Gene profiling on mixed embryonic stem cell populations reveals a biphasic role for beta-catenin in osteogenic differentiation

The differentiation of embryonic stem cells (ESCs) into osteoblasts is enhanced to 60% when exposed to vitamin D3 (VD3) but leaves a remainder of one half of the cell population unidentified. To increase differentiation outcome, the known osteoinducers retinoic acid (RA) and bone morphogenetic protein-2 (BMP-2) were evaluated. Initial studies using RA and BMP-2 during early osteogenesis in addition to VD3 increased osteogenic yield in the case of RA, but surprisingly decreased osteogenesis when BMP-2 was administered together with VD3 or RA. This paper describes a comprehensive microarray study examining the gene expression profile of differentiating osteoblasts in these mixed ESC populations. In addition to five other families of signaling molecules (insulin growth factors, prostaglandin, follistatin, TGFbeta2, and Wnt molecules), we identified an endogenous expression pattern for BMPs and RA that differed from our previous exogenous administration of these molecules. By mimicking the change in expression of the RA and BMP-2 families with exogenous supplementation at the correct time, it was then possible to increase the number of ESC-derived osteoblasts to 90%. This effect was mediated through alteration in beta-catenin (CatnB) expression levels and nuclear CatnB activity, both of which are modulated by VD3, RA, and BMP-2. Our results suggest that blockage of CatnB activity by VD3 and RA is opposed by induction of CatnB activity through BMP-2 when administered together. Hence, osteoinduction, in vitro, is an intricate process involving both temporal and quantitative changes in gene expression and CatnB activity.