Establishment of Assay System for Studying Osteoblastic Function and Differentiation Using Newly Isolated Osteoblast-Like Cell Lines

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In order to establish the suitable assay system for studying osteoblastic phenotypes and differentiation, we isolated various kinds of clonal osteoblast-like cell lines from neonatal rat calvariae and human osteosarcoma.I. Establishment of osteoblast-like cell lines1) Rat osteoblast-like cell lines isolated from rat calvariaeFive clonal osteoblast-like cell lines (ROB-C8a, Cll, C20. C23. C26) were isolated from 1-day old rat calvariae. Cll, C20 and C23 cell lines expressed high degree of osteoblastic properties but C26 cells expressed very low level of that properties. Expression degree of the osteoblastic properties in C8a was intermediate. More importantly, C26 cells expressed differentiation potential into not only osteoblasts but also myotubes and adipocytes. These indicated that C26 cells are useful cell line for studying the differentiation pathway of osteoblasts, muscle cells and adipocytes from undifferentiated mesenchymal cells.2) Human osteoblast-like cell lines isolated from … More human osteosarcomaTwo clonal osteoblast-like cell lines (YR-2 and YR-6) were isolated from human osteosarcoma. Osteoblastic expression of these cell lines were compared with those of other established osteosarcoma cell lines (HOS, SAOS-2 and Mg63). This study demonstrated the diversity of osteoblastic phenotypes among various human osteosarcoma cell lines tested, and suggested that much understanding of the osteoblastic characteristics in each cell line is important when human osteosarcoma cell lines are used for studying osteoblastic phenotypes.II. Regulatory mechanism of osteoblast differentiationWe demonstrated that bone morphogenetic protein (BMP) and retinoic acid play an important role in osteoblast differentiation.1) BMP : Recombinant human bone morphogenetic protein-2 (rhBMP-2 ; provided from Dr. Wozney, Genetics Institute, MA, USA) strongly stimulated osteoblastic differentiation and inhibited myogenic differentiation in the immature osteoblast-like cell line, C26. rhBMP-2 also induced non-osteogenic fibroblasts, C3H1OT1/2 cells, to differentiate into osteoblastic cells. These results strongly suggest that BMP-2 plays an important role in osteoblastic differentiation from undifferentiated mesenchymal cells.2) Retinoic acid : Retinoic acid stimulated ALP activity and PTH responsiveness, and inhibited myogenic and adipose differentiation in C26 cells. These results suggest that retinoic acid regulates differentiation pathway into osteoblasts, muscle cells and adipocytes from undifferentiated mesenchymal cells. Less