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

Abstract

Background: Human embryos grow naturally in vivo in lower oxygen (O(2)) tension environments than atmospheric O(2) tension. Therefore, human embryonic stem cells (hESC), a derivative of embryos, will likely grow more favorably in a reduced O(2) tension. This study aimed to compare the behavior of hESC under reduced O(2) tension (5%) versus normoxia (21%).

Methods: hESC lines were cultured in different O(2) tensions and then examined for morphology, apoptosis and gene expression profiles.

Results: hESC grown in 5% O(2) 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 O(2) tensions showed no major difference in representative stemness genes (Oct-3/4, Nanog and Cripto), differentiation genes (Desmin, Nestin, alpha-fetoprotein and GDF-9) and hypoxia-related genes (HIF-1alpha 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% O(2).

Conclusions: This study suggests that for routine culture of hESC with a short splitting interval (7 days), a low O(2) tension (5% O(2)) 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.