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. 2020 Jun 6;11(7):483–504. doi: 10.1007/s13238-020-00728-4

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© The Author(s) 2020

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Generation and characterization of SIRT7-deficient hESCs. (A) Left, Western blot analysis of SIRT7 protein in replicatively senescent (RS) hMSCs at early (EP, P3) and late passages (LP, P8) with β-Tubulin as loading control. Right, statistical analysis of relative SIRT7 protein expression levels. Data are presented as the means ± SEM. n = 3. *, P < 0.05 (t test). (B) Left, Western blot analysis of SIRT7 protein in WT and HGPS-specific (LMNA^G608G/+ or LMNA^G608G/G608G) hMSCs at LP (P8) with β-Tubulin used as loading control. Right, statistical analysis of relative SIRT7 protein expression levels. Data are presented as the means ± SEM. n = 3. *, P < 0.05, **, P < 0.01 (t test). (C) Statistical analysis of relative SIRT7 protein expression levels in young and old primary hMSCs. Data are presented as the means ± SEM. n = 4 samples. *, P < 0.05 (t test). (D) Left, schematic illustration of SIRT7 gene editing (exon 4) using CRISPR/Cas9-mediated non-homologous end joining (NHEJ) in hESCs. Right, DNA sequence chromatogram showing the introduction of termination codon TAA by gene editing. (E) Schematic workflow showing the generation of SIRT7^+/+ and SIRT7^−/− hMSCs from hESCs. (F) Genome-wide analysis of copy number variations (CNVs) in SIRT7^+/+ and SIRT7^−/− hMSCs at middle passage (MP, P6). (G) Western blot analysis of SIRT7 protein in SIRT7^+/+ and SIRT7^−/− hMSCs with β-Tubulin used as loading control. (H) Growth curves of SIRT7^+/+ and SIRT7^−/− hMSCs. Data are presented as the means ± SEM. n = 3. (I) SA-β-gal staining of SIRT7^+/+ and SIRT7^−/− hMSCs at EP (P3) and LP (P8). Scale bar, 125 μm. Data are presented as the means ± SEM. n = 3. ns, not significant, **, P < 0.01 (t test). (J) Clonal expansion analysis of SIRT7^+/+ and SIRT7^−/− hMSCs at EP (P3) and LP (P8). Data are presented as the means ± SEM. n = 3. ns, not significant, **, P < 0.01 (t test). (K) Immunostaining of Ki67 in SIRT7^+/+ and SIRT7^−/− hMSCs at EP (P3) and LP (P8). Scale bar, 25 μm. Data are presented as the means ± SEM. n = 3. **, P < 0.01 (t test). (L) Bar plot showing the percentages of cells in S-phase of cell cycle in SIRT7^+/+ and SIRT7^−/− hMSCs at EP (P3) and LP (P8). Data are presented as the means ± SEM. n = 3. **, P < 0.01, ***, P < 0.001 (t test). (M) ROS levels were determined by staining with the free radical sensor H2DCFDA and measured by FACS in SIRT7^+/+ and SIRT7^−/− hMSCs at MP (P6). Data are presented as the means ± SEM. n = 3. (N) Heatmap showing quantitative RT-PCR analysis of aging-related genes in SIRT7^+/+ and SIRT7^−/− hMSCs at EP (P3) and LP (P8) hMSCs. Expression levels of the indicated genes in each cell type were normalized to those in SIRT7^+/+ hMSCs at EP (P3). (O) Western blot analysis of cyclin-dependent kinase inhibitors P16 and P21 proteins in SIRT7^+/+ and SIRT7^−/− hMSCs at MP (P6) with GAPDH used as loading control. (P) Analysis of luciferase activities in TA muscles of immunodeficient mice transplanted with SIRT7^+/+ (left) or SIRT7^−/− hMSCs (right) at MP (P6) in Day 0, 3 and 5 after implantation. Data calculated by the ratios of log₂ (SIRT7^−/−/SIRT7^+/+) are presented as the means ± SEM. n = 6. ns, not significant, ***, P < 0.001 (t test)