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Understanding the mechanisms that control the proliferation and commitment of human stem cells into cells of the osteogenic lineage for the preservation of skeletal structure is of basic importance in bone physiology. This study examines some aspects of the differentiation in vitro of human bone marrow fibroblastic cells cultured in the absence (basal media) or presence of 1nM dexamethasone and 50 micrograms/ml ascorbate for 6, 10, 14, and 21 days. Northern blot analysis and in situ hybridisation with digoxygenin-labelled riboprobes for Type I collagen, osteocalcin, bone morphogenetic proteins 2 (BMP-2), and 4 (BMP-4) and the estrogen receptor alpha (ERalpha), together with immunocytochemical analysis of ERalpha expression and histochemical staining of alkaline phosphatase was performed. In basal media, alkaline phosphatase activity and collagen expressions were detected at day 6, ERalpha from day 10 and osteocalcin from day 10. In the presence of dexamethasone and ascorbate, cell proliferation and alkaline phosphatase were markedly stimulated over 10 to 14 days with a dramatic increase in the temporal expression of Type I collagen, ERalpha, and osteocalcin mRNAs in these cultures. Northern blot analysis showed cells cultured in basal media, expressed the highest levels of the mRNA for each marker protein at day 14, whereas in the presence of ascorbate and dexamethasone, the highest levels for alkaline phosphatase, ERalpha, osteocalcin, BMP-2, and BMP-4 were observed at day 21. ERalpha, BMP-2, and BMP-4 expression were found to correlate temporally with induction of the osteoblast phenotype as determined by alkaline phosphatase, collagen, and osteocalcin expression. These results give additional information on the development of the osteoblast phenotype from early fibroblastic stem cells and on the biological factors involved in this process. These studies suggest a role for estrogen and BMP-2 and -4 in the differentiation of osteoprogenitor cells.

Original publication

DOI

10.1002/(sici)1097-4644(19991201)75:3<382::aid-jcb4>3.3.co;2-e

Type

Journal article

Journal

Journal of cellular biochemistry

Publication Date

12/1999

Volume

75

Pages

382 - 392

Addresses

MRC Bone Research Laboratory, Nuffield Department of Orthopaedic Surgery, University of Oxford, Oxford OX3 7LD, England.

Keywords

Hematopoietic Stem Cells, Cells, Cultured, Osteoblasts, Humans, Collagen, Alkaline Phosphatase, Transforming Growth Factor beta, Osteocalcin, Bone Morphogenetic Proteins, Receptors, Estrogen, Estrogen Receptor alpha, RNA, Messenger, In Situ Hybridization, Cell Differentiation, Gene Expression, Aged, Aged, 80 and over, Bone Morphogenetic Protein 2, Bone Morphogenetic Protein 4