Human embryonic stem cells (hESCs) may offer an unlimited supply of cells that can be directed to differentiate into all cell types within the body and used in regenerative medicine for tissue and cell replacement therapies. Previous work has shown that exposing hESCs to exogenous factors such as dexamethasone, ascorbic acid and β-glycerophosphate can induce osteogenesis. The specific factors that induce osteogenic differentiation of hESCs have not been identified yet, however, it is possible that differentiated human bone marrow stromal cells (hMBSCs) may secrete factors within the local microenvironment that promote osteogenesis. Objective: To determine the lineage progression of hESCs to osteoblasts in the presence of soluble signaling factors derived from differentiated hBMSCs. Methods: For 28 days, hESCs were grown in transwell co-culture with hBMSCs that had been previously differentiated in growth medium containing defined osteogenic supplements for 7-24 days. As a control, hESCs were co-cultured with undifferentiated hBMSCs and alone. Results: Von Kossa and Alizarin Red staining as well as inmmunohistochemistry confirmed that the hESCs co-cultured with differentiated hBMSCs formed mineralized bone nodules and secreted extracellular matrix protein osteocalcin. Quantitative Alizarin Red assays showed increased mineralization as compared to the controls. RT-PCR revealed the loss of pluripotent hESC markers with the concomitant gain of osteoblastic markers such as type I collagen, Runx2, and osterix. Conclusion: We demonstrate that osteogenic growth factors derived from differentiated hBMSCs within the local microenvironment may help to promote hESC osteogenic differentiation. 3 RO1 DE016530-01S1

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