Osteoprogenitor cell frequency in rat bone marrow stromal populations: Role for heterotypic cell-cell interactions in osteoblast differentiation

Glucocorticoids, notably dexamethasone (Dex), have been reported to be a re quirement for osteoprogenitor cell differentiation in young adult rat bone marrow stromal cell populations. We have reinvestigated the requirement for Dex and analyzed the frequency of osteoprogenitor cells present. Stromal c ells were grown as primary or first subcultures in the presence or absence of Dex and their expression of osteogenic markers (alkaline phosphatase act ivity hormone responsiveness, and matrix molecules, including type I collag en, osteopontin, bone sialoprotein, and osteocalcin), as well as their func tional capacity to differentiate to form a mineralized bone nodule, were as sessed. Dex increased, but was not an absolute requirement for, the express ion of osteogenic markers. Bone nodule formation was plating cell density d ependent and occurred under all combinations of treatment With or without D ex but was maximal when Dex was present in both the primary and secondary c ultures. Dex increased CFU-F by similar to 2-fold, but increased CFU-O (ost eoprogenitor cells; bone nodule forming cells) by 5- to 50-fold depending o n the cell density and duration of treatment. Neither CFU-F nor CFU-O expre ssion followed a linear relationship in limiting dilution analysis until ve ry high cell densities were reached, suggesting cooperativity of cell types within the population and a multitarget phenomenon leading to osteoprogeni tor differentiation. When a large number of nonadherent bone marrow cells o r their conditioned medium was added to the stromal cells, osteoprogenitors comprised approximately 1/100 of plated adherent cells and their expressio n followed a linear, single-hit relationship. By contrast, rat skin fibrobl asts or their conditioned medium totally inhibited bone nodule formation. T hese data support the hypothesis that in marrow stroma, as in other bone ce ll populations such as those from calvaria, there are at least two classes of osteoprogenitor cells: those differentiating in the absence of added glu cocorticoid and those requiring glucocorticoid to differentiate, that more than one cell type is limiting for stromal osteoprogenitor differentiation suggesting a role for heterotypic cell-cell interactions in osteogenesis in this tissue, and that Dex may be acting directly and/or indirectly through accessory cells in the bone marrow to alter osteoprogenitor cell expressio n. J. Cell. Biochem. 72:396-410, 1999. (C) 1999 Wiley-Liss, Inc.