OSTEOGENIC DIFFERENTIATION OF PURIFIED, CULTURE-EXPANDED HUMAN MESENCHYMAL STEM-CELLS IN-VITRO

Human bone marrow contains a population of cells capable of differenti ating along multiple mesenchymal cell lineages. Recently, techniques f or the purification and culture-expansion of these human marrow-derive d Mesenchymal Stem Cells (MSCs) have been developed. The goals of the current study were to establish a reproducible system for the in vitro osteogenic differentiation of human MSCs, and to characterize the eff ect of changes in the microenvironment upon the process. MSCs derived from 2nd or 3rd passage were cultured for 16 days in various base medi a containing 1 to 1000 nM dexamethasone (Dex), 0.01 to 4 mM L-ascorbic acid-2-phosphate (AsAP) or 0.25 mM ascorbic acid, and 1 to 10 mM beta -glycerophosphale (beta GP). Optimal osteogenic differentiation, as de termined by osteoblastic morphology, expression of alkaline phosphatas e (APase), reactivity with anti-osteopenic cell surface monoclonal ant ibodies, modulation of osteocalcin mRNA production, and the formation of a mineralized extracellular matrix containing hydroxyapatite was ac hieved with DMEM base medium plus 100 nM Dex, 0.05 mM AsAP, and 10 mM beta GP. The formation of a continuously interconnected network of APa se-positive cells and mineralized matrix supports the characterization of this progenitor population as homogeneous. White higher initial se eding densities did not affect cell number or APase activity, signific antly more mineral was deposited in these cultures, suggesting that ev ents which occur early in the differentiation process are linked to en d-stage phenotypic expression. Furthermore, cultures allowed to concen trate their soluble products in the media produced more mineralized ma trix, thereby implying a role for autocrine or paracrine factors synth esized by human MSCs undergoing osteoblastic lineage progression. This culture system is responsive to subtle manipulations including the ba sal nutrient medium, dose of physiologic supplements, cell seeding den sity, and volume tissue culture medium. Cultured human MSCs provide a useful model for evaluating the multiple factors responsible for the s tep-wise progression of cells from undifferentiated precursors to secr etory osteoblasts, and eventually terminally differentiated osteocytes . (C) 1997 Wiley-Liss, Inc.