Altered Electrical, Biomolecular, and Immunologic Phenotypes in a Novel Patient-Derived Stem Cell Model of Desmoglein-2 Mutant ARVC

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a progressive heart condition which causes fibro-fatty myocardial scarring, ventricular arrhythmias, and sudden cardiac death. Most cases of ARVC can be linked to pathogenic mutations in the cardiac desmosome, but the pathophysiology is not well understood, particularly in early phases when arrhythmias can develop prior to structural changes. Here, we created a novel human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) model of ARVC from a patient with a c.2358delA variant in desmoglein-2 (DSG2). These DSG2-mutant (DSG2Mut) hiPSC-CMs were compared against two wildtype hiPSC-CM lines via immunostaining, RT-qPCR, Western blot, RNA-Seq, cytokine expression and optical mapping. Mutant cells expressed reduced DSG2 mRNA and had altered localization of desmoglein-2 protein alongside thinner, more disorganized myofibrils. No major changes in other desmosomal proteins were noted. There was increased pro-inflammatory cytokine expression that may be linked to canonical and non-canonical NFκB signaling. Action potentials in DSG2Mut CMs were shorter with increased upstroke heterogeneity, while time-to-peak calcium and calcium decay rate were reduced. These were accompanied by changes in ion channel and calcium handling gene expression. Lastly, suppressing DSG2 in control lines via siRNA allowed partial recapitulation of electrical anomalies noted in DSG2Mut cells. In conclusion, the aberrant cytoskeletal organization, cytokine expression, and electrophysiology found DSG2Mut hiPSC-CMs could underlie early mechanisms of disease manifestation in ARVC patients.

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Abstract 342: Serum Albumin Hydrogels Alter Excitation-Contraction Coupling in Neonatal Rat Myocytes and Human Induced Pluripotent Stem Cell Derived Cardiomyocytes

Circulation Research ◽

10.1161/res.121.suppl_1.342 ◽

2017 ◽

Vol 121 (suppl_1) ◽

Author(s):

Eleanor J Humphrey ◽

Manuel M Mazo ◽

Nadav Amdursky ◽

Nicholas S Peters ◽

Molly M Stevens ◽

...

Keyword(s):

Gene Expression ◽

Stem Cell ◽

Serum Albumin ◽

Pluripotent Stem Cell ◽

Induced Pluripotent Stem Cell ◽

Neonatal Rat ◽

Calcium Handling ◽

Time To Peak ◽

Induced Pluripotent ◽

Reduced Time

Tissue engineering provides a promising method of introducing functional cardiomyocytes (CMs) to damaged myocardium after myocardial infarction; however, finding a biocompatible construct with the chemical and mechanical properties capable of supporting CM function is challenging. Serum albumin hydrogels are novel autogenic scaffolds with elastic properties that can be tailored to mimic the stiffness of native adult myocardium. We assessed the hypothesis that culturing immature CMs on these serum albumin hydrogels would affect CM gene expression and calcium handling. Neonatal cardiomyocyte (NRVM) viability was maintained for at least 14 days on the hydrogels, with clear sarcomeric striations. Cardiac gene expression was quantified using RT-qPCR and demonstrated an up regulation in many genes of cells cultured on hydrogels compared to glass (e.g. relative expression (log 2-ΔΔCt) of ryanodine receptor 2: glass= -2.3±0.5, hydrogel= -0.3±0.1,p<0.01; connexin 43:glass= -1.7±0.5, hydrogel= 0.3±0.1,p<0.01,n=4-6). Compared to glass, NRVMs on hydrogels have an increased time to peak of the calcium transients measured using Fluo-4AM and field stimulated at 1 Hz (tp glass=38±3 ms, tp hydrogel= 54±2 ms, p<0.01,n=4-6). Compared to glass the hydrogels also have a reduced time 50% decay (t50 glass=108±13 ms, t50 hydrogel=78±6 ms, p<0.05,n=4-6) and 80% decay (t80 glass=217±19 ms, t80 hydrogel= 152±10 ms,p<0.05,n=4-6). Human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) were cultured on the hydrogels for up to 28 days. Calcium handling was faster in the iPSC-CMs cultured on the hydrogels in comparison to glass with a reduced time to peak (tp glass=281±43 ms, tp hydrogel= 186±8 ms, p<0.05, n=4) and time to 50% decay (t50 glass=269±15 ms, t50 hydrogel=204±10 ms,p<0.01,n=4) and 90% decay (t90 glass=535±33 ms, t90 hydrogel=397±19 ms, p<0.01,n=4). The serum albumin hydrogels are compatible with NRVM and iPSC-CM culture for at least 28 days. We demonstrate that the serum albumin hydrogels have significant effects on CM calcium cycling and have the potential for use in myocardial repair. Further study is required to determine the mechanisms involved in calcium handling alterations and then assess this engineered patch in vivo for cardiac repair.

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Development of a fundamental cancer therapeutic agent using an induced pluripotent stem cell derived cancer stem cell model

Impact ◽

10.21820/23987073.2017.6.93 ◽

2017 ◽

Vol 2017 (6) ◽

pp. 93-95

Author(s):

Masaharu Seno

Keyword(s):

Stem Cell ◽

Cancer Stem Cell ◽

Pluripotent Stem Cell ◽

Therapeutic Agent ◽

Cell Model ◽

Induced Pluripotent Stem Cell ◽

Stem Cell Model ◽

Induced Pluripotent ◽

Cancer Stem Cell Model

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Environmentally Relevant Developmental Methylmercury Exposures Alter Neuronal Differentiation in a Human-Induced Pluripotent Stem Cell Model

Food and Chemical Toxicology ◽

10.1016/j.fct.2021.112178 ◽

2021 ◽

pp. 112178

Author(s):

Lisa M. Prince ◽

M. Diana Neely ◽

Emily B. Warren ◽

Morgan G. Thomas ◽

Madeline R. Henley ◽

...

Keyword(s):

Stem Cell ◽

Neuronal Differentiation ◽

Pluripotent Stem Cell ◽

Cell Model ◽

Induced Pluripotent Stem Cell ◽

Stem Cell Model ◽

Induced Pluripotent

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Abstract 161: Induced Pluripotent Stem Cell-based Model of Cardiac Arrhythmia: New Platform for Drug Screen

Circulation Research ◽

10.1161/res.117.suppl_1.161 ◽

2015 ◽

Vol 117 (suppl_1) ◽

Author(s):

LouJin Song ◽

Masayuki Yazawa

Keyword(s):

Stem Cell ◽

Action Potentials ◽

Pluripotent Stem Cell ◽

Induced Pluripotent Stem Cell ◽

Calcium Handling ◽

Drug Screen ◽

Cardiac Diseases ◽

Timothy Syndrome ◽

Human Ipscs ◽

Induced Pluripotent

Human induced pluripotent stem cell (iPSC)-based model of cardiac diseases has been proved to be useful and valuable for identifying new therapeutics. However, the use of human iPSC-based model of cardiac diseases for drug screen is hampered by the high-cost and complexity of methods used for reprogramming, in vitro differentiation, and phenotyping. To address the limitations, we first optimized a protocol for reprogramming of human fibroblasts and keratinocytes into pluripotency using single lipofection and the episomal vectors in a 24-well plate format. This method allowed us to generate multiple lines of integration-free and feeder-free iPSCs from seven patients with cardiac diseases and three controls. Second, we differentiated human iPSCs derived from Timothy syndrome patients into cardiomyocytes using a monolayer differentiation method. We found that Timothy syndrome cardiomyocytes showed slower, irregular contractions and abnormal calcium handling compared to controls, which were consistent with previous reports using a retroviral method for reprogramming and using an embryoid body-based method for cardiac differentiation. Third, we developed an efficient approach for recording action potentials and calcium transients simultaneously in control and patient cardiomyocytes using genetically encoded fluorescent indicators, ArcLight and R-GECO1. The dual optical recordings enabled us to observe prolonged action potentials and abnormal calcium handling in Timothy syndrome cardiomyocytes. We confirmed that roscovitine rescued the phenotypes in Timothy syndrome cardiomyocytes and these findings were consistent with previous studies using conventional electrophysiological recordings and calcium imaging with dyes. The approaches using our optimized methods and dual optical recordings will improve iPSC applicability for disease modeling to test potential therapeutics. With those new approaches in hand, next we plan to use the iPSC-based model of Timothy syndrome to investigate novel molecules involved in the pathogenesis of Timothy syndrome and to screen and identify new therapeutic compounds for Timothy syndrome patients.

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Abstract 14908: Anatomic Validation of Induced Pluripotent Stem Cell-derived Aortic Smooth Muscle Cell Model of Loeys Dietz Syndrome

Circulation ◽

10.1161/circ.142.suppl_3.14908 ◽

2020 ◽

Vol 142 (Suppl_3) ◽

Author(s):

Albert J Pedroza ◽

Samantha Churovich ◽

Nobu Yokoyama ◽

Ken Nakamura ◽

Cristiana Iosef Husted ◽

...

Keyword(s):

Smooth Muscle ◽

Stem Cell ◽

Aortic Root ◽

Pluripotent Stem Cell ◽

Cell Model ◽

Expression Profiles ◽

Induced Pluripotent Stem Cell ◽

Aortic Root Aneurysm ◽

Ligand Expression ◽

Induced Pluripotent

Introduction: Mutations in TGF-beta (TGF-ß) signaling genes lead to aortic root aneurysm in Loeys Dietz syndrome (LDS). Smooth muscle cells (SMCs) in the proximal aorta develop from two embryologic origins: second heart field (SHF) and neural crest (NC). Induced pluripotent stem cell (iPSC) models simulate these lineages, but direct correlation to clinical disease is lacking. Hypothesis: iPSC-derived SMCs accurately model lineage-specific aortopathy in LDS. Methods: We generated SMC lines from root and ascending aortic surgical tissue and iPSC-derived SMCs through SHF and NC-specific pathways from an LDS patient ( TGFBR1 mutation). Lineage-specific TGF-ß responses were determined by western blot/ELISA. RNA sequencing and RT-PCR identified SMC transcriptomes. Results: Aortic root SMCs showed greater canonical TGF-ß activation (p-SMAD2/3) versus ascending at baseline and with TGF-ß stimulation ( Figure ). Synonymous results were seen in SHF versus NC SMCs from the iPSC pathway. RNAseq identified 1,600 differentially expressed genes between iPSC lineages, including altered TGF-ß receptor and ligand expression profiles. Primary aortic lines validated iPSC data: root SMCs showed enriched TGF-ß receptor 1/2/3 expression (1.7-, 3.9- and 5.9-fold) while ascending SMCs overexpressed TGFB1 and TGFB2 ligands (1.8- and 3.5-fold). Despite discordant TGF-ß activation, SMC contractile gene expression was similar between lineages in aortic and iPSC-SMCs, suggesting alternative downstream effects in LDS aneurysm. Conclusion: iPSC-derived SMCs effectively model lineage-specific aortic root aneurysm pathology, validating this model as a tool for mechanistic testing and therapy discovery.

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