Morpho-functional comparison of differentiation protocols to create iPSC-derived cardiomyocytes
Year: 2022
Type: article
Abstract: Cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs) offer an attractive platform for cardiovascular research. Patient-specific iPSC-CMs are very useful for studying disease development, and bear potential for disease diagnostics, prognosis evaluation and development of personalized treatment. Several monolayer-based serum-free protocols have been described for the differentiation of iPSCs into cardiomyocytes, but data on their performance are scarce. In this study, we evaluated two protocols that are based on temporal modulation of the Wnt/β-catenin pathway for iPSC-CM differentiation from four iPSC lines, including two control individuals and two patients carrying an SCN5A mutation. The SCN5A gene encodes the cardiac voltage-gated sodium channel (Nav1.5) and loss-of-function mutations can cause the cardiac arrhythmia Brugada syndrome. We performed molecular characterization of the obtained iPSC-CMs by immunostaining for cardiac specific markers and by expression analysis of selected cardiac structural and ionic channel protein-encoding genes with qPCR. We also investigated cell growth morphology, contractility and survival of the iPSC-CMs after dissociation. Finally, we performed electrophysiological characterization of the cells, focusing on the action potential (AP) and calcium transient (CT) characteristics using patch-clamping and optical imaging, respectively. Based on our comprehensive morpho-functional analysis, we concluded that both tested protocols result in a high percentage of contracting CMs. Moreover, they showed acceptable survival and cell quality after dissociation (>50% of cells with a smooth cell membrane, possible to seal during patch-clamping). Both protocols generated cells presenting with typical iPSC-CM AP and CT characteristics, although one protocol (that involves sequential addition of CHIR99021 and Wnt-C59) rendered iPSC-CMs, which were more accessible for patch-clamp and calcium transient experiments and showed an expression pattern of cardiac-specific markers more similar to this observed in human heart left ventricle samples.
Source: Biology Open
DOI: 10.1242/bio.059016
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $1,995
Awards: Optical mapping of in vivo cardiac mechanics in zebrafish: exploring the pathogenesis and mode of inheritance in catecholaminergic polymorphic ventricular tachycardia., Functional assessment and therapeutic targeting of a novel aortopathy syndrome caused by recessive IPO8 mutations, G1708367646, Genomics and innovative induced pluripotent stem cell (iPSC) modeling to improve understanding of pathomechanisms underlying Brugada syndrome (BrS)., Elucidating the pathogenicity of genetic variants of uncertain significance in Brugada syndrome patients by functional modelling in hiPSC-derived cardiomyocytes and zebrafish, Identification of novel therapeutic targets for Brugada Syndrome through discovery and characterization of genetic modifiers., G5559060178, Molecular exploration of a new aortopathy syndrome with strong potential to inform the pathogenesis and treatment of heritable thoracic aortic aneurysm, G5970416178, G6127554328, G6290902372, Pathomechanistic study of biglycan mutations in aortopathy and skeletal dysplasia, G7763247486, Genome-wide Epistasis for cardiovascular severity in Marfan Study
OpenAlex ID: W4213422772