Hypoxia inhibits senescence and maintains mesenchymal stem cell properties through down-regulation of E2A-p21 by HIF-TWIST.
Although low-density culture provides an efficient method for rapid expansion of human mesenchymal stem cells (MSCs), MSCs enriched by this method undergo senescence and lose their stem cell properties, which could be preserved by combining low-density and hypoxic culture. The mechanism was mediated through direct down-regulation of E2A-p21 by the hypoxia-inducible factor-1α (HIF-1α)-TWIST axis. Expansion under normoxia induced E2A and p21 expression, which were abrogated by overexpression of TWIST, whereas siRNA against TWIST up-regulated E2A and p21 in hypoxic cells. Furthermore, siRNA against p21 in normoxic cells enhanced proliferation and increased differentiation potential, whereas overexpression of p21 in hypoxic cells induced a decrease in proliferation and a loss of differentiation capacity. More importantly, MSCs expanded under hypoxic conditions by up to 100 population doublings, exhibited telomerase activity with maintained telomere length, normal karyotyping, and intact genetic integrity, and did not form tumors. These results support low-density hypoxic culture as a method for efficiently expanding MSCs without losing stem cell properties or increasing tumorigenicity.
Tsai
Chih-Chien
CC
Institute of Pharmacology, National Yang-Ming University, Taipei, Taiwan.
Chen
Yann-Jang
YJ
Yew
Tu-Lai
TL
Chen
Ling-Lan
LL
Wang
Jir-You
JY
Chiu
Chao-Hua
CH
Hung
Shih-Chieh
SC
D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism*; D015153:Blotting, Western; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D015687:Cell Hypoxia / Q000502:physiology; D016922:Cellular Senescence / Q000502:physiology*; D047369:Chromatin Immunoprecipitation; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism*; D015536:Down-Regulation; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D059630:Mesenchymal Stem Cells / Q000378:metabolism*; D051379:Mice; D016513:Mice, SCID; D010012:Osteogenesis / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051879:Twist-Related Protein 1 / Q000378:metabolism*
D051792:Basic Helix-Loop-Helix Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D051879:Twist-Related Protein 1; C493956:CDKN1A protein, human; C407909:TCF3 protein, human