Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 26
Filtrar
1.
Stem Cell Res Ther ; 10(1): 203, 2019 07 08.
Artículo en Inglés | MEDLINE | ID: mdl-31286988

RESUMEN

BACKGROUND: Friedreich's ataxia (FRDA) is an autosomal recessive disease caused by a non-coding mutation in the first intron of the frataxin (FXN) gene that suppresses its expression. Compensatory hypertrophic cardiomyopathy, dilated cardiomyopathy, and conduction system abnormalities in FRDA lead to cardiomyocyte (CM) death and fibrosis, consequently resulting in heart failure and arrhythmias. Murine models have been developed to study disease pathology in the past two decades; however, differences between human and mouse physiology and metabolism have limited the relevance of animal studies in cardiac disease conditions. To bridge this gap, we aimed to generate species-specific, functional in vitro experimental models of FRDA using 2-dimensional (2D) and 3-dimensional (3D) engineered cardiac tissues from FXN-deficient human pluripotent stem cell-derived ventricular cardiomyocytes (hPSC-hvCMs) and to compare their contractile and electrophysiological properties with healthy tissue constructs. METHODS: Healthy control and FRDA patient-specific hPSC-hvCMs were derived by directed differentiation using a small molecule-based protocol reported previously. We engineered the hvCMs into our established human ventricular cardiac tissue strip (hvCTS) and human ventricular cardiac anisotropic sheet (hvCAS) models, and functional assays were performed on days 7-17 post-tissue fabrication to assess the electrophysiology and contractility of FRDA patient-derived and FXN-knockdown engineered tissues, in comparison with healthy controls. To further validate the disease model, forced expression of FXN was induced in FXN-deficient tissues to test if disease phenotypes could be rescued. RESULTS: Here, we report for the first time the generation of human engineered tissue models of FRDA cardiomyopathy from hPSCs: FXN-deficient hvCTS displayed attenuated developed forces (by 70-80%) compared to healthy controls. High-resolution optical mapping of hvCAS with reduced FXN expression also revealed electrophysiological defects consistent with clinical observations, including action potential duration prolongation and maximum capture frequency reduction. Interestingly, a clear positive correlation between FXN expression and contractility was observed (ρ > 0.9), and restoration of FXN protein levels by lentiviral transduction rescued contractility defects in FXN-deficient hvCTS. CONCLUSIONS: We conclude that human-based in vitro cardiac tissue models of FRDA provide a translational, disease-relevant biomimetic platform for the evaluation of novel therapeutics and to provide insight into FRDA disease progression.


Asunto(s)
Ataxia de Friedreich/metabolismo , Proteínas de Unión a Hierro/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Potenciales de Acción/fisiología , Cardiomiopatías/metabolismo , Diferenciación Celular/fisiología , Células Cultivadas , Insuficiencia Cardíaca/metabolismo , Humanos , Frataxina
2.
Am J Physiol Heart Circ Physiol ; 317(5): H1105-H1115, 2019 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-31347915

RESUMEN

Human pluripotent stem cell (hPSCs)-derived ventricular (V) cardiomyocytes (CMs) display immature Ca2+-handing properties with smaller transient amplitudes and slower kinetics due to such differences in crucial Ca2+-handling proteins as the poor sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump but robust Na+-Ca2+ exchanger (NCX) activities in human embryonic stem cell (ESC)-derived VCMs compared with adult. Despite their fundamental importance in excitation-contraction coupling, the relative contribution of SERCA and NCX to Ca2+-handling of hPSC-VCMs remains unexplored. We systematically altered the activities of SERCA and NCX in human embryonic stem cell-derived ventricular cardiomyocytes (hESC-VCMs) and their engineered microtissues, followed by examining the resultant phenotypic consequences. SERCA overexpression in hESC-VCMs shortened the decay of Ca2+ transient at low frequencies (0.5 Hz) without affecting the amplitude, SR Ca2+ content and Ca2+ baseline. Interestingly, short hairpin RNA-based NCX suppression did not prolong the transient decay, indicating a compensatory response for Ca2+ removal. Although hESC-VCMs and their derived microtissues exhibited negative frequency-transient/force responses, SERCA overexpression rendered them less negative at high frequencies (>2 Hz) by accelerating Ca2+ sequestration. We conclude that for hESC-VCMs and their microtissues, SERCA, rather than NCX, is the main Ca2+ remover during diastole; poor SERCA expression is the leading cause for immature negative-frequency/force responses, which can be partially reverted by forced expression. Combinatorial approach to mature calcium handling in hESC-VCMs may help shed further mechanistic insights.NEW & NOTEWORTHY In this study of human pluripotent stem cell-derived cardiomyocytes, we studied the role of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and Na+-Ca2+ exchanger (NCX) in Ca2+ handling. Our data support the notion that SERCA is more effective in cytosolic calcium removal than the NCX.


Asunto(s)
Señalización del Calcio , Calcio/metabolismo , Células Madre Embrionarias Humanas/enzimología , Contracción Miocárdica , Miocitos Cardíacos/enzimología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Diferenciación Celular , Linaje de la Célula , Células Cultivadas , Humanos , Fenotipo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , Intercambiador de Sodio-Calcio/genética , Factores de Tiempo
3.
Clin Pharmacol Ther ; 106(2): 402-414, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-30723889

RESUMEN

Traditional drug discovery is an inefficient process. Human pluripotent stem cell-derived cardiomyocytes can potentially fill the gap between animal and clinical studies, but conventional two-dimensional cultures inadequately recapitulate the human cardiac phenotype. Here, we systematically examined the pharmacological responses of engineered human ventricular-like cardiac tissue strips (hvCTS) and organoid chambers (hvCOC) to 25 cardioactive compounds covering various drug classes. While hvCTS effectively detected negative and null inotropic effects, the sensitivity to positive inotropes was modest. We further quantified the predictive capacity of hvCTS in a blinded screening, with accuracies for negative, positive, and null inotropic effects at 100%, 86%, and 80%, respectively. Interestingly, hvCOC, with a pro-maturation milieu that yields physiologically complex parameters, displayed enhanced positive inotropy. Based on these results, we propose a two-tiered screening system for avoiding false positives and negatives. Such an approach would facilitate drug discovery by leading to better overall success.


Asunto(s)
Cardiotónicos/farmacología , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos , Organoides , Fármacos Cardiovasculares/farmacología , Células Cultivadas , Depresión Química , Descubrimiento de Drogas/métodos , Evaluación Preclínica de Medicamentos/métodos , Humanos , Células Madre Pluripotentes Inducidas , Modelos Cardiovasculares , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/fisiología , Organoides/efectos de los fármacos , Organoides/fisiología , Estimulación Química , Ingeniería de Tejidos/métodos
4.
Toxicol Lett ; 294: 61-72, 2018 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-29758359

RESUMEN

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are emerging tools for applications such as drug discovery and screening for pro-arrhythmogenicity and cardiotoxicity as leading causes for drug attrition. Understanding the electrophysiology (EP) of hPSC-CMs is essential but conventional manual patch-clamping is highly laborious and low-throughput. Here we adapted hPSC-CMs derived from two human embryonic stem cell (hESC) lines, HES2 and H7, for a 16-channel automated planar-recording approach for single-cell EP characterization. Automated current- and voltage-clamping, with an overall success rate of 55.0 ±â€¯11.3%, indicated that 90% of hPSC-CMs displayed ventricular-like action potential (AP) and the ventricular cardiomyocytes (VCMs) derived from the two hESC lines expressed similar levels of INa, ICaL, Ikr and If and similarly lacked Ito and IK1. These well-characterized hPSC-VCMs could also be readily adapted for automated assays of pro-arrhythmic drug screening. As an example, we showed that flecainide (FLE) induced INa blockade, leftward steady-state inactivation shift, slowed recovery from inactivation in our hPSC-VCMs. Since single-cell EP assay is insufficient to predict drug-induced reentrant arrhythmias, hPSC-VCMs were further reassembled into 2D human ventricular cardiac monolayers (hvCMLs) for multi-cellular electrophysiological assessments. Indeed, FLE significantly slowed the conduction velocity while causing AP prolongation. Our RNA-seq data suggested that cell-cell interaction enhanced the maturity of hPSC-VCMs. Taken collectively, a combinatorial approach using single-cell EP and hvCMLs is needed to comprehensively assess drug-induced arrhythmogenicity.


Asunto(s)
Evaluación Preclínica de Medicamentos , Flecainida/efectos adversos , Ventrículos Cardíacos/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento , Miocitos Cardíacos/efectos de los fármacos , Bloqueadores del Canal de Sodio Activado por Voltaje/efectos adversos , Canales de Sodio Activados por Voltaje/metabolismo , Potenciales de Acción/efectos de los fármacos , Automatización de Laboratorios , Diferenciación Celular , Línea Celular , Células Cultivadas , Fenómenos Electrofisiológicos/efectos de los fármacos , Estudios de Factibilidad , Sistema de Conducción Cardíaco/citología , Sistema de Conducción Cardíaco/efectos de los fármacos , Sistema de Conducción Cardíaco/metabolismo , Ventrículos Cardíacos/citología , Ventrículos Cardíacos/metabolismo , Humanos , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Técnicas de Placa-Clamp , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/efectos de los fármacos , Células Madre Pluripotentes/metabolismo , Reproducibilidad de los Resultados , Análisis de la Célula Individual , Canales de Sodio Activados por Voltaje/química
5.
Biomaterials ; 163: 116-127, 2018 05.
Artículo en Inglés | MEDLINE | ID: mdl-29459321

RESUMEN

Tissue engineers and stem cell biologists have made exciting progress toward creating simplified models of human heart muscles or aligned monolayers to help bridge a longstanding gap between experimental animals and clinical trials. However, no existing human in vitro systems provide the direct measures of cardiac performance as a pump. Here, we developed a next-generation in vitro biomimetic model of pumping human heart chamber, and demonstrated its capability for pharmaceutical testing. From human pluripotent stem cell (hPSC)-derived ventricular cardiomyocytes (hvCM) embedded in collagen-based extracellular matrix hydrogel, we engineered a three-dimensional (3D) electro-mechanically coupled, fluid-ejecting miniature human ventricle-like cardiac organoid chamber (hvCOC). Structural characterization showed organized sarcomeres with myofibrillar microstructures. Transcript and RNA-seq analyses revealed upregulation of key Ca2+-handling, ion channel, and cardiac-specific proteins in hvCOC compared to lower-order 2D and 3D cultures of the same constituent cells. Clinically-important, physiologically complex contractile parameters such as ejection fraction, developed pressure, and stroke work, as well as electrophysiological properties including action potential and conduction velocity were measured: hvCOC displayed key molecular and physiological characteristics of the native ventricle, and showed expected mechanical and electrophysiological responses to a range of pharmacological interventions (including positive and negative inotropes). We conclude that such "human-heart-in-a-jar" technology could facilitate the drug discovery process by providing human-specific preclinical data during early stage drug development.


Asunto(s)
Materiales Biomiméticos/química , Ventrículos Cardíacos/citología , Miocardio/citología , Células Madre Pluripotentes/citología , Potenciales de Acción , Materiales Biomiméticos/metabolismo , Técnicas de Cultivo de Célula , Diferenciación Celular , Colágeno/química , Fenómenos Electrofisiológicos , Humanos , Hidrogeles , Contracción Miocárdica , Miocitos Cardíacos/citología , Ingeniería de Tejidos , Función Ventricular
6.
Stem Cells ; 36(4): 501-513, 2018 04.
Artículo en Inglés | MEDLINE | ID: mdl-29271023

RESUMEN

Autophagy is a process essential for cell survival under stress condition. The patients with autosomal dominant polycystic kidney disease, which is caused by polycystin-1 or polycystin-2 (PKD2) mutation, display cardiovascular abnormalities and dysregulation in autophagy. However, it is unclear whether PKD2 plays a role in autophagy. In the present study, we explored the functional role of PKD2 in autophagy and apoptosis in human embryonic stem cell-derived cardiomyocytes. HES2 hESC line-derived cardiomyocytes (HES2-CMs) were transduced with adenoviral-based PKD2-shRNAs (Ad-PKD2-shRNAs), and then cultured with normal or glucose-free medium for 3 hours. Autophagy was upregulated in HES2-CMs under glucose starvation, as indicated by increased microtubule-associated protein 1 light chain 3-II level in immunoblots and increased autophagosome and autolysosome formation. Knockdown of PKD2 reduced the autophagic flux and increased apoptosis under glucose starvation. In Ca2+ measurement, Ad-PKD2-shRNAs reduced caffeine-induced cytosolic Ca2+ rise. Co-immunoprecipitation and in situ proximity ligation assay demonstrated an increased physical interaction of PKD2 with ryanodine receptor 2 (RyR2) under glucose starvation condition. Furthermore, Ad-PKD2-shRNAs substantially attenuated the starvation-induced activation of AMP-activated protein kinase (AMPK) and inactivation of mammalian target of rapamycin (mTOR). The present study for the first time demonstrates that PKD2 functions to promote autophagy under glucose starvation, thereby protects cardiomyocytes from apoptotic cell death. The mechanism may involve PKD2 interaction with RyR2 to alter Ca2+ release from sarcoplasmic reticulum, consequently modulating the activity of AMPK and mTOR, resulting in alteration of autophagy and apoptosis. Stem Cells 2018;36:501-513.


Asunto(s)
Autofagia , Glucosa/metabolismo , Células Madre Embrionarias Humanas/metabolismo , Miocitos Cardíacos/metabolismo , Canales Catiónicos TRPP/biosíntesis , Apoptosis , Línea Celular , Glucosa/genética , Células Madre Embrionarias Humanas/citología , Humanos , Miocitos Cardíacos/citología , Canales Catiónicos TRPP/genética
7.
Stem Cell Reports ; 9(5): 1560-1572, 2017 11 14.
Artículo en Inglés | MEDLINE | ID: mdl-29033305

RESUMEN

Accurately predicting cardioactive effects of new molecular entities for therapeutics remains a daunting challenge. Immense research effort has been focused toward creating new screening platforms that utilize human pluripotent stem cell (hPSC)-derived cardiomyocytes and three-dimensional engineered cardiac tissue constructs to better recapitulate human heart function and drug responses. As these new platforms become increasingly sophisticated and high throughput, the drug screens result in larger multidimensional datasets. Improved automated analysis methods must therefore be developed in parallel to fully comprehend the cellular response across a multidimensional parameter space. Here, we describe the use of machine learning to comprehensively analyze 17 functional parameters derived from force readouts of hPSC-derived ventricular cardiac tissue strips (hvCTS) electrically paced at a range of frequencies and exposed to a library of compounds. A generated metric is effective for then determining the cardioactivity of a given drug. Furthermore, we demonstrate a classification model that can automatically predict the mechanistic action of an unknown cardioactive drug.


Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Aprendizaje Automático , Contracción Miocárdica , Miocitos Cardíacos/citología , Células Madre Pluripotentes/citología , Cardiotoxicidad/etiología , Diferenciación Celular , Células Cultivadas , Humanos , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/fisiología
8.
Int J Mol Med ; 39(3): 519-526, 2017 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-28204831

RESUMEN

Sick sinus syndrome (SSS) encompasses a group of disorders whereby the heart is unable to perform its pacemaker function, due to genetic and acquired causes. Tachycardia­bradycardia syndrome (TBS) is a complication of SSS characterized by alternating tachycardia and bradycardia. Techniques such as genetic screening and molecular diagnostics together with the use of pre-clinical models have elucidated the electrophysiological mechanisms of this condition. Dysfunction of ion channels responsible for initiation or conduction of cardiac action potentials may underlie both bradycardia and tachycardia; bradycardia can also increase the risk of tachycardia, and vice versa. The mainstay treatment option for SSS is pacemaker implantation, an effective approach, but has disadvantages such as infection, limited battery life, dislodgement of leads and catheters to be permanently implanted in situ. Alternatives to electronic pacemakers are gene­based bio­artificial sinoatrial node and cell­based bio­artificial pacemakers, which are promising techniques whose long-term safety and efficacy need to be established. The aim of this article is to review the different ion channels involved in TBS, examine the three­way relationship between ion channel dysfunction, tachycardia and bradycardia in TBS and to consider its current and future therapies.


Asunto(s)
Bradicardia/etiología , Bradicardia/metabolismo , Taquicardia/etiología , Taquicardia/metabolismo , Animales , Bradicardia/fisiopatología , Bradicardia/terapia , Calcio/metabolismo , Fenómenos Electrofisiológicos , Uniones Comunicantes/metabolismo , Humanos , Canales Iónicos/metabolismo , Síndrome del Seno Enfermo/complicaciones , Nodo Sinoatrial/metabolismo , Nodo Sinoatrial/fisiopatología , Síndrome , Taquicardia/fisiopatología , Taquicardia/terapia
9.
Adv Mater ; 29(1)2017 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-27805726

RESUMEN

A novel cardiomimetic biohybrid material, termed as the human ventricular cardiac anisotropic sheet (hvCAS) is reported. Well-characterized human pluripotent stem-cell-derived ventricular cardiomyocytes are strategically aligned to reproduce key electrophysiological features of native human ventricle, which, along with specific selection criteria, allows for a direct visualization of arrhythmic spiral re-entry and represents a revolutionary tool to assess preclinical drug-induced arrhythmogenicity.


Asunto(s)
Células Madre Pluripotentes , Diferenciación Celular , Ventrículos Cardíacos , Humanos , Miocitos Cardíacos
10.
Stem Cells ; 33(12): 3452-67, 2015 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-26175298

RESUMEN

Adult mesenchymal stem cells (MSCs) are immunoprivileged cells due to the low expression of major histocompatibility complex (MHC) II molecules. However, the expression of MHC molecules in human-induced pluripotent stem cells (iPSCs)-derived MSCs has not been investigated. Here, we examined the expression of human leukocyte antigen (HLA) in human MSCs derived from iPSCs, fetuses, and adult bone marrow (BM) after stimulation with interferon-γ (IFN-γ), compared their repair efficacy, cell retention, inflammation, and HLA II expression in immune humanized NOD Scid gamma (NSG) mice of hind limb ischemia. In the absence of IFN-γ stimulation, HLA-II was expressed only in BM-MSCs after 7 days. Two and seven days after stimulation, high levels of HLA-II were observed in BM-MSCs, intermediate levels were found in fetal-MSCs, and very low levels in iPSC-MSCs. The levels of p-STAT1, interferon regulatory factor 1, and class II transactivator exhibited similar phenomena. Moreover, p-STAT1 antagonist significantly reversed the high expression of HLA-II in BM-MSCs. Compared to adult BM-MSCs, transplanting iPSC-MSCs into hu-PBMNC NSG mice revealed markedly more survival iPSC-MSCs, less inflammatory cell accumulations, and better recovery of hind limb ischemia. The expression of HLA-II in MSCs in the ischemia limbs was detected in BM-MSCs group but not in iPSC-MSCs group at 7 and 21 days after transplantation. Our results demonstrate that, compared to adult MSCs, human iPSC-MSCs are insensitive to proinflammatory IFN-γ-induced HLA-II expression and iPSC-MSCs have a stronger immune privilege after transplantation. It may attribute to a better therapeutic efficacy in allogeneic transplantation.


Asunto(s)
Miembro Posterior/irrigación sanguínea , Antígenos de Histocompatibilidad Clase II/biosíntesis , Células Madre Pluripotentes Inducidas/metabolismo , Interferón gamma/farmacología , Isquemia/terapia , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/metabolismo , Animales , Xenoinjertos , Humanos , Isquemia/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID
11.
Circ Arrhythm Electrophysiol ; 8(1): 193-202, 2015 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-25504561

RESUMEN

BACKGROUND: Human (h) embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) serve as a potential unlimited ex vivo source of cardiomyocytes (CMs). However, a well-accepted roadblock has been their immature phenotype. hESC/iPSC-derived ventricular (v) CMs and their engineered cardiac microtissues (hvCMTs) similarly displayed positive chronotropic but null inotropic responses to ß-adrenergic stimulation. Given that phospholamban (PLB) is robustly present in adult but poorly expressed in hESC/iPSC-vCMs and its defined biological role in ß-adrenergic signaling, we investigated the functional consequences of PLB expression in hESC/iPSC-vCMs and hvCMTs. METHODS AND RESULTS: First, we confirmed that PLB protein was differentially expressed in hESC (HES2, H9)- and iPSC-derived and adult vCMs. We then transduced hES2-vCMs with the recombinant adenoviruses (Ad) Ad-PLB or Ad-S16E-PLB to overexpress wild-type PLB or the pseudophosphorylated point-mutated variant, respectively. As anticipated from the inhibitory effect of unphosphorylated PLB on sarco/endoplasmic reticulum Ca2+-ATPase, Ad-PLB transduction significantly attenuated electrically evoked Ca2+ transient amplitude and prolonged the 50% decay time. Importantly, Ad-PLB-transduced hES2-vCMs uniquely responded to isoproterenol. Ad-S16E-PLB-transduced hES2-vCMs displayed an intermediate phenotype. The same trends were observed with H9- and iPSC-vCMs. Directionally, similar results were also seen with Ad-PLB-transduced and Ad-S16E-transduced hvCMTs. However, Ad-PLB altered neither the global transcriptome nor ICa,L, implicating a PLB-specific effect. CONCLUSIONS: Engineered upregulation of PLB expression in hESC/iPSC-vCMs restores a positive inotropic response to ß-adrenergic stimulation. These results not only provide a better mechanistic understanding of the immaturity of hESC/iPSC-vCMs but will also lead to improved disease models and transplantable prototypes with adult-like physiological responses.


Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Diferenciación Celular , Células Madre Embrionarias/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Contracción Miocárdica , Miocitos Cardíacos/metabolismo , Ingeniería de Tejidos/métodos , Agonistas Adrenérgicos beta/farmacología , Señalización del Calcio , Proteínas de Unión al Calcio/genética , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Células Madre Embrionarias/efectos de los fármacos , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Isoproterenol/farmacología , Mutación , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Fenotipo , Retículo Sarcoplasmático/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Transducción de Señal , Transducción Genética , Transfección , Regulación hacia Arriba
12.
Macromol Biosci ; 15(3): 426-36, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25420623

RESUMEN

Microgrooved thin PLGA film (≈30 µm) is successfully fabricated on a Teflon mold, which could be readily peeled off and is used for the construction of a biomimetic cardiac patch. The contraction of it is studied with optical mapping on transmembrane action potential. Our results suggest that steady-state contraction could be easily established on it under regular electrical stimuli. Besides, the biomimetic cardiac patch recapitulates the anisotropic electrophysiological feature of native cardiac tissue and is much more refractory to premature stimuli than the random one constructed with non-grooved PLGA film, as proved by the reduced incidence of arrhythmia. Considering the good biocompatibility of PLGA as demonstrated in our study and the biodegradability of it, our biomimetic cardiac patch may find applications in the treatment of myocardial infarction. Moreover, the Teflon mold could be applied in the fabrication of various scaffolds with fine features for other tissues.


Asunto(s)
Biomimética/métodos , Ventrículos Cardíacos/citología , Células Madre Embrionarias Humanas/citología , Ácido Láctico/química , Miocitos Cardíacos/citología , Ácido Poliglicólico/química , Trasplante de Células Madre , Andamios del Tejido/química , Potenciales de Acción , Arritmias Cardíacas/terapia , Estimulación Eléctrica , Humanos , Microscopía Confocal , Miocardio/patología , Copolímero de Ácido Poliláctico-Ácido Poliglicólico
13.
Stem Cells Dev ; 23(14): 1704-16, 2014 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-24564569

RESUMEN

Self-renewable human pluripotent stem cells (hPSCs) serve as a potential unlimited ex vivo source of human cardiomyocytes (CMs) for cell-based disease modeling and therapies. Although recent advances in directed differentiation protocols have enabled more efficient derivation of hPSC-derived CMs with an efficiency of ∼50%-80% CMs and a final yield of ∼1-20 CMs per starting undifferentiated hPSC, these protocols are often not readily transferrable across lines without first optimizing multiple parameters. Further, the resultant populations are undefined for chamber specificity or heterogeneous containing mixtures of atrial, ventricular (V), and pacemaker derivatives. Here we report a highly cost-effective and reproducibly efficient system for deriving hPSC-ventricular cardiomyocytes (VCMs) from all five human embryonic stem cell (HES2, H7, and H9) and human induced PSC (hiPSC) (reprogrammed from human adult peripheral blood CD34(+) cells using nonintegrating episomal vectors) lines tested. Cardiogenic embryoid bodies could be formed by the sequential addition of BMP4, Rho kinase inhibitor, activin-A, and IWR-1. Spontaneously contracting clusters appeared as early as day 8. At day 16, up to 95% of cells were cTnT(+). Of which, 93%, 94%, 100%, 92%, and 92% of cardiac derivatives from HES2, H7, H9, and two iPSC lines, respectively, were VCMs as gauged by signature ventricular action potential and ionic currents (INa(+)/ICa,L(+)/IKr(+)/IKATP(+)); Ca(2+) transients showed positive chronotropic responses to ß-adrenergic stimulation. Our simple, cost-effective protocol required the least amounts of reagents and time compared with others. While the purity and percentage of PSC-VCMs were comparable to a recently published protocol, the present yield and efficiency with a final output of up to 70 hPSC-VCMs per hPSC was up to 5-fold higher and without the need of performing line-specific optimization. These differences were discussed. The results may lead to mass production of hPSC-VCMs in bioreactors.


Asunto(s)
Células Madre Embrionarias/citología , Ventrículos Cardíacos/citología , Células Madre Pluripotentes Inducidas/citología , Miocitos Cardíacos/citología , Reactores Biológicos , Proteína Morfogenética Ósea 4/biosíntesis , Diferenciación Celular/genética , Humanos
14.
Biomaterials ; 34(35): 8878-86, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-23942210

RESUMEN

Human (h) pluripotent stem cells (PSC) such as embryonic stem cells (ESC) can be directed into cardiomyocytes (CMs), representing a potential unlimited cell source for disease modeling, cardiotoxicity screening and myocardial repair. Although the electrophysiology of single hESC-CMs is now better defined, their multi-cellular arrhythmogenicity has not been thoroughly assessed due to the lack of a suitable experimental platform. Indeed, the generation of ventricular (V) fibrillation requires single-cell triggers as well as sustained multi-cellular reentrant events. Although native VCMs are aligned in a highly organized fashion such that electrical conduction is anisotropic for coordinated contractions, hESC-derived CM (hESC-CM) clusters are heterogenous and randomly organized, and therefore not representative of native conditions. Here, we reported that engineered alignment of hESC-VCMs on biomimetic grooves uniquely led to physiologically relevant responses. Aligned but not isotropic control preparations showed distinct longitudinal (L) and transverse (T) conduction velocities (CV), resembling the native human V anisotropic ratio (AR = LCV/TCV = 1.8-2.0). Importantly, the total incidence of spontaneous and inducible arrhythmias significantly reduced from 57% in controls to 17-23% of aligned preparations, thereby providing a physiological baseline for assessing arrhythmogenicity. As such, promotion of pro-arrhythmic effect (e.g., spatial dispersion by ß adrenergic stimulation) could be better predicted. Mechanistically, such anisotropy-induced electrical stability was not due to maturation of the cellular properties of hESC-VCMs but their physical arrangement. In conclusion, not only do functional anisotropic hESC-VCMs engineered by multi-scale topography represent a more accurate model for efficacious drug discovery and development as well as arrhythmogenicity screening (of pharmacological and genetic factors), but our approach may also lead to future transplantable prototypes with improved efficacy and safety against arrhythmias.


Asunto(s)
Arritmias Cardíacas/prevención & control , Ventrículos Cardíacos/patología , Miocitos Cardíacos/citología , Células Madre Pluripotentes/metabolismo , Ingeniería de Tejidos , Anisotropía , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/terapia , Materiales Biomiméticos , Diferenciación Celular , Células Cultivadas , Fenómenos Electrofisiológicos , Humanos , Miocitos Cardíacos/metabolismo
15.
PLoS One ; 7(11): e50238, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-23189191

RESUMEN

Severe trauma such as burn injury is often associated with a systemic inflammatory syndrome characterized by a hyperactive innate immune response and suppressed adaptive immune function. Dendritic cells (DCs), which sense pathogens via their Toll-like receptors (TLRs), play a pivotal role in protecting the host against infections. The effect of burn injury on TLR-mediated DC function is a debated topic and the mechanism controlling the purported immunosuppressive response remains to be elucidated. Here we examined the effects of burn injury on splenic conventional DC (cDC) and plasmacytoid DC (pDC) responses to TLR9 activation. We demonstrate that, following burn trauma, splenic cDCs' cytokine production profile in response to TLR9 activation became anti-inflammatory dominant, with high production of IL-10 (>50% increase) and low production of IL-6, TNF-α and IL-12p70 (∼25-60% reduction). CD4+ T cells activated by these cDCs were defective in producing Th1 and Th17 cytokines. Furthermore, burn injury had a more accentuated effect on pDCs than on cDCs. Following TLR9 activation, pDCs displayed an immature phenotype with an impaired ability to secrete pro-inflammatory cytokines (IFN-α, IL-6 and TNF-α) and to activate T cell proliferation. Moreover, cDCs and pDCs from burn-injured mice had low transcript levels of TLR9 and several key molecules of the TLR signaling pathway. Although hyperactive innate immune response has been associated with severe injury, our data show to the contrary that DCs, as a key player in the innate immune system, had impaired TLR9 reactivity, an anti-inflammatory phenotype, and a dysfunctional T cell-priming ability. We conclude that burn injury induced impairments in DC immunobiology resulting in suppression of adaptive immune response. Targeted DC immunotherapies to promote their ability in triggering T cell immunity may represent a strategy to improve immune defenses against infection following burn injury.


Asunto(s)
Quemaduras/inmunología , Quemaduras/metabolismo , Células Dendríticas/inmunología , Linfocitos T/inmunología , Receptor Toll-Like 9/metabolismo , Animales , Quemaduras/genética , Diferenciación Celular , Citocinas/biosíntesis , Citocinas/inmunología , Células Dendríticas/metabolismo , Modelos Animales de Enfermedad , Femenino , Mediadores de Inflamación/inmunología , Mediadores de Inflamación/metabolismo , Activación de Linfocitos/inmunología , Ratones , Transducción de Señal , Bazo/citología , Bazo/inmunología , Bazo/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Linfocitos T/metabolismo , Células TH1/citología , Células TH1/inmunología , Células TH1/metabolismo , Células Th17/citología , Células Th17/inmunología , Células Th17/metabolismo , Receptor Toll-Like 9/genética
16.
Birth Defects Res C Embryo Today ; 96(1): 98-108, 2012 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-22457181

RESUMEN

Heart diseases such as myocardial infarction cause massive loss of cardiomyocytes, but the human heart lacks the innate ability to regenerate. In the adult mammalian heart, a resident progenitor cell population, termed epicardial progenitors, has been identified and reported to stay quiescent under uninjured conditions; however, myocardial infarction induces their proliferation and de novo differentiation into cardiac cells. It is conceivable to develop novel therapeutic approaches for myocardial repair by targeting such expandable sources of cardiac progenitors, thereby giving rise to new muscle and vasculatures. Human pluripotent stem cells such as embryonic stem cells and induced pluripotent stem cells can self-renew and differentiate into the three major cell types of the heart, namely cardiomyocytes, smooth muscle, and endothelial cells. In this review, we describe our current knowledge of the therapeutic potential and challenges associated with the use of pluripotent stem cell and progenitor biology in cell therapy. An emphasis is placed on the contribution of paracrine factors in the growth of myocardium and neovascularization as well as the role of immunogenicity in cell survival and engraftment.


Asunto(s)
Células Madre Pluripotentes Inducidas/trasplante , Miocitos Cardíacos/citología , Medicina Regenerativa/métodos , Ingeniería de Tejidos , Corazón/crecimiento & desarrollo , Humanos , Células Madre Pluripotentes Inducidas/inmunología , Infarto del Miocardio/inmunología , Infarto del Miocardio/patología , Infarto del Miocardio/terapia , Miocardio/inmunología , Miocardio/patología , Miocitos Cardíacos/inmunología , Comunicación Paracrina/inmunología , Pericardio/citología , Regeneración/inmunología , Trasplante de Células Madre
17.
PLoS One ; 6(11): e27417, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-22110643

RESUMEN

BACKGROUND: MicroRNAs (miRs) negatively regulate transcription and are important determinants of normal heart development and heart failure pathogenesis. Despite the significant knowledge gained in mouse studies, their functional roles in human (h) heart remain elusive. METHODS AND RESULTS: We hypothesized that miRs that figure prominently in cardiac differentiation are differentially expressed in differentiating, developing, and terminally mature human cardiomyocytes (CMs). As a first step, we mapped the miR profiles of human (h) embryonic stem cells (ESCs), hESC-derived (hE), fetal (hF) and adult (hA) ventricular (V) CMs. 63 miRs were differentially expressed between hESCs and hE-VCMs. Of these, 29, including the miR-302 and -371/372/373 clusters, were associated with pluripotency and uniquely expressed in hESCs. Of the remaining miRs differentially expressed in hE-VCMs, 23 continued to express highly in hF- and hA-VCMs, with miR-1, -133, and -499 displaying the largest fold differences; others such as miR-let-7a, -let-7b, -26b, -125a and -143 were non-cardiac specific. Functionally, LV-miR-499 transduction of hESC-derived cardiovascular progenitors significantly increased the yield of hE-VCMs (to 72% from 48% of control; p<0.05) and contractile protein expression without affecting their electrophysiological properties (p>0.05). By contrast, LV-miR-1 transduction did not bias the yield (p>0.05) but decreased APD and hyperpolarized RMP/MDP in hE-VCMs due to increased I(to), I(Ks) and I(Kr), and decreased I(f) (p<0.05) as signs of functional maturation. Also, LV-miR-1 but not -499 augmented the immature Ca(2+) transient amplitude and kinetics. Molecular pathway analyses were performed for further insights. CONCLUSION: We conclude that miR-1 and -499 play differential roles in cardiac differentiation of hESCs in a context-dependent fashion. While miR-499 promotes ventricular specification of hESCs, miR-1 serves to facilitate electrophysiological maturation.


Asunto(s)
Diferenciación Celular , Células Madre Embrionarias/citología , Ventrículos Cardíacos/citología , MicroARNs/metabolismo , Miocitos Cardíacos/citología , Función Ventricular/genética , Adulto , Animales , Calcio/metabolismo , Línea Celular , Fenómenos Electrofisiológicos/genética , Feto/citología , Ventrículos Cardíacos/metabolismo , Humanos , Ratones , Miocitos Cardíacos/metabolismo
18.
Am J Physiol Cell Physiol ; 298(3): C486-95, 2010 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-19955484

RESUMEN

Human embryonic stem cells (hESCs) can self-renew while maintaining their pluripotency. Direct reprogramming of adult somatic cells to induced pluripotent stem cells (iPSCs) has been reported. Although hESCs and human iPSCs have been shown to share a number of similarities, such basic properties as the electrophysiology of iPSCs have not been explored. Previously, we reported that several specialized ion channels are functionally expressed in hESCs. Using transcriptomic analyses as a guide, we observed tetraethylammonium (TEA)-sensitive (IC(50) = 3.3 +/- 2.7 mM) delayed rectifier K(+) currents (I(KDR)) in 105 of 110 single iPSCs (15.4 +/- 0.9 pF). I(KDR) in iPSCs displayed a current density of 7.6 +/- 3.8 pA/pF at +40 mV. The voltage for 50% activation (V(1/2)) was -7.9 +/- 2.0 mV, slope factor k = 9.1 +/- 1.5. However, Ca(2+)-activated K(+) current (I(KCa)), hyperpolarization-activated pacemaker current (I(f)), and voltage-gated sodium channel (Na(V)) and voltage-gated calcium channel (Ca(V)) currents could not be measured. TEA inhibited iPSC proliferation (EC(50) = 7.8 +/- 1.2 mM) and viability (EC(50) = 5.5 +/- 1.0 mM). By contrast, 4-aminopyridine (4-AP) inhibited viability (EC(50) = 4.5 +/- 0.5 mM) but had less effect on proliferation (EC(50) = 0.9 +/- 0.5 mM). Cell cycle analysis further revealed that K(+) channel blockers inhibited proliferation primarily by arresting the mitotic phase. TEA and 4-AP had no effect on iPSC differentiation as gauged by ability to form embryoid bodies and expression of germ layer markers after induction of differentiation. Neither iberiotoxin nor apamin had any function effects, consistent with the lack of I(KCa) in iPSCs. Our results reveal further differences and similarities between human iPSCs and hESCs. A better understanding of the basic biology of iPSCs may facilitate their ultimate clinical application.


Asunto(s)
Canales de Potasio de Tipo Rectificador Tardío/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Potasio/metabolismo , Canales de Calcio/metabolismo , Ciclo Celular , Diferenciación Celular , Línea Celular , Proliferación Celular , Supervivencia Celular , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Canales de Potasio de Tipo Rectificador Tardío/antagonistas & inhibidores , Canales de Potasio de Tipo Rectificador Tardío/genética , Relación Dosis-Respuesta a Droga , Células Madre Embrionarias/metabolismo , Perfilación de la Expresión Génica , Humanos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Cinética , Potenciales de la Membrana , Células Madre Mesenquimatosas/metabolismo , Proteínas Musculares/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio , Canales de Potasio Calcio-Activados/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Canales de Sodio/metabolismo
19.
Biochem Biophys Res Commun ; 377(1): 46-51, 2008 Dec 05.
Artículo en Inglés | MEDLINE | ID: mdl-18823947

RESUMEN

Gap junctions, encoded by the connexin (Cx) multi-gene family, couple adjacent cells and underlie cell-cell communications. Previous mouse studies suggest that Cxs play an important role in development but their role in human cardiogenesis is undefined. Human embryonic stem cells (hESC) provide a unique model for studying human differentiation. Lentivirus-mediated stable overexpression of Cx43 in hESC (Cx43-hESC) did not affect colony morphology, karyotype and expression of pluripotency genes such as Oct4 but completely suppressed the formation of spontaneously beating, cardiomyocyte-containing clusters in embryoid bodies (EBs). Unlike control hEBs, the transcripts of several mesodermal markers (kallikrein, delta-globin, and CMP), ventricular myosin light chain and cardiac troponin I were absent or delayed. Transcriptomic and pathway analyses showed that 194 genes crucial for movement, growth, differentiation and maintenance were differentially expressed in Cx43-hESC. We conclude that Cx43 mediates the expression of an array of genes involved in human cardiogenesis, in addition to intercellular communication.


Asunto(s)
Conexina 43/metabolismo , Células Madre Embrionarias/fisiología , Uniones Comunicantes/metabolismo , Regulación del Desarrollo de la Expresión Génica , Corazón/embriología , Organogénesis/genética , Células Madre Pluripotentes/fisiología , Animales , Comunicación Celular/genética , Diferenciación Celular/genética , Línea Celular , Conexina 43/genética , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Humanos , Miocitos Cardíacos/citología , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Ratas , Transcripción Genética
20.
Stem Cells Dev ; 17(2): 315-24, 2008 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-18447646

RESUMEN

Self-renewing pluripotent human embryonic stem (hES) cells are capable of regenerating such non-dividing cells as neurons and cardiomyocytes for therapies and can serve as an excellent experimental model for studying early human development. Both the spatial and temporal relationships of gene expression play a crucial role in determining differentiation; to obtain a better understanding of hES cell differentiation, it will be necessary to establish an inducible system in hES cells that enables specific transgene(s) to reversibly and conditionally express (1) at specific levels and (2) at particular time points during development. Using lentivirus (LV)-mediated gene transfer and a tetracycline-controlled trans-repressor (TR), we first established in hES cells a doxycycline (DOX)-inducible expression system of green fluorescent protein (GFP) to probe its reversibility and kinetics. Upon the addition of DOX, the percentage of GFP(+) hES cells increased time dependently: The time at which 50% of all green cells appeared (T(50)(on)) was 119.5+/-3.2 h; upon DOX removal, GFP expression declined with a half-time (T(50)(off)) of 127.7+/-3.9 h and became completely silenced at day 8. Both the proportion and total mean fluorescence intensity (MFI) were dose-dependent (EC(50)=24.5+/-2.2 ng/ml). The same system when incorporated into murine (m) ES cells similarly exhibited reversible dose-dependent responses with a similar sensitivity (EC(50)=49.5+/-8.5 ng/ml), but the much faster kinetics (T(50)(on)=35.5+/-5.5 h, T(50)(off) = 71.5+/-2.4 hours). DOX-induced expression of the Kir2.1 channels in mES and hES cells led to robust expression of the inwardly rectifying potassium (K(+)) current and thereby hyperpolarized the resting membrane potential (RMP). We conclude that the LV-inducible system established presents a unique tool for probing differentiation.


Asunto(s)
Diferenciación Celular/genética , Células Madre Embrionarias/fisiología , Regulación de la Expresión Génica , Transgenes , Animales , Células Cultivadas , Clonación Molecular , Relación Dosis-Respuesta a Droga , Doxiciclina/farmacología , Electrofisiología , Células Madre Embrionarias/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Potenciales de la Membrana/genética , Ratones , Modelos Biológicos , Fenotipo , Canales de Potasio de Rectificación Interna/genética , Canales de Potasio de Rectificación Interna/metabolismo
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA
...