Cell sorting enriches osteogenic populations in rat bone marrow stromal cell cultures

The presence of multiple cell types in bone marrow stromal populations complicates interpretation of cytokine and hormone effects on the osteoprogenitors present, indicating a need for a method for purification of the osteoprogenitor population. Flow cytometric sorting of 7 day primary rat bone marrow stromal cell cultures was performed on the basis of alkaline phosphatase (AP) expression with an antibody against AP (RBM 211.13). The resultant APhigh, APlow, or control cells were plated to determine osteoprogenitor, macrophage, and adipocyte distribution and frequency. Approximately 50% of osteoprogenitor/bone nodule-forming cells were lost during processing/sorting when compared with unsorted controls. Nevertheless, within the APhigh fraction, the numbers of AP-positive colonies and osteoprogenitors (bone nodules) were significantly enriched compared with the unfractionated control; the increase in osteoprogenitor frequency ranged from approximately 2 to 100-fold. There were few assayable osteoprogenitors in either the APhigh or APlow fractions in the absence of dexamethasone (dex), suggesting that RBM stroma contains largely dex-dependent osteoprogenitor populations, and that dex may regulate osteoprogenitors subsequent to the upregulation of AP. Osteoprogenitor/bone nodule numbers in either the APhigh or APlow fraction did not follow a linear relationship with decreasing plating density. The APhigh fraction of cells was depleted for adipocyte and macrophage colonies. In contrast, within the APlow fraction of cells, adipocyte and macrophage colonies were consistently enriched. We conclude that flow-cytometric sorting of RBM stromal populations according to high or low AP expression is an effective technique for enrichment of AP-positive colonies and osteoprogenitors/bone nodule-forming cells.