Hum. Reprod. Advance Access originally published online on September 26, 2008
Human Reproduction 2009 24(1):71-80; doi:10.1093/humrep/den345
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A reduced oxygen tension (5%) is not beneficial for maintaining human embryonic stem cells in the undifferentiated state with short splitting intervals
1 Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, College of Medicine and the Hospital, National Taiwan University, No. 7 Chung-Shan S. Rd, Taipei 100, Taiwan 2 Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan 3 Institute of Cellular and Organismic Biology and Genomics Research Center, Academia Sinica, Taipei, Taiwan 4 Biomedical Engineering Research Laboratories, Industrial Technology Research Institute, Taipei, Taiwan 5 Graduate Institute of Immunology, College of Medicine, National Taiwan University, Taipei, Taiwan
6 Correspondence address. Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, College of Medicine and the Hospital, National Taiwan University, No. 7 Chung-Shan S. Rd, Taipei 100, Taiwan. Tel: +886-2-23123456 ext. 5161; Fax: +886-2-23418557; E-mail: hnho{at}ntu.edu.tw
BACKGROUND: Human embryos grow naturally in vivo in lower oxygen (O2) 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% O2, 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, 
-fetoprotein and GDF-9) and hypoxia-related genes (HIF-1 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% O2 tension for the maintenance of an undifferentiated state by the criteria used in this study.
Key words: embryonic stem cells/hypoxia/undifferentiated/oxygen/cell culture
Submitted on January 23, 2008; resubmitted on August 20, 2008; accepted on August 22, 2008.