The SIRT6 activator MDL-800 improves genomic stability and pluripotency of old murine-derived iPS cells

Abstract

Cellular reprogramming is an emerging strategy for delaying the aging processes. However, a number of challenges, including the impaired genome integrity and decreased pluripotency of induced pluripotent stem cells (iPSCs) derived from old donors, may hinder their potential clinical applications. The longevity gene, Sirtuin 6 (SIRT6), functions in multiple biological processes such as the maintenance of genome integrity and the regulation of somatic cell reprogramming. Here, for the first time, we demonstrate that MDL-800, a recently developed selective SIRT6 activator, improved genomic stability by activating two DNA repair pathways-nonhomologous end joining (NHEJ) and base excision repair (BER) in old murine-derived iPSCs. More interestingly, we found that pretreating old murine iPSCs, which normally exhibit a restricted differentiation potential, with MDL-800 promoted the formation of teratomas comprised of all three germ layers and robustly stimulated chimera generation. Our findings suggest that pharmacological activation of SIRT6 holds great promise in treating aging-associated diseases with iPSC-based cell therapy.

Keywords: DNA repair; MDL-800; SIRT6; aging; genome integrity; pluripotency.

FIGURE 1

MDL‐800 promotes genome integrity by enhancing NHEJ and BER in old murine‐derived iPSCs. (a) Chemical structure of MDL‐800. (b) H3K56Ac levels in old murine‐derived iPSCs treated with the indicated doses of MDL‐800 for 24 hr. (c) H3K56Ac levels in old murine‐derived iPSCs treated with 20 μΜ MDL‐800 for indicated time period. (d‐e) Analysis of genome integrity in old murine‐derived iPSCs treated with the indicated doses of MDL‐800 for 5 passages by alkaline comet assay. Data in (d) and (e) are from the same experiment. The average tail moments are shown in (d), and the percentage of DNA content in tails are shown in (e). At least 50 cells per group were included for analysis. Error bars represent SEM. p value in (e) was determined by ANOVA. (f) Western blot analysis of γH2AX level. Old murine‐derived iPSCs were treated with 20 μΜ MDL‐800 for 5 passages before X‐ray irradiation at 8 Gy. Cells were lysed for protein extraction at the indicated time point post‐X‐ray treatment. (g) The schematic depictions of NHEJ and BER efficiency assay. For NHEJ efficiency analysis, the NHEJ reporter was linearized by I‐SceI endonuclease in vitro to mimic DSBs. For BER efficiency analysis, the pEGFP‐N1 plasmid was mixed with methylene blue and exposed to visible light produced by a 100‐W bulb for 120 min to induce base damage. The purified linearized NHEJ reporter (0.4 μg) or damaged pEGFP‐N1 reporter (0.2 μg), along with 0.1 μg pCAG‐DsRed vector, was transfected into 2 × 10⁵ mouse iPSCs. FACS analysis was performed at 48‐hr post‐transfection. (h‐i) Analysis of NHEJ and BER efficiency of old murine‐derived iPSCs treated with indicated doses of MDL‐800 for 5 passages. Error bars represent s.d.. All experiments were repeated at least three times. **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s. not significant

FIGURE 2

MDL‐800 improves the differentiation potential of old murine‐derived iPSCs. (a) MDL‐800 promotes the formation of teratomas comprised of all three germ layers from old murine‐derived iPSCs while teratomas in the control group show a neuroectoderm‐skewed differentiation phenotype. Old murine iPSCs were treated with 20 μΜ MDL‐800 for 5 passages before injection. Teratomas were dissected for HE staining. Ectoderm: neural tube, keratinized epithelium; Mesoderm: myofiber, adipocyte; Endoderm: cylindrical epithelium. (b‐c) MDL‐800 promotes chimera formation from GFP‐tagged old murine‐derived iPSCs. GFP‐tagged old murine‐derived iPSCs were treated with 20 μΜ MDL‐800 for 5 passages before blastocyst microinjection. Representative fluorescent images of E14.5 chimeric mouse embryos are shown in (b). Scale bar: 2 mm. The percentage of GFP positive cells in E14.5 embryos (left panel) and the representative FACS traces (right panel) are shown in (c). (d‐e) MDL‐800 promotes the generation of mice with higher chimerism from old murine‐derived iPSCs. iPSCs were treated with 20 μΜ MDL‐800 for 5 passages before blastocyst microinjection. Representative images of adult chimeric mice (upper panel) and the rate of chimera formation (lower panel) are shown in (d). The degree of chimerism was evaluated by the coat color in (e). Error bars represent SEM, *p < 0.05