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Patient-specific induced pluripotent stem-cell-derived models of LEOPARD syndrome.

ABSTRACT: The generation of reprogrammed induced pluripotent stem cells (iPSCs) from patients with defined genetic disorders holds the promise of increased understanding of the aetiologies of complex diseases and may also facilitate the development of novel therapeutic interventions. We have generated iPSCs from patients with LEOPARD syndrome (an acronym formed from its main features; that is, 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-mitogen-activated protein kinase signalling diseases, which also includes Noonan syndrome, with pleomorphic effects on several tissues and organ systems. The patient-derived cells have a mutation in the PTPN11 gene, which encodes the SHP2 phosphatase. The iPSCs 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 LEOPARD syndrome iPSCs are larger, have a higher degree of sarcomeric organization and preferential localization of NFATC4 in the nucleus when compared with cardiomyocytes derived from human embryonic stem cells or wild-type iPSCs derived from a healthy brother of one of the LEOPARD syndrome patients. These features correlate with a potential hypertrophic state. We also provide molecular insights into signalling pathways that may promote the disease phenotype.

SUBMITTER: Carvajal-Vergara X

PROVIDER: S-EPMC2885001 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

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Patient-specific induced pluripotent stem-cell-derived models of LEOPARD syndrome.

Carvajal-Vergara Xonia X Sevilla Ana A D'Souza Sunita L SL Ang Yen-Sin YS Schaniel Christoph C Lee Dung-Fang DF Yang Lei L Kaplan Aaron D AD Adler Eric D ED Rozov Roye R Ge Yongchao Y Cohen Ninette N Edelmann Lisa J LJ Chang Betty B Waghray Avinash A Su Jie J Pardo Sherly S Lichtenbelt Klaske D KD Tartaglia Marco M Gelb Bruce D BD Lemischka Ihor R IR

Nature 20100601 7299

The generation of reprogrammed induced pluripotent stem cells (iPSCs) from patients with defined genetic disorders holds the promise of increased understanding of the aetiologies of complex diseases and may also facilitate the development of novel therapeutic interventions. We have generated iPSCs from patients with LEOPARD syndrome (an acronym formed from its main features; that is, lentigines, electrocardiographic abnormalities, ocular hypertelorism, pulmonary valve stenosis, abnormal genitali ...[more]

PMID: 20535210

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Project description:BackgroundWhile mutations in the cardiac type 2 ryanodine receptor (RyR2) have been linked to exercise-induced or catecholaminergic polymorphic ventricular tachycardia (CPVT), its association with polymorphic ventricular tachycardia (PMVT) occurring at rest is unclear. We aimed at constructing a patient-specific human-induced pluripotent stem cell (hiPSC) model of PMVT occurring at rest linked to a single point mutation in RyR2.MethodsBlood samples were obtained from a patient with PMVT at rest due to a heterozygous RyR2-H29D mutation. Patient-specific hiPSCs were generated from the blood samples, and the hiPSC-derived cardiomyocytes (CMs) were generated via directed differentiation. Using CRIPSR/Cas9 technology, isogenic controls were generated by correcting the RyR2-H29D mutation. Using patch-clamp, fluorescent confocal microscopy and video-image-based analysis, the molecular and functional properties of RyR2-H29D hiPSCCMs and control hiPSCCMs were compared.FindingsRyR2-H29D hiPSCCMs exhibit intracellular sarcoplasmic reticulum (SR) Ca2+ leak through RyR2 under physiological pacing. RyR2-H29D enhances the contribution of inositol 1,4,5-trisphosphate receptors to excitation-contraction coupling (ECC) that exacerbates abnormal Ca2+ release in RyR2-H29D hiPSCCMs. RyR2-H29D hiPSCCMs exhibit shorter action potentials, delayed afterdepolarizations, arrhythmias and aberrant contractile properties compared to isogenic controls. The RyR2-H29D mutation causes post-translational remodeling that is fully reversed with isogenic controls.InterpretationTo conclude, in a model based on a RyR2 point mutation that is associated with short-coupled PMVT at rest, RyR2-H29D hiPSCCMs exhibited aberrant intracellular Ca2+ homeostasis, shortened action potentials, arrhythmias and abnormal contractile properties.FundingFrench Muscular Dystrophy Association (AFM; project 16,073, MNM2 2012 and 20,225), "Fondation de la Recherche Médicale" (FRM; SPF20130526710), "Institut National pour la Santé et la Recherche Médicale" (INSERM), National Institutes of Health (ARM; R01 HL145473) and New York State Department of Health (NYSTEM C029156).

Project description:Background Optogenetics, using light-sensitive proteins, emerged as a unique experimental paradigm to modulate cardiac excitability. We aimed to develop high-resolution optogenetic approaches to modulate electrical activity in 2- and 3-dimensional cardiac tissue models derived from human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes. Methods and Results To establish light-controllable cardiac tissue models, opsin-carrying HEK293 cells, expressing the light-sensitive cationic-channel CoChR, were mixed with hiPSC-cardiomyocytes to generate 2-dimensional hiPSC-derived cardiac cell-sheets or 3-dimensional engineered heart tissues. Complex illumination patterns were designed with a high-resolution digital micro-mirror device. Optical mapping and force measurements were used to evaluate the tissues' electromechanical properties. The ability to optogenetically pace and shape the tissue's conduction properties was demonstrated by using single or multiple illumination stimulation sites, complex illumination patterns, or diffuse illumination. This allowed to establish in vitro models for optogenetic-based cardiac resynchronization therapy, where the electrical activation could be synchronized (hiPSC-derived cardiac cell-sheets and engineered heart tissue models) and contractile properties improved (engineered heart tissues). Next, reentrant activity (rotors) was induced in the hiPSC-derived cardiac cell-sheets and engineered heart tissue models through optogenetics programmed- or cross-field stimulations. Diffuse illumination protocols were then used to terminate arrhythmias, demonstrating the potential to study optogenetics cardioversion mechanisms and to identify optimal illumination parameters for arrhythmia termination. Conclusions By combining optogenetics and hiPSC technologies, light-controllable human cardiac tissue models could be established, in which tissue excitability can be modulated in a functional, reversible, and localized manner. This approach may bring a unique value for physiological/pathophysiological studies, for disease modeling, and for developing optogenetic-based cardiac pacing, resynchronization, and defibrillation approaches.

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Patient-specific induced pluripotent stem-cell-derived models of LEOPARD syndrome.

Publications

Patient-specific induced pluripotent stem-cell-derived models of LEOPARD syndrome.

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