Simultaneous detection of multiple bone-related mRNAs and protein expression during osteoblast differentiation: polymerase chain reaction and immunocytochemical studies at the single cell level.

Messenger RNA expression analyzed by in situ hybridization and Northern analysis and protein expression analyzed biochemically or immunocytochemically have been used to study the developmental expression of various osteoblast (OB)-associated molecules. These approaches have shown that over a time course of OB differentiation in vivo and in vitro, the expression of macromolecules associated with OB cells changes. However, ambiguities in data from different approaches and in populations representative of cells at different developmental stages are extant. To begin to discriminate differentiation stages with more precision and to address intercellular heterogeneity, fetal rat calvaria cells were grown at low densities under conditions in which bone nodules form and mineralize and colonies were classified morphologically as fibroblastic or osteoblastic (early, intermediate, or mature). Whole discrete colonies and single cells from individual colonies were analyzed molecularly by a random amplification poly(A)-polymerase chain reaction (PCR) and for protein expression by immunocytochemistry; we analyzed the expression of known bone-related macromolecules (collagen type I, alkaline phosphatase, osteopontin, bone sialoprotein, and osteocalcin). Both PCR and immunocytochemistry revealed that different colony types were reproducibly distinguishable in their expression of either general (collagen type I) or bone-associated (alkaline phosphatase, osteopontin, bone sialoprotein, and osteocalcin) macromolecules, such that fibroblastic colonies were distinguishable from osteoblastic colonies and the latter could be subdivided into less mature or more mature osteoblastic colonies. While some aspects of the temporal differentiation sequence defined earlier were confirmed, several additional features were evident from these single cell-single colony studies. First, different repertoires of OB-associated markers were expressed in different cells, suggesting variation in the switch-on of the OB differentiation program and heterogeneity in the OB phenotype. Second, among colonies classified as fibroblastic on the basis of morphology heterogeneity was also evident and there were some cells expressing features consistent with their being osteoprogenitor cells. Our data support the hypothesis that individual fibroblastic and osteoblastic cells are heterogeneous in expression of marker molecules. We also conclude that individual cells and colonies analyzed by poly(A)-PCR will be useful in lieu of mass populations to extend investigation of stages in the progression of OB differentiation.