CRISPR/Cas9-mediated engineering of subtype-specific cell lines for iPSC-derived cardiomyocyte phenotyping

Thesis

Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) represent a useful tool for cardiovascular research. However, the mixed subtypes, including atrial-like, ventricular-like and nodal-like, that result from differentiation protocols present a challenge for selective studies. This thesis exploited the emerging genome engineering technology “CRISPR/Cas9” to generate subtype-specific fluorescent reporter systems to label specific CM subtypes, facilitating subsequent isolation for use in subtype-specific disease modelling.

Atrial-specific myosin light chain 7 (MYL7)-mClover and pituitary homeobox 2 (PITX2)-mClover, ventricular-specific myosin light chain 2 (MYL2)-mClover and nodal-specific hyperpolarisation activated cyclic nucleotide-gated potassium channel 4 (HCN4)-mScarlet iPSCs were established by targeted insertion of the fluorophores at the stop codons of the target genes. Each fluorophore was preceded by a P2A linker, allowing bicistronic expression. Unfortunately, the PITX2-mClover and MYL2-mClover lines failed quality control and could not be used for fluorescent reporter iPSC-CM derivation. The MYL7-mClover and HCN4-mScarlet lines, however, exhibited faithful reporter systems where the expression of the fluorophores mirrored that of their respective target genes.

Heterozygous TTN c.59926+1 G>A, a truncating mutation of the titin gene, is associated with atrial fibrillation and dilated cardiomyopathy. This mutation was introduced into parental iPSCs for preliminary characterisations. Subsequently, the mutation was engineered in the MYL7-mClover iPSCs for exploring the application of subtype-specific reporter systems in disease studies. The fluorescent sorting of the cells could not be optimised in the available time, however, the engineered mutant MYL7-mClover line was differentiated towards the atrial and the ventricular lineages.

No prominent consequences in heart failure-associated foetal gene reprogramming, overall TTN expression, myofibrillar protein isoform switching, sarcomere organisation, sarcomeric protein localisation were identified in the heterozygous mutant model. Interestingly, the Ca2+ transients were altered specifically in the atrial-like heterozygous model. This alteration may underlie irregular, fast atrial rhythm in atrial fibrillation. The atrial-specific phenotype of TTN c.59926+1 G>A may be further clarified when using purer atrial-like iPSC-CMs obtainable by fluorescence-activated cell sorting of the differentiated MYL7-mClover iPSC-CMs.

Authors: Sontayananon, N

• DOI: 10.5287/ora-rbywgxjvn
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: selective disease modelling; iPSC-CM subtype purification; iPSC-CM population's heterogeneity; TTNtv; CRISPR/Cas9-assisted genome editing; fluorescent reporter line generation
• Subjects: Gene editing; Cardiovascular system--Diseases