Positive and negative immunoselection for enrichment of two classes of osteoprogenitor cells

The number of identifiable stages and expression of differentiation markers in cells of the osteoblast lineage are not well understood. In the present study, a mAb, designated rat bone marrow (RBM) 211.13, was prepared that stained selectively the osteogenic and preosteoblastic cells along the surfaces of bone in calvariae, femurs, and metatarsals. The staining was cell surface associated and coincided with that for alkaline phosphatase (APase) detected histochemically. Only cells positive for APase activity by biochemical assay and not those without APase activity (e.g., fetal rat skin) stained with RBM 211.13. By immunoblotting, RBM 211.13 recognized a band coinciding with APase activity on nonreducing/nondenaturing gels, and RBM 211.13 precipitated a protein which on reduced gels migrated with an apparent molecular mass of approximately 80 kD. RBM 211.13 labeling was abolished by phosphatidylinosital-specific phospholipase C, known to release APase from the cell surface. All of these data support the concept that RBM 211.13 recognizes the bone isoenzyme of APase. RBM 211.13 was used to sort by flow cytometry the APase-positive and APase-negative cells from mixed fetal rat calvaria (RC) cell populations. The osteoprogenitors we identified earlier that form bone nodules in vitro (Bellows, C. G., J. E. Aubin, J. N. M. Heersche, and M. E. Antosz. 1986. Calcif. Tissue Int. 36:143-154; Bellows, C. J., J. N. M. Heersche, and J. E. Aubin. 1990. Dev. Biol. 140:132-138) were found within the APase-positive pool. By immunopanning, RC cells were separated into APase-enriched (APase-positive, adherent) and APase-depleted (APase-negative, nonadherent) populations. The APase-positive fraction was enriched two-to-threefold for bone-forming osteoprogenitors compared to unfractionated cells, while the APase-negative population formed very few nodules under the same conditions. Both populations responded to the glucocorticoid dexamethasone (DEX) with an increase in bone nodule formation. However, the fold stimulation in bone formation in the APase-negative population was approximately 30-fold, while the fold stimulation in the APase-positive population was only approximately 5-fold. These data suggest that APase expression can be used for immunoselection to fractionate osteoblastic populations into an APase-positive population and a population initially APase-negative, that virtually all osteoprogenitors forming bone in vitro in the absence of added glucocorticoids reside in the APase-positive pool, and that the only osteoprogenitors present in the APase-negative pool are those requiring DEX to differentiate.