Osteoblastic differentiation and mRNA analysis of STRO-1-positive human bone marrow stromal cells using primary in vitro culture and poly (A) PCR

Investigation of osteoblast dysfunction in osteoporosis has been hampered b y a poor understanding of normal early osteoblast differentiation, due to a relative lack of markers for the earliest cells in the lineage. Attempts t o identify such markers have used cultures of animal or immortalized human cells, of uncertain relevance to human biology, or heterogeneous cultures i n which genetic variability precludes the isolation of stage-specific genot ypic markers. Primary in vitro generation of clonal populations of human bo ne marrow stromal cells was used in order to overcome these problems. Fibro blast-like stromal cells were isolated from human sternal bone marrow, They showed differentiation to an osteoblastic phenotype when stimulated with d examethasone (10(-7) M) and fluorescence activated cell analysis demonstrat ed immunopositivity for STRO-1 (an antibody that recognizes osteoprogenitor stem cells of the colony-forming unit-fibroblastic) in from 8 to 40 per ce nt of the cells, dependent on time post-harvest. Cells positive for STRO-1 were immunoselected using magnetic activated cell sorting and seeded at low density (10 cells/cm(2)) to product clones. Each clone was subpassaged, os teoblastic differentiation stimulated with dexamethasone. and mRNA-extracte d at time points post-stimulation (0 h and 1-14 days). A novel poly (A) rev erse transcriptase-polymerase chain reaction (RT-PCR) was used to amplify c DNA representative of all transcripts expressed at each time point. Differe ntial gene expression within the amplified cDNA was assessed using 3' end c DNA probes to osteocalcin, osteopontin, and collagen type I (positive), dem onstrating the acquisition of an osteoblastic phenotype. Time-specific gene products for early osteoblast differentiation have been generated from pri mary human cultures, utilizing very low density seeding and poly (A) RT-PCR , These products overcome the problems associated with animal, immortalized or heterogeneous culture and can be used to study normal and altered early osteoblast differentiation, indicating the possibility of using the same s ystem to study other disease states. Copyright (C) 1999 John Wiley & Sons, Ltd.