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Bone matrix production and mineralization involves sophisticated mechanisms, including the initial formation of an organic extracellular matrix into which inorganic hydroxyapatite crystals are later deposited. Human embryonic stem (hES) cells offer a potential to study early developmental processes and provide an unlimited source of cells. In this study, four different hES cell lines were used, and two different approaches to differentiate hES cells into the osteogenic lineage were taken. Undifferentiated cells were cultured either in suspension, facilitating the formation of embryoid bodies (EBs), or in monolayer, and both methods were in the presence of osteogenic supplements. Novel to our osteogenic differentiation study was the use of commercially available human foreskin fibroblasts to support the undifferentiated growth of the hES cell colonies, and their propagation in serum replacement-containing medium. Characterization of the osteogenic phenotype revealed that all hES cell lines differentiated toward the mesenchymal lineage, because T-Brachyury, Flt-1, and bone morphogenetic protein-4 could be detected. Main osteoblastic marker genes Runx2, osterix, bone sialoprotein, and osteocalcin were up-regulated. Alizarin Red S staining demonstrated the formation of bone-like nodules, and bone sialoprotein and osteocalcin were localized to these foci by immunohistochemistry. Cells differentiated in monolayer conditions exhibited greater osteogenic potential compared to those from EB-derived cells. We conclude that in vitro hES cells can produce a mineralized matrix possessing all the major bone markers, the differentiation of pluripotent hES cells to an osteogenic lineage does not require initiation via EB formation, and that lineage potential is not dependent on the mode of differentiation induction but on a cell line itself.

No abstract

The variation of HoxB4 expression levels might be a key regulatory mechanism in the differentiation of human embryonic stem cell (hESC)-derived hematopoietic stem cells (HSCs). In this study, hESCs ectopically expressing high and low levels of HoxB4 were obtained using lentiviral gene transfer. Quantification throughout differentiation revealed a steady increase in transcription levels from our constructs. The effects of the two expression levels of HoxB4 were compared regarding the differentiation potential into HSCs. High levels of HoxB4 expression correlated to an improved yield of cells expressing CD34, CD38, the stem cell leukemia gene, and vascular epitheliumcadherin. However, no improvement in myeloid cell maturation was observed, as determined by colony formation assays. In contrast, hESCs with low HoxB4 levels did not show any elevated hematopoietic development. In addition, we found that the total population of HoxB4-expressing cells, on both levels, decreased in developing embryoid bodies. Notably, a high HoxB4 expression in hESCs also seemed to interfere with the formation of germ layers after xenografting into immunodeficient mice. These data suggest that HoxB4-induced effects on hESC-derived HSCs are concentrationdependent during in vitro development and reduce proliferation of other cell types in vitro and in vivo. The application of the transcription factor HoxB4 during early hematopoiesis from hESCs might provide new means for regenerative medicine, allowing efficient differentiation and engraftment of genetically modified hESC clones. Our study highlights the importance of HoxB4 dosage and points to the need for experimental systems allowing controlled gene 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.