Department of Biotechnology and Bioinformatics, Jaypee University of Information Technology, Waknaghat 173234, India.
Department of Biotechnology and Bioinformatics, Jaypee University of Information Technology, Waknaghat 173234, India.
Exp Cell Res. 2014 Apr 1;322(2):389-401. doi: 10.1016/j.yexcr.2014.02.008. Epub 2014 Feb 18.
The efficient utilization of embryonic stem cells for applications like cell based therapy, transplantation and drug discovery largely depends upon the culturing conditions of these cells. In this report, we have analyzed gene, protein expression and morphological changes of embryonic stem cells when subjected to lowered CO2 levels i.e. hypocapnia. We studied the quantitative expression of pluripotent genes, Oct3/4, Nanog and Sox2 and genes involved in the differentiation to the three lineages, under varying CO2 levels. Enhanced expression of these genes was seen at cultures maintained at 1.5% CO2 as compared to those maintained at 5% CO2. The cells exposed to hypocapnic conditions when subjected to immunocytochemical analysis stained positive for Oct-3/4, Nanog and Sox2 transcription factors. Flow cytometry and western blot further showed that the pluripotent proteins in the 1.5% CO2 maintained cultures have higher levels of expression as compared to the ES cells at 5% CO2. In addition, there was enhanced differentiation particularly towards the mesodermal and endodermal lineages at cultures maintained and differentiated at 1.5% CO2 at all the time periods analyzed i.e. day 10 (5+5d), 12 (5+7d) and day 15 (5+10d). These results, which we feel are the first of their kind, indicate that lowered CO2 levels seem to be preferred for the maintenance of pluripotency and the subsequent differentiation.
胚胎干细胞的高效利用，例如在细胞治疗、移植和药物发现方面的应用，在很大程度上取决于这些细胞的培养条件。在本报告中，我们分析了胚胎干细胞在 CO2 水平降低（即低碳酸血症）时的基因、蛋白质表达和形态变化。我们研究了在不同 CO2 水平下，多能基因 Oct3/4、Nanog 和 Sox2 以及涉及到三个谱系分化的基因的定量表达。与在 5% CO2 下培养的细胞相比，在 1.5% CO2 下培养的细胞中这些基因的表达增强。暴露于低碳酸血症条件下的细胞在进行免疫细胞化学分析时，Oct-3/4、Nanog 和 Sox2 转录因子呈阳性染色。流式细胞术和 Western blot 进一步表明，与在 5% CO2 下培养的 ES 细胞相比，在 1.5% CO2 下培养的多能蛋白表达水平更高。此外，在所有分析的时间点（第 10 天（5+5d）、第 12 天（5+7d）和第 15 天（5+10d）），在 1.5% CO2 下维持和分化的培养物中，向中胚层和内胚层谱系的分化增强。这些结果，我们认为是此类研究的首次结果，表明降低 CO2 水平似乎更有利于维持多能性和随后的分化。
