Transient Expression of Telomerase Reverse Transcriptase in Aged Human Stromal Vascular Fraction Improves Function and Regenerative Capacity

Objective: Autologous adult stem cell therapy for cardiovascular disease has shown minimal or no benefit in clinical trials. This may in part be explained by aging-related alteration of secretory function and loss of replicative capacity of autologous cell preparations. Previously we have shown that functional and replicative capacity of human aortic endothelial cells can be increased by retroviral overexpression of telomerase. However, there is a concern for immortalization and insertional mutagenesis with this approach. More recently, we have developed methods for transient expression of human telomerase reverse transcriptase (TERT). We hypothesize that this method will increase telomere length (TL) sufficiently to enhance the regenerative capacity of adult stem cells. In the current study we assessed the effects of this approach on adipose-derived stromal vascular fraction (SVF).

Methods: We transfected human SVF from elderly donors with nucleoside-modified mRNA (mmRNA) encoding human TERT using a cationic lipid mediated approach. Successive measurements of TERT activity and TL, replicative capacity and secretory response to inflammatory mediators and hypoxia are performed in treated and untreated SVF from elderly and untreated SVF from young donors. In vivo angiogenesis and regenerative capacity are assessed with telomere-extended SVF cells in a mouse model of limb ischemia.

Results: Our initial results show that TERT mmRNA successfully increased telomerase activity and TL (untreated: TL 85% of young donor control; treated: 94% of young donor control) in aged SVF. Additionally, TERT treatment leads to significantly increased secretory capacity for IL-6, IL-8, RANTES and VEGF (Fig. 1).

Conclusion: Our preliminary data indicates that transient expression of TERT is sufficient in lengthening telomeres and enhancing the secretory profile of SVF cells. We intend to assess the regenerative capacity of mmRNA TERT-treated cells in murine models of limb ischemia.

Fig. 1: (A) Telomerase activity measured by TRAP-assay. (B) Relative telomere length measurement by monochrome multiplex real-time PCR. All samples are normalized to telomere length of young donor (31 years/o). (C) Old donor cells were challenged with LPS/ Poly I:C to evaluate their secretory capacity before and after TERT-treatment. 13 cytokinens were evaluated. Changes in RANTES secretion upon challenges are shown in Fig. 1C (* p < 0.05; ** p < 0.01).