Characterization of a growth-elevated cell line of human bone marrow-derived mesenchymal stem cells by SV40 T-antigen

Abstract

Human bone marrow-derived mesenchymal stem cells (hMSCs) are capable of self-renewal and differentiation into various tissue lineages, attracting attention as tools for use in cell therapy. However, hMSCs have very poor proliferative capacity and a short life span in culture. To overcome this problem, we expressed the T antigen of SV40 in hMSCs because it is known to have the ability to elevate the growth rate of various primary animal cells. We obtained several hMSCs lines (hMSCs-T) known for stable expression of T antigen. Cells expressing T antigen proliferated on the monolayer of hMSCs, forming high density foci. hMSCs-T showed changed morphology and improved growth rate and life span, and demonstrated preservation of the potential for differentiation into osteoblasts. In addition, hMSCs-T did not proliferate in soft agar culture, indicating that the cells did not transform into tumor cells. In order to evaluate metabolic change of amino acids in hMSCs-T compared to primary hMSCs, we investigated altered amino acids (AA) with gas chromatography-mass spectrometry (GC-MS) in selected ion monitoring (SIM) mode (GC-SIM-MS). A total of 14 AAs were positively measured. Results from the Student's t-test on the hMSCs group mean of the hMSCs-T group showed significantly elevated levels of glycine, proline, pipecolic acid, aspartic acid, lysine and tryptophan, whereas valine, leucine and isoleucine as branched-chain amino acids (BCAAs), and phenylalanine showed a significant decrease. Altered AAs metabolic pattern in the hMSCs-T may explain the disturbance of AA metabolism related to the expression of SV40 T antigen in hMSCs.

Usefulness of Mesenchymal Cell Lines for Bone and Cartilage Regeneration Research

“…In four out of the thirty iMSC lines which displayed osteogenic potential, this differentiation ability was increased in comparison with primary MSCs, in terms of mineralization [14,85] and osteogenesic-related gene expression [44,51]. This increment of the osteogenic potential neither seems to be related to tissue's origin, since it has been detected in iMSCs from bone marrow [14,44], cranial periosteum [51], and adipose tissue origin [85], nor seems to be related to immortalization protocol, as two of these cell lines were transduced with SV40LT [44,51], one was transduced with hTERT alone [14] and the other one with hTERT and E6/E7 [85].…”

“…Positive for VKS and osteocalcin upregulation (increased compared with primary MSCs) [44] Not tested Not tested…”

“…SV40LT expression increases the lifespan of MSCs and usually raises its proliferation rate as well, but it has also been observed that this increased lifespan is not unlimited. Lee et al (2015) reported that after more than 80 passages, SV40LT-transduced MSCs decreased their growth rate and entered senescence, indicating that this antigen is not enough for complete immortalization of MSCs [44].…”

Generation of Mesenchymal Cell Lines Derived from Aged Donors

“…Since cartilage and bone disorders like osteoarthritis (OA) are more frequent in the aged population, some efforts have been made to overcome MSCs predisposition to senescence in vitro [ 16 , 17 , 18 , 19 ]. Different strategies have been applied to give an unlimited growth capacity to MSCs, mainly involving the transduction of viral genes and/or human telomerase reverse transcriptase (hTERT) [ 16 , 17 , 18 , 20 , 21 , 22 ].…”

“…Since cartilage and bone disorders like osteoarthritis (OA) are more frequent in the aged population, some efforts have been made to overcome MSCs predisposition to senescence in vitro [ 16 , 17 , 18 , 19 ]. Different strategies have been applied to give an unlimited growth capacity to MSCs, mainly involving the transduction of viral genes and/or human telomerase reverse transcriptase (hTERT) [ 16 , 17 , 18 , 20 , 21 , 22 ]. Simian virus 40 large T antigen (SV40LT), as well as other viral genes, avoids cell cycle detention by meddling with Rb- and p53-mediated pathways [ 23 ], whilst hTERT avoids telomere shortening and therefore the DNA damage-induced senescence [ 24 , 25 ].…”