Methanol fixed feeder layers altered the pluripotency and metabolism of bovine pluripotent stem cells

The pluripotency maintenance of pluripotent stem cells (PSCs) requires the suitable microenvironment, which commonly provided by feeder layers. However, the preparation of feeder layers is time consuming and labor exhaustive, and the feeder cells treated with mitomycin C or gamma-ray irradiation bring heterologous contamination. In this study, mouse embryonic fibroblasts (MEFs) were treated by methanol to generate chemical fixed feeder cells, and bovine embryonic stem cells F7 (bESC-F7) cultured on this feeder layer. Then the pluripotency and metabolism of bESC-F7 cultured on methanol-fixed MEFs (MT-MEFs) named MT-F7 was compared with mitomycin C treated MEFs (MC-MEFs). The results showed that bESC-F7 formed alkaline phosphatase positive colonies on MT-MEFs, the relative expression of pluripotent markers of these cells was different from the bESCs cultured on the MC-MEFs (MC-F7). The long-term cultured MT-F7 formed embryoid bodies, showed the ability to differentiate into three germ layers similar to MC-F7. The analyses of RNA-seq data showed that MT-MEFs lead bESCs to novel steady expression patterns of genes regulating pluripotency and metabolism. Furthermore, the bovine expanded pluripotent stem cells (bEPSCs) cultured on MT-MEFs formed classical colonies, maintained pluripotency, and elevated metabolism. In conclusion, MT-MEFs were efficient feeder layer that maintain the distinctive pluripotency and metabolism of PSCs.