Developmental pattern of human embryonic stem cells in optimized hypoxic culture condition

Human embryonic stem cells(hESCs) derived from inner cell mass of the preimplanted blastocyte. Because hESCs has a pluripotent potential, it has been thought to a good source for studying human development. Early mammalian embryogenesis and development generally takes place in a low O₂ environment(hypoxia). However, the most of the differentiation studies carried out using hESCs have been achieved in a normoxic condition (5%CO ₂/21%O₂). Such conditions may not be the suitable to mimic in vivo environment. Recently, various hypoxia induction methods such as gas pack or hypoxic chamber were developed by many researchers, but still they did not control of dissolved oxygen(DO) in media. Here, we newly have developed stable hypoxic condition and shown differentiation pattern of hESCs in that condition. For stable hypoxic condition, we removed DO in media using a N ₂ gas injection technique which was more quickly adjusted to the media with low DO value compared with non-bubbled media in hypoxia incubator. After inducing optimized hypoxic condition, we analyzed differentiation pattern of hESCs as compared with normoxic condition using RT-PCR, Real time-PCR and immuno-staining. As a result, in hypoxic condition, we shown that expression of pluripotancy marker, Oct4 was rapidly decreased and expression of mature neuronal marker, MAP2 and endothelial junction protein, VECad were increased in comparison to normoxic condition. These results suggest that hypoxic condition should promote hESCs differentiation to neuronal lineage and vessel lineage. Furthermore, the optimized hypoxic condition developed in this study will be a useful technique for study of human development via hESCs.