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Biochemical and morphological effects of hypoxic environment on human embryonic stem cells in long-term culture and differentiating embryoid bodies

Authors
Lim, Hee-JoungHan, JiyouWoo, Dong-HunKim, Sung-EunKim, Suel-KeeKang, Hee-GyooKim, Jong-Hoon
Issue Date
2월-2011
Publisher
KOREAN SOC MOLECULAR & CELLULAR BIOLOGY
Keywords
differentiation; embryoid body; germ-layer; human embryo stem cell; hypoxia
Citation
MOLECULES AND CELLS, v.31, no.2, pp.123 - 132
Indexed
SCIE
SCOPUS
KCI
Journal Title
MOLECULES AND CELLS
Volume
31
Number
2
Start Page
123
End Page
132
URI
https://scholar.korea.ac.kr/handle/2021.sw.korea/113172
DOI
10.1007/s10059-011-0016-8
ISSN
1016-8478
Abstract
The mammalian reproductive tract is known to contain 1.5-5.3% oxygen (O-2), but human embryonic stem cells (hESCs) derived from preimplantation embryos are typically cultured under 21% O-2 tension. The aim of this study was to investigate the effects of O-2 tension on the long-term culture of hESCs and on cell-fate determination during early differentiation. hESCs and embryoid bodies (EBs) were grown under different O-2 tensions (3, 12, and 21% O-2). The expression of markers associated with pluripotency, embryonic germ layers, and hypoxia was analyzed using RTPCR, immunostaining, and Western blotting. Proliferation, apoptosis, and chromosomal aberrations were examined using BrdU incorporation, caspase-3 immunostaining, and karyotype analysis, respectively. Structural and morphological changes of EBs under different O-2 tensions were comparatively examined using azan- and hematoxylineosin staining, and scanning and transmission electron microscopy. Mild hypoxia (12% O-2) increased the number of cells expressing Oct4/Nanog and reduced BrdU incorporation and aneuploidy. The percentage of cells positive for active caspase-3, which was high during normoxia (21% O-2), gradually decreased when hESCs were continuously cultured under mild hypoxia. EBs subjected to hypoxia (3% O-2) exhibited well-differentiated microvilli on their surface, secreted high levels of collagen, and showed enhanced differentiation into primitive endoderm. These changes were associated with increased expression of Foxa2, Sox17, AFP, and GATA4 on the EB periphery. Our data suggest that mild hypoxia facilitates the slow mitotic division of hESCs in long-term culture and reduces the frequency of chromosomal abnormalities and apoptosis. In addition, hypoxia promotes the differentiation of EBs into extraembryonic endoderm.
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