Differentiation of human embryonic stem cells into osteogenic or hematopoietic lineages: a dose-dependent effect of osterix over-expression.

Enhanced differentiation of human embryonic stem cells (HESCs), induced by genetic modification could potentially generate a vast number of diverse cell types. Such genetic modifications have frequently been achieved by over-expression of individual regulatory proteins. However, careful evaluation of the expression levels is critical, since this might have important implications for the differentiation potential of HESCs. To date, attempts to promote osteogenesis by means of gene transfer into HESCs using the early bone "master" transcription factor osterix (Osx) have not been reported. In this study, we attained HESC subpopulations expressing two significantly different levels of Osx, following lentiviral gene transfer. Both subpopulations exhibited spontaneous differentiation and reduced expression of markers characteristic of the pluripotent phenotype, such as SSEA3, Tra1-60, and Nanog, In order to promote bone differentiation, the cells were treated with ascorbic acid, beta-glycerophosphate and dexamethasone. The high level of Osx, compared to endogenous levels found in primary human osteoblasts, did not enhance osteogenic differentiation, and did not up-regulate collagen I expression. We show that the high Osx levels instead induced the commitment towards the hematopoietic-endothelial lineage-by up-regulating the expression of CD34 and Gata1. However, low levels of Osx up-regulated collagen I, bone sialoprotein and osteocalcin. Conversely, forced high level expression of the homeobox transcription factor HoxB4, a known regulator for early hematopoiesis, promoted osteogenesis in HESCs, while low levels of HoxB4 lead to hematopoietic gene expression.