Fig. 7

Functional assays in endothelial cells derived from CRISPR-edited patient's iPSC. (a) Schematic presentation of CRISPR/Cas9-mediated homology-directed repair of patient's TOMM7 p.P29L with a single-strand oligodeoxynucleotide (ssODN) template carrying WT TOMM7 and two silent mutations (cyan). Asterisks represent phosphorothioate modifications. eCas9, enhanced Cas9 protein containing K484A, K1003A, and R1060A mutations. (b) Sanger sequencing confirmed gene-edited iPSCs with heterozygous and homozygous TOMM7 p.P29L. (c) Immunoblotting in whole-cell lysate from iEndo cells derived from patient's iPSCs with anti-TOM7 and anti-TOM40 antibodies. (d) Representative transmission electron microscopy images of iEndo cells derived from patient's iPSCs and variant-corrected WT iPSCs (edited WT). Scale bars, 200 nm. (e) Representative confocal images of immunofluorescent staining with anti-TOM40 (Red; staining mitochondria) and DAPI (Blue) in patient's iEndo cells. White arrows indicate large, round mitochondria. Cyan arrows indicate elongated mitochondria. (f) Quantitative analyses of mitochondrial morphology in the images of immunofluorescent staining by Mitochondrial Network Analysis (MiNA) toolset showing counts of individual mitochondria, counts of network, mean mitochondrial branch length, mean network branches, and mitochondrial footprint in patient's iEndo cells from (e). Statistical analysis was performed using Kruskal–Wallis test with Dunn's multiple comparisons test. (g) Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) measured by the Seahorse Mito Stress Test with 3 × 104 iEndo cells. One representative result was shown. Oligo., oligomycin; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; Rot, rotenone; AA, antimycin A. (h) Basal respiration, ATP-coupled respiration, percentage of respiration used for ATP synthesis, maximal mitochondrial respiratory capacity, and proton leak-linked respiration in iEndo cells derived from patient's iPSCs were calculated from (g). Statistical analysis was performed using Mann–Whitney U-tests. (i) ATP production rates from mitochondrial respiration and glycolysis. Statistical analysis of mitochondrial ATP and glycolytic ATP was performed using Mann–Whitney U-tests, with comparisons made to WT cells for each parameter. (j) Quantitative RT-PCR confirmed the differentially expressed genes in patient's iEndos, and statistical analysis was performed using Friedman test followed by Dunn's post hoc test, with comparisons made to WT cells. (k) Immunoblotting in whole-cell lysate from iEndo cells derived from patient's iPSCs with anti-HK2 antibody. (l) Basal glycolysis and compensatory glycolysis in iEndo cells derived from patient's iPSCs measured by glycolytic proton efflux rate (PER) in Seahorse assay. Statistical analysis was performed using Mann–Whitney U-tests. Edited WT, wild type TOMM7 p.P29L; Edited Het, heterozygous TOMM7 p.P29L; Patient, patient 1-III, homozygous TOMM7 p.P29L; Edited KO, TOMM7 knockout. Data were presented as mean ± S.E.M. ∗P < 0.05, ∗∗P < 0.01.