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BACKGROUND AND OBJECTIVE: Elexacaftor/tezacaftor/ivacaftor is highly effective in treating people with cystic fibrosis (pwCF) who have ≥ 1 responsive mutation. Liver disease occurs in approximately 10%-20% of pwCF. The objective of this study was to assess the safety and pharmacokinetics of elexacaftor/tezacaftor/ivacaftor in people with moderate hepatic impairment, which is necessary to inform on its use and guide dosing recommendations. METHODS: The safety and pharmacokinetics of elexacaftor/tezacaftor/ivacaftor were evaluated in subjects without CF with moderate hepatic impairment versus matched healthy controls. Twenty-two subjects (11 with moderate hepatic impairment and 11 healthy subjects) received half the standard adult daily dose of elexacaftor/tezacaftor/ivacaftor (elexacaftor 100 mg/tezacaftor 50 mg/ivacaftor 150 mg) orally for 10 days. RESULTS: Elexacaftor/tezacaftor/ivacaftor was safe and well tolerated in subjects with moderate hepatic impairment and healthy controls. On day 10, the mean values of the area under the curve during the dosing interval (AUCτ) for total (bound and unbound) elexacaftor and its major active metabolite M23-elexacaftor were increased 1.25-fold (95% CI 1.01, 1.54) and 1.73-fold (95% CI 1.27, 2.35), respectively, in subjects with moderate hepatic impairment compared with matched healthy subjects. The mean values of AUCτ for ivacaftor and tezacaftor were increased 1.50-fold (95% CI 1.09, 2.06) and 1.20-fold (95% CI 1.00, 1.43), respectively, while the mean value of AUCτ for the active metabolite M1-tezacaftor was 1.29-fold lower [ratio of moderate hepatic impairment to healthy subjects (95% CI): 0.778 (0.655, 0.924)] in subjects with moderate hepatic impairment. CONCLUSIONS: A dose reduction of elexacaftor/tezacaftor/ivacaftor is warranted in people with moderate hepatic impairment. (Trial registry number 2018-002570-40; registered 2 July 2018.).
Elexacaftor/tezacaftor/ivacaftor is a combination product (made up of the three drugs elexacaftor, tezacaftor, and ivacaftor) that can effectively treat cystic fibrosis (CF). About 10%20% of people with CF have liver disease, and the liver plays an important role in breaking down these drugs. Thus, it is important to understand how liver disease or reduced liver function affects the amounts of these drugs in the body over time. This can help determine how much of the drug (i.e., what dose) people should take.We gave people with reduced liver function and healthy people (with normal liver function) elexacaftor/tezacaftor/ivacaftor for 10 days. We looked at the safety of the combination and measured the amounts of elexacaftor, tezacaftor, and ivacaftor in the body over time.We found that when people with moderately reduced liver function take elexacaftor/tezacaftor/ivacaftor, they have higher amounts of the drugs elexacaftor, tezacaftor, and ivacaftor in their bodies compared with healthy people with normal liver function. These findings mean that people with moderately reduced liver function should take a lower dose of elexacaftor/tezacaftor/ivacaftor.
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Fibrosis Quística , Hepatopatías , Adulto , Humanos , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/genética , Fibrosis Quística/inducido químicamente , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/uso terapéutico , Hepatopatías/tratamiento farmacológicoRESUMEN
The goal of this investigation was to examine clinical translation of glucose responsiveness of MK-2640, which is a novel insulin saccharide conjugate that can bind the insulin receptor or mannose receptor C type 1 (MRC1), the latter dependent upon glucose concentration. In a rising dose study in 36 healthy adults under euglycemic clamp conditions, rising exposures revealed saturation of MK-2640 clearance, likely due to saturation of clearance by MRC1. Potency of MK-2640 was ~25-fold reduced relative to regular human insulin. In a randomized, 2-period crossover trial in 16 subjects with type 1 diabetes mellitus to evaluate glucose-responsiveness of i.v. administered MK-2640, we were unable to demonstrate a glucose-dependent change in MK-2640 clearance, although a significant glucose-dependent augmentation of glucose infusion rate was observed. These pharmacokinetic (PK) and pharmacodynamic (PD) data provide crucial insights into next steps for developing an insulin saccharide conjugate as a clinically effective glucose-responsive insulin analog.
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Glucemia/efectos de los fármacos , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Hipoglucemiantes/uso terapéutico , Insulina/análogos & derivados , Administración Intravenosa , Adolescente , Adulto , Antígenos CD/efectos de los fármacos , Estudios Cruzados , Diabetes Mellitus Tipo 1/sangre , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Femenino , Técnica de Clampeo de la Glucosa , Humanos , Hipoglucemiantes/efectos adversos , Hipoglucemiantes/farmacocinética , Insulina/efectos adversos , Insulina/farmacocinética , Insulina/uso terapéutico , Masculino , Persona de Mediana Edad , Receptor de Insulina/efectos de los fármacos , Adulto JovenRESUMEN
Biomarkers of heart failure in adults have been extensively studied. However, biomarkers to monitor the progression of heart failure in children with univentricular physiology are less well understood. We proposed that as mediators of diverse pathophysiology, miRNAs contained within circulating microvesicles could serve as biomarkers for the presence and progression of heart failure in univentricular patients. To test this, we studied the association of heart failure with elevations in specific miRNAs isolated from circulating microvesicles in a cohort of children with univentricular heart disease and heart failure. We conducted a single site cross-sectional observational study of 71 children aged 1 month-7 years with univentricular heart disease and heart failure. We demonstrated that levels of miR129-5p isolated from plasma microvesicles were inversely related to the degree of clinical heart failure as assessed by Ross score. We then showed that miR129-5p levels are downregulated in HL1 cells and human embryonic stem cell-derived cardiomyocytes exposed to oxidative stress. We demonstrated that bone morphogenetic protein receptor 2, which has been implicated in the development of pulmonary vascular disease, is a target of miR129-5p, and conversely regulated in response to oxidative stress in cell culture. Levels of miR129-5p were inversely related to the degree of clinical heart failure in patients with univentricular heart disease. This study demonstrates that miR129-5p is a sensitive and specific biomarker for heart failure in univentricular heart disease independent of ventricular morphology or stage of palliation. Further study is warranted to understand the targets affected by miR129-5p with the development of heart failure in patients with univentricular physiology.
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Biomarcadores/sangre , Insuficiencia Cardíaca/sangre , Ventrículos Cardíacos/fisiopatología , MicroARNs/sangre , Micropartículas Derivadas de Células/metabolismo , Micropartículas Derivadas de Células/patología , Niño , Preescolar , Estudios Transversales , Progresión de la Enfermedad , Femenino , Insuficiencia Cardíaca/fisiopatología , Ventrículos Cardíacos/metabolismo , Humanos , Lactante , Recién Nacido , MasculinoRESUMEN
The increasing burden and the continued suboptimal outcomes for patients with heart failure underlines the importance of continued research to develop novel therapeutics for this disorder. This can only be accomplished with successful translation of basic science discoveries into direct human application through effective clinical trial design and execution that results in a substantially improved clinical course and outcomes. In this respect, phase II clinical trials play a pivotal role in determining which of the multitude of potential basic science discoveries should move to the large and expansive registration trials in humans. A critical examination of the phase II trials in heart failure reveals multiple shortcomings in their concept, design, execution, and interpretation. To further a dialogue on the challenges and potential for improvement and the role of phase II trials in patients with heart failure, the Food and Drug Administration facilitated a meeting on October 17, 2016, represented by clinicians, researchers, industry members, and regulators. This document summarizes the discussion from this meeting and provides key recommendations for future directions.
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Fármacos Cardiovasculares/uso terapéutico , Ensayos Clínicos Fase II como Asunto/normas , Insuficiencia Cardíaca/tratamiento farmacológico , Proyectos de Investigación/normas , Fármacos Cardiovasculares/efectos adversos , Ensayos Clínicos Fase II como Asunto/métodos , Consenso , Insuficiencia Cardíaca/diagnóstico , Insuficiencia Cardíaca/mortalidad , Insuficiencia Cardíaca/fisiopatología , Humanos , Resultado del Tratamiento , Estados Unidos , United States Food and Drug AdministrationRESUMEN
AIM: Patients with elevated levels of B-type natriuretic peptide (BNP) and/or NT-proBNP as measured by clinical tests have an elevated risk of heart failure (HF). Despite utility in large clinical studies, both assays are plagued by large biological variability and specificity issues. To address these concerns and further investigate BNP in the HF setting, we developed an LC/MS assay to characterize the ratio of active to total BNP. RESULTS: We have developed and validated a novel immunoaffinity LC/MS assay to measure BNP-derived fragments, as well as 'total BNP' in human plasma. The ratio of active BNP1-32 to total BNP in 11 HF subjects was found to be <8%, and the sum of detectable BNP fragments contributed approximately 20% of total BNP. CONCLUSION: We developed an assay with the specificity to measure the active form of BNP, which may aid in the accurate diagnosis and better management of HF.
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The epidemiological, clinical, and societal implications of the heart failure (HF) epidemic cannot be overemphasized. Approximately half of all HF patients have HF with preserved ejection fraction (HFpEF). HFpEF is largely a syndrome of the elderly, and with aging of the population, the proportion of patients with HFpEF is expected to grow. Currently, there is no drug known to improve mortality or hospitalization risk for these patients. Besides mortality and hospitalization, it is imperative to realize that patients with HFpEF have significant impairment in their functional capacity and their quality of life on a daily basis, underscoring the need for these parameters to ideally be incorporated within a regulatory pathway for drug approval. Although attempts should continue to explore therapies to reduce the risk of mortality or hospitalization for these patients, efforts should also be directed to improve other patient-centric concerns, such as functional capacity and quality of life. To initiate a dialogue about the compelling need for and the challenges in developing such alternative endpoints for patients with HFpEF, the US Food and Drug Administration on November 12, 2015, facilitated a meeting represented by clinicians, academia, industry, and regulatory agencies. This document summarizes the discussion from this meeting.
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Insuficiencia Cardíaca/terapia , Hospitalización , Mortalidad , Medición de Resultados Informados por el Paciente , Volumen Sistólico , Congresos como Asunto , Aprobación de Drogas , Descubrimiento de Drogas , Prueba de Esfuerzo , Insuficiencia Cardíaca/fisiopatología , Humanos , Evaluación de Resultado en la Atención de Salud , Consumo de Oxígeno , Calidad de Vida , Estados Unidos , United States Food and Drug Administration , Prueba de PasoRESUMEN
Mechanisms of initial cell fate decisions differ among species. To gain insights into lineage allocation in humans, we derived ten human embryonic stem cell lines (designated UCSFB1-10) from single blastomeres of four 8-cell embryos and one 12-cell embryo from a single couple. Compared with numerous conventional lines from blastocysts, they had unique gene expression and DNA methylation patterns that were, in part, indicative of trophoblast competence. At a transcriptional level, UCSFB lines from different embryos were often more closely related than those from the same embryo. As predicted by the transcriptomic data, immunolocalization of EOMES, T brachyury, GDF15 and active ß-catenin revealed differential expression among blastomeres of 8- to 10-cell human embryos. The UCSFB lines formed derivatives of the three germ layers and CDX2-positive progeny, from which we derived the first human trophoblast stem cell line. Our data suggest heterogeneity among early-stage blastomeres and that the UCSFB lines have unique properties, indicative of a more immature state than conventional lines.
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Blastómeros/citología , Técnicas de Cultivo de Embriones , Células Madre Embrionarias/citología , Trofoblastos/citología , Blastocisto/citología , Diferenciación Celular , Línea Celular , Linaje de la Célula , Metilación de ADN , Endodermo/metabolismo , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Factor 15 de Diferenciación de Crecimiento/metabolismo , Humanos , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Células-Madre Neurales/citología , Análisis de Secuencia por Matrices de Oligonucleótidos , Transcripción Genética , Transcriptoma , beta Catenina/metabolismoRESUMEN
BACKGROUND: We previously reported the generation of a reporter line of human embryonic stem cells (hESCs) with enhanced green fluorescent protein (eGFP) expression driven by the α-myosin heavy chain (αMHC) promoter. The GFP+/αMHC+ cells derived from this cell line behave as multipotent, human myocardial precursors (hMPs) in vitro. In this study, we evaluated the therapeutic effects of GFP+/αMHC+ cells isolated from the reporter line in a mouse model of myocardial infarction (MI). METHODS: MI was generated in immunodeficient mice. hMPs were injected into murine infarcted hearts under ultrasound guidance at 3 days post-MI. Human fetal skin fibroblasts (hFFs) were injected as control. Cardiac function was evaluated by echocardiography. Infarct size, angiogenesis, apoptosis, cell fate, and teratoma formation were analyzed by immunohistochemical staining. RESULTS: Compared with control, hMPs resulted in improvement of cardiac function post-MI with smaller infarct size, induced endogenous angiogenesis, and reduced apoptosis of host cardiomyocytes at the peri-infarct zone at 28 days post-MI. CONCLUSION: Intramyocardial injection of hMPs improved cardiac function post-MI. The engraftment rate of these cells in the myocardium post-MI was low, suggesting that the majority of effect occurs via paracrine mechanisms.
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Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Células Madre Embrionarias/trasplante , Células Madre Multipotentes/trasplante , Infarto del Miocardio/terapia , Miocitos Cardíacos/citología , Animales , Apoptosis/fisiología , Células Cultivadas , Ecocardiografía , Femenino , Proteínas Fluorescentes Verdes/genética , Corazón/fisiopatología , Pruebas de Función Cardíaca , Humanos , Ratones , Ratones SCID , Neovascularización FisiológicaRESUMEN
A biomarker is a characteristic that can be used as an indicator of a biological state. A biomarker can be a clinical observation, laboratory test or an imaging parameter. In this review, we discuss the use of biomarkers in differentiating cardiac from noncardiac disease; predicting the prognosis of patients with heart failure, pulmonary hypertension and dilated cardiomyopathy; diagnosing subclinical cardiac involvement in muscular dystrophy and postchemotherapy cancer patients; detecting acute rejection following heart transplantation; diagnosing Kawasaki disease; aiding the management of postoperative cardiac patients; and managing both common (tetralogy of Fallot) and complex (single-ventricle physiology) congenital heart diseases.
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Biomarcadores/sangre , Cardiopatías/diagnóstico , Pediatría , Adolescente , Niño , Preescolar , Cardiopatías/sangre , HumanosRESUMEN
INTRODUCTION: Posttranscriptional control of mRNA by microRNA (miRNA) has been implicated in the regulation of diverse biologic processes from directed differentiation of stem cells through organism development. We describe a unique pathway by which miRNA regulates the specialized differentiation of cardiomyocyte (CM) subtypes. METHODS: We differentiated human embryonic stem cells (hESCs) to cardiac progenitor cells and functional CMs, and characterized the regulated expression of specific miRNAs that target transcriptional regulators of left/right ventricular-subtype specification. RESULTS: From >900 known human miRNAs in hESC-derived cardiac progenitor cells and functional CMs, a subset of differentially expressed cardiac miRNAs was identified, and in silico analysis predicted highly conserved binding sites in the 3'-untranslated regions (3'UTRs) of Hand-and-neural-crest-derivative-expressed (HAND) genes 1 and 2 that are involved in left and right ventricular development. We studied the temporal and spatial expression patterns of four miRNAs in differentiating hESCs, and found that expression of miRNA (miR)-363, miR-367, miR-181a, and miR-181c was specific for stage and site. Further analysis showed that miR-363 overexpression resulted in downregulation of HAND1 mRNA and protein levels. A dual luciferase reporter assay demonstrated functional interaction of miR-363 with the full-length 3'UTR of HAND1. Expression of anti-miR-363 in-vitro resulted in enrichment for HAND1-expressing CM subtype populations. We also showed that BMP4 treatment induced the expression of HAND2 with less effect on HAND1, whereas miR-363 overexpression selectively inhibited HAND1. CONCLUSIONS: These data show that miR-363 negatively regulates the expression of HAND1 and suggest that suppression of miR-363 could provide a novel strategy for generating functional left-ventricular CMs.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/biosíntesis , Células Madre Embrionarias/citología , Regulación del Desarrollo de la Expresión Génica/genética , Ventrículos Cardíacos/citología , MicroARNs/genética , Miocitos Cardíacos/citología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Diferenciación Celular/genética , Línea Celular , Humanos , Immunoblotting , Hibridación Fluorescente in Situ , Células Madre Pluripotentes Inducidas/citología , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Stem cell antigen-1 (Sca-1) is a member of the Ly-6 multigene family encoding highly homologous, glycosyl-phosphatidylinositol-anchored membrane proteins. Sca-1 is expressed on muscle-derived stem cells and myogenic precursors recruited to sites of muscle injury. We previously reported that inhibition of Sca-1 expression stimulated myoblast proliferation in vitro and regulated the tempo of muscle repair in vivo. Despite its function in myoblast expansion during muscle repair, a role for Sca-1 in normal, post-natal muscle has not been thoroughly investigated. We systematically compared Sca-1-/- (KO) and Sca-1+/+ (WT) mice and hindlimb muscles to elucidate the tissue, contractile, and functional effects of Sca-1 in young and aging animals. Comparison of muscle volume, fibrosis, myofiber cross-sectional area, and Pax7+ myoblast number showed little differences between ages or genotypes. Exercise protocols, however, demonstrated decreased stamina in KO versus WT mice, with young KO mice achieving results similar to aging WT animals. In addition, KO mice did not improve with practice, while WT animals demonstrated conditioning over time. Surprisingly, myomechanical analysis of isolated muscles showed that KO young muscle generated more force and experienced less fatigue. However, KO muscle also demonstrated incomplete relaxation with fatigue. These findings suggest that Sca-1 is necessary for muscle conditioning with exercise, and that deficient conditioning in Sca-1 KO animals becomes more pronounced with age.
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Muscular dystrophy comprises a group of genetic diseases that cause progressive weakness and degeneration of skeletal muscle resulting from defective proteins critical to muscle structure and function. This leads to premature exhaustion of the muscle stem cell pool that maintains muscle integrity during normal use and exercise. Stem cell therapy holds promise as a treatment for muscular dystrophy by providing cells that can both deliver functional muscle proteins and replenish the stem cell pool. Here, we review the current state of research on myogenic stem cells and identify the important challenges that must be addressed as stem cell therapy is brought to the clinic.
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Distrofias Musculares/terapia , Células Madre Pluripotentes/trasplante , Células Satélite del Músculo Esquelético/trasplante , Trasplante de Células Madre , División Celular , Ensayos Clínicos como Asunto , Humanos , Desarrollo de Músculos , Músculo Esquelético/citología , Músculo Esquelético/fisiología , RegeneraciónRESUMEN
Advances in directed differentiation of human embryonic stem cells (hESCs) toward cardiac lineages have generated much interest within the myocardial therapy field. Beyond the promise that hESCs would provide a supply of new cardiomyocytes to the damaged heart, recent studies have also shown that paracrine effects of stem cell therapy may facilitate myocardial healing. This review describes the advantages of hESCs for these purposes, current methods for directing differentiation of hESCs toward cardiac fates, approaches to purification and engineered selection of hESC-derived cardiomyocytes and cardiac precursors, as well as animal studies that have shed light on the therapeutic uses of hESCs in cardiac regenerative medicine.
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Células Madre Embrionarias/citología , Miocardio/patología , Medicina Regenerativa , Cicatrización de Heridas , Ensayos Clínicos como Asunto , Células Madre Embrionarias/trasplante , Humanos , Miocitos Cardíacos/citologíaRESUMEN
Human embryonic stem cells have the capacity for self-renewal and pluripotency and thus are a primary candidate for tissue engineering and regenerative therapies. These cells also provide an opportunity to study the development of human tissues ex vivo. To date, numerous human embryonic stem cell lines have been derived and characterized. In this review, we will detail the strategies used to direct tissue-specific differentiation of embryonic stem cells. We also will discuss how these strategies have produced new sources of tissue-specific progenitor cells. Finally, we will describe the next generation of methods being developed to identify and select stem cell-derived tissue precursors for experimental study and clinical use.
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Diferenciación Celular , Medios de Cultivo/química , Células Madre Embrionarias/citología , Estratos Germinativos/citología , Células Madre Pluripotentes/citología , Técnicas de Cultivo de Célula/métodos , Línea Celular , Células Madre Embrionarias/química , Epigénesis Genética , Citometría de Flujo/métodos , Transferencia Resonante de Energía de Fluorescencia/métodos , Expresión Génica , Técnicas de Transferencia de Gen , Genes Reporteros , Estratos Germinativos/química , Estratos Germinativos/efectos de los fármacos , Humanos , Células Madre Pluripotentes/química , Receptores de Superficie Celular/químicaRESUMEN
Unlike other essential organs, the heart does not undergo tissue repair following injury. Human embryonic stem cells (hESCs) grow indefinitely in culture while maintaining the ability to differentiate into many tissues of the body. As such, they provide a unique opportunity to explore the mechanisms that control human tissue development, as well as treat diseases characterized by tissue loss, including heart failure. MicroRNAs are small, non-coding RNAs that are known to play critical roles in the regulation of gene expression. We profiled the expression of microRNAs during hESC differentiation into myocardial precursors and cardiomyocytes (CMs), and determined clusters of human microRNAs that are specifically regulated during this process. We determined that miR-125b overexpression results in upregulation of the early cardiac transcription factors, GATA4 and Nkx2-5, and accelerated progression of hESC-derived myocardial precursors to an embryonic CM phenotype. We used an in silico approach to identify Lin28 as a target of miR-125b, and validated this interaction using miR-125b knockdown. Anti-miR-125b inhibitor experiments also showed that miR-125b controls the expression of miRNA let-7d, likely through the negative regulatory effects of Lin28 on let-7. We then determined that miR-125b overexpression inhibits the expression of Nanog and Oct4 and promotes the onset of Brachyury expression, suggesting that miR-125b controls the early events of human CM differentiation by inhibiting hESC pluripotency and promoting mesodermal differentiation. These studies identified miR-125b as an important regulator of hESC differentiation in general, and the development of hESC-derived mesoderm and cardiac muscle in particular. Manipulation of miR-125b-mediated pathways may provide a novel approach to directing the differentiation of hESC-derived CMs for cell therapy applications.
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Células Madre Embrionarias/citología , Regulación del Desarrollo de la Expresión Génica , Mesodermo/citología , MicroARNs/genética , Proteínas de Unión al ARN/genética , Animales , Secuencia de Bases , Diferenciación Celular , Línea Celular , Células Madre Embrionarias/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Ratones , MicroARNs/metabolismo , Datos de Secuencia Molecular , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Proteínas de Unión al ARN/metabolismo , Regulación hacia ArribaRESUMEN
Congenital heart disease occurs in 1% of liveborn infants, making it the most common birth defect worldwide. Many of these children develop heart failure. In addition, both genetic and acquired forms of dilated cardiomyopathy are a significant source of heart failure in the pediatric population. Heart failure occurs when the myocardium is unable to meet the body's metabolic demands. Unlike some organs, the heart has limited, if any, capacity for repair after injury. Heart transplantation remains the ultimate approach to treating heart failure, but this is costly and excludes patients who are poor candidates for transplantation given their comorbidities, or for whom a donor organ is unavailable. Stem cell therapy represents the first realistic strategy for reversing the effects of what has until now been considered terminal heart damage. We will discuss potential sources of cardiac-specific stem cells, including mesenchymal, resident cardiac, embryonic, and induced pluripotent stem cells. We will consider efforts to enhance cardiac stem cell engraftment and survival in damaged myocardium, the incorporation of cardiac stem cells into tissue patches, and techniques for creating bioartificial myocardial tissue as well as whole organs. Finally, we will review progress being made in assessing functional improvement in animals and humans after cellular transplant.
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Cardiopatías/terapia , Insuficiencia Cardíaca/prevención & control , Miocardio/citología , Pediatría/métodos , Trasplante de Células Madre/métodos , Células Madre/fisiología , Animales , Técnicas Electrofisiológicas Cardíacas , Hemodinámica , Humanos , Recién Nacido , Pediatría/tendenciasRESUMEN
While the pathologies associated with in utero smoke exposure are well established, their underlying molecular mechanisms are incompletely understood. We differentiated human embryonic stem cells in the presence of physiological concentrations of tobacco smoke and nicotine. Using post hoc microarray analysis, quantitative PCR, and immunoblot analysis, we demonstrated that tobacco smoke has lineage- and stage-specific effects on human embryonic stem cell differentiation, through both nicotine-dependent and -independent pathways. We show that three major stem cell pluripotency/differentiation pathways, Notch, canonical Wnt, and transforming growth factor-ß, are affected by smoke exposure, and that Nodal signaling through SMAD2 is specifically impacted by effects on Lefty1, Nodal, and FoxH1. These events are associated with upregulation of microRNA-302a, a post-transcriptional silencer of Lefty1. The described studies provide insight into the mechanisms by which tobacco smoke influences fetal development at the cellular level, and identify specific transcriptional, post-transcriptional, and signaling pathways by which this likely occurs.
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Diferenciación Celular/fisiología , Células Madre Embrionarias/citología , Nicotiana , Proteína Nodal/fisiología , Humo , Factor de Crecimiento Transformador beta/fisiología , Western Blotting , Humanos , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
BACKGROUND: Endogenous cardiac progenitor cells are a promising option for cell-therapy for myocardial infarction (MI). However, obtaining adequate numbers of cardiac progenitors after MI remains a challenge. Cardiospheres (CSs) have been proposed to have cardiac regenerative properties; however, their cellular composition and how they may be influenced by the tissue milieu remains unclear. METHODOLOGY/PRINCIPAL FINDING: Using "middle aged" mice as CSs donors, we found that acute MI induced a dramatic increase in the number of CSs in a mouse model of MI, and this increase was attenuated back to baseline over time. We also observed that CSs from post-MI hearts engrafted in ischemic myocardium induced angiogenesis and restored cardiac function. To determine the role of Sca-1(+)CD45(-) cells within CSs, we cloned these from single cell isolates. Expression of Islet-1 (Isl1) in Sca-1(+)CD45(-) cells from CSs was 3-fold higher than in whole CSs. Cloned Sca-1(+)CD45(-) cells had the ability to differentiate into cardiomyocytes, endothelial cells and smooth muscle cells in vitro. We also observed that cloned cells engrafted in ischemic myocardium induced angiogenesis, differentiated into endothelial and smooth muscle cells and improved cardiac function in post-MI hearts. CONCLUSIONS/SIGNIFICANCE: These studies demonstrate that cloned Sca-1(+)CD45(-) cells derived from CSs from infarcted "middle aged" hearts are enriched for second heart field (i.e., Isl-1(+)) precursors that give rise to both myocardial and vascular tissues, and may be an appropriate source of progenitor cells for autologous cell-therapy post-MI.
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Antígenos Ly/metabolismo , Proteínas con Homeodominio LIM/metabolismo , Proteínas de la Membrana/metabolismo , Infarto del Miocardio/metabolismo , Miocardio/metabolismo , Células Madre/metabolismo , Factores de Transcripción/metabolismo , Animales , Apoptosis , Diferenciación Celular , Células Cultivadas , Células Endoteliales/metabolismo , Citometría de Flujo , Corazón/fisiopatología , Inmunohistoquímica , Antígenos Comunes de Leucocito/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Infarto del Miocardio/fisiopatología , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/fisiopatología , Miocardio/patología , Miocitos Cardíacos/metabolismo , Miocitos del Músculo Liso/metabolismo , Neovascularización Fisiológica , Trasplante de Células Madre , Factores de TiempoRESUMEN
BACKGROUND AIMS: We have shown previously that inhibition of the p38 mitogen-activated protein kinase (p38MAPK) directs the differentiation of human embryonic stem cell (hESC)-derived cardiomyocytes (hCM). We investigated the therapeutic benefits of intramyocardial injection of hCM differentiated from hESC by p38MAPK inhibition using closed-chest ultrasound-guided injection at a clinically relevant time post-myocardial infarction (MI) in a mouse model. METHODS: MI was induced in mice and the animals treated at day 3 with: (a) hCM, (b) human fetal fibroblasts (hFF) as cell control, or (c) medium control (n = 10 animals/group). Left ventricular ejection fraction (LVEF) was evaluated post-MI prior to therapy, and at days 28 and 60 post-cell therapy. Hearts were analyzed at day 60 for infarct size, angiogenesis, cell fate and teratoma formation. RESULTS: LVEF was improved in the hCM-treated animals compared with both hFF and medium control-treated animals at day 28 (39.03 ± 1.79% versus 27.89 ± 1.27%, P < 0.05, versus 32.90 ± 1.46%, P < 0.05, respectively), with sustained benefit until day 60. hCM therapy resulted in significantly smaller scar size, increased capillary bed area, increased number of arterioles, less native cardiomyocyte (CM) apoptosis, and increased CM proliferation compared with the other two groups. These benefits were achieved despite a very low retention rate of the injected cells at day 60, as assessed by immunohistochemistry and quantitative real-time polymerase chain reaction (qPCR). Therapy with hCM did not result in intramyocardial teratoma formation at day 60. CONCLUSIONS: This study demonstrates that hCM derived from p38MAPK-treated hESC have encouraging therapeutic potential.