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Published Online: 16 September 2014

Hypoxia Enhances Differentiation of Mouse Embryonic Stem Cells into Definitive Endoderm and Distal Lung Cells

Publication: Stem Cells and Development
Volume 24, Issue Number 5


We investigated the effects of hypoxia on spontaneous (SP)- and activin A (AA)-induced definitive endoderm (DE) differentiation of mouse embryonic stem cells (mESCs) and their subsequent differentiation into distal pulmonary epithelial cells. SP differentiation for 6 days of mESCs toward endoderm at hypoxia of 1% O2, but not at 3% or 21% (normoxia), increased the expression of Sox17 and Foxa2 by 31- and 63-fold above maintenance culture, respectively. Treatment of mESCs with 20 ng/mL AA for 6 days under hypoxia further increased the expression of DE marker genes Sox17, Foxa2, and Cxcr4 by 501-, 1,483-, and 126-fold above maintenance cultures, respectively. Transient exposure to hypoxia, as short as 24 h, was sufficient to enhance AA-induced endoderm formation. The involvement of hypoxia-inducible factor (HIF)-1α and reactive oxygen species (ROS) in the AA-induced endoderm enrichment was assessed using HIF-1α−/− mESCs and the ROS scavenger N-acetylcysteine (NAC). Under SP conditions, HIF-1α−/− mESCs failed to increase the expression of endodermal marker genes but rather shifted toward ectoderm. Hypoxia induced only a marginal potentiation of AA-induced endoderm differentiation in HIF-1α−/− mESCs. Treatment of mESCs with AA and NAC led to a dose-dependent decrease in Sox17 and Foxa2 expression. In addition, the duration of exposure to hypoxia in the course of a recently reported lung differentiation protocol resulted in differentially enhanced expression of distal lung epithelial cell marker genes aquaporin 5 (Aqp5), surfactant protein C (Sftpc), and secretoglobin 1a1 (Scgb1a1) for alveolar epithelium type I, type II, and club cells, respectively. Our study is the first to show the effects of in vitro hypoxia on efficient formation of DE and lung lineages. We suggest that the extent of hypoxia and careful timing may be important components of in vitro differentiation bioprocesses for the differential generation of distal lung epithelial cells from pluripotent progenitors.

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Published In

cover image Stem Cells and Development
Stem Cells and Development
Volume 24Issue Number 5March 1, 2015
Pages: 663 - 676
PubMed: 25226206


Published in print: March 1, 2015
Published ahead of print: 27 October 2014
Published online: 16 September 2014
Accepted: 15 September 2014
Received: 10 July 2014


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Pimchanok Pimton*
Department of Biology, School of Science, Walailak University, Nakhon Si Thammarat, Thailand.
Department of Bioengineering, College of Engineering, Temple University, Philadelphia, Pennsylvania.
Shimon Lecht*
Department of Bioengineering, College of Engineering, Temple University, Philadelphia, Pennsylvania.
Collin T. Stabler
Department of Bioengineering, College of Engineering, Temple University, Philadelphia, Pennsylvania.
Gregg Johannes
Department of Pathology, College of Medicine, Drexel University, Philadelphia, Pennsylvania.
Edward S. Schulman
Division of Pulmonary, Critical Care and Sleep Medicine, Drexel University College of Medicine, Philadelphia, Pennsylvania.
Peter I. Lelkes
Department of Bioengineering, College of Engineering, Temple University, Philadelphia, Pennsylvania.


These two authors contributed equally to this work.
Address correspondence to:Peter I. Lelkes, PhDDepartment of BioengineeringCollege of EngineeringTemple UniversityEngineering Building Room 8111947 North 12th StreetPhiladelphia, PA 19122E-mail: [email protected]

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No competing financial interests exist.

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