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

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) ba sed on the cell surface expression of the stromal precursor cell marker STR O-1 and the osteoblastic marker alkaline phosphatase (ALP), Cells expressin g the STRO-1 antigen exclusively (STRO-1(+)/ALP(-)), were found to exhibit qualities preosteoblastic in nature both functionally by their reduced abil ity to form a mineralized bone matrix over time, as measured by calcium rel ease assay, and in the lack of their expression of various bone-related mar kers including bone sialoprotein, osteopontin, and parathyroid hormone rece ptor 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 osteoblast s, while the STRO-1(+)/ALP(-) subset represented an intermediate preosteobl astic 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 prod uct 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 c ultures. The data presented in this study demonstrate a hierarchy of bone c ell development in vitro and facilitate the study of bone cell differentiat ion and function.