Differentiation of osteoblasts and in vitro bone formation from murine embryonic stem cells

Pluripotent embryonic stem (ES) cells have the potential to differentiate t o all fetal and adult cell types and might represent a useful cell source f or tissue engineering and repair. Here we show that differentiation of ES c ells toward the osteoblast lineage can be enhanced by supplementing serum-c ontaining media with ascorbic acid, beta -glycerophosphate, and/or dexameth asone/retinoic acid or by co-culture with fetal murine osteoblasts. ES cell differentiation into osteoblasts was characterized by the formation of dis crete mineralized bone nodules that consisted of 50-100 cells within an ext racellular matrix of collagen-1 and osteocalcin. Dexamethasone in combinati on with ascorbic acid and beta -glycerophosphate induced the greatest numbe r of bone nodules and was dependent on time of stimulation with a seven-fol d increase when added to ES cultures after, but not before, 14 days. Co-cul ture with fetal osteoblasts also provided a potent stimulus for osteogenic differentiation inducing a five-fold increase in nodule number relative to ES cells cultured alone. These data demonstrate the application of a quanti tative assay for the derivation of osteoblast lineage progenitors from plur ipotent ES cells. This could be applied to obtain purified osteoblasts to a nalyze mechanisms of osteogenesis and for use of ES cells in skeletal tissu e repair.