Objective: As a first step in refining current in vivo and in vitro models of osteoblastic differentiation using human mesenchymal stem cells (hMSCs), the aim of project was to define transcriptional changes that occur in during dexamethasone-induced in vitro differentiation. Methods: Bone marrow derived hMSCs from three individual donors (Osiris Therapeutics; cryopreserved) were rapidly thawed, plated and grown in DMEM containing 10% fetal bovine serum and antibiotics (basal media, BM). At confluence (Day 0) cells were grown in either BM or BM plus ascorbic acid, b glycerophosphate and dexamethasone (OS). At 0, 3, 7 and 14 days, total RNA was isolated from BM and OS cultured cell layers using TriSol and 32P-labelled probes were synthesized using Superscript II. Atlas 1.2 gene arrays (Clontech) were hybridized overnight at 65oC using donor-specific cDNA probes, washed at high stringency and exposed using phosphoimaging screens. Acquired images were analyzed using Genespring software enabling normalization and averaging of donor-specific data sets(n=3). Genes displaying average increased or decreased expression of >3-fold were tabulated. mRNA from ectopically formed bone at 3 – 21 days following hMSC implantation in SCID mice was isolated and used to program RT-PCR assays to confirm in vitro gene expression events. Results: Both IGF-II and Leptin receptor encoding mRNA levels were consistently elevated. RORa, a transcription factor acting at the BSP gene was also induced after 7 days. RNAs from HA/TCP adherent hMSCs ectopically engrafted in SCID mouse were isolated and RT-PCR analysis confirmed induction of IGF-II, Leptin Receptor and RORa gene transcription during hMSC osteogenesis in vivo. Conclusion: The in vivo and in vitro hMSC differentiation should be compared using larger, more comprehensive gene arrays.

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