Expression of Telomerase Extends the Lifespan and Enhances Osteogenic Differentiation of Adipose Tissue–Derived Stromal Cells

Abstract

Expression of TERT, the catalytic protein subunit of the telomerase complex, can be used to generate cell lines that expand indefinitely and retain multilineage potential. We have created immortal adipose stromal cell lines (ATSCs) by stably transducing nonhuman primate-derived ATSCs with a retroviral vector expressing TERT. Transduced cells (ATSC-TERT) had an increased level of telomerase activity and increased mean telomere length in the absence of malignant cellular transformation. Long-term culture of the ATSC-TERT cells demonstrated that the cells retain the ability to undergo differentiation along multiple lineages such as adipogenic, chondrogenic, and neurogenic. Untransduced cells demonstrated markedly reduced multilineage and selfrenewal potentials after 12 passages in vitro. To determine the functional role of telomerase during osteogenesis, we examined osteogenic differentiation potential of ATSC-TERT cells in vitro. Compared with naive ATSCs, which typically begin to accumulate calcium after 3-4 weeks of induction by osteogenic differentiation medium, ATSC-TERT cells were found to accumulate significant amounts of calcium after only 1 week of culture in osteogenic induction medium. The cells have increased production of osteoblastic markers, such as AP2, osteoblast-specific factor 2, chondroitin sulfate proteoglycan 4, and the tumor necrosis factor receptor superfamily, compared with control ATSCs, indicating that telomerase expression may aid in maintaining the osteogenic stem cell pool during in vitro expansion. These results show that ectopic expression of the telomerase gene in nonhuman primate ATSCs prevents senescenceassociated impairment of osteoblast functions and that telomerase therapy may be a useful strategy for bone regeneration and repair.

Generation and Characterization of an Immortalized Human Mesenchymal Stromal Cell Line

“…We show that the capacity to differentiate into the adipogenic and osteogenic lineage was maintained in iMSC#3 after immortalization, which is in line with previous studies [14][15][16][17]. In addition, a genome-wide profiling of mRNAs during adipogenesis, followed by IPA showed that numerous highly relevant biological processes were affected.…”

“…Several studies have also demonstrated that long-term culturing of hMSCs affects their capacity to proliferate and differentiate, limiting their use in therapeutic applications and basic research. Immortalization can be achieved by several methods, including introduction of telomerase reverse transcriptase (TERT), human papillomavirus-16 E6 and E7 genes or simian virus 40 large antigen [14][15][16][17][18]. Previous studies have demonstrated that the phenotype of hMSCs is maintained after immortalization [19][20][21] and their differentiation potential has even been reported to improve thereafter [21].…”

Novel Polypyrrole-Coated Polylactide Scaffolds Enhance Adipose Stem Cell Proliferation and Early Osteogenic Differentiation

“…This needs elucidation since CS fragments have well reported biological activity both in vitro and in vivo. 38,39 Nevertheless, our results showed that the effect of PPy coating on hASC response was as strong with and without ES, suggesting that the surface chemistry of PPy plays a more important role in triggering cell response than in the electrical conductivity of PPy.…”

Mitochondria in stem cells

“…The results were compared to a rhesus adult bone marrow stromal cell line (BMSC) and the rhesus ATSC stromal adult stem line, as well as an immortalized version of the ATSC line created by transfection with a retroviral vector resulting in the expression of telomerase activity (ATSC-TERT) (Kang et al, 2004). The common deletion was detected, but not quantified, in the in vivo-derived R4 cell line and the three ORMES cell lines derived from IVF-derived embryos.…”