Role of Heme-Oxygenase-1 in Biology of Cardiomyocytes Derived from Human Induced Pluripotent Stem Cells.
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ABSTRACT:
SUBMITTER: Jez M
PROVIDER: S-EPMC8000937 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Human induced pluripotent stem (iPS) cells hold great promise for cardiovascular research and therapeutic applications, but the ability of human iPS cells to differentiate into functional cardiomyocytes has not yet been demonstrated. The aim of this study was to characterize the cardiac differentiation potential of human iPS cells generated using OCT4, SOX2, NANOG, and LIN28 transgenes compared to human embryonic stem (ES) cells. The iPS and ES cells were differentiated using the embryoid body (EB) method. The time course of developing contracting EBs was comparable for the iPS and ES cell lines, although the absolute percentages of contracting EBs differed. RT-PCR analyses of iPS and ES cell-derived cardiomyocytes demonstrated similar cardiac gene expression patterns. The pluripotency genes OCT4 and NANOG were downregulated with cardiac differentiation, but the downregulation was blunted in the iPS cell lines because of residual transgene expression. Proliferation of iPS and ES cell-derived cardiomyocytes based on 5-bromodeoxyuridine labeling was similar, and immunocytochemistry of isolated cardiomyocytes revealed indistinguishable sarcomeric organizations. Electrophysiology studies indicated that iPS cells have a capacity like ES cells for differentiation into nodal-, atrial-, and ventricular-like phenotypes based on action potential characteristics. Both iPS and ES cell-derived cardiomyocytes exhibited responsiveness to beta-adrenergic stimulation manifest by an increase in spontaneous rate and a decrease in action potential duration. We conclude that human iPS cells can differentiate into functional cardiomyocytes, and thus iPS cells are a viable option as an autologous cell source for cardiac repair and a powerful tool for cardiovascular research.
| S-EPMC2741334 | biostudies-literature
Project description:RationaleCardiomyocytes generated from human induced pluripotent stem cells (hiPSCs) are suggested as the most promising candidate to replenish cardiomyocyte loss in regenerative medicine. Little is known about their calcium homeostasis, the key process underlying excitation-contraction coupling.ObjectiveWe investigated the calcium handling properties of hiPSC-derived cardiomyocytes and compared with those from human embryonic stem cells (hESCs).Methods and resultsWe differentiated cardiomyocytes from hiPSCs (IMR90 and KS1) and hESCs (H7 and HES3) with established protocols. Beating outgrowths from embryoid bodies were typically observed 2 weeks after induction. Cells in these outgrowths were stained positively for tropomyosin and sarcomeric alpha-actinin. Reverse-transcription polymerase chain reaction studies demonstrated the expressions of cardiac-specific markers in both hiPSC- and hESC-derived cardiomyocytes. Calcium handling properties of 20-day-old hiPSC- and hESC-derived cardiomyocytes were investigated using fluorescence confocal microscopy. Compared with hESC-derived cardiomyocytes, spontaneous calcium transients from both lines of hiPSC-derived cardiomyocytes were of significantly smaller amplitude and with slower maximal upstroke velocity. Better caffeine-induced calcium handling kinetics in hESC-CMs indicates a higher sacroplasmic recticulum calcium store. Furthermore, in contrast with hESC-derived cardiomyocytes, ryanodine did not reduce the amplitudes, maximal upstroke and decay velocity of calcium transients of hiPSC-derived cardiomyocytes. In addition, spatial inhomogeneity in temporal properties of calcium transients across the width of cardiomyocytes was more pronounced in hiPSC-derived cardiomyocytes than their hESC counterpart as revealed line-scan calcium imaging. Expressions of the key calcium-handling proteins including ryanodine recptor-2 (RyR2), sacroplasmic recticulum calcium-ATPase (SERCA), junction (Jun) and triadin (TRDN), were significantly lower in hiPSC than in hESCs.ConclusionsThe results indicate the calcium handling properties of hiPSC-derived cardiomyocytes are relatively immature to hESC counterparts.
| S-EPMC3226695 | biostudies-literature
Project description:BackgroundThe ability to establish human induced pluripotent stem cells (hiPSCs) by reprogramming of adult fibroblasts and to coax their differentiation into cardiomyocytes opens unique opportunities for cardiovascular regenerative and personalized medicine. In the current study, we investigated the Ca(2+)-handling properties of hiPSCs derived-cardiomyocytes (hiPSC-CMs).Methodology/principal findingsRT-PCR and immunocytochemistry experiments identified the expression of key Ca(2+)-handling proteins. Detailed laser confocal Ca(2+) imaging demonstrated spontaneous whole-cell [Ca(2+)](i) transients. These transients required Ca(2+) influx via L-type Ca(2+) channels, as demonstrated by their elimination in the absence of extracellular Ca(2+) or by administration of the L-type Ca(2+) channel blocker nifedipine. The presence of a functional ryanodine receptor (RyR)-mediated sarcoplasmic reticulum (SR) Ca(2+) store, contributing to [Ca(2+)](i) transients, was established by application of caffeine (triggering a rapid increase in cytosolic Ca(2+)) and ryanodine (decreasing [Ca(2+)](i)). Similarly, the importance of Ca(2+) reuptake into the SR via the SR Ca(2+) ATPase (SERCA) pump was demonstrated by the inhibiting effect of its blocker (thapsigargin), which led to [Ca(2+)](i) transients elimination. Finally, the presence of an IP3-releasable Ca(2+) pool in hiPSC-CMs and its contribution to whole-cell [Ca(2+)](i) transients was demonstrated by the inhibitory effects induced by the IP3-receptor blocker 2-Aminoethoxydiphenyl borate (2-APB) and the phospholipase C inhibitor U73122.Conclusions/significanceOur study establishes the presence of a functional, SERCA-sequestering, RyR-mediated SR Ca(2+) store in hiPSC-CMs. Furthermore, it demonstrates the dependency of whole-cell [Ca(2+)](i) transients in hiPSC-CMs on both sarcolemmal Ca(2+) entry via L-type Ca(2+) channels and intracellular store Ca(2+) release.
| S-EPMC3069979 | biostudies-literature
Project description:Cyclophosphamide (CP) is an anticancer drug, an alkylating agent. Cardiotoxicity of CP is associated with one of its metabolites, acrolein, and clinical cardiotoxicity manifestations are described for cases of taking CP in high doses. Nevertheless, modern arrhythmogenicity prediction assays in vitro include evaluation of beat rhythm and rate as well as suppression of cardiac late markers after acute exposure to CP, but not its metabolites. The mechanism of CP side effects when taken at low doses (i.e., < 100 mg/kg), especially at the cellular level, remains unclear. In this study conduction properties and cytoskeleton structure of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) obtained from a healthy donor under CP were evaluated. Arrhythmogenicity testing including characterization of 3 values: conduction velocity, maximum capture rate (MCR) measurements and number of occasions of re-entry on a standard linear obstacle was conducted and revealed MCR decrease of 25% ± 7% under CP. Also, conductivity area reduced by 34 ± 15%. No effect of CP on voltage-gated ion channels was found. Conduction changes (MCR and conductivity area decrease) are caused by exposure time-dependent alpha-actinin disruption detected both in hiPSC-CMs and neonatal ventricular cardiomyocytes in vitro. Deviation from the external stimulus frequency and appearance of non-conductive areas in cardiac tissue under CP is potentially arrhythmogenic and could develop arrhythmic effects in vivo.
| S-EPMC7841168 | biostudies-literature
Project description:Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are being used as an in vitro model system in cardiac biology and in drug discovery (e.g., cardiotoxicity testing). Qualification of these cells for use in mechanistic investigations will require detailed evaluations of cardiomyocyte signaling pathways and cellular responses. ErbB signaling and the ligand neuregulin play critical roles in survival and functional integrity of cardiac myocytes. As such, we sought to characterize the expression and activity of the ErbB family of receptors. Antibody microarray analysis performed on cell lysates derived from maturing hiPSC-CMs detected expression of ∼570 signaling proteins. EGFR/ErbB1, HER2/ErbB2, and ErbB4, but not ErbB3 receptors, of the epidermal growth factor receptor family were confirmed by Western blot. Activation of ErbB signaling by neuregulin-1β (NRG, a natural ligand for ErbB4) and its modulation by trastuzumab (a monoclonal anti-ErbB2 antibody) and lapatinib (a small molecule ErbB2 tyrosine kinase inhibitor) were evaluated through assessing phosphorylation of AKT and Erk1/2, two major downstream kinases of ErbB signaling, using nanofluidic proteomic immunoassay. Downregulation of ErbB2 expression by siRNA silencing attenuated NRG-induced AKT and Erk1/2 phosphorylation. Activation of ErbB signaling with NRG, or inhibition with trastuzumab, alleviated or aggravated doxorubicin-induced cardiomyocyte damage, respectively, as assessed by a real-time cellular impedance analysis and ATP measurement. Collectively, these results support the expanded use of hiPSC-CMs to examine mechanisms of cardiotoxicity and support the value of using these cells in early assessments of cardiotoxicity or efficacy.
| S-EPMC4200050 | biostudies-literature
Project description:Cadmium, a highly ubiquitous toxic heavy metal, has been widely recognized as an environmental and industrial pollutant, which confers serious threats to human health. The molecular mechanisms of the cadmium-induced cardiotoxicity (CIC) have not been studied in human cardiomyocytes at the cellular level. Here we showed that human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) can recapitulate the CIC at the cellular level. The cadmium-treated hPSC-CMs exhibited cellular phenotype including reduced cell viability, increased apoptosis, cardiac sarcomeric disorganization, elevated reactive oxygen species, altered action potential profile and cardiac arrhythmias. RNA-sequencing analysis revealed a differential transcriptome profile and activated MAPK signalling pathway in cadmium-treated hPSC-CMs, and suppression of P38 MAPK but not ERK MAPK or JNK MAPK rescued CIC phenotype. We further identified that suppression of PI3K/Akt signalling pathway is sufficient to reverse the CIC phenotype, which may play an important role in CIC. Taken together, our data indicate that hPSC-CMs can serve as a suitable model for the exploration of molecular mechanisms underlying CIC and for the discovery of CIC cardioprotective drugs.
| S-EPMC6111808 | biostudies-other
Project description:Causative mutations and variants associated with cardiac diseases have been found in genes encoding cardiac ion channels, accessory proteins, cytoskeletal components, junctional proteins, and signaling molecules. In most cases the functional evaluation of the genetic alteration has been carried out by expressing the mutated proteins in in-vitro heterologous systems. While these studies have provided a wealth of functional details that have greatly enhanced the understanding of the pathological mechanisms, it has always been clear that heterologous expression of the mutant protein bears the intrinsic limitation of the lack of a proper intracellular environment and the lack of pathological remodeling. The results obtained from the application of the next generation sequencing technique to patients suffering from cardiac diseases have identified several loci, mostly in non-coding DNA regions, which still await functional analysis. The isolation and culture of human embryonic stem cells has initially provided a constant source of cells from which cardiomyocytes (CMs) can be obtained by differentiation. Furthermore, the possibility to reprogram cellular fate to a pluripotent state, has opened this process to the study of genetic diseases. Thus induced pluripotent stem cells (iPSCs) represent a completely new cellular model that overcomes the limitations of heterologous studies. Importantly, due to the possibility to keep spontaneously beating CMs in culture for several months, during which they show a certain degree of maturation/aging, this approach will also provide a system in which to address the effect of long-term expression of the mutated proteins or any other DNA mutation, in terms of electrophysiological remodeling. Moreover, since iPSC preserve the entire patients' genetic context, the system will help the physicians in identifying the most appropriate pharmacological intervention to correct the functional alteration. This article summarizes the current knowledge of cardiac genetic diseases modelled with iPSC.
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Project description:Doxorubicin is a highly efficacious anti-cancer drug but causes cardiotoxicity in many patients. The mechanisms of doxorubicin-induced cardiotoxicity (DIC) remain incompletely understood. We investigated the characteristics and molecular mechanisms of DIC in human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). We found that doxorubicin causes dose-dependent increases in apoptotic and necrotic cell death, reactive oxygen species production, mitochondrial dysfunction and increased intracellular calcium concentration. We characterized genome-wide changes in gene expression caused by doxorubicin using RNA-seq, as well as electrophysiological abnormalities caused by doxorubicin with multi-electrode array technology. Finally, we show that CRISPR-Cas9-mediated disruption of TOP2B, a gene implicated in DIC in mouse studies, significantly reduces the sensitivity of hPSC-CMs to doxorubicin-induced double stranded DNA breaks and cell death. These data establish a human cellular model of DIC that recapitulates many of the cardinal features of this adverse drug reaction and could enable screening for protective agents against DIC as well as assessment of genetic variants involved in doxorubicin response.
| S-EPMC4855185 | biostudies-literature
Project description:The current inability to derive mature cardiomyocytes from human pluripotent stem cells has been the limiting step for transitioning this powerful technology into clinical therapies. To address this, scaffold-based tissue engineering approaches have been utilized to mimic heart development in vitro and promote maturation of cardiomyocytes derived from human pluripotent stem cells. While scaffolds can provide 3D microenvironments, current scaffolds lack the matched physical/chemical/biological properties of native extracellular environments. On the other hand, scaffold-free, 3D cardiac spheroids (i.e., spherical-shaped microtissues) prepared by seeding cardiomyocytes into agarose microwells were shown to improve cardiac functions. However, cardiomyocytes within the spheroids could not assemble in a controlled manner and led to compromised, unsynchronized contractions. Here, we show, for the first time, that incorporation of a trace amount (i.e., ∼0.004% w/v) of electrically conductive silicon nanowires (e-SiNWs) in otherwise scaffold-free cardiac spheroids can form an electrically conductive network, leading to synchronized and significantly enhanced contraction (i.e., >55% increase in average contraction amplitude), resulting in significantly more advanced cellular structural and contractile maturation.
| S-EPMC4431939 | biostudies-literature
Project description:Despite a substantial appreciation for the critical role of macrophages in cardiometabolic diseases, understanding of human macrophage biology has been hampered by the lack of reliable and scalable models for cellular and genetic studies. Human induced pluripotent stem cell (iPSC)-derived macrophages (IPSDM), as an unlimited source of subject genotype-specific cells, will undoubtedly play an important role in advancing our understanding of the role of macrophages in human diseases. In this review, we summarize current literature in the differentiation and characterization of IPSDM at phenotypic, functional, and transcriptomic levels. We emphasize the progress in differentiating iPSC to tissue resident macrophages, and in understanding the ontogeny of in vitro differentiated IPSDM that resembles primitive hematopoiesis, rather than adult definitive hematopoiesis. We review the application of IPSDM in modeling both Mendelian genetic disorders and host-pathogen interactions. Finally, we highlighted the potential areas of research using IPSDM in functional validation of coronary artery disease loci in genome-wide association studies, functional genomic analyses, drug testing, and cell therapeutics in cardiovascular diseases.
| S-EPMC5687272 | biostudies-literature