Inducible immortality in hTERT-human mesenchymal stem cells.
Human mesenchymal stem cells (hMSCs) are attractive candidates for tissue engineering and cell-based therapy because of their multipotentiality and availability in adult donors. However, in vitro expansion and differentiation of these cells is limited by replicative senescence. The proliferative capacity of hMSCs can be enhanced by ectopic expression of telomerase, allowing for long-term culture. However, hMSCs with constitutive telomerase expression demonstrate unregulated growth and even tumor formation. To address this problem, we used an inducible Tet-On gene expression system to create hMSCs in which ectopic telomerase expression can be induced selectively by the addition of doxycycline (i-hTERT hMSCs). i-hTERT hMSCs have inducible hTERT expression and telomerase activity, and are able to proliferate significantly longer than wild type hMSCs when hTERT expression is induced. They stop proliferating when hTERT expression is turned off and can be rescued when expression is re-induced. They retain multipotentiality in vitro even at an advanced age. We also used a selective inhibitor of telomere elongation to show that the mechanism driving immortalization of hMSCs by hTERT is dependent upon maintenance of telomere length. Thanks to their extended lifespan, preserved multipotentiality and controlled growth, i-hTERT hMSCs may prove to be a useful tool for the development and testing of novel stem cell therapies.
Department of Orthopaedic Surgery, University of California San Francisco, 500 Parnassus Avenue, San Francisco, California 94143, USA.
Samantha L
SL
Piper
Miqi
M
Wang
Akira
A
Yamamoto
Farbod
F
Malek
Andrew
A
Luu
Alfred C
AC
Kuo
Hubert T
HT
Kim
D050156:Adipogenesis; D000328:Adult; D001854:Bone Marrow Cells / Q000166:cytology; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D049109:Cell Proliferation; D016922:Cellular Senescence; D020219:Chondrogenesis; D004318:Doxycycline / Q000494:pharmacology; D005786:Gene Expression Regulation; D006801:Humans; D008297:Male; D059630:Mesenchymal Stem Cells / Q000378:metabolism*; D010012:Osteogenesis; D010957:Plasmids / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology; D019098:Telomerase / Q000235:genetics* / Q000378:metabolism*; D016615:Telomere / Q000648:ultrastructure; D023822:Tissue Engineering / Q000379:methods*
C509186:TERT protein, human; D019098:Telomerase; D004318:Doxycycline