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1.
Int J Mol Sci ; 22(6)2021 Mar 10.
Artículo en Inglés | MEDLINE | ID: mdl-33802229

RESUMEN

Brugada syndrome (BrS) is an inherited cardiac arrhythmia that predisposes to ventricular fibrillation and sudden cardiac death. It originates from oligogenic alterations that affect cardiac ion channels or their accessory proteins. The main hurdle for the study of the functional effects of those variants is the need for a specific model that mimics the complex environment of human cardiomyocytes. Traditionally, animal models or transient heterologous expression systems are applied for electrophysiological investigations, each of these models having their limitations. The ability to create induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), providing a source of human patient-specific cells, offers new opportunities in the field of cardiac disease modelling. Contemporary iPSC-CMs constitute the best possible in vitro model to study complex cardiac arrhythmia syndromes such as BrS. To date, thirteen reports on iPSC-CM models for BrS have been published and with this review we provide an overview of the current findings, with a focus on the electrophysiological parameters. We also discuss the methods that are used for cell derivation and data acquisition. In the end, we critically evaluate the knowledge gained by the use of these iPSC-CM models and discuss challenges and future perspectives for iPSC-CMs in the study of BrS and other arrhythmias.


Asunto(s)
Síndrome de Brugada/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Modelos Cardiovasculares , Miocitos Cardíacos/metabolismo , Síndrome de Brugada/patología , Humanos , Células Madre Pluripotentes Inducidas/patología , Miocitos Cardíacos/patología
2.
Int J Mol Sci ; 22(5)2021 Mar 03.
Artículo en Inglés | MEDLINE | ID: mdl-33802405

RESUMEN

Histone deacetylase 2 (HDAC2) is a major HDAC protein in the adult brain and has been shown to regulate many neuronal genes. The aberrant expression of HDAC2 and subsequent dysregulation of neuronal gene expression is implicated in neurodegeneration and brain aging. Human induced pluripotent stem cell-derived neurons (hiPSC-Ns) are widely used models for studying neurodegenerative disease mechanisms, but the role of HDAC2 in hiPSC-N differentiation and maturation has not been explored. In this study, we show that levels of HDAC2 progressively decrease as hiPSCs are differentiated towards neurons. This suppression of HDAC2 inversely corresponds to an increase in neuron-specific isoforms of Endophilin-B1, a multifunctional protein involved in mitochondrial dynamics. Expression of neuron-specific isoforms of Endophilin-B1 is accompanied by concomitant expression of a neuron-specific alternative splicing factor, SRRM4. Manipulation of HDAC2 and Endophilin-B1 using lentiviral approaches shows that the knock-down of HDAC2 or the overexpression of a neuron-specific Endophilin-B1 isoform promotes mitochondrial elongation and protects against cytotoxic stress in hiPSC-Ns, while HDAC2 knock-down specifically influences genes regulating mitochondrial dynamics and synaptogenesis. Furthermore, HDAC2 knock-down promotes enhanced mitochondrial respiration and reduces levels of neurotoxic amyloid beta peptides. Collectively, our study demonstrates a role for HDAC2 in hiPSC-neuronal differentiation, highlights neuron-specific isoforms of Endophilin-B1 as a marker of differentiating hiPSC-Ns and demonstrates that HDAC2 regulates key neuronal and mitochondrial pathways in hiPSC-Ns.


Asunto(s)
Péptidos beta-Amiloides/metabolismo , Histona Desacetilasa 2/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Mitocondrias/metabolismo , Dinámicas Mitocondriales/fisiología , Neuronas/metabolismo , Neuronas/fisiología , Aciltransferasas/metabolismo , Biomarcadores/metabolismo , Encéfalo/metabolismo , Encéfalo/fisiología , Diferenciación Celular/fisiología , Células Cultivadas , Humanos , Mitocondrias/fisiología , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Isoformas de Proteínas/metabolismo
3.
Int J Mol Sci ; 22(6)2021 Mar 23.
Artículo en Inglés | MEDLINE | ID: mdl-33806803

RESUMEN

Several studies have shown that human induced pluripotent stem cell (iPSC)-derivatives are essentially fetal in terms of their maturational status. Inducing ageing in iPSC-motor neuron (MN) models of amyotrophic lateral sclerosis (ALS) has the potential to capture pathology with higher fidelity and consequently improve translational success. We show here that the telomerase inhibitor BIBR1532, hypothesised to recapitulate the telomere attrition hallmark of ageing in iPSC-MNs, was in fact cytotoxic to feeder-free iPSCs when used at doses previously shown to be effective in iPSCs grown on a layer of mouse embryonic fibroblasts. Toxicity in feeder-free cultures was not rescued by co-treatment with Rho Kinase (ROCK) inhibitor (Y-27632). Moreover, the highest concentration of BIBR1532 compatible with continued iPSC culture proved insufficient to induce detectable telomerase inhibition. Our data suggest that direct toxicity by BIBR1532 is the most likely cause of iPSC death observed, and that culture methods may influence enhanced toxicity. Therefore, recapitulation of ageing hallmarks in iPSC-MNs, which might reveal novel and relevant human disease targets in ALS, is not achievable in feeder-free culture through the use of this small molecule telomerase inhibitor.


Asunto(s)
Aminobenzoatos/farmacología , Inhibidores Enzimáticos/farmacología , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Neuronas Motoras/citología , Naftalenos/farmacología , Neurogénesis/efectos de los fármacos , Telomerasa/antagonistas & inhibidores , Telomerasa/genética , Diferenciación Celular/efectos de los fármacos , Línea Celular , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Neuronas Motoras/metabolismo
4.
Int J Mol Sci ; 22(8)2021 Apr 13.
Artículo en Inglés | MEDLINE | ID: mdl-33924373

RESUMEN

A common pathological hallmark of several neurodegenerative diseases, including amyotrophic lateral sclerosis, is cytoplasmic mislocalization and aggregation of nuclear RNA-binding protein TDP-43. Perry disease, which displays inherited atypical parkinsonism, is a type of TDP-43 proteinopathy. The causative gene DCTN1 encodes the largest subunit of the dynactin complex. Dynactin associates with the microtubule-based motor cytoplasmic dynein and is required for dynein-mediated long-distance retrograde transport. Perry disease-linked missense mutations (e.g., p.G71A) reside within the CAP-Gly domain and impair the microtubule-binding abilities of DCTN1. However, molecular mechanisms by which such DCTN1 mutations cause TDP-43 proteinopathy remain unclear. We found that DCTN1 bound to TDP-43. Biochemical analysis using a panel of truncated mutants revealed that the DCTN1 CAP-Gly-basic supradomain, dynactin domain, and C-terminal region interacted with TDP-43, preferentially through its C-terminal region. Remarkably, the p.G71A mutation affected the TDP-43-interacting ability of DCTN1. Overexpression of DCTN1G71A, the dynactin-domain fragment, or C-terminal fragment, but not the CAP-Gly-basic fragment, induced cytoplasmic mislocalization and aggregation of TDP-43, suggesting functional modularity among TDP-43-interacting domains of DCTN1. We thus identified DCTN1 as a new player in TDP-43 cytoplasmic-nuclear transport, and showed that dysregulation of DCTN1-TDP-43 interactions triggers mislocalization and aggregation of TDP-43, thus providing insights into the pathological mechanisms of Perry disease and other TDP-43 proteinopathies.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Complejo Dinactina/metabolismo , Agregado de Proteínas , Secuencia de Aminoácidos , Animales , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Complejo Dinactina/química , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Modelos Biológicos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Neuronas/metabolismo , Señales de Localización Nuclear/metabolismo , Mutación Puntual/genética , Unión Proteica , Fracciones Subcelulares/metabolismo
5.
Int J Mol Sci ; 22(8)2021 Apr 13.
Artículo en Inglés | MEDLINE | ID: mdl-33924575

RESUMEN

Niemann-Pick type C2 (NP-C2) disease is a rare hereditary disease caused by mutations in the NPC2 gene. NPC2 is a small, soluble protein consisting of 151 amino acids, primarily expressed in late endosomes and lysosomes (LE/LY). Together with NPC1, a transmembrane protein found in these organelles, NPC2 accomplishes the exclusion of cholesterol; thus, both proteins are essential to maintain cellular cholesterol homeostasis. Consequently, mutations in the NPC2 or NPC1 gene result in pathophysiological accumulation of cholesterol and sphingolipids in LE/LY. The vast majority of Niemann-Pick type C disease patients, 95%, suffer from a mutation of NPC1, and only 5% display a mutation of NPC2. The biochemical phenotype of NP-C1 and NP-C2 appears to be indistinguishable, and both diseases share several commonalities in the clinical manifestation. Studies of the pathological mechanisms underlying NP-C2 are mostly based on NP-C2 animal models and NP-C2 patient-derived fibroblasts. Recently, we established induced pluripotent stem cells (iPSCs), derived from a donor carrying the NPC2 mutations c.58G>T/c.140G>T. Here, we present a profile of pathophysiological in vitro features, shared by NP-C1 and NP-C2, of neural differentiated cells obtained from the patient specific iPSCs. Profiling comprised a determination of the NPC2 protein level, detection of cholesterol accumulation by filipin staining, analysis of oxidative stress, and determination of autophagy. As expected, the NPC2-deficient cells displayed a significantly reduced amount of NPC2 protein, and, accordingly, we observed a significantly increased amount of cholesterol. Most notably, NPC2-deficient cells displayed only a slight increase of reactive oxygen species (ROS), suggesting that they do not suffer from oxidative stress and express catalase at a high level. As a site note, comparable NPC1-deficient cells suffer from a lack of catalase and display an increased level of ROS. In summary, this cell line provides a valuable tool to gain deeper understanding, not only of the pathogenic mechanism of NP-C2, but also of NP-C1.


Asunto(s)
Diferenciación Celular , Células Madre Pluripotentes Inducidas/patología , Mutación/genética , Neuronas/patología , Enfermedad de Niemann-Pick Tipo C/patología , Enfermedad de Niemann-Pick Tipo C/fisiopatología , Proteínas de Transporte Vesicular/genética , Antioxidantes/metabolismo , Autofagia , Colesterol/metabolismo , Células HEK293 , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Neuroglía/metabolismo , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo
6.
Int J Mol Sci ; 22(7)2021 Apr 03.
Artículo en Inglés | MEDLINE | ID: mdl-33916879

RESUMEN

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the gene encoding the methyl-CpG-binding protein 2 (MeCP2). Among many different roles, MeCP2 has a high phenotypic impact during the different stages of brain development. Thus, it is essential to intensively investigate the function of MeCP2, and its regulated targets, to better understand the mechanisms of the disease and inspire the development of possible therapeutic strategies. Several animal models have greatly contributed to these studies, but more recently human pluripotent stem cells (hPSCs) have been providing a promising alternative for the study of RTT. The rapid evolution in the field of hPSC culture allowed first the development of 2D-based neuronal differentiation protocols, and more recently the generation of 3D human brain organoid models, a more complex approach that better recapitulates human neurodevelopment in vitro. Modeling RTT using these culture platforms, either with patient-specific human induced pluripotent stem cells (hiPSCs) or genetically-modified hPSCs, has certainly contributed to a better understanding of the onset of RTT and the disease phenotype, ultimately allowing the development of high throughput drugs screening tests for potential clinical translation. In this review, we first provide a brief summary of the main neurological features of RTT and the impact of MeCP2 mutations in the neuropathophysiology of this disease. Then, we provide a thorough revision of the more recent advances and future prospects of RTT modeling with human neural cells derived from hPSCs, obtained using both 2D and organoids culture systems, and its contribution for the current and future clinical trials for RTT.


Asunto(s)
Células Madre Pluripotentes Inducidas , Proteína 2 de Unión a Metil-CpG , Modelos Neurológicos , Mutación , Organoides , Síndrome de Rett , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Proteína 2 de Unión a Metil-CpG/genética , Proteína 2 de Unión a Metil-CpG/metabolismo , Organoides/metabolismo , Organoides/patología , Síndrome de Rett/genética , Síndrome de Rett/metabolismo , Síndrome de Rett/patología
7.
Int J Mol Sci ; 22(9)2021 Apr 22.
Artículo en Inglés | MEDLINE | ID: mdl-33922276

RESUMEN

Sialidosis, caused by a genetic deficiency of the lysosomal sialidase gene (NEU1), is a systemic disease involving various tissues and organs, including the nervous system. Understanding the neurological dysfunction and pathology associated with sialidosis remains a challenge, partially due to the lack of a human model system. In this study, we have generated two types of induced pluripotent stem cells (iPSCs) with sialidosis-specific NEU1G227R and NEU1V275A/R347Q mutations (sialidosis-iPSCs), and further differentiated them into neural precursor cells (iNPCs). Characterization of NEU1G227R- and NEU1V275A/R347Q- mutated iNPCs derived from sialidosis-iPSCs (sialidosis-iNPCs) validated that sialidosis-iNPCs faithfully recapitulate key disease-specific phenotypes, including reduced NEU1 activity and impaired lysosomal and autophagic function. In particular, these cells showed defective differentiation into oligodendrocytes and astrocytes, while their neuronal differentiation was not notably affected. Importantly, we found that the phenotypic defects of sialidosis-iNPCs, such as impaired differentiation capacity, could be effectively rescued by the induction of autophagy with rapamycin. Our results demonstrate the first use of a sialidosis-iNPC model with NEU1G227R- and NEU1V275A/R347Q- mutation(s) to study the neurological defects of sialidosis, particularly those related to a defective autophagy-lysosome pathway, and may help accelerate the development of new drugs and therapeutics to combat sialidosis and other LSDs.


Asunto(s)
Astrocitos/patología , Células Madre Pluripotentes Inducidas/patología , Mucolipidosis/patología , Células-Madre Neurales/patología , Neuraminidasa/metabolismo , Oligodendroglía/patología , Teratoma/patología , Astrocitos/metabolismo , Autofagia , Diferenciación Celular , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Lisosomas , Mucolipidosis/genética , Mucolipidosis/metabolismo , Mutación , Células-Madre Neurales/metabolismo , Neuraminidasa/genética , Oligodendroglía/metabolismo , Fenotipo , Teratoma/genética , Teratoma/metabolismo
8.
Int J Mol Sci ; 22(4)2021 Feb 18.
Artículo en Inglés | MEDLINE | ID: mdl-33670788

RESUMEN

Epigenetic mechanisms are emerging key players for the regulation of brain function, synaptic activity, and the formation of neuronal engrams in health and disease. As one important epigenetic mechanism of transcriptional control, DNA methylation was reported to distinctively modulate synaptic activity in excitatory and inhibitory cortical neurons in mice. Since DNA methylation signatures are responsive to neuronal activity, DNA methylation seems to contribute to the neuron's capacity to adapt to and integrate changing activity patterns, being crucial for the plasticity and functionality of neuronal circuits. Since most studies addressing the role of DNA methylation in the regulation of synaptic function were conducted in mice or murine neurons, we here asked whether this functional implication applies to human neurons as well. To this end, we performed calcium imaging in human induced pluripotent stem cell (iPSC)-derived excitatory cortical neurons forming synaptic contacts and neuronal networks in vitro. Treatment with DNMT1 siRNA that diminishs the expression of the DNA (cytosine-5)-methyltransferase 1 (DNMT1) was conducted to investigate the functional relevance of DNMT1 as one of the main enzymes executing DNA methylations in the context of neuronal activity modulation. We observed a lowered proportion of actively firing neurons upon DNMT1-knockdown in these iPSC-derived excitatory neurons, pointing to a correlation of DNMT1-activity and synaptic transmission. Thus, our experiments suggest that DNMT1 decreases synaptic activity of human glutamatergic neurons and underline the relevance of epigenetic regulation of synaptic function also in human excitatory neurons.


Asunto(s)
Corteza Cerebral/citología , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Glutamatos/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Neuronas/enzimología , Animales , Señalización del Calcio , Diferenciación Celular , Humanos , Ratones
9.
Int J Mol Sci ; 22(4)2021 Feb 18.
Artículo en Inglés | MEDLINE | ID: mdl-33670616

RESUMEN

Arrhythmogenic Right Ventricular cardiomyopathy (ARVC) is an inherited cardiac muscle disease linked to genetic deficiency in components of the desmosomes. The disease is characterized by progressive fibro-fatty replacement of the right ventricle, which acts as a substrate for arrhythmias and sudden cardiac death. The molecular mechanisms underpinning ARVC are largely unknown. Here we propose a mathematical model for investigating the molecular dynamics underlying heart remodeling and the loss of cardiac myocytes identity during ARVC. Our methodology is based on three computational models: firstly, in the context of the Wnt pathway, we examined two different competition mechanisms between ß-catenin and Plakoglobin (PG) and their role in the expression of adipogenic program. Secondly, we investigated the role of RhoA-ROCK pathway in ARVC pathogenesis, and thirdly we analyzed the interplay between Wnt and RhoA-ROCK pathways in the context of the ARVC phenotype. We conclude with the following remark: both Wnt/ß-catenin and RhoA-ROCK pathways must be inactive for a significant increase of PPARγ expression, suggesting that a crosstalk mechanism might be responsible for mediating ARVC pathogenesis.


Asunto(s)
Células Madre Pluripotentes Inducidas/metabolismo , Miocitos Cardíacos/metabolismo , Vía de Señalización Wnt , beta Catenina/metabolismo , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Adipogénesis/genética , Algoritmos , Displasia Ventricular Derecha Arritmogénica/genética , Displasia Ventricular Derecha Arritmogénica/metabolismo , Displasia Ventricular Derecha Arritmogénica/patología , Células Cultivadas , Simulación por Computador , Regulación de la Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/citología , Modelos Teóricos , PPAR gamma/genética , PPAR gamma/metabolismo , gamma Catenina/metabolismo
10.
Nat Commun ; 12(1): 1929, 2021 03 26.
Artículo en Inglés | MEDLINE | ID: mdl-33771987

RESUMEN

Leigh syndrome (LS) is a severe manifestation of mitochondrial disease in children and is currently incurable. The lack of effective models hampers our understanding of the mechanisms underlying the neuronal pathology of LS. Using patient-derived induced pluripotent stem cells and CRISPR/Cas9 engineering, we developed a human model of LS caused by mutations in the complex IV assembly gene SURF1. Single-cell RNA-sequencing and multi-omics analysis revealed compromised neuronal morphogenesis in mutant neural cultures and brain organoids. The defects emerged at the level of neural progenitor cells (NPCs), which retained a glycolytic proliferative state that failed to instruct neuronal morphogenesis. LS NPCs carrying mutations in the complex I gene NDUFS4 recapitulated morphogenesis defects. SURF1 gene augmentation and PGC1A induction via bezafibrate treatment supported the metabolic programming of LS NPCs, leading to restored neuronal morphogenesis. Our findings provide mechanistic insights and suggest potential interventional strategies for a rare mitochondrial disease.


Asunto(s)
Células Madre Pluripotentes Inducidas/metabolismo , Enfermedad de Leigh/genética , Proteínas de la Membrana/genética , Proteínas Mitocondriales/genética , Mutación , Neuronas/metabolismo , Organoides/metabolismo , Células Cultivadas , Preescolar , Humanos , Células Madre Pluripotentes Inducidas/citología , Enfermedad de Leigh/metabolismo , Masculino , Metabolómica/métodos , Mitocondrias/genética , Mitocondrias/metabolismo , Morfogénesis/genética , Neuronas/citología , Proteómica/métodos , Análisis de la Célula Individual/métodos , Secuenciación del Exoma Completo
11.
Int J Mol Sci ; 22(4)2021 Feb 11.
Artículo en Inglés | MEDLINE | ID: mdl-33670118

RESUMEN

Inherited ichthyoses represent a large heterogeneous group of skin disorders characterised by impaired epidermal barrier function and disturbed cornification. Current knowledge about disease mechanisms has been uncovered mainly through the use of mouse models or human skin organotypic models. However, most mouse lines suffer from severe epidermal barrier defects causing neonatal death and human keratinocytes have very limited proliferation ability in vitro. Therefore, the development of disease models based on patient derived human induced pluripotent stem cells (hiPSCs) is highly relevant. For this purpose, we have generated hiPSCs from patients with congenital ichthyosis, either non-syndromic autosomal recessive congenital ichthyosis (ARCI) or the ichthyosis syndrome trichothiodystrophy (TTD). hiPSCs were successfully differentiated into basal keratinocyte-like cells (hiPSC-bKs), with high expression of epidermal keratins. In the presence of higher calcium concentrations, terminal differentiation of hiPSC-bKs was induced and markers KRT1 and IVL expressed. TTD1 hiPSC-bKs showed reduced expression of FLG, SPRR2B and lipoxygenase genes. ARCI hiPSC-bKs showed more severe defects, with downregulation of several cornification genes. The application of hiPSC technology to TTD1 and ARCI demonstrates the successful generation of in vitro models mimicking the disease phenotypes, proving a valuable system both for further molecular investigations and drug development for ichthyosis patients.


Asunto(s)
Regulación de la Expresión Génica , Ictiosis/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Queratinocitos/metabolismo , Modelos Biológicos , Niño , Preescolar , Femenino , Humanos , Ictiosis/patología , Células Madre Pluripotentes Inducidas/patología , Lactante , Queratinocitos/patología , Masculino
12.
J Vis Exp ; (168)2021 02 14.
Artículo en Inglés | MEDLINE | ID: mdl-33645588

RESUMEN

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson's disease and Alzheimer's disease. Microglia clear up dead and dying neurons through the process of efferocytosis, a specialized form of phagocytosis. The phagocytosis function can be disrupted by environmental or genetic risk factors that affect microglia. This paper presents a rapid and simple in vitro microscopy protocol for studying microglial efferocytosis in an induced pluripotent stem cell (iPSC) model of microglia, using a human neuroblastoma cell line (SH-SY5Y) labeled with a pH-sensitive dye for the phagocytic cargo. The procedure results in a high yield of dead neuroblastoma cells, which display surface phosphatidylserine, recognized as an "eat-me" signal by phagocytes. The 96-well plate assay is suitable for live-cell time-lapse imaging, or the plate can be successfully fixed prior to further processing and quantified by high-content microscopy. Fixed-cell high-content microscopy enables the assay to be scaled up for screening of small molecule inhibitors or assessing the phagocytic function of genetic variant iPSC lines. While this assay was developed to study phagocytosis of whole dead neuroblastoma cells by iPSC-macrophages, the assay can be easily adapted for other cargoes relevant to neurodegenerative diseases, such as synaptosomes and myelin, and other phagocytic cell types.


Asunto(s)
Bioensayo/métodos , Células Madre Pluripotentes Inducidas/metabolismo , Macrófagos/metabolismo , Neuroblastoma/patología , Fagocitosis , Animales , Muerte Celular , Línea Celular Tumoral , Análisis de Datos , Colorantes Fluorescentes/química , Células Madre Embrionarias Humanas/citología , Humanos , Concentración de Iones de Hidrógeno , Células Madre Pluripotentes Inducidas/citología , Control de Calidad , Reproducibilidad de los Resultados , Imagen de Lapso de Tiempo
13.
J Vis Exp ; (169)2021 03 14.
Artículo en Inglés | MEDLINE | ID: mdl-33779590

RESUMEN

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided a unique opportunity to study the complex interplay among different cardiovascular cell types that drives tissue development and disease. In the area of cardiac tissue models, several sophisticated three-dimensional (3D) approaches use induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to mimic physiological relevance and native tissue environment with a combination of extracellular matrices and crosslinkers. However, these systems are complex to fabricate without microfabrication expertise and require several weeks to self-assemble. Most importantly, many of these systems lack vascular cells and cardiac fibroblasts that make up over 60% of the nonmyocytes in the human heart. Here we describe the derivation of all three cardiac cell types from iPSCs to fabricate cardiac microtissues. This facile replica molding technique allows cardiac microtissue culture in standard multi-well cell culture plates for several weeks. The platform allows user-defined control over microtissue sizes based on initial seeding density and requires less than 3 days for self-assembly to achieve observable cardiac microtissue contractions. Furthermore, the cardiac microtissues can be easily digested while maintaining high cell viability for single-cell interrogation with the use of flow cytometry and single-cell RNA sequencing (scRNA-seq). We envision that this in vitro model of cardiac microtissues will help accelerate validation studies in drug discovery and disease modeling.


Asunto(s)
Células Endoteliales/metabolismo , Fibroblastos/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Miocitos Cardíacos/metabolismo , Técnicas de Cultivo de Célula , Diferenciación Celular , Humanos , Células Madre Pluripotentes Inducidas/citología , Miocitos Cardíacos/citología
14.
Nat Commun ; 12(1): 1648, 2021 03 12.
Artículo en Inglés | MEDLINE | ID: mdl-33712605

RESUMEN

Cardiomyocytes undergo significant structural and functional changes after birth, and these fundamental processes are essential for the heart to pump blood to the growing body. However, due to the challenges of isolating single postnatal/adult myocytes, how individual newborn cardiomyocytes acquire multiple aspects of the mature phenotype remains poorly understood. Here we implement large-particle sorting and analyze single myocytes from neonatal to adult hearts. Early myocytes exhibit wide-ranging transcriptomic and size heterogeneity that is maintained until adulthood with a continuous transcriptomic shift. Gene regulatory network analysis followed by mosaic gene deletion reveals that peroxisome proliferator-activated receptor coactivator-1 signaling, which is active in vivo but inactive in pluripotent stem cell-derived cardiomyocytes, mediates the shift. This signaling simultaneously regulates key aspects of cardiomyocyte maturation through previously unrecognized proteins, including YAP1 and SF3B2. Our study provides a single-cell roadmap of heterogeneous transitions coupled to cellular features and identifies a multifaceted regulator controlling cardiomyocyte maturation.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Miocitos Cardíacos/metabolismo , Receptores Activados del Proliferador del Peroxisoma/metabolismo , Factores de Empalme de ARN/metabolismo , Factores de Transcripción/metabolismo , Animales , Calcio/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular , Redes Reguladoras de Genes , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Receptores Activados del Proliferador del Peroxisoma/genética , Células Madre Pluripotentes/metabolismo , Transducción de Señal , Factores de Transcripción/genética , Transcriptoma
15.
Nat Med ; 27(4): 632-639, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33649496

RESUMEN

Degeneration of dopamine (DA) neurons in the midbrain underlies the pathogenesis of Parkinson's disease (PD). Supplement of DA via L-DOPA alleviates motor symptoms but does not prevent the progressive loss of DA neurons. A large body of experimental studies, including those in nonhuman primates, demonstrates that transplantation of fetal mesencephalic tissues improves motor symptoms in animals, which culminated in open-label and double-blinded clinical trials of fetal tissue transplantation for PD1. Unfortunately, the outcomes are mixed, primarily due to the undefined and unstandardized donor tissues1,2. Generation of induced pluripotent stem cells enables standardized and autologous transplantation therapy for PD. However, its efficacy, especially in primates, remains unclear. Here we show that over a 2-year period without immunosuppression, PD monkeys receiving autologous, but not allogenic, transplantation exhibited recovery from motor and depressive signs. These behavioral improvements were accompanied by robust grafts with extensive DA neuron axon growth as well as strong DA activity in positron emission tomography (PET). Mathematical modeling reveals correlations between the number of surviving DA neurons with PET signal intensity and behavior recovery regardless autologous or allogeneic transplant, suggesting a predictive power of PET and motor behaviors for surviving DA neuron number.


Asunto(s)
Conducta Animal , Depresión/complicaciones , Trasplante de Tejido Fetal , Actividad Motora , Enfermedad de Parkinson/fisiopatología , Enfermedad de Parkinson/terapia , Animales , Dopamina/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Inflamación/patología , Modelos Lineales , Macaca mulatta , Masculino , Mesencéfalo/trasplante , Ratones , Enfermedad de Parkinson/complicaciones , Tomografía de Emisión de Positrones , Trasplante Autólogo , Trasplante Homólogo , Tirosina 3-Monooxigenasa/metabolismo
16.
Comput Biol Med ; 131: 104293, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33662681

RESUMEN

BACKGROUND: Coronavirus disease 2019 (COVID-19) is an emerging infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Up to 20%-30% of patients hospitalized with COVID-19 have evidence of cardiac dysfunction. Xuebijing injection is a compound injection containing five traditional Chinese medicine ingredients, which can protect cells from SARS-CoV-2-induced cell death and improve cardiac function. However, the specific protective mechanism of Xuebijing injection on COVID-19-induced cardiac dysfunction remains unclear. METHODS: The therapeutic effect of Xuebijing injection on COVID-19 was validated by the TCM Anti COVID-19 (TCMATCOV) platform. RNA-sequencing (RNA-seq) data from GSE150392 was used to find differentially expressed genes (DEGs) from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) infected with SARS-CoV-2. Data from GSE151879 was used to verify the expression of Angiotensin I Converting Enzyme 2 (ACE2) and central hub genes in both human embryonic-stem-cell-derived cardiomyocytes (hESC-CMs) and adult human CMs with SARS-CoV-2 infection. RESULTS: A total of 97 proteins were identified as the therapeutic targets of Xuebijing injection for COVID-19. There were 22 DEGs in SARS-CoV-2 infected hiPSC-CMs overlapped with the 97 therapeutic targets, which might be the therapeutic targets of Xuebijing injection on COVID-19-induced cardiac dysfunction. Based on the bioinformatics analysis, 7 genes (CCL2, CXCL8, FOS, IFNB1, IL-1A, IL-1B, SERPINE1) were identified as central hub genes and enriched in pathways including cytokines, inflammation, cell senescence and oxidative stress. ACE2, the receptor of SARS-CoV-2, and the 7 central hub genes were differentially expressed in at least two kinds of SARS-CoV-2 infected CMs. Besides, FOS and quercetin exhibited the tightest binding by molecular docking analysis. CONCLUSION: Our study indicated the underlying protective effect of Xuebijing injection on COVID-19, especially on COVID19-induced cardiac dysfunction, which provided the theoretical basis for exploring the potential protective mechanism of Xuebijing injection on COVID19-induced cardiac dysfunction.


Asunto(s)
/metabolismo , Medicamentos Herbarios Chinos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Miocitos Cardíacos/metabolismo , RNA-Seq , /metabolismo , /tratamiento farmacológico , Línea Celular , Células Madre Embrionarias Humanas/metabolismo , Células Madre Embrionarias Humanas/patología , Células Madre Embrionarias Humanas/virología , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Células Madre Pluripotentes Inducidas/virología , Miocitos Cardíacos/patología , Miocitos Cardíacos/virología
17.
Toxicol Appl Pharmacol ; 418: 115480, 2021 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-33689843

RESUMEN

Drug-induced cardiotoxicity is a major barrier to drug development and a main cause of withdrawal of marketed drugs. Drugs can strongly alter the spontaneous functioning of the heart by interacting with the cardiac membrane ion channels. If these effects only surface during in vivo preclinical tests, clinical trials or worse after commercialization, the societal and economic burden will be significant and seriously hinder the efficient drug development process. Hence, cardiac safety pharmacology requires in vitro electrophysiological screening assays of all drug candidates to predict cardiotoxic effects before clinical trials. In the past 10 years, microelectrode array (MEA) technology began to be considered a valuable approach in pharmaceutical applications. However, an effective tool for high-throughput intracellular measurements, compatible with pharmaceutical standards, is not yet available. Here, we propose laser-induced optoacoustic poration combined with CMOS-MEA technology as a reliable and effective platform to detect cardiotoxicity. This approach enables the acquisition of high-quality action potential recordings from large numbers of cardiomyocytes within the same culture well, providing reliable data using single-well MEA devices and single cardiac syncytia per each drug. Thus, this technology could be applied in drug safety screening platforms reducing times and costs of cardiotoxicity assessments, while simultaneously improving the data reliability.


Asunto(s)
Potenciales de Acción/efectos de los fármacos , Arritmias Cardíacas/inducido químicamente , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Rayos Láser , Microelectrodos , Miocitos Cardíacos/efectos de los fármacos , Técnicas Fotoacústicas/instrumentación , Pruebas de Toxicidad/instrumentación , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatología , Cardiotoxicidad , Ahorro de Costo , Análisis Costo-Beneficio , Frecuencia Cardíaca/efectos de los fármacos , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Microelectrodos/economía , Miocitos Cardíacos/metabolismo , Técnicas Fotoacústicas/economía , Reproducibilidad de los Resultados , Medición de Riesgo , Factores de Tiempo , Pruebas de Toxicidad/economía , Flujo de Trabajo
18.
Nat Genet ; 53(4): 467-476, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33731941

RESUMEN

Gene regulatory divergence is thought to play a central role in determining human-specific traits. However, our ability to link divergent regulation to divergent phenotypes is limited. Here, we utilized human-chimpanzee hybrid induced pluripotent stem cells to study gene expression separating these species. The tetraploid hybrid cells allowed us to separate cis- from trans-regulatory effects, and to control for nongenetic confounding factors. We differentiated these cells into cranial neural crest cells, the primary cell type giving rise to the face. We discovered evidence of lineage-specific selection on the hedgehog signaling pathway, including a human-specific sixfold down-regulation of EVC2 (LIMBIN), a key hedgehog gene. Inducing a similar down-regulation of EVC2 substantially reduced hedgehog signaling output. Mice and humans lacking functional EVC2 show striking phenotypic parallels to human-chimpanzee craniofacial differences, suggesting that the regulatory divergence of hedgehog signaling may have contributed to the unique craniofacial morphology of humans.


Asunto(s)
Quimera/genética , Síndrome de Ellis-Van Creveld/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Cresta Neural/metabolismo , Pan troglodytes/genética , Cráneo/metabolismo , Animales , Evolución Biológica , Diferenciación Celular , Quimera/metabolismo , Síndrome de Ellis-Van Creveld/metabolismo , Síndrome de Ellis-Van Creveld/patología , Femenino , Expresión Génica , Genotipo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/deficiencia , Masculino , Ratones , Ratones Noqueados , Cresta Neural/patología , Pan troglodytes/anatomía & histología , Pan troglodytes/metabolismo , Fenotipo , Transducción de Señal , Cráneo/anatomía & histología , Especificidad de la Especie , Tetraploidía
19.
Methods Mol Biol ; 2269: 25-33, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33687669

RESUMEN

In an increasingly geriatric population, in which elderly people frequently face chronic diseases and degenerative conditions, cell therapies as part of novel regenerative medicine approaches are of great interest. Even though today's cell therapies mostly rely on adult stem cells like the mesenchymal stem cells or primary somatic cells, pluripotent stem cells represent an enormously versatile cell model to explore possible new avenues in the field of regenerative medicine due to their capacity to grow indefinitely and to differentiate into the desired cell types. The discovery of reprogramming somatic cells into induced pluripotent stem cells augmented the pool of applicable cell entities so that researchers nowadays can resort to embryonic stem cells, but also to a plethora of patient- and disease-specific induced pluripotent stem cells. The ease of targeted genome engineering is an additional benefit that allows using pluripotent stem cells for disease modeling, drug discovery, and the development of cell therapies. However, the task is still demanding as the generation of subpopulations and a sufficient cell maturation for some cell entities have yet to be achieved. Likewise, even though for some applications the cells of interest can be produced in the large-scale dimensions and purity that are required for clinical purposes, proper integration, and function in the host tissue remain challenging. Nonetheless, the immense progress that has been made over the last decades warrants the prominent role of pluripotent stem cells in regenerative medicine as in vitro models to broaden our knowledge of disease onset/progression and treatment as well as in vivo as a substitution of damaged/aged tissue.


Asunto(s)
Tratamiento Basado en Trasplante de Células y Tejidos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Mesenquimatosas/metabolismo , Células Madre Pluripotentes/metabolismo , Medicina Regenerativa , Animales , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Mesenquimatosas/citología , Células Madre Pluripotentes/citología
20.
Methods Mol Biol ; 2269: 93-105, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33687674

RESUMEN

Mesenchymal stem cells (MSCs) have emerged as an attractive candidate for cell-based therapy. In the past decade, many animal and pilot clinical studies have demonstrated that MSCs are therapeutically beneficial for the treatment of obstructive lung diseases such as asthma and chronic obstructive pulmonary disease (COPD). However, due to the scarcity of adult human MSCs, human-induced pluripotent stem cells mesenchymal stem cells (iPSCs) are now increasingly used as a source of MSCs. iPSCs are derived by reprogramming somatic cells from a wide variety of tissues such as skin biopsies and then differentiating them into iPSC-MSCs. One of the mechanisms through which MSCs exert their protective effects is mitochondrial transfer. Specifically, transfer of mitochondria from iPSC-MSCs to lung cells was shown to protect lung cells against oxidative stress-induced mitochondrial dysfunction and apoptosis and to reduce lung injury and inflammation in in vivo models of lung disease. In this chapter, we detail our methods to visualize and quantify iPSC-MSC-mediated mitochondrial transfer and to study its effects on oxidant-induced airway epithelial and smooth muscle cell models of acute airway cell injury.


Asunto(s)
Células Epiteliales Alveolares/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Mesenquimatosas/metabolismo , Mitocondrias , Miocitos del Músculo Liso/metabolismo , Estrés Oxidativo , Células Epiteliales Alveolares/patología , Línea Celular , Humanos , Células Madre Pluripotentes Inducidas/patología , Células Madre Mesenquimatosas/patología , Mitocondrias/metabolismo , Mitocondrias/patología , Mitocondrias/trasplante , Miocitos del Músculo Liso/patología
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