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 can be assayed by limiting dilution analysis of "cobblestone area" (CA) formation on stromal layers in microlong-term bone marrow cultures. Cobblestone area forming cell (CAFC) frequency determined at early time points (day 7) correlates with mature stem cells measured as day 8 CFU-S, whereas CAFC frequency determined at day 28 correlates more closely with long-term marrow repopulating ability. Here we report a modification of the CAFC assay in which a clonal bone marrow stromal cell line, GB1/6, is substituted for fresh marrow-derived stromal layers. This modification simplifies the initial culture setup, eliminates inhomogeneities 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 completely replace the heterogeneous microenvironment of fresh stroma for in vitro stem cell support.