Abstract
Human induced pluripotent stem cells (iPSCs) represent a powerful human model to study cardiac disease in vitro, notably channelopathies and sarcomeric cardiomyopathies. Different protocols for cardiac differentiation of iPSCs have been proposed either based on embroid body formation (3D) or, more recently, on monolayer culture (2D). We performed a direct comparison of the characteristics of the derived cardiomyocytes (iPSC-CMs) on day 27 ± 2 of differentiation between 3D and 2D differentiation protocols with two different Wnt-inhibitors were compared: IWR1 (inhibitor of Wnt response) or IWP2 (inhibitor of Wnt production). We firstly found that the level of Troponin T (TNNT2) expression measured by FACS was significantly higher for both 2D protocols as compared to the 3D protocol. In the three methods, iPSC-CM show sarcomeric structures. However, iPSC-CM generated in 2D protocols constantly displayed larger sarcomere lengths as compared to the 3D protocol. In addition, mRNA and protein analyses reveal higher cTNi to ssTNi ratios in the 2D protocol using IWP2 as compared to both other protocols, indicating a higher sarcomeric maturation. Differentiation of cardiac myocytes with 2D monolayer-based protocols and the use of IWP2 allows the production of higher yield of cardiac myocytes that have more suitable characteristics to study sarcomeric cardiomyopathies.
Highlights
The ability to generate human induced pluripotent stem cells from patients and the capacity to differentiate these iPSCs into disease-relevant cell types represent a breakthrough for human diseases modeling, preclinical evaluations and drug discovery
We investigated three differentiation protocols that differed in terms of architectural and pharmacological environment (Figure 1A) in a total of four different iPSC clones (Figure S1)
As we found significant differences in sarcomere formation and maturation, we asked whether differentiation protocols influenced the electrophysiogical characteristics of generated cardiomyocytes
Summary
The ability to generate human induced pluripotent stem cells (hiPSC) from patients and the capacity to differentiate these iPSCs into disease-relevant cell types represent a breakthrough for human diseases modeling, preclinical evaluations and drug discovery. In the field of cardiology, the hiPSC technology has been successfully used to model a number of inherited heart diseases, including monogenic channelopathies (notably long QT syndrome [1,2], drug-induced long QT [3] and catecholaminergic polymorphic tachycardia [4]), cardiomyopathies due to mutations in mitochondrial (Friedreich’s ataxia [5]) or desmosomal proteins (ARVC [6,7]) and other rare genetic disorders (LEOPARD [8], pompe disease [9], laminopathie s [10]). Monolayer-based methods, in which cells are seeded and differentiated on a culture substrate, have been developed as they achieve higher efficiency in terms of quantity of generated cardiomyocytes [20,21]. Some small molecules (such as IWR1) block the canonical Wnt β-catenin signaling pathway while others (such as IWP2) block the Wnt-mediated mechanisms irrespective of pathway mechanisms
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