TY - JOUR
T1 - Biochemical and morphological effects of hypoxic environment on human embryonic stem cells in long-term culture and differentiating embryoid bodies
AU - Lim, Hee Joung
AU - Han, Jiyou
AU - Woo, Dong Hun
AU - Kim, Sung Eun
AU - Kim, Suel Kee
AU - Kang, Hee Gyoo
AU - Kim, Jong Hoon
N1 - Funding Information:
This research was supported by a grant (SC-3130) from the Stem Cell Research Center of the 21st Century Frontier Research Program funded by the Ministry of Education, Science and Technology, Republic of Korea.
PY - 2011/2
Y1 - 2011/2
N2 - 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.
AB - 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.
KW - differentiation
KW - embryoid body
KW - germ-layer
KW - human embryo stem cell
KW - hypoxia
UR - http://www.scopus.com/inward/record.url?scp=79952986775&partnerID=8YFLogxK
U2 - 10.1007/s10059-011-0016-8
DO - 10.1007/s10059-011-0016-8
M3 - Article
C2 - 21347709
AN - SCOPUS:79952986775
SN - 1016-8478
VL - 31
SP - 123
EP - 132
JO - Molecules and cells
JF - Molecules and cells
IS - 2
ER -