Induced pluripotent stem cell–derived cardiomyocytes in studies of inherited arrhythmias
Silvia G. Priori, Carlo Napolitano, Elisa Di Pasquale, Gianluigi Condorelli
Silvia G. Priori, Carlo Napolitano, Elisa Di Pasquale, Gianluigi Condorelli
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Induced pluripotent stem cell–derived cardiomyocytes in studies of inherited arrhythmias

Abstract

The discovery of the genetic basis of inherited arrhythmias has paved the way for an improved understanding of arrhythmogenesis in a wide spectrum of life-threatening conditions. In vitro expression of mutations and transgenic animal models have been instrumental in enhancing this understanding, but the applicability of results to the human heart remains unknown. The ability to differentiate induced pluripotent stem cells (iPSs) into cardiomyocytes enables the potential to generate patient-specific myocytes, which could be used to recapitulate the features of inherited arrhythmias in the context of the patient’s genetic background. Few studies have been reported on iPS-derived myocytes obtained from patients with heritable arrhythmias, but they have demonstrated the applicability of this innovative approach to the study of inherited arrhythmias. Here we review the results achieved by iPS investigations in arrhythmogenic syndromes and discuss the existing challenges to be addressed before the use of iPS-derived myocytes can become a part of personalized management of inherited arrhythmias.

Authors

Silvia G. Priori, Carlo Napolitano, Elisa Di Pasquale, Gianluigi Condorelli

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Figure 1

iPS generation, characterization, and derivation of beating cardiomyocytes.

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iPS generation, characterization, and derivation of beating cardiomyocyt...
(A) iPS generation strategy. Skin fibroblasts obtained from patients are expanded and transduced with a combination of embryonic genes (Oct4, Sox2, Klf4, Nanog, Linn28, and Myc). Transduced fibroblasts are maintained in an embryonic-specific medium supplemented with bFGF until iPS colonies appear. Clones are isolated manually based on their ESC-like morphology and expanded for further characterization. (B) iPS characterization. Pluripotency marker expression (left) and developmental competence (right) should be evaluated to prove pluripotency of derived iPS lines. Analysis of the expression of typical markers of pluripotency in iPS progenitors includes expression of the “stemness” markers alkaline phosphatase, SSEA4, TRA-1-80, and OCT4 using FACS and immunofluorescence. Developmental competence is assessed both in vivo and in vitro. For in vivo analysis, teratoma formation assay is used: bona fide iPSs, when injected subcutaneously into immunodeficient mice, can give rise to a teratoma containing the three germ layers (ectoderm, mesoderm, and endoderm). In vitro, iPSs are induced to differentiate through EB aggregation and give rise to cells of all three germ layers. Neuronal (β3-tubulin positive) and intestinal (AFP positive) markers indicate differentiation to ectodermal and endodermal lineages, respectively. (C) Generation of iPS-derived beating cardiomyocytes. Cardiac differentiation is induced by aggregation of EBs. The inductive process is enhanced in the presence of specific medium and ascorbic acid. Appearance of beating areas within the EBs is indicative of cardiomyocyte differentiation; cells are dissociated into single cells, which are analyzed for cardiac markers and electrophysiology.