Generation of bi-allelic MYBPC3 truncating mutant and isogenic control from an iPSC line of a patient with hypertrophic cardiomyopathy

Chronic activation of tubulin tyrosination in HCM mice and in human iPSC-engineered heart tissues improves heart function

“…To translate our mouse data to humans, we used a HCM patient-derived heterozygous mutant MYBPC3 (MYBPC3het; UKEi070-A) and its isogenic control (MYBPC3ic; UKEi070-A-1) hiPSC lines that were previously created. 12 MYBPC3het carries the c.2308G>A genetic variant (p.Asp770Serfs98X), resulting in MYBPC3 protein haploinsufficiency in cardiomyocytes. 12 We first evaluated the dose-response of AAV9-TTL in MYBPC3ic hiPSC-derived cardiomyocytes and found that a MOI of 100,000 gave rise to 19-fold higher TTL protein level than in non-transduced or AAV9-Empty-transduced cardiomyocytes (Figure 6A,B).…”

“…12 MYBPC3het carries the c.2308G>A genetic variant (p.Asp770Serfs98X), resulting in MYBPC3 protein haploinsufficiency in cardiomyocytes. 12 We first evaluated the dose-response of AAV9-TTL in MYBPC3ic hiPSC-derived cardiomyocytes and found that a MOI of 100,000 gave rise to 19-fold higher TTL protein level than in non-transduced or AAV9-Empty-transduced cardiomyocytes (Figure 6A,B). We then evaluated the impact of a 10-day gene transfer of AAV9-TTL and AAV9-Empty (MOI 100,000) in both genotypes in the presence of H 2 O (Ctrl) or 100 nM endothelin-1 (ET1) for the last 3 days (Figure 6C-H), as previously described.…”

“…The HCM patient carrying the MYBPC3 (c.2308G>A; p.Asp770Serfs98X) mutation was recruited in the outpatient HCM clinic at the University Heart and Vascular Center Hamburg and provided written informed consent for genetic analysis and the use of skin fibroblasts 12 . All procedures were in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki).…”

“…[22][23][24] We also used a patient-derived heterozygous MYBPC3 (MYBPC3het; UKEi070-A) and its isogenic control (MYBPC3ic; UKEi070-A-1) hiPSC lines that were previously created. 12 MYBPC3het and MYBPC3ic hiPSC-cardiomyocytes (3-4 batches of differentiation of triplicates) were transduced with AAV9-TTL or AAV9-Empty (MOI 100,000) for 10 days and treated with 100 nM endothelin-1 (ET1) or vehicle (H 2 O) the last 3 days as previously described. 25 Cardiomyocytes were then stained for -actinin (Sigma, SAB2108642, 1:800) and N-cadherin (Sigma, C2542, 1:500), followed by the secondary antibodies Alexa Fluor TM 488 (mouse) and Alexa Fluor TM 546 (rabbit) (Invitrogen, A11001; A11035, 1:800), and cell area was analysed by confocal microscopy and ImageJ.…”

Susceptibility to innate immune activation in genetically mediated myocarditis

“…DSP +/+ and DSP –/– cell lines were generated from human skin fibroblasts (Coriell, sample GM03348) that were reprogrammed using electroporation with pCXLE-hOCT3/4-shp53-F (Addgene plasmid 27077), pCXLE-hSK (Addgene plasmid 27078), and pCXLE-hUL (Addgene plasmid 27080) as described previously ( 62 ). Successful reprogramming was assessed via cell morphology, ability to differentiate, and purity as measured with SSEA3 staining (BD, catalog 560879) via flow cytometry ( 63 ). HiPSCs were maintained in mTeSR-1 (STEMCELL Technologies, catalog 85850) and mTeSR-Plus (STEMCELL Technologies, 100-0276) on Matrigel-coated, 6-well plates (Corning, catalog 354230).…”