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

Hee Joung Lim, Jiyou Han, Dong Hun Woo, Sung Eun Kim, Suel Kee Kim, Hee Gyoo Kang, Jong-Hoon Kim

Research output: Contribution to journalArticle

23 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)123-132
Number of pages10
JournalMolecules and Cells
Volume31
Issue number2
DOIs
Publication statusPublished - 2011 Feb 1

Fingerprint

Embryoid Bodies
Endoderm
Bromodeoxyuridine
Chromosome Aberrations
Caspase 3
Apoptosis
Germ Layers
Scanning Transmission Electron Microscopy
Blastocyst
Aneuploidy
Microvilli
Karyotype
Human Embryonic Stem Cells
Hypoxia
Collagen
Cell Count
Western Blotting
Staining and Labeling
Oxygen

Keywords

  • differentiation
  • embryoid body
  • germ-layer
  • human embryo stem cell
  • hypoxia

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

Cite this

Biochemical and morphological effects of hypoxic environment on human embryonic stem cells in long-term culture and differentiating embryoid bodies. / Lim, Hee Joung; Han, Jiyou; Woo, Dong Hun; Kim, Sung Eun; Kim, Suel Kee; Kang, Hee Gyoo; Kim, Jong-Hoon.

In: Molecules and Cells, Vol. 31, No. 2, 01.02.2011, p. 123-132.

Research output: Contribution to journalArticle

Lim, Hee Joung ; Han, Jiyou ; Woo, Dong Hun ; Kim, Sung Eun ; Kim, Suel Kee ; Kang, Hee Gyoo ; Kim, Jong-Hoon. / Biochemical and morphological effects of hypoxic environment on human embryonic stem cells in long-term culture and differentiating embryoid bodies. In: Molecules and Cells. 2011 ; Vol. 31, No. 2. pp. 123-132.
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