Effects of sustalned hypoxia on proliferation of mouse embryonic fibroblasts and preparation of feeder layers / 中国组织工程研究

ABSTRACT

BACKGROUND: In traditional culture systems for embryonic stem cells, feeder cell preparation and embryonic stem cell culture are mostly performed under normoxic conditions. Changes in oxygen culture conditions are likely to influence feeder cells, thereby altering the growth characteristics or differentiation ability of embryonic stem cells, but there is still no relevant systematic report until now. OBJECTIVE: To investigate the effects of sustained hypoxia culture on the pluripotency of mouse embryonic stem cells cultured on mouse embryonic fibroblast feeder layers. METHODS: Primary mouse embryonic fibroblasts were persistently subcultured under normoxia (20% O2) and hypoxia (5% O2) conditions. Cell proliferation was measured for drawing growth curve. Reactive oxygen species level and mitochondria membrane potential of the feeder cells were detected respectively. Mouse embryonic stem cells were divided into two groups normoxia group (plated on mouse embryonic fibroblast feeder layers under 20% O2), and hypoxia group (plated on mouse embryonic fibroblast feeder layers under 5% O2). The cell morphology was observed and the pluripotency of embryonic stem cells were detected by measurement of Oct4 and Sox2 expressions. Hypoxia inducible factor-1α mRNA expression was also tested in the four groups. RESULTS AND CONCLUSION: As compared to the normoxia group, mouse embryonic fibroblasts in the hypoxia group proliferated faster, reactive oxygen species significantly declined, and the mitochondria membrane potential level increased significantly (P < 0.05). Embryonic stem cells were positive for alkaline phosphatase, and highly expressed Oct4 and Sox2 mRNA. Much more median- or small-sized colonies formed in the hypoxia group than the normoxia group (P < 0.05). The mRNA expression of hypoxia inducible factor-1α in embryonic stem cells had a significant difference between the hypoxia and normoxia groups (P < 0.05). These findings indicate that a sustained hypoxia environment can significantly promote the viability of mouse embryonic fibroblasts as feeder layers and maintain the pluripotency of embryonic stem cells under 5% O2.