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      Differential Sarcomere and Electrophysiological Maturation of Human iPSC-Derived Cardiac Myocytes in Monolayer vs. Aggregation-Based Differentiation Protocols

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          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.

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          Patient-specific induced pluripotent stem cells as a model for familial dilated cardiomyopathy.

          Ning Sun, Masayuki Yazawa, Jianwei Liu … (2012)
          Characterized by ventricular dilatation, systolic dysfunction, and progressive heart failure, dilated cardiomyopathy (DCM) is the most common form of cardiomyopathy in patients. DCM is the most common diagnosis leading to heart transplantation and places a significant burden on healthcare worldwide. The advent of induced pluripotent stem cells (iPSCs) offers an exceptional opportunity for creating disease-specific cellular models, investigating underlying mechanisms, and optimizing therapy. Here, we generated cardiomyocytes from iPSCs derived from patients in a DCM family carrying a point mutation (R173W) in the gene encoding sarcomeric protein cardiac troponin T. Compared to control healthy individuals in the same family cohort, cardiomyocytes derived from iPSCs from DCM patients exhibited altered regulation of calcium ion (Ca(2+)), decreased contractility, and abnormal distribution of sarcomeric α-actinin. When stimulated with a β-adrenergic agonist, DCM iPSC-derived cardiomyocytes showed characteristics of cellular stress such as reduced beating rates, compromised contraction, and a greater number of cells with abnormal sarcomeric α-actinin distribution. Treatment with β-adrenergic blockers or overexpression of sarcoplasmic reticulum Ca(2+) adenosine triphosphatase (Serca2a) improved the function of iPSC-derived cardiomyocytes from DCM patients. Thus, iPSC-derived cardiomyocytes from DCM patients recapitulate to some extent the morphological and functional phenotypes of DCM and may serve as a useful platform for exploring disease mechanisms and for drug screening.
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            Patient-specific induced pluripotent stem cell derived models of LEOPARD syndrome

            Xonia Carvajal-Vergara, Ana Sevilla, Sunita L. D’Souza … (2010)
            Generation of reprogrammed induced pluripotent stem cells (iPSC) from patients with defined genetic disorders promises important avenues to understand the etiologies of complex diseases, and the development of novel therapeutic interventions. We have generated iPSC from patients with LEOPARD syndrome (LS; acronym of its main features: Lentigines, Electrocardiographic abnormalities, Ocular hypertelorism, Pulmonary valve stenosis, Abnormal genitalia, Retardation of growth and Deafness), an autosomal dominant developmental disorder belonging to a relatively prevalent class of inherited RAS-MAPK signaling diseases, which also includes Noonan syndrome (NS), with pleiomorphic effects on several tissues and organ systems1,2. The patient-derived cells have a mutation in the PTPN11 gene, which encodes the SHP2 phosphatase. The iPSC have been extensively characterized and produce multiple differentiated cell lineages. A major disease phenotype in patients with LEOPARD syndrome is hypertrophic cardiomyopathy. We show that in vitro-derived cardiomyocytes from LS-iPSC are larger, have a higher degree of sarcomeric organization and preferential localization of NFATc4 in the nucleus when compared to cardiomyocytes derived from human embryonic stem cells (HESC) or wild type (wt) iPSC derived from a healthy brother of one of the LS patients. These features correlate with a potential hypertrophic state. We also provide molecular insights into signaling pathways that may promote the disease phenotype.
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              Biphasic role for Wnt/beta-catenin signaling in cardiac specification in zebrafish and embryonic stem cells.

              Edward Golob, Gilbert Weidinger, Shuichi Ueno … (2007)
              Understanding pathways controlling cardiac development may offer insights that are useful for stem cell-based cardiac repair. Developmental studies indicate that the Wnt/beta-catenin pathway negatively regulates cardiac differentiation, whereas studies with pluripotent embryonal carcinoma cells suggest that this pathway promotes cardiogenesis. This apparent contradiction led us to hypothesize that Wnt/beta-catenin signaling acts biphasically, either promoting or inhibiting cardiogenesis depending on timing. We used inducible promoters to activate or repress Wnt/beta-catenin signaling in zebrafish embryos at different times of development. We found that Wnt/beta-catenin signaling before gastrulation promotes cardiac differentiation, whereas signaling during gastrulation inhibits heart formation. Early treatment of differentiating mouse embryonic stem (ES) cells with Wnt-3A stimulates mesoderm induction, activates a feedback loop that subsequently represses the Wnt pathway, and increases cardiac differentiation. Conversely, late activation of beta-catenin signaling reduces cardiac differentiation in ES cells. Finally, constitutive overexpression of the beta-catenin-independent ligand Wnt-11 increases cardiogenesis in differentiating mouse ES cells. Thus, Wnt/beta-catenin signaling promotes cardiac differentiation at early developmental stages and inhibits it later. Control of this pathway may promote derivation of cardiomyocytes for basic research and cell therapy applications.
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                Author and article information

                Contributors
                Maurizio Muraca: Role: Academic Editor
                Journal
                Journal ID (nlm-ta): Int J Mol Sci
                Journal ID (iso-abbrev): Int J Mol Sci
                Journal ID (publisher-id): ijms
                Title: International Journal of Molecular Sciences
                Publisher: MDPI
                ISSN (Electronic): 1422-0067
                Publication date (Electronic): 01 June 2017
                Publication date Collection: June 2017
                Volume: 18
                Issue: 6
                Electronic Location Identifier: 1173
                Affiliations
                [1 ]Sorbonne Universités, UPMC Univ Paris 06, AP-HP, INSERM, Pitié-Salpêtrière Hospital, F-75013 Paris, France; d.jeziorowska@ 123456ican-institute.org (D.J.); v.fontaine@ 123456ican-institute.org (V.F.); c.jouve@ 123456ican-institute.org (C.J.); eric.villard@ 123456upmc.fr (E.V.); sebastien.dussaud@ 123456upmc.fr (S.D.)
                [2 ]Institute of Cardiometabolism and Nutrition (ICAN), F-75013 Paris, France
                [3 ]Institut du Cerveau et de la Moelle épinière, ICM, CNRS UMR 7225, Inserm U 1127, UPMC-P6 UMR S 1127, Plateforme d’exploration cellulaire, CELIS-Culture Cellulaire, F-75013 Paris, France; d.akbar-ihu@ 123456icm-institute.org
                [4 ]Sanofi Recherche et Développement, F-91380 Chilly-Mazarin, France; valerie.letang@ 123456sanofi.com (V.L.); pauline.cervello@ 123456sanofi.com (P.C.); marie-pierre.pruniaux@ 123456sanofi.com (M.-P.P.)
                [5 ]Sanofi Recherche et Développement, F-94403 Vitry, France; jean-michel.itier@ 123456sanofi.com
                Author notes
                [* ]Correspondence: Jean-sebastien.hulot@ 123456upmc.fr ; Tel.: +33-1-40-77-96-81; Fax: +33-1-40-77-95-84
                Article
                Publisher ID: ijms-18-01173
                DOI: 10.3390/ijms18061173
                PMC ID: 5485997
                PubMed ID: 28587156
                SO-VID: 1b7fec8f-ee40-415e-b45a-c38ff4fbe740
                Copyright © © 2017 by the authors.
                License:

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                Date received : 20 April 2017
                Date accepted : 26 May 2017
                Categories
                Subject: Article

                ScienceOpen disciplines: Molecular biology
                Keywords: induced pluripotent stem cells,differentiation,cardiomyocytes,sarcomere,cardiomyopathies
                Data availability:
                ScienceOpen disciplines: Molecular biology
                Keywords: induced pluripotent stem cells, differentiation, cardiomyocytes, sarcomere, cardiomyopathies

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