Differential cell surface expression of the STRO-1 and alkaline phosphatase antigens on discrete developmental stages in primary cultures of human bone cells

Human osteoblast-like cells can be readily cultured from explants of trabecular bone, reproducibly expressing the characteristics of cells belonging to the osteoblastic lineage. Dual-color fluorescence-activated cell sorting was employed to develop a model of bone cell development in primary cultures of normal human bone cells (NHBCs) based on the cell surface expression of the stromal precursor cell marker STRO-1 and the osteoblastic marker alkaline phosphatase (ALP). Cells expressing the STRO-1 antigen exclusively (STRO-I⁺/ALP-), were found to exhibit qualities preosteoblastic in nature both functionally by their reduced ability to form a mineralized bone matrix over time, as measured by calcium release assay, and in the lack of their expression of various bone-related markers including bone sialoprotein, osteopontin, and parathyroid hormone receptor based on reverse trancriptase polymerase chain reaction (PCR) analysis. The majority of the NHBCs which expressed the STRO-1^-/ALP⁺ and STRO-1^-/ALP^- phenotypes appeared to represent fully differentiated osteoblasts, while the STRO- 1⁺/ALP⁺ subset represented an intermediate preosteoblastic stage of development. All STRO-1/ALP NHBC subsets were also found to express the DNA- binding transcription factor CBFA-1, confirming that these cultures represent committed osteogenic cells. In addition, our primer sets yielded four distinct alternative splice variants of the expected PCR product for CBFA-1 in each of the STRO-1/ALP subsets, with the exception of the proposed preosteoblastic STRO-1⁺/ALP^- subpopulation. Furthermore, upon re-culture of the four different STRO-1/ALP subsets only the STRO-1⁺/ALP^- subpopulation was able to give rise to all of the four subsets yielding the same proportions of STRO-1/ALP expression as in the original primary cultures. The data presented in this study demonstrate a hierarchy of bone cell development in vitro and facilitate the study of bone cell differentiation and function.