Introducing a single-cell-derived human mesenchymal stem cell line expressing hTERT after lentiviral gene transfer.
Human mesenchymal stem cells (hMSCs) can be readily isolated from bone marrow and differentiate into multiple tissues, making them a promising target for future cell and gene therapy applications. The low frequency of hMSCs in bone marrow necessitates their isolation and expansion in vitro prior to clinical use, but due to senescence-associated growth arrest during culture, limited cell numbers can be generated. The lifespan of hMSCs has been extended by ectopic expression of human telomerase reverse transcriptase (hTERT) using retroviral vectors. Since malignant transformation was observed in hMSCs and retroviral vectors cause insertional mutagenesis, we ectopically expressed hTERT using lentiviral gene transfer. Single-cell-derived hMSC clones expressing hTERT did not show malignant transformation in vitro and in vivo after extended culture periods. There were no changes observed in the expression of tumour suppressor genes and karyotype. Cultured hMSCs lack telomerase activity, but it was significantly increased by ectopic expression of hTERT. HTERT expression prevented hMSC senescence and the cells showed significantly higher and unlimited proliferation capacity. Even after an extended culture period, hMSCs expressing hTERT preserved their stem cells character as shown by osteogenic, adipogenic and chondrogenic differentiation. In summary, extending the lifespan of human mesenchymal stem cells by ectopic expression of hTERT using lentiviral gene transfer may be an attractive and safe way to generate appropriate cell numbers for cell and gene therapy applications.
Böcker
Wolfgang
W
Experimental Surgery and Regenerative Medicine, Department of Surgery, Ludwig-Maximilians-University, Nussbaumstrasse 20, 80336 Munich, Germany.
Yin
Zhanhai
Z
Drosse
Inga
I
Haasters
Florian
F
Rossmann
Oliver
O
Wierer
Matthias
M
Popov
Cvetan
C
Locher
Melanie
M
Mutschler
Wolf
W
Docheva
Denitsa
D
Schieker
Matthias
M
D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D049109:Cell Proliferation; D048430:Cell Shape; D002471:Cell Transformation, Neoplastic; D016922:Cellular Senescence; D002999:Clone Cells; D006801:Humans; D007621:Karyotyping; D007700:Kinetics; D016086:Lentivirus / Q000235:genetics*; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000201:enzymology*; D051379:Mice; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D010957:Plasmids / Q000235:genetics; D019098:Telomerase / Q000235:genetics*; D014161:Transduction, Genetic
C509186:TERT protein, human; D019098:Telomerase