Evaluation of in vivo and in vitro repopulation assays for murine haemopoietic stem cells

Abstract

Three assays to determine the repopulation potential of stem cells in murine bone marrow grafts were evaluated on their reliability with regard to the ranges in graft size. In vivo, marrow repopulating ability, as calculated from the number of in vitro clonable progenitors (colony-forming units in culture, CFU-C) generated by the graft in the femur of an irradiated recipient, appeared to be independent of the input over a wide range of cell numbers grafted. A second assay, erythroid repopulating ability is a measure of the number of new reticulocytes or erythrocytes in the blood generated by the graft, and significantly underestimates stem cell activity of the graft. The third assay measures the long-term repopulating ability of bone marrow cells in vitro on pre-established stromal cell layers by determination of the number of CFU-C produced in these cultures. Calculations of short-term in vitro repopulating ability, done from measurements of the production of non-adherent CFU-C in the first week of culture, appeared to be independent of cell input. Long-term in vitro repopulating potential, measured by the CFU-C content of the adherent layer at 4 weeks, is also independent of the numbers of cell input.