Kärner Elerin, Unger Christian, Cerny Radim, Ahrlund-Richter Lars, Ganss Bernhard, Dilber M Sirac, Wendel Mikael
Center for Oral Biology, Institute of Odontology, Karolinska Institutet, Stockholm, Sweden.
J Cell Physiol. 2009 Feb;218(2):323-33. doi: 10.1002/jcp.21605.
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.
通过基因改造诱导人类胚胎干细胞（HESC）增强分化，有可能产生大量多样的细胞类型。这种基因改造常常通过单个调节蛋白的过表达来实现。然而，仔细评估表达水平至关重要，因为这可能对HESC的分化潜能具有重要影响。迄今为止，尚未有关于使用早期骨“主控”转录因子osterix（Osx）通过基因转移到HESC中来促进成骨的报道。在本研究中，我们在慢病毒基因转移后获得了表达两种显著不同水平Osx的HESC亚群。两个亚群均表现出自发分化以及多能表型特征性标志物（如SSEA3、Tra1-60和Nanog）的表达降低。为了促进骨分化，细胞用抗坏血酸、β-甘油磷酸酯和地塞米松进行处理。与原代人成骨细胞中发现的内源性水平相比，高水平的Osx并未增强成骨分化，也未上调I型胶原的表达。我们发现，高水平的Osx反而通过上调CD34和Gata1的表达诱导细胞向造血内皮谱系分化。然而，低水平的Osx上调了I型胶原、骨唾液蛋白和骨钙素的表达。相反，同源盒转录因子HoxB4（一种已知的早期造血调节因子）的强制高水平表达促进了HESC的成骨，而低水平的HoxB4则导致造血基因表达。
