A reduced oxygen tension (5%) is not beneficial for maintaining human embryonic stem cells in the undifferentiated state with short splitting intervals

BACKGROUND: Human embryos grow naturally in vivo in lower oxygen (O ₂) tension environments than atmospheric O₂ tension. Therefore, human embryonic stem cells (hESC), a derivative of embryos, will likely grow more favorably in a reduced O₂ tension. This study aimed to compare the behavior of hESC under reduced O₂ tension (5%) versus normoxia (21%). METHODS: hESC lines were cultured in different O₂ tensions and then examined for morphology, apoptosis and gene expression profiles. RESULTS: hESC grown in 5% O₂ tension were not morphologically different from hESC grown in normoxia on day 7 of the first and fourth passages. However, after prolonged culture without splitting (10-14 days), hESC colonies were thinner and looked better morphologically in 5% O ₂, but the cells proliferated more slowly and their sizes were larger. At most time points, the gene expression profiles in both O₂ tensions showed no major difference in representative stemness genes (Oct-3/4, Nanog and Cripto), differentiation genes (Desmin, Nestin, α-fetoprotein and GDF-9) and hypoxia-related genes (HIF-1α and VEGF). A lower level of cyclin-D1 mRNA (suggestive of less Wnt pathway signaling on day 7 of the fourth passage) and a higher level of Desmin (suggestive of more differentiation to mesoderm, at day 7 of the first passage) were detected in 5% O₂. CONCLUSIONS: This study suggests that for routine culture of hESC with a short splitting interval (7 days), a low O₂ tension (5% O₂) probably does not provide significant advantages over the standard 21% O ₂ tension for the maintenance of an undifferentiated state by the criteria used in this study.