Fig. 5

Rescue of premature senescence in mutation-rectified HGPS-MSCs. a Schematic illustration of the strategy to rectify LMNA mutation in HGPS-iPSCs by CRISPR/Cas9-mediated gene targeting. The small guide RNA targets the downstream of 3’UTR of LMNA gene with two homologous arms adjacent to sgRNA covering 1.5 kp upstream and downstream of genomic DNA, respectively. The double selective markers (puromycin resistant gene and fluorescent protein mCherry) are flanked by the homologous arms. The mutation corrected clones were screened and selected and confirmed by Sanger sequencing. Selection markers and other foreign DNA sequences were removed by transient expression of Flpase. b Sanger sequencing to confirm mutation rectification of LMNA gene in a HGPS-CiPSCs clone, in comparison to its parental HGPS-iPSCs. c Immunostaining of pluripotency markers SSEA-4, TRA60-1, NANOG and OCT3/4 (Scar bar 10 μm) in HGPS-CiPSCs. Scale bar, 10 μm. d Karyotyping of HGPS-CiPSCs showing normal diploid male karyotype. e Flow cytometry analyses of surface markers in MSCs generated from HGPS-CiPSCs. Note that more than 95% of the cells are positive for CD73, CD90, CD105 and CD44, and negative for CD45, CD34, CD11b, CD19 or HDL. f Heatmap of gene expressions of progerin, p16, p21 and SASP-related genes (IL1a, IL1b, IL6, IL8 and PAI), based on mean value in the MSCs derived from HGPS-iPSCs and HGPS-CiPSCs. Data represent mean ± S.D.; n = 4; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, not significant; one-way ANOVA coupled with Tukey’s post hoc test is employed for statistical analyses. g Comparison of the In vivo bioluminescence between MSCs derived from mutation rectified HGPS-CiPSCs and MSCs derived from HGPS-iPSCs. Bioluminescent intensities were quantified according to the luciferase activities. Data represent mean ± S.D.; n = 3; **p < 0.01; one-way ANOVA coupled with Tukey’s post hoc test is employed for statistical analyses