Vittavat Termglinchan

Human Induced Pluripotent Stem Cell–Derived Cardiomyocytes Insights Into Molecular, Cellular, and Functional Phenotypes

Disease models are essential for understanding cardiovascular disease pathogenesis and developing new therapeutics. The human induced pluripotent stem cell (iPSC) technology has generated significant enthusiasm for its potential application in basic and translational cardiac research. Patient-specific iPSC-derived cardiomyocytes offer an attractive experimental platform to model cardiovascular diseases, study the earliest stages of human development, accelerate predictive drug toxicology tests, and advance potential regenerative therapies. Harnessing the power of iPSC-derived cardiomyocytes could eliminate confounding species-specific and interpersonal variations and ultimately pave the way for the development of personalized medicine for cardiovascular diseases. However, the predictive power of iPSC-derived cardiomyocytes as a valuable model is contingent on comprehensive and rigorous molecular and functional characterization.

Correction of human phospholamban R14del mutation associated with cardiomyopathy using targeted nucleases and combination therapy

A number of genetic mutations is associated with cardiomyopathies. A mutation in the coding region of the phospholamban (PLN) gene (R14del) is identified in families with hereditary heart failure. Heterozygous patients exhibit left ventricular dilation and ventricular arrhythmias. Here we generate induced pluripotent stem cells (iPSCs) from a patient harbouring the PLN R14del mutation and differentiate them into cardiomyocytes (iPSC-CMs). We find that the PLN R14del mutation induces Ca2+ handling abnormalities, electrical instability, abnormal cytoplasmic distribution of PLN protein and increases expression of molecular markers of cardiac hypertrophy in iPSC-CMs. Gene correction using transcription activator-like effector nucleases (TALENs) ameliorates the R14del-associated disease phenotypes in iPSC-CMs. In addition, we show that knocking down the endogenous PLN and simultaneously expressing a codon-optimized PLN gene reverses the disease phenotype in vitro. Our findings offer novel strategies for targeting the pathogenic mutations associated with cardiomyopathies.

Human-induced pluripotent stem cell models of inherited cardiomyopathies.

Purpose of review This article provides an overview of the latest advances in in-vitro modeling of inherited cardiomyopathies using human-induced pluripotent stem cells (iPSCs). Recent findings Inherited cardiomyopathies have been recently modeled by generating iPSCs from patients harboring mutations in genes associated with the pathogenesis of hypertrophic cardiomyopathy, dilated cardiomyopathy, and arrhythmogenic right ventricular cardiomyopathy/dysplasia. Summary Patient-specific iPSCs and their differentiated cardiomyocytes (induced pluripotent stem cell-derived cardiomyocytes) now provide a novel model to study the underlying molecular mechanism of the pathogenesis of familial cardiomyopathies as well as for in-vitro drug screening and drug discovery.

A Comprehensive TALEN-Based Knockout Library for Generating Human-Induced Pluripotent Stem Cell–Based Models for Cardiovascular Diseases

Rationale: Targeted genetic engineering using programmable nucleases such as transcription activator–like effector nucleases (TALENs) is a valuable tool for precise, site-specific genetic modification in the human genome. Objective: The emergence of novel technologies such as human induced pluripotent stem cells (iPSCs) and nuclease-mediated genome editing represent a unique opportunity for studying cardiovascular diseases in vitro. Methods and Results: By incorporating extensive literature and database searches, we designed a collection of TALEN constructs to knockout 88 human genes that are associated with cardiomyopathies and congenital heart diseases. The TALEN pairs were designed to induce double-strand DNA break near the starting codon of each gene that either disrupted the start codon or introduced a frameshift mutation in the early coding region, ensuring faithful gene knockout. We observed that all the constructs were active and disrupted the target locus at high frequencies. To illustrate the utility of the TALEN–mediated knockout technique, 6 individual genes (TNNT2, LMNA/C, TBX5, MYH7, ANKRD1, and NKX2.5) were knocked out with high efficiency and specificity in human iPSCs. By selectively targeting a pathogenic mutation (TNNT2 p.R173W) in patient-specific iPSC-derived cardiac myocytes, we demonstrated that the knockout strategy ameliorates the dilated cardiomyopathy phenotype in vitro. In addition, we modeled the Holt–Oram syndrome in iPSC-cardiac myocytes in vitro and uncovered novel pathways regulated by TBX5 in human cardiac myocyte development. Conclusions: Collectively, our study illustrates the powerful combination of iPSCs and genome editing technologies for understanding the biological function of genes, and the pathological significance of genetic variants in human cardiovascular diseases. The methods, strategies, constructs, and iPSC lines developed in this study provide a validated, readily available resource for cardiovascular research.

Concise Review: Mending a Broken Heart: The Evolution of Biological Therapeutics

Heart failure (HF), a common sequela of cardiovascular diseases, remains a staggering clinical problem, associated with high rates of morbidity and mortality worldwide. Advances in pharmacological, interventional, and operative management have improved patient care, but these interventions are insufficient to halt the progression of HF, particularly the end‐stage irreversible loss of functional cardiomyocytes. Innovative therapies that could prevent HF progression and improve the function of the failing heart are urgently needed. Following successful preclinical studies, two main strategies have emerged as potential solutions: cardiac gene therapy and cardiac regeneration through stem and precursor cell transplantation. Many potential gene‐ and cell‐based therapies have entered into clinical studies, intending to ameliorate cardiac dysfunction in patients with advanced HF. In this review, we focus on the recent advances in cell‐ and gene‐based therapies in the context of cardiovascular disease, emphasizing the most advanced therapies. The principles and mechanisms of action of gene and cell therapies for HF are discussed along with the limitations of current approaches. Finally, we highlight the emerging technologies that hold promise to revolutionize the biological therapies for cardiovascular diseases. Stem Cells 2017;35:1131–1140

Current Status of Genome Editing in Cardiovascular Medicine

During the past decades, numerous genetic mutations have been implicated in the pathogenesis of cardiovascular diseases (CVDs). With the launching of the Precision Medicine Initiative in January 2015, emerging technologies such as induced pluripotent stem cells (iPSCs) and genome editing are well positioned to provide powerful tools to correlate genotypes with phenotypes. These new technologies are helping to identify specific mutations associated with human CVDs. Patient-specific iPSC-derived cardiomyocytes (iPSC-CMs) offer an exciting experimental platform to model CVDs, study the molecular basis of cardiovascular biology, accelerate predictive drug toxicology tests, and advance potential regenerative therapies. By harnessing the power of genome engineering, scientists are uncovering the molecular mechanisms underlying the pathogenesis of CVDs, which will pave the way for the development of new personalized prediction, prevention, and treatment of diseases.

Telomere shortening is a hallmark of genetic cardiomyopathies

Significance We find that telomere shortening, which usually accompanies cell division in the course of aging, occurs in cardiomyocytes (CMs) of individuals with genetic hypertrophic cardiomyopathy (HCM) or dilated cardiomyopathy (DCM). HCM and DCM CMs differentiated from human-induced pluripotent stem cells (hiPSCs) also exhibit significant telomere shortening relative to healthy controls. By contrast, no telomere shortening was detected in vascular smooth muscle cells in tissue or hiPSC-derived cells, a cell type that does not express the mutant proteins. Our findings provide evidence for accelerated aging in CMs with familial cardiomyopathy. The potential to monitor the dynamics of telomere attrition in hiPSC-CMs over time will enable future mechanistic studies and screens for novel therapeutic agents to arrest telomere shortening and disease progression. This study demonstrates that significantly shortened telomeres are a hallmark of cardiomyocytes (CMs) from individuals with end-stage hypertrophic cardiomyopathy (HCM) or dilated cardiomyopathy (DCM) as a result of heritable defects in cardiac proteins critical to contractile function. Positioned at the ends of chromosomes, telomeres are DNA repeats that serve as protective caps that shorten with each cell division, a marker of aging. CMs are a known exception in which telomeres remain relatively stable throughout life in healthy individuals. We found that, relative to healthy controls, telomeres are significantly shorter in CMs of genetic HCM and DCM patient tissues harboring pathogenic mutations: TNNI3, MYBPC3, MYH7, DMD, TNNT2, and TTN. Quantitative FISH (Q-FISH) of single cells revealed that telomeres were significantly reduced by 26% in HCM and 40% in DCM patient CMs in fixed tissue sections compared with CMs from age- and sex-matched healthy controls. In the cardiac tissues of the same patients, telomere shortening was not evident in vascular smooth muscle cells that do not express or require the contractile proteins, an important control. Telomere shortening was recapitulated in DCM and HCM CMs differentiated from patient-derived human-induced pluripotent stem cells (hiPSCs) measured by two independent assays. This study reveals telomere shortening as a hallmark of genetic HCM and DCM and demonstrates that this shortening can be modeled in vitro by using the hiPSC platform, enabling drug discovery.

Recent advances in cardiac gene therapy strategies targeting advanced heart failure

Heart failure (HF) is the common sequela of many cardiovascular diseases. Although device‐based treatments and pharmacotherapies have improved patient survival, current standard therapies are far from optimal, and do not address the molecular mechanisms underlying HF pathogenesis. In the last decade, cardiac gene therapy has emerged as an innovative experimental therapeutic strategy for the treatment of advanced HF. Increasing insights into the molecular pathophysiology and improvements in gene targeting approaches provide novel therapeutic modalities. In this review we focus on the recent developments in cardiac gene therapy strategies targeting advanced HF. We emphasize ongoing clinical trials, and discuss the limitations and future evolution of the field.