Hypocapnia leads to enhanced expression of pluripotency and meso-endodermal differentiation genes in mouse embryonic stem cells.

Abstract 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 CO 2 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 CO 2 levels. Enhanced expression of these genes was seen at cultures maintained at 1.5% CO 2 as compared to those maintained at 5% CO 2 . 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% CO 2 maintained cultures have higher levels of expression as compared to the ES cells at 5% CO 2 . In addition, there was enhanced differentiation particularly towards the mesodermal and endodermal lineages at cultures maintained and differentiated at 1.5% CO 2 at all the time periods analyzed i.e . day 10 (5+5 d), 12 (5+7 d) and day 15 (5+10 d). These results, which we feel are the first of their kind, indicate that lowered CO 2 levels seem to be preferred for the maintenance of pluripotency and the subsequent differentiation.