Generation of Mesenchymal Cell Lines Derived from Aged Donors

Abstract

Background: Mesenchymal stromal cells (MSCs) have the capacity for self-renewal and multi-differentiation, and for this reason they are considered a potential cellular source in regenerative medicine of cartilage and bone. However, research on this field is impaired by the predisposition of primary MSCs to senescence during culture expansion. Therefore, the aim of this study was to generate and characterize immortalized MSC (iMSC) lines from aged donors. Methods: Primary MSCs were immortalized by transduction of simian virus 40 large T antigen (SV40LT) and human telomerase reverse transcriptase (hTERT). Proliferation, senescence, phenotype and multi-differentiation potential of the resulting iMSC lines were analyzed. Results: MSCs proliferate faster than primary MSCs, overcome senescence and are phenotypically similar to primary MSCs. Nevertheless, their multi-differentiation potential is unbalanced towards the osteogenic lineage. There are no clear differences between osteoarthritis (OA) and non-OA iMSCs in terms of proliferation, senescence, phenotype or differentiation potential. Conclusions: Primary MSCs obtained from elderly patients can be immortalized by transduction of SV40LT and hTERT. The high osteogenic potential of iMSCs converts them into an excellent cellular source to take part in in vitro models to study bone tissue engineering.

Generation of an Immortalized Human Adipose-Derived Mesenchymal Stromal Cell Line Suitable for Wound Healing Therapy

“…However, the development of such treatment requires the in vitro expansion of cells for a long period of time, which has the disadvantage that the biological material is quickly depleted and additional isolation of primary ADSC is needed, which could negatively impact the variability of the cell products. This depletion is due to the cell senescence occurring during in vitro propagation due to telomere shortening which leads to the decline of cell growth and modification of their properties [ 13 , 14 ]. This issue could be overcome by immortalization of cells, which can be attained by transduction of genes, such as the human telomerase reverse transcriptase (hTERT) [ 15 , 16 , 17 ].…”

Generation of human immortalized chondrocytes from osteoarthritic and healthy cartilage

“…There were no significant differences between OA and non-OA iACs regarding proliferation capacity. However, the generation time of both OA and non-OA iACs was significantly higher in comparison with previously immortalized MSCs (mean 2.0 days (SE 0.7)) 26,33 (p = 0.001). Immortalized chondrocytes were grown over 40 PDs, and regression analysis showed a constant proliferation rate, with a multiple correlation coefficient R > 0.95 (Figure 2a) for both OA and non-OA iACs, and a p-value < 0.001.…”

“…Although intranuclear localization of hTERT is regulated by the cell cycle phase, SV40LT transduction has been described to promote the release of telomerase from nucleoli to the nucleoplasm. 28 Immortalization with these transgenes allowed chondrocytes to bypass senescence, as evidenced by the absence of granular content related to SA-β-Gal activity, similarly to immortalized MSCs, 33,42,43 and unlike primary articular chondrocytes derived from aged donors and hTERT-transduced chondrocytes. 23 However, mean generation time of immortalized chondrocytes was higher than that previously shown for immortalized MSCs, 33 suggesting that immortalized chondrocytes retain a higher degree of differentiation than immortalized MSCs.…”

Application of telomere biology and telomerase in mesenchymal stem cells

“…Additionally, it affects the direction of differentiation, making MSCs an ideal cell source. Diverse immortalized MSCs cell lines based on TERT have been developed (Table 1) [54,[84][85][86][87][88][89][90][91][92][93][94][95][96][97][98][99][100] to improve their convenience and feasibility for clinical use.…”