrow-derived stromal layers. T his modification simplifies the initial culture setup, eliminates inho mogeneities in the stromal layer and reduces the need for mice. Normal bone marrow CAFC frequencies were the same for both types of stromal cell underlayer, demonstrating the ability of a clonal cell line to co mpletely replace the heterogeneous microenvironment of fresh stroma fo r in vitro stem cell support.