CRISPR/Cas9-mediated generation of cardiomyocytes derived from human induced pluripotent stem cells to model SCN5A deficiency - 18/05/21
Résumé |
Introduction |
The cardiac voltage-gated sodium channel α-subunit (Nav1.5), encoded by SCN5A, is a key determinant of cardiac excitability. Variants in SCN5A have been clearly identified as responsible for cardiac arrythmias, highlighting its critical role in cardiac physiology and heart diseases, such as Lev's disease or Brugada syndrome, two rare inherited cardiac arrhythmias associated with SCN5A loss-of-function mutations.
Aim |
Here, we aim at developing a human cellular model of SCN5A deficiency to provide a new resource for in vitro understanding of sodium channelopathies and screening of novel therapies.
Methods |
We engineered SCN5A heterozygous knockout in a commercial wild-type human induced pluripotent stem cell (hiPSC) line. Using the CRISPR/Cas9 strategy, we generated frameshifts in exon 2 of SCN5A, which resulted in premature stop codon. Edited hiPSCs were then characterized and differentiated into functional cardiomyocytes for further molecular and electrophysiological investigations. Differentiation was performed according to a published protocol based on small molecules modulation of Wnt/β-catenin pathway.
Results |
SCN5A heterozygous knockout mutations were identified in two selected clones by DNA sequencing. In parallel, conservation of genome integrity of these cell lines was assessed by m-fish karyotyping and exome sequencing. Their pluripotency was validated using immunofluorescence staining and RT-qPCR of pluripotency markers, phosphatase alkaline test and scorecard analysis. Additionally, flow cytometry and immunocytochemistry of sarcomeric cardiac markers demonstrated their ability to differentiate efficiently into cardiomyocytes.
Conclusion |
In conclusion, we have generated two lines of SCN5A+/− cardiomyocytes derived from hiPSC that could provide a valuable model to explore pathophysiology of SCN5A deficiency-related diseases and to test new therapies.
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Vol 13 - N° 2
P. 232-233 - mai 2021 Retour au numéroBienvenue sur EM-consulte, la référence des professionnels de santé.