RESUMO
Human induced pluripotent stem cells (hiPSCs) have revolutionized the field of human disease modeling, with an enormous potential to serve as paradigm shifting platforms for preclinical trials, personalized clinical diagnosis, and drug treatment. In this review, we describe how hiPSCs could transition cardiac healthcare away from simple disease diagnosis to prediction and prevention, bridging the gap between basic and clinical research to bring the best science to every patient.
Assuntos
Doenças Cardiovasculares/terapia , Células-Tronco Pluripotentes Induzidas , Medicina de Precisão , HumanosRESUMO
The ability to differentiate human pluripotent stem cells (hPSCs) into cardiomyocytes (CMs) makes them an attractive source for repairing injured myocardium, disease modeling, and drug testing. Although current differentiation protocols yield hPSC-CMs to >90% efficiency, hPSC-CMs exhibit immature characteristics. With the goal of overcoming this limitation, we tested the effects of varying passive stretch on engineered heart muscle (EHM) structural and functional maturation, guided by computational modeling. Human embryonic stem cells (hESCs, H7 line) or human induced pluripotent stem cells (IMR-90 line) were differentiated to hPSC-derived cardiomyocytes (hPSC-CMs) in vitro using a small molecule based protocol. hPSC-CMs were characterized by troponin+ flow cytometry as well as electrophysiological measurements. Afterwards, 1.2 × 106 hPSC-CMs were mixed with 0.4 × 106 human fibroblasts (IMR-90 line) (3:1 ratio) and type-I collagen. The blend was cast into custom-made 12-mm long polydimethylsiloxane reservoirs to vary nominal passive stretch of EHMs to 5, 7, or 9 mm. EHM characteristics were monitored for up to 50 days, with EHMs having a passive stretch of 7 mm giving the most consistent formation. Based on our initial macroscopic observations of EHM formation, we created a computational model that predicts the stress distribution throughout EHMs, which is a function of cellular composition, cellular ratio, and geometry. Based on this predictive modeling, we show cell alignment by immunohistochemistry and coordinated calcium waves by calcium imaging. Furthermore, coordinated calcium waves and mechanical contractions were apparent throughout entire EHMs. The stiffness and active forces of hPSC-derived EHMs are comparable with rat neonatal cardiomyocyte-derived EHMs. Three-dimensional EHMs display increased expression of mature cardiomyocyte genes including sarcomeric protein troponin-T, calcium and potassium ion channels, ß-adrenergic receptors, and t-tubule protein caveolin-3. Passive stretch affects the structural and functional maturation of EHMs. Based on our predictive computational modeling, we show how to optimize cell alignment and calcium dynamics within EHMs. These findings provide a basis for the rational design of EHMs, which enables future scale-up productions for clinical use in cardiovascular tissue engineering. Stem Cells 2018;36:265-277.
Assuntos
Biologia Computacional/métodos , Miocárdio/citologia , Linhagem Celular , Citometria de Fluxo , Humanos , Miocárdio/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Engenharia Tecidual/métodosRESUMO
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.
Assuntos
Doenças Cardiovasculares/genética , Técnicas de Inativação de Genes/métodos , Biblioteca Gênica , Engenharia Genética/métodos , Células-Tronco Pluripotentes Induzidas/fisiologia , Sequência de Bases , Doenças Cardiovasculares/terapia , Células Cultivadas , Marcação de Genes/métodos , Humanos , Células-Tronco Pluripotentes Induzidas/transplanteRESUMO
Existing methods for human induced pluripotent stem cell (hiPSC) cardiac differentiation are efficient but require complex, undefined medium constituents that hinder further elucidation of the molecular mechanisms of cardiomyogenesis. Using hiPSCs derived under chemically defined conditions on synthetic matrices, we systematically developed an optimized cardiac differentiation strategy, using a chemically defined medium consisting of just three components: the basal medium RPMI 1640, L-ascorbic acid 2-phosphate and rice-derived recombinant human albumin. Along with small molecule-based induction of differentiation, this protocol produced contractile sheets of up to 95% TNNT2(+) cardiomyocytes at a yield of up to 100 cardiomyocytes for every input pluripotent cell and was effective in 11 hiPSC lines tested. This chemically defined platform for cardiac specification of hiPSCs will allow the elucidation of cardiomyocyte macromolecular and metabolic requirements and will provide a minimal system for the study of maturation and subtype specification.
Assuntos
Miócitos Cardíacos/citologia , Diferenciação Celular , Meios de Cultura , Humanos , Células-Tronco Pluripotentes Induzidas/citologiaRESUMO
Cardiac regeneration strategies and de novo generation of cardiomyocytes have long been significant areas of research interest in cardiovascular medicine. In this review, we outline a variety of common cell sources and methods used to regenerate cardiomyocytes and highlight the important role that key Circulation Research articles have played in this flourishing field.
Assuntos
Coração/fisiologia , Miócitos Cardíacos/citologia , Regeneração , Transplante de Células-Tronco , Animais , Diferenciação Celular , Cardiopatias/cirurgia , História do Século XX , História do Século XXI , Humanos , Pesquisa com Células-Tronco/história , Transplante de Células-Tronco/história , Transplante de Células-Tronco/tendências , Células-Tronco/classificação , Células-Tronco/citologiaRESUMO
RATIONALE: Viral myocarditis is a life-threatening illness that may lead to heart failure or cardiac arrhythmias. A major causative agent for viral myocarditis is the B3 strain of coxsackievirus, a positive-sense RNA enterovirus. However, human cardiac tissues are difficult to procure in sufficient enough quantities for studying the mechanisms of cardiac-specific viral infection. OBJECTIVE: This study examined whether human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) could be used to model the pathogenic processes of coxsackievirus-induced viral myocarditis and to screen antiviral therapeutics for efficacy. METHODS AND RESULTS: hiPSC-CMs were infected with a luciferase-expressing coxsackievirus B3 strain (CVB3-Luc). Brightfield microscopy, immunofluorescence, and calcium imaging were used to characterize virally infected hiPSC-CMs for alterations in cellular morphology and calcium handling. Viral proliferation in hiPSC-CMs was quantified using bioluminescence imaging. Antiviral compounds including interferonß1, ribavirin, pyrrolidine dithiocarbamate, and fluoxetine were tested for their capacity to abrogate CVB3-Luc proliferation in hiPSC-CMs in vitro. The ability of these compounds to reduce CVB3-Luc proliferation in hiPSC-CMs was consistent with reported drug effects in previous studies. Mechanistic analyses via gene expression profiling of hiPSC-CMs infected with CVB3-Luc revealed an activation of viral RNA and protein clearance pathways after interferonß1 treatment. CONCLUSIONS: This study demonstrates that hiPSC-CMs express the coxsackievirus and adenovirus receptor, are susceptible to coxsackievirus infection, and can be used to predict antiviral drug efficacy. Our results suggest that the hiPSC-CM/CVB3-Luc assay is a sensitive platform that can screen novel antiviral therapeutics for their effectiveness in a high-throughput fashion.
Assuntos
Antivirais/uso terapêutico , Enterovirus Humano B/isolamento & purificação , Infecções por Enterovirus/tratamento farmacológico , Modelos Cardiovasculares , Miocardite/tratamento farmacológico , Miócitos Cardíacos/patologia , Células-Tronco Pluripotentes/patologia , Antivirais/farmacologia , Cálcio/metabolismo , Proliferação de Células , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Infecções por Enterovirus/metabolismo , Humanos , Técnicas In Vitro , Miocardite/metabolismo , Miocardite/virologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/virologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/virologia , RNA Viral/metabolismo , Resultado do TratamentoRESUMO
The emphasis in human pluripotent stem cell (hPSC) technologies has shifted from cell therapy to in vitro disease modelling and drug screening. This review examines why this shift has occurred, and how current technological limitations might be overcome to fully realise the potential of hPSCs. Details are provided for all disease-specific human induced pluripotent stem cell lines spanning a dozen dysfunctional organ systems. Phenotype and pharmacology have been examined in only 17 of 63 lines, primarily those that model neurological and cardiac conditions. Drug screening is most advanced in hPSC-cardiomyocytes. Responses for almost 60 agents include examples of how careful tests in hPSC-cardiomyocytes have improved on existing in vitro assays, and how these cells have been integrated into high throughput imaging and electrophysiology industrial platforms. Such successes will provide an incentive to overcome bottlenecks in hPSC technology such as improving cell maturity and industrial scalability whilst reducing cost.
Assuntos
Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Células-Tronco Pluripotentes/metabolismo , Animais , Ensaios de Triagem em Larga Escala , Humanos , Fenótipo , Células-Tronco Pluripotentes/citologia , Transplante de Células-TroncoRESUMO
AIMS: Long-QT syndromes (LQTS) are mostly autosomal-dominant congenital disorders associated with a 1:1000 mutation frequency, cardiac arrest, and sudden death. We sought to use cardiomyocytes derived from human-induced pluripotency stem cells (hiPSCs) as an in vitro model to develop and evaluate gene-based therapeutics for the treatment of LQTS. METHODS AND RESULTS: We produced LQTS-type 2 (LQT2) hiPSC cardiomyocytes carrying a KCNH2 c.G1681A mutation in a IKr ion-channel pore, which caused impaired glycosylation and channel transport to cell surface. Allele-specific RNA interference (RNAi) directed towards the mutated KCNH2 mRNA caused knockdown, while leaving the wild-type mRNA unaffected. Electrophysiological analysis of patient-derived LQT2 hiPSC cardiomyocytes treated with mutation-specific siRNAs showed normalized action potential durations (APDs) and K(+) currents with the concurrent rescue of spontaneous and drug-induced arrhythmias (presented as early-afterdepolarizations). CONCLUSIONS: These findings provide in vitro evidence that allele-specific RNAi can rescue diseased phenotype in LQTS cardiomyocytes. This is a potentially novel route for the treatment of many autosomal-dominant-negative disorders, including those of the heart.
Assuntos
Canais de Potássio Éter-A-Go-Go/genética , Síndrome do QT Longo/genética , Miócitos Cardíacos/fisiologia , Células-Tronco Pluripotentes/fisiologia , Interferência de RNA/fisiologia , Canal de Potássio ERG1 , Fenômenos Eletrofisiológicos/genética , Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Terapia Genética , Humanos , Síndrome do QT Longo/fisiopatologia , Síndrome do QT Longo/terapia , Mutação de Sentido Incorreto/genética , Fenótipo , TransfecçãoRESUMO
AIMS: Congenital long QT syndromes (LQTSs) are associated with prolonged ventricular repolarization and sudden cardiac death. Limitations to existing clinical therapeutic management strategies prompted us to develop a novel human in vitro drug-evaluation system for LQTS type 2 (LQT2) that will complement the existing in vitro and in vivo models. METHODS AND RESULTS: Skin fibroblasts from a patient with a KCNH2 G1681A mutation (encodes I(Kr) potassium ion channel) were reprogrammed to human induced pluripotent stem cells (hiPSCs), which were subsequently differentiated to functional cardiomyocytes. Relative to controls (including the patient's mother), multi-electrode array and patch-clamp electrophysiology of LQT2-hiPSC cardiomyocytes showed prolonged field/action potential duration. When LQT2-hiPSC cardiomyocytes were exposed to E4031 (an I(Kr) blocker), arrhythmias developed and these presented as early after depolarizations (EADs) in the action potentials. In contrast to control cardiomyocytes, LQT2-hiPSC cardiomyocytes also developed EADs when challenged with the clinically used stressor, isoprenaline. This effect was reversed by ß-blockers, propranolol, and nadolol, the latter being used for the patient's therapy. Treatment of cardiomyocytes with experimental potassium channel enhancers, nicorandil and PD118057, caused action potential shortening and in some cases could abolish EADs. Notably, combined treatment with isoprenaline (enhancers/isoprenaline) caused EADs, but this effect was reversed by nadolol. CONCLUSIONS: Findings from this paper demonstrate that patient LQT2-hiPSC cardiomyocytes respond appropriately to clinically relevant pharmacology and will be a valuable human in vitro model for testing experimental drug combinations.
Assuntos
Cromossomos Humanos Par 7/genética , Canais de Potássio Éter-A-Go-Go/genética , Células-Tronco Pluripotentes Induzidas/fisiologia , Síndrome do QT Longo/genética , Mutação Puntual/genética , Adolescente , Agonistas Adrenérgicos beta/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Antiarrítmicos/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Canal de Potássio ERG1 , Eletrocardiografia , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Isoproterenol/farmacologia , Síndrome do QT Longo/tratamento farmacológico , Miócitos Cardíacos/fisiologia , Nicorandil/farmacologiaRESUMO
Safety pharmacology is an essential part of drug development aiming to identify, evaluate and investigate undesirable pharmacodynamic properties of a drug primarily prior to clinical trials. In particular, cardiovascular adverse drug reactions (ADR) have halted many drug development programs. Safety pharmacology has successfully implemented a screening strategy to detect cardiovascular liabilities, but there is room for further refinement. In this setting, we present the INSPIRE project, a European Training Network in safety pharmacology for Early Stage Researchers (ESRs), funded by the European Commission's H2020-MSCA-ITN programme. INSPIRE has recruited 15 ESR fellows that will conduct an individual PhD-research project for a period of 36 months. INSPIRE aims to be complementary to ongoing research initiatives. With this as a goal, an inventory of collaborative research initiatives in safety pharmacology was created and the ESR projects have been designed to be complementary to this roadmap. Overall, INSPIRE aims to improve cardiovascular safety evaluation, either by investigating technological innovations or by adding mechanistic insight in emerging safety concerns, as observed in the field of cardio-oncology. Finally, in addition to its hands-on research pillar, INSPIRE will organize a number of summer schools and workshops that will be open to the wider community as well. In summary, INSPIRE aims to foster both research and training in safety pharmacology and hopes to inspire the future generation of safety scientists.
Assuntos
Sistema Cardiovascular/efeitos dos fármacos , Desenvolvimento de Medicamentos/métodos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/prevenção & controle , Farmacologia/métodos , Humanos , SegurançaRESUMO
Chagas disease (ChD) is one of the most neglected tropical diseases, with cardiomyopathy being the main cause of death in Trypanosoma cruzi-infected patients. As the parasite actively replicates in cardiomyocytes (CMs), the heart remains a key target organ in the pathogenesis of ChD. Here we modeled ChD using human induced pluripotent stem cell-derived CMs (iPSC-CMs) to understand the complex interplay between the parasite and host cells. We showed that iPSC-CMs can get infected with the T. cruzi Y strain and that all parasite cycle stages can be identified in our model system. Importantly, characterization of T. cruzi-infected iPSC-CMs showed significant changes in their gene expression profile, cell contractility, and distribution of key cardiac markers. Moreover, these infected iPSC-CMs exhibited a pro-inflammatory profile as indicated by significantly elevated cytokine levels and cell-trafficking regulators. We believe our iPSC-CM model is a valuable platform to explore new treatment strategies for ChD.
Assuntos
Cardiomiopatia Chagásica/metabolismo , Células-Tronco Pluripotentes Induzidas , Modelos Biológicos , Miócitos Cardíacos , Trypanosoma cruzi/metabolismo , Cardiomiopatia Chagásica/patologia , Cardiomiopatia Chagásica/terapia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/parasitologia , Células-Tronco Pluripotentes Induzidas/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/parasitologia , Miócitos Cardíacos/patologiaRESUMO
The measurement of the electrophysiology of human pluripotent stem cell-derived cardiomyocytes is critical for their biomedical applications, from disease modeling to drug screening. Yet, a method that enables the high-throughput intracellular electrophysiology measurement of single cardiomyocytes in adherent culture is not available. To address this area, we have fabricated vertical nanopillar electrodes that can record intracellular action potentials from up to 60 single beating cardiomyocytes. Intracellular access is achieved by highly localized electroporation, which allows for low impedance electrical access to the intracellular voltage. Herein, we demonstrate that this method provides the accurate measurement of the shape and duration of intracellular action potentials, validated by patch clamp, and can facilitate cellular drug screening and disease modeling using human pluripotent stem cells. This study validates the use of nanopillar electrodes for myriad further applications of human pluripotent stem cell-derived cardiomyocytes such as cardiomyocyte maturation monitoring and electrophysiology-contractile force correlation.
RESUMO
There is growing interest in using embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) derivatives for tissue regeneration. However, an increased understanding of human immune responses to stem cell-derived allografts is necessary for maintaining long-term graft persistence. To model this alloimmunity, humanized mice engrafted with human hematopoietic and immune cells could prove to be useful. In this study, an in-depth analysis of graft-infiltrating human lymphocytes and splenocytes revealed that humanized mice incompletely model human immune responses toward allogeneic stem cells and their derivatives. Furthermore, using an "allogenized" mouse model, we show the feasibility of reconstituting immunodeficient mice with a functional mouse immune system and describe a key role of innate immune cells in the rejection of mouse stem cell allografts.
Assuntos
Transplante de Células-Tronco Hematopoéticas/métodos , Imunidade Inata/imunologia , Células-Tronco Pluripotentes/metabolismo , Condicionamento Pré-Transplante/métodos , Animais , Modelos Animais de Doenças , Rejeição de Enxerto , Humanos , CamundongosRESUMO
Tyrosine kinase inhibitors (TKIs), despite their efficacy as anticancer therapeutics, are associated with cardiovascular side effects ranging from induced arrhythmias to heart failure. We used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), generated from 11 healthy individuals and 2 patients receiving cancer treatment, to screen U.S. Food and Drug Administration-approved TKIs for cardiotoxicities by measuring alterations in cardiomyocyte viability, contractility, electrophysiology, calcium handling, and signaling. With these data, we generated a "cardiac safety index" to reflect the cardiotoxicities of existing TKIs. TKIs with low cardiac safety indices exhibit cardiotoxicity in patients. We also derived endothelial cells (hiPSC-ECs) and cardiac fibroblasts (hiPSC-CFs) to examine cell type-specific cardiotoxicities. Using high-throughput screening, we determined that vascular endothelial growth factor receptor 2 (VEGFR2)/platelet-derived growth factor receptor (PDGFR)-inhibiting TKIs caused cardiotoxicity in hiPSC-CMs, hiPSC-ECs, and hiPSC-CFs. With phosphoprotein analysis, we determined that VEGFR2/PDGFR-inhibiting TKIs led to a compensatory increase in cardioprotective insulin and insulin-like growth factor (IGF) signaling in hiPSC-CMs. Up-regulating cardioprotective signaling with exogenous insulin or IGF1 improved hiPSC-CM viability during cotreatment with cardiotoxic VEGFR2/PDGFR-inhibiting TKIs. Thus, hiPSC-CMs can be used to screen for cardiovascular toxicities associated with anticancer TKIs, and the results correlate with clinical phenotypes. This approach provides unexpected insights, as illustrated by our finding that toxicity can be alleviated via cardioprotective insulin/IGF signaling.
Assuntos
Cardiotoxicidade/patologia , Ensaios de Triagem em Larga Escala/métodos , Células-Tronco Pluripotentes Induzidas/metabolismo , Inibidores de Proteínas Quinases/toxicidade , Biomarcadores/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Insulina/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Modelos Biológicos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Sarcômeros/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
The advent of human induced pluripotent stem cell (hiPSC) technology has revitalized the efforts in the past decade to realize more fully the potential of human embryonic stem cells for scientific research. Adding to the possibility of generating an unlimited amount of any cell type of interest, hiPSC technology now enables the derivation of cells with patient-specific phenotypes. Given the introduction and implementation of the large-scale Precision Medicine Initiative, hiPSC technology will undoubtedly have a vital role in the advancement of cardiovascular research and medicine. In this Review, we summarize the progress that has been made in the field of hiPSC technology, with particular emphasis on cardiovascular disease modelling and drug development. The growing roles of hiPSC technology in the practice of precision medicine will also be discussed.
Assuntos
Doenças Cardiovasculares/tratamento farmacológico , Células-Tronco Pluripotentes Induzidas , Modelos Cardiovasculares , Medicina de Precisão , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/prevenção & controle , Diferenciação Celular , Técnicas de Reprogramação Celular , Descoberta de Drogas , Células Endoteliais/citologia , Humanos , Miócitos Cardíacos/citologia , Miócitos de Músculo Liso/citologiaRESUMO
The generation of cardiomyocytes from human induced pluripotent stem cells (hiPSCs) provides a source of cells that accurately recapitulate the human cardiac pathophysiology. The application of these cells allows for modeling of cardiovascular diseases, providing a novel understanding of human disease mechanisms and assessment of therapies. Here, we describe a stepwise protocol developed in our laboratory for the generation of hiPSCs from patients with a specific disease phenotype, long-term hiPSC culture and cryopreservation, differentiation of hiPSCs to cardiomyocytes, and assessment of disease phenotypes. Our protocol combines a number of innovative tools that include a codon-optimized mini intronic plasmid (CoMiP), chemically defined culture conditions to achieve high efficiencies of reprogramming and differentiation, and calcium imaging for assessment of cardiomyocyte phenotypes. Thus, this protocol provides a complete guide to use a patient cohort on a testable cardiomyocyte platform for pharmacological drug assessment.
Assuntos
Cardiomiopatia Dilatada/patologia , Reprogramação Celular , Fibroblastos/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Modelos Biológicos , Miócitos Cardíacos/citologia , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Biomarcadores/metabolismo , Cálcio/metabolismo , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/metabolismo , Diferenciação Celular , Criopreservação , Derme/citologia , Derme/metabolismo , Fibroblastos/metabolismo , Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Imagem Molecular , Mutação , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Cultura Primária de Células , Proteoglicanas/genética , Proteoglicanas/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Inoculações Seriadas , Troponina T/genética , Troponina T/metabolismoRESUMO
Doxorubicin is an anthracycline chemotherapy agent effective in treating a wide range of malignancies, but it causes a dose-related cardiotoxicity that can lead to heart failure in a subset of patients. At present, it is not possible to predict which patients will be affected by doxorubicin-induced cardiotoxicity (DIC). Here we demonstrate that patient-specific human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) can recapitulate the predilection to DIC of individual patients at the cellular level. hiPSC-CMs derived from individuals with breast cancer who experienced DIC were consistently more sensitive to doxorubicin toxicity than hiPSC-CMs from patients who did not experience DIC, with decreased cell viability, impaired mitochondrial and metabolic function, impaired calcium handling, decreased antioxidant pathway activity, and increased reactive oxygen species production. Taken together, our data indicate that hiPSC-CMs are a suitable platform to identify and characterize the genetic basis and molecular mechanisms of DIC.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Doxorrubicina/farmacologia , Insuficiência Cardíaca/induzido quimicamente , Mitocôndrias Cardíacas/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Adulto , Idoso , Antibióticos Antineoplásicos/efeitos adversos , Cálcio/metabolismo , Cardiotoxicidade/genética , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Suscetibilidade a Doenças , Doxorrubicina/efeitos adversos , Feminino , Citometria de Fluxo , Imunofluorescência , Insuficiência Cardíaca/genética , Humanos , Células-Tronco Pluripotentes Induzidas , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Pessoa de Meia-Idade , Mitocôndrias Cardíacas/metabolismo , Miócitos Cardíacos/metabolismo , Polimorfismo de Nucleotídeo Único , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , TranscriptomaRESUMO
Understanding individual susceptibility to drug-induced cardiotoxicity is key to improving patient safety and preventing drug attrition. Human induced pluripotent stem cells (hiPSCs) enable the study of pharmacological and toxicological responses in patient-specific cardiomyocytes (CMs) and may serve as preclinical platforms for precision medicine. Transcriptome profiling in hiPSC-CMs from seven individuals lacking known cardiovascular disease-associated mutations and in three isogenic human heart tissue and hiPSC-CM pairs showed greater inter-patient variation than intra-patient variation, verifying that reprogramming and differentiation preserve patient-specific gene expression, particularly in metabolic and stress-response genes. Transcriptome-based toxicology analysis predicted and risk-stratified patient-specific susceptibility to cardiotoxicity, and functional assays in hiPSC-CMs using tacrolimus and rosiglitazone, drugs targeting pathways predicted to produce cardiotoxicity, validated inter-patient differential responses. CRISPR/Cas9-mediated pathway correction prevented drug-induced cardiotoxicity. Our data suggest that hiPSC-CMs can be used in vitro to predict and validate patient-specific drug safety and efficacy, potentially enabling future clinical approaches to precision medicine.
Assuntos
Perfilação da Expressão Gênica , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/citologia , Tacrolimo/efeitos adversos , Tiazolidinedionas/efeitos adversos , Sistemas CRISPR-Cas/genética , Morte Celular/efeitos dos fármacos , Edição de Genes , Genoma Humano , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/metabolismo , Humanos , Sequências Repetidas Invertidas/genética , Miócitos Cardíacos/metabolismo , Rosiglitazona , Resultado do TratamentoRESUMO
ß-adrenergic signaling pathways mediate key aspects of cardiac function. Its dysregulation is associated with a range of cardiac diseases, including dilated cardiomyopathy (DCM). Previously, we established an iPSC model of familial DCM from patients with a mutation in TNNT2, a sarcomeric protein. Here, we found that the ß-adrenergic agonist isoproterenol induced mature ß-adrenergic signaling in iPSC-derived cardiomyocytes (iPSC-CMs) but that this pathway was blunted in DCM iPSC-CMs. Although expression levels of several ß-adrenergic signaling components were unaltered between control and DCM iPSC-CMs, we found that phosphodiesterases (PDEs) 2A and PDE3A were upregulated in DCM iPSC-CMs and that PDE2A was also upregulated in DCM patient tissue. We further discovered increased nuclear localization of mutant TNNT2 and epigenetic modifications of PDE genes in both DCM iPSC-CMs and patient tissue. Notably, pharmacologic inhibition of PDE2A and PDE3A restored cAMP levels and ameliorated the impaired ß-adrenergic signaling of DCM iPSC-CMs, suggesting therapeutic potential.
Assuntos
Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/metabolismo , Células-Tronco Pluripotentes Induzidas/fisiologia , Receptores Adrenérgicos beta/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Cardiomiopatia Dilatada/patologia , Diferenciação Celular , Células Cultivadas , Epigênese Genética , Frequência Cardíaca/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/patologia , Isoproterenol/farmacologia , Modelos Cardiovasculares , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Miócitos Cardíacos/fisiologia , Transdução de Sinais , Troponina T/genética , Troponina T/metabolismo , Regulação para CimaRESUMO
A major research focus in the field of cardiovascular medicine is the prospect of using stem cells and progenitor cells for cardiac regeneration. With the advent of induced pluripotent stem cell (iPSC) technology, major efforts are also underway to use iPSCs to model heart disease, to screen for new drugs, and to test candidate drugs for cardiotoxicity. Here, we discuss recent advances in the exciting fields of stem cells and cardiovascular disease.