Qu Su, Yan Liang, Fang Bo, Ye Shoudong, Li Ping, Ge Shengyang, Wu Jian, Qu Di, Song Houyan
The Key Laboratory of Molecular Medicine of Ministry of Education, School of Basic Medical Sciences, Fudan University, Shanghai, China.
Key Laboratory of Medical Molecular Virology of Ministry of Education and Ministry of Health, School of Basic Medical Sciences, Fudan University, Shanghai, China.
Life Sci. 2017 Apr 15;175:37-46. doi: 10.1016/j.lfs.2017.03.017. Epub 2017 Mar 22.
To enhance survival and generation of definitive endoderm cells from human embryonic stem cells in a simple and reproducible system.
Definitive endoderm (DE) differentiation from human embryonic stem cells (hESCs) was induced under a chemical-defined condition withdrawn insulin supplement and serum albumin. We dissected influence of "alternative growth factors", WNT3A, BMP4 and bFGF in activin A-driven differentiation by detection of DE-associated genes expression and cell viability. Expression of DE-associated SOX17 and FOXA2 genes was analyzed by real time reverse transcription polymerase chain reaction (RT-PCR) and Western blot assays. Quantitative evaluation of DE efficiency was performed by flow cytometry analysis of CXCR4-expressed cell population. Cell viability during DE differentiation was analyzed by an Annexin V/PI double staining test.
Supplementation with WNT3A, BMP4 or bFGF promoted DE generation in a dose- and time-dependent manner. Cell apoptosis elicited by activin A was significantly ameliorated by a cocktail with WNT3A, BMP4 and bFGF. This allowed for sustained cell viability without insulin-containing supplements, thereby indirectly improving the efficiency of DE generation. Therefore, the cocktail containing is optimal for efficient DE generation in the presence of activin A and an insulin/albumin-free condition.
This optimal condition facilitates the balance between the productivity and the viability maintenance, and could be valuable for mass production of DE with minimal variation.
在一个简单且可重复的系统中提高人胚胎干细胞中确定内胚层细胞的存活和生成。
在去除胰岛素补充剂和血清白蛋白的化学成分确定的条件下，诱导人胚胎干细胞（hESCs）向确定内胚层（DE）分化。我们通过检测DE相关基因表达和细胞活力，剖析了“替代生长因子”WNT3A、BMP4和bFGF在激活素A驱动的分化中的影响。通过实时逆转录聚合酶链反应（RT-PCR）和蛋白质免疫印迹分析，分析DE相关的SOX17和FOXA2基因的表达。通过对CXCR4表达细胞群体的流式细胞术分析，对DE效率进行定量评估。通过Annexin V/PI双染试验分析DE分化过程中的细胞活力。
添加WNT3A、BMP4或bFGF以剂量和时间依赖性方式促进DE生成。激活素A引发的细胞凋亡通过与WNT3A、BMP4和bFGF的混合物得到显著改善。这使得在不含胰岛素补充剂的情况下细胞活力得以维持，从而间接提高了DE生成的效率。因此，在激活素A存在且无胰岛素/白蛋白的条件下，该混合物对于高效生成DE是最优的。
这种最优条件有助于在生产力和活力维持之间取得平衡，对于以最小变异大规模生产DE可能具有重要价值。
