A Combination of Culture Conditions and Gene Expression Analysis Can Be Used to Investigate and Predict hES Cell Differentiation Potential towards Male Gonadal Cells

Human embryonic stem cell differentiation towards various cell types belonging to ecto-, endo- and mesodermal cell lineages has been demonstrated, with high efficiency rates using standardized differentiation protocols. However, germ cell differentiation from human embryonic stem cells has been very inefficient so far. Even though the influence of various growth factors has been evaluated, the gene expression of different cell lines in relation to their differentiation potential has not yet been extensively examined. In this study, the potential of three male human embryonic stem cell lines to differentiate towards male gonadal cells was explored by analysing their gene expression profiles. The human embryonic stem cell lines were cultured for 14 days as monolayers on supporting human foreskin fibroblasts or as spheres in suspension, and were differentiated using BMP7, or spontaneous differentiation by omitting exogenous FGF2. TLDA analysis revealed that in the undifferentiated state, these cell lines have diverse mRNA profiles and exhibit significantly different potentials for differentiation towards the cell types present in the male gonads. This potential was associated with important factors directing the fate of the male primordial germ cells in vivo to form gonocytes, such as SOX17 or genes involved in the NODAL/ACTIVIN pathway, for example. Stimulation with BMP7 in suspension culture resulted in up-regulation of cytoplasmic SOX9 protein expression in all three lines. The observation that human embryonic stem cells differentiate towards germ and somatic cells after spontaneous and BMP7-induced stimulation in suspension emphasizes the important role of somatic cells in germ cell differentiation in vitro.

本研究揭示了人类胚胎干细胞向胚外、内胚层和中胚层细胞谱系内多种细胞类型分化的可能性，并采用了标准化的分化方案，实现了高效率。然而，人类胚胎干细胞向生殖细胞分化的效率至今仍然较低。尽管已评估了多种生长因子的影响，但关于不同细胞系在分化潜能方面的基因表达尚未得到充分的探讨。在本研究中，通过分析三株男性人类胚胎干细胞的基因表达谱，探讨了它们向男性生殖细胞分化的潜力。这些人类胚胎干细胞系在支持性人类包皮成纤维细胞上以单层形式或悬浮球状形式培养14天，并使用BMP7进行分化，或通过省略外源性FGF2来实现自发分化。TLDA分析显示，在未分化状态下，这些细胞系具有多样的mRNA谱，并显示出向男性生殖腺中存在的细胞类型分化的显著不同潜能。这种潜能与诸如SOX17或参与NODAL/ACTIVIN途径的基因等重要因素相关，这些因素可指导男性原始生殖细胞在体内的命运，以形成精原细胞。例如，悬浮培养中BMP7的刺激导致所有三株细胞系中细胞质SOX9蛋白表达的上调。人类胚胎干细胞在悬浮培养中经自发和由BMP7诱导的刺激后分化为生殖细胞和体细胞，这一观察强调了体细胞在体外生殖细胞分化中的重要作用。