Supplementary MaterialsSupplementary Material 41598_2019_55837_MOESM1_ESM. implying an extended duration of phase 3. Treatment with the IKr inhibitor E4031 only caused APD prolongation in the control line. Patch clamp showed a reduction of IKr on LQTS2 CM-iPSC compared to control, but channel activation was not significantly affected. Immunofluorescence for Rabbit polyclonal to CD80 hERG demonstrated perinuclear staining in LQTS2 CM-iPSC. In conclusion, CM-iPSC recapitulated the LQTS2 phenotype and our findings suggest that the R534C mutation in KCNH2 leads to a channel trafficking defect to the plasma membrane. using oocytes or HEK293 cells to dissect the underlying genetic causes of hERG dysfunction20C23. However, these exogenous expression systems do not recapitulate the complex interactions between the various types of ion channels present in a human cardiomyocyte. Current gene editing technologies make it possible to correct or introduce mutations in iPSC, controlling for patient genetic background and epigenetic variability24. In this study, we have generated iPSC from two LQTS2 patients with c.1600C?>?T, p.R534C mutation and introduced this same mutation in a control iPSC line. These cell lines were differentiated into cardiomyocytes and characterized by electrophysiology. Results Generation of induced pluripotent stem cells and genome editing Peripheral blood mononuclear cells (PBMNC) were isolated from a healthy male donor (24 years old, CTRL-iPSC) and 2 donors with a diagnosis of familial LQTS2 with a heterozygous R534C mutation (female, 44 years old, LQTS2-iPSC1; and male, 17 years of age, LQTS2-iPSC2). PBMNC had been enriched for erythroblasts and, after 12 times, cells had been reprogrammed (Supplementary Fig.?S1a). The initial colonies with pluripotent features emerged ~15 times post-transduction. iPSCs had been selected predicated on morphology (curved colonies, well-defined colony sides, and high nucleus-to-cytoplasm proportion) (Supplementary Fig.?S1b), expanded and characterized (Supplementary Fig.?S1c-e and S2). These clones got a standard karyotype (Supplementary Fig.?S1c) and, to verify the current presence of the mutation following reprogramming, exon 7 of KCNH2 was genotyped. We noticed a normal series inside our CTRL-iPSC and discovered the idea mutation (c.1600C?>?T) in heterozygosis (Supplementary Fig.?S1d) in LQTS2-iPSC1 and LQTS2-iPSC2. To research the effect from the R534C KCNH2 mutation within an similar hereditary background, a homologous recombination technique was found in our CTRL-iPSC to put in this mutation. Using the CRISPR/Cas9 program, we designed an individual information RNA (sgRNA) to precede a 5-NGG PAM area to cleave the mark (Supplementary Fig.?S3a) and cloned the sgRNA within a plasmid that contained CRISPR/Cas9 (Supplementary Fig.?S3b). The fix template utilized was a single-stranded DNA oligonucleotide (ssODN) formulated with the KCNH2 one nucleotide mutation (Supplementary Fig.?S3c). The plasmid as well as YZ9 the ssODN were nucleofected into the CTRL-iPSC and puromycin-resistant colonies were isolated manually (Supplementary Fig.?S1b). Homologous recombination in homozygosis was confirmed by DNA sequencing of one clone (Supplementary Fig.?S1d). The clone maintained its normal karyotype (46 XY) (Supplementary Fig.?S1c) after homologous recombination. Cells expressed pluripotency markers (Supplementary Fig.?S1e and S2a) and differentiated spontaneously into the three YZ9 embryonic germ layers (Supplementary Fig.?S2b). We observed characteristic nuclear staining for OCT4, SOX2 and NANOG and cytoplasmic staining for LIN28, TRA1-60 and TRA1-81 in all of our iPSC lines (Supplementary Fig.?S2a). Spontaneous differentiation resulted in the expression of Nestin (ectoderm), Brachyury (mesoderm) and alpha-fetoprotein (AFP, endoderm), providing additional evidence of YZ9 pluripotency (Supplementary Fig.?S2b). LQTS2 cardiomyocytes exhibit prolonged action potential duration After confirming that iPSC lines were pluripotent, they were submitted to cardiac differentiation (Fig.?1a). On day 7, we observed the first beating areas. Cells were cultured for 30 days before electrophysiology experiments. Open in a separate windows Physique 1 Differentiation and electrophysiology of iPSC-derived cardiomyocytes. (a) Schematic diagram demonstrating the main steps of the differentiation procedure. (b) Representative action potential recordings of spontaneously contracting ventricular-like cardiomyocytes. Note the red line that marks the end of phase 3 for CTRL-iPSC and the green line that marks the end of phase 3 for LQTS2-iPSC1 and LQTS2-iPSC2. (c,d) Our analysis demonstrates that action potential duration of LQTS2-iPSC1, 2 and CRISPR was significantly longer than that of CTRL-iPSC, as.