ISOLATION AND CHARACTERIZATION OF OSTEOBLAST PRECURSOR CELLS FROM HUMAN BONE-MARROW

Osteoblasts are derived from precursor cells present in low frequency in the stromal element of bone marrow, Because of the lack of a practi cal procedure to isolate osteoblast precursors from early cultures of plastic adherent cells from bone marrow previous studies of marrow str omal cells have been made in confluent cultures of bone marrow when th e osteoblast (OB) precursors are already differentiated, Also these st udies utilized cultures containing mixed populations of cells includin g hematopoietic cells, Thus we have employed a negative immunoselectio n procedure to remove contaminating hematopoietic cells and to isolate nearly homogeneous populations of early human stromal cells derived f rom the plastic-adherent mononuclear marrow cells cultured in the pres ence of serum, By reverse transcriptase polymerase chain reaction (RT- PCR) analysis for mRNA, and by immunocytochemical study for protein, w e studied the sequential expression in culture of multiple markers of the osteoblast phenotype-alkaline phosphatase, osteopontin, parathyroi d hormone receptor, types I and III procollagen, and osteocalcin-as we ll as lipoprotein lipase (LPL), a marker of the adipocyte phenotype, A t an early stage of culture (7-9 days), human OB precursors formed col onies of variable sizes that expressed low levels of mRNA and protein concentrations of OB markers, and their concentration increased on gro wth to a confluent monolayer (approximately 14 days), LPL mRNA was exp ressed at high levels in the colony stage, and its level decreased upo n confluency, suggesting a loss of potential for commitment to the adi pocyte lineage, Interestingly, treatment with dexamethasone at 10(-8) M increased the expression for some of the osteoblast markers and for the LPL gene and was required for the deposition of mineralized matrix and for the formation of adipocytes containing cytoplasmic lipid drop lets in confluent cultures, Cloned single early colonies were able to coexpress the osteoblast and adipocyte markers (as assessed by RT-PCR) , Thus these immunoselected marrow stromal cells have the characterist ics of authentic human osteoblast precursor cells which also are capab le of differentiating into adipocytes.