Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 6.732
Filtrar
1.
Int J Mol Sci ; 22(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34360966

RESUMO

Neurodegenerative diseases affect millions of people worldwide and are characterized by the chronic and progressive deterioration of neural function. Neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and Huntington's disease (HD), represent a huge social and economic burden due to increasing prevalence in our aging society, severity of symptoms, and lack of effective disease-modifying therapies. This lack of effective treatments is partly due to a lack of reliable models. Modeling neurodegenerative diseases is difficult because of poor access to human samples (restricted in general to postmortem tissue) and limited knowledge of disease mechanisms in a human context. Animal models play an instrumental role in understanding these diseases but fail to comprehensively represent the full extent of disease due to critical differences between humans and other mammals. The advent of human-induced pluripotent stem cell (hiPSC) technology presents an advantageous system that complements animal models of neurodegenerative diseases. Coupled with advances in gene-editing technologies, hiPSC-derived neural cells from patients and healthy donors now allow disease modeling using human samples that can be used for drug discovery.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Descoberta de Drogas/métodos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Medicina de Precisão/métodos
2.
Nat Commun ; 12(1): 4730, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354063

RESUMO

Brain organoids derived from human pluripotent stem cells provide a highly valuable in vitro model to recapitulate human brain development and neurological diseases. However, the current systems for brain organoid culture require further improvement for the reliable production of high-quality organoids. Here, we demonstrate two engineering elements to improve human brain organoid culture, (1) a human brain extracellular matrix to provide brain-specific cues and (2) a microfluidic device with periodic flow to improve the survival and reduce the variability of organoids. A three-dimensional culture modified with brain extracellular matrix significantly enhanced neurogenesis in developing brain organoids from human induced pluripotent stem cells. Cortical layer development, volumetric augmentation, and electrophysiological function of human brain organoids were further improved in a reproducible manner by dynamic culture in microfluidic chamber devices. Our engineering concept of reconstituting brain-mimetic microenvironments facilitates the development of a reliable culture platform for brain organoids, enabling effective modeling and drug development for human brain diseases.


Assuntos
Encéfalo/crescimento & desenvolvimento , Encéfalo/fisiologia , Dispositivos Lab-On-A-Chip , Neurogênese/fisiologia , Organoides/crescimento & desenvolvimento , Organoides/fisiologia , Animais , Encéfalo/citologia , Meios de Cultura , Fenômenos Eletrofisiológicos , Matriz Extracelular/fisiologia , Estudos de Viabilidade , Perfilação da Expressão Gênica , Humanos , Hidrogéis , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Modelos Anatômicos , Modelos Neurológicos , Neurogênese/genética , Neuroglia/citologia , Neuroglia/fisiologia , Técnicas de Cultura de Órgãos/instrumentação , Técnicas de Cultura de Órgãos/métodos , Organoides/citologia , Suínos
3.
Int J Mol Sci ; 22(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34360992

RESUMO

Several protocols exist for generating megakaryocytes (MKs) and platelets from human induced pluripotent stem cells (hiPSCs) with limited efficiency. We observed previously that mesoderm induction improved endothelial and stromal differentiation. We, therefore, hypothesized that a protocol modification prior to hemogenic endothelial cell (HEC) differentiation will improve MK progenitor (MKP) production and increase platelet output. We further asked if basic media composition affects MK maturation. In an iterative process, we first compared two HEC induction protocols. We found significantly more HECs using the modified protocol including activin A and CHIR99021, resulting in significantly increased MKs. MKs released comparable platelet amounts irrespective of media conditions. In a final validation phase, we obtained five-fold more platelets per hiPSC with the modified protocol (235 ± 84) compared to standard conditions (51 ± 15; p < 0.0001). The regenerative potency of hiPSC-derived platelets was compared to adult donor-derived platelets by profiling angiogenesis-related protein expression. Nineteen of 24 angiogenesis-related proteins were expressed equally, lower or higher in hiPSC-derived compared to adult platelets. The hiPSC-platelet's coagulation hyporeactivity compared to adult platelets was confirmed by thromboelastometry. Further stepwise improvement of hiPSC-platelet production will, thus, permit better identification of platelet-mediated regenerative mechanisms and facilitate manufacture of sufficient amounts of functional platelets for clinical application.


Assuntos
Plaquetas/citologia , Diferenciação Celular , Técnicas de Reprogramação Celular/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Megacariócitos/citologia , Células Cultivadas , Meios de Cultura/química , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo
4.
Int J Mol Sci ; 22(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34360991

RESUMO

The possibility to reproduce key tissue functions in vitro from induced pluripotent stem cells (iPSCs) is offering an incredible opportunity to gain better insight into biological mechanisms underlying development and disease, and a tool for the rapid screening of drug candidates. This review attempts to summarize recent strategies for specification of iPSCs towards hepatobiliary lineages -hepatocytes and cholangiocytes-and their use as platforms for disease modeling and drug testing. The application of different tissue-engineering methods to promote accurate and reliable readouts is discussed. Space is given to open questions, including to what extent these novel systems can be informative. Potential pathways for improvement are finally suggested.


Assuntos
Técnicas de Reprogramação Celular/métodos , Doenças do Sistema Digestório/terapia , Descoberta de Drogas/métodos , Hepatócitos/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Medicina de Precisão/métodos , Animais , Linhagem da Célula , Doenças do Sistema Digestório/metabolismo , Doenças do Sistema Digestório/patologia , Hepatócitos/metabolismo , Humanos , Engenharia Tecidual/métodos
5.
Int J Mol Sci ; 22(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34360950

RESUMO

The Bruch's membrane (BrM) is a five-layered extracellular matrix (ECM) that supports the retinal pigment epithelium (RPE). Normal age-related changes in the BrM may lead to RPE cell damage and ultimately to the onset and progression of age-related macular degeneration (AMD), which is the most common cause of visual loss among the elderly. A role for the complement system in AMD pathology has been established, but the disease mechanisms are poorly understood, which hampers the design of efficient therapies to treat millions of patients. In an effort to identify the mechanisms that lead from normal aging to pathology, we have developed a cell-based model using complement deficient human induced pluripotent stem cell (iPSC)-derived RPE cells cultured on an AMD-like ECM that mimics BrM. The data present evidence that changes in the ECM result in loss of differentiation and promote epithelial mesenchymal transition (EMT) of healthy RPE cells. This pathological process is mediated by complement activation and involves the formation of a randomly oriented collagen meshwork that drives the dedifferentiation of the RPE monolayer. Genetic ablation of complement component 3 has a protective effect against EMT but does not prevent the abnormal deposition of collagens. These findings offer new insights into the sequence of events that initiate AMD and may guide the design of efficient therapies to treat this disease with unmet medical needs.


Assuntos
Complemento C3/metabolismo , Transição Epitelial-Mesenquimal , Degeneração Macular/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Linhagem Celular , Colágeno/metabolismo , Ativação do Complemento , Complemento C3/genética , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Matriz Extracelular/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Epitélio Pigmentado da Retina/citologia
6.
Nat Commun ; 12(1): 4897, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34385432

RESUMO

Precise control of mammalian gene expression is facilitated through epigenetic mechanisms and nuclear organization. In particular, insulated chromosome structures are important for regulatory control, but the phenotypic consequences of their boundary disruption on developmental processes are complex and remain insufficiently understood. Here, we generated deeply sequenced Hi-C data for human pluripotent stem cells (hPSCs) that allowed us to identify CTCF loop domains that have highly conserved boundary CTCF sites and show a notable enrichment of individual developmental regulators. Importantly, perturbation of such a boundary in hPSCs interfered with proper differentiation through deregulated distal enhancer-promoter activity. Finally, we found that germline variations affecting such boundaries are subject to purifying selection and are underrepresented in the human population. Taken together, our findings highlight the importance of developmental gene isolation through chromosomal folding structures as a mechanism to ensure their proper expression.


Assuntos
Diferenciação Celular/genética , Perfilação da Expressão Gênica/métodos , Genoma Humano/genética , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Elementos Reguladores de Transcrição/genética , Sítios de Ligação/genética , Western Blotting , Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , Linhagem Celular , Elementos Facilitadores Genéticos/genética , Células-Tronco Embrionárias Humanas/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Regiões Promotoras Genéticas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA/métodos
7.
Methods Mol Biol ; 2320: 23-27, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302644

RESUMO

Regenerative medicine using human-induced pluripotent stem cells (hiPSCs) is a promising approach to treat heart failure. However, a large number of cells are required to achieve the desired therapeutic effect. The stirring-type suspension culture method allows a large-scale production of hiPSC-derived cardiomyocytes (more than 1 × 108 cells/100 mL), leading to a stable cell supply. Here, we describe a method to scale-up hiPSC-derived cardiomyocyte production with a high differentiation efficiency.


Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Animais , Técnicas de Cultura de Células/métodos , Células Cultivadas , Humanos , Camundongos
8.
Methods Mol Biol ; 2320: 29-33, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302645

RESUMO

The human adult heart consists of approximately four billion cardiomyocytes, which do not possess self-renewal abilities. Severe myocardial infarction and dilated cardiomyopathy result in the loss of more than a billion cardiomyocytes. Induced pluripotent stem cells (iPSCs) can differentiate into various types of cells. Due to this ability, these cells could potentially serve as a new resource for cell therapy. Many studies have utilized cardiomyocytes derived from iPSCs for myocardial regeneration therapy. To obtain large number of cardiomyocytes for transplantation, we need to develop effective methods that would allow us to dissociate multiple cardiomyocyte aggregates simultaneously. Here, we describe a method to efficiently dissociate large number of iPSC-derived cardiomyocyte aggregates.


Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Diferenciação Celular/fisiologia , Células Cultivadas , Humanos , Infarto do Miocárdio/terapia
9.
Methods Mol Biol ; 2320: 53-63, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302647

RESUMO

The fabrication of three-dimensional (3D) cardiac tissue using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) is useful not only for regenerative medicine, but also for drug discovery. Here, we report a bio-3D printer that can fabricate tubular cardiac constructs using only human iPSC-CMs. Protocols to evaluate the contractile force and response to electrical stimulation in the cardiac constructs are described. We confirmed that the constructs can be applied for transplantation or drug response testing. In the near future, we expect that the constructs will be used as alternatives for heart transplantation and in animal experiments for new drug development.


Assuntos
Miócitos Cardíacos/citologia , Engenharia Tecidual/métodos , Células Cultivadas , Transplante de Coração , Células Endoteliais da Veia Umbilical Humana , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Impressão Tridimensional , Medicina Regenerativa/métodos , Tecidos Suporte
10.
Methods Mol Biol ; 2320: 81-88, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302650

RESUMO

The present protocol describes a method to generate cylindrical engineered cardiac tissues (ECTs) composed of cardiovascular cell lineages induced from human induced pluripotent stem cells (hiPSCs). Cardiomyocytes, endothelial cells, and vascular mural cells induced from hiPSCs are mixed with gel matrix and poured into a tissue mold with posts. By culture day 14, the mixed culture matures into a cylindrical ECT which beats spontaneously and synchronously. Cardiomyocytes align to the long axis of the ECT. The ECTs generated by the present method may be regarded as a surrogate of human myocardium and be served as researches in cardiac regenerative medicine, disease modeling, drug discovery, and cardiac toxicity tests.


Assuntos
Linhagem da Célula/fisiologia , Células-Tronco Pluripotentes Induzidas/citologia , Miocárdio/citologia , Miócitos Cardíacos/citologia , Diferenciação Celular/fisiologia , Células Cultivadas , Células Endoteliais/citologia , Humanos , Engenharia Tecidual/métodos
11.
Methods Mol Biol ; 2320: 91-100, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302651

RESUMO

Induced pluripotent stem cells (iPSCs) have been utilized to study physiological development and also the pathogenesis of heart diseases. iPS-derived cardiomyocytes and engineered cardiac tissues provide a promising capacity for investigating cardiac development and disease modeling. In addition to protocols for cardiac differentiation and 3D cardiac tissue construction, the establishment of protocols for the comprehensive evaluation of the physiological and/or pathophysiological properties for the iPS-derived cells/tissues are indispensable.


Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Diferenciação Celular/fisiologia , Células Cultivadas , Cardiopatias/terapia , Humanos , Fenótipo , Engenharia Tecidual/métodos
12.
Methods Mol Biol ; 2320: 101-110, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302652

RESUMO

FluoVolt, a membrane potential dye, has been used to depict the action potentials of cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CMs) in order to classify the cardiac cell subtype, evaluate long QT syndrome, and conduct cardiotoxic drug-responsive tests. To apply FluoVolt, users must prepare the hiPSC-CMs, assess the dye loadings, and apply the excitation light. Here we describe the steps to measure action potentials from single hiPSC-CMs and hiPSC-CM monolayers using this dye.


Assuntos
Potenciais de Ação/fisiologia , Diferenciação Celular/fisiologia , Corantes/química , Corpos Embrioides/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Células Cultivadas , Humanos , Síndrome do QT Longo/terapia
13.
Methods Mol Biol ; 2320: 111-119, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302653

RESUMO

Induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs) have been shown to have great potential to play a key role in investigating cardiac diseases in vitro. Multielectrode array (MEA) system is sometimes preferable to patch-clamp in electrophysiological experiments in terms of several advantages. Here we show our protocol of electrophysiological examinations using MEA.


Assuntos
Bioensaio/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Análise Serial de Tecidos/métodos , Células Cultivadas , Fenômenos Eletrofisiológicos/fisiologia , Humanos , Técnicas de Patch-Clamp/métodos
14.
Methods Mol Biol ; 2320: 121-133, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302654

RESUMO

Electrophysiological analysis of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) using a patch-clamp technique enables the most precise evaluation of electrophysiological properties in single cells. Compared to multielectrode array (MEA) and membrane voltage imaging, patch-clamp recordings offer quantitative measurements of action potentials, and the relevant ionic currents which are essential for the research of disease modeling of inherited arrhythmias, safety pharmacology, and drug discovery using hiPSC-CMs. In this chapter, we describe the detail flow of patch-clamp recordings in hiPSC-CMs.


Assuntos
Fenômenos Eletrofisiológicos/fisiologia , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Técnicas de Patch-Clamp/métodos , Análise Serial de Tecidos/métodos , Potenciais de Ação/fisiologia , Arritmias Cardíacas/terapia , Células Cultivadas , Humanos
15.
Methods Mol Biol ; 2320: 135-149, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302655

RESUMO

Human iPSC-derived cardiomyocytes (hiPSC-CMs) are expected to be used in regenerative therapies and drug discovery for heart failure. hiPSC-CMs are a mixture of mainly ventricular CMs (VCMs) and also of atrial CMs (ACMs) and pacemaker cells. Here we describe a method to enrich VCM and ACM differentiation and to characterize these subtypes by gene expression analysis using qRT-PCR and by electrophysiological properties using the patch-clamp method. The differentiated VCMs and ACMs highly express VCM and ACM marker genes, respectively. Furthermore, both subtypes show specific properties of action potentials.


Assuntos
Ventrículos do Coração/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Potenciais de Ação/fisiologia , Diferenciação Celular/fisiologia , Células Cultivadas , Fenômenos Eletrofisiológicos/fisiologia , Átrios do Coração/citologia , Humanos , Técnicas de Patch-Clamp/métodos
16.
Methods Mol Biol ; 2320: 151-160, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302656

RESUMO

Human-induced pluripotent stem cell (iPSC) technology paves the way for next-generation drug-safety assessment. In particular, human iPSC-derived cardiomyocytes, which exhibit electrical activity, are useful as a human cell model for assessing QT-interval prolongation and the risk of the lethal arrhythmia Torsade de Pointes (TdP). In addition to proarrhythmia assay, contractile behavior has received increased attention in drug development. In this study, we developed a novel high-throughput in vitro assay system using motion vectors to evaluate the contractile activity of iPSC-derived cardiomyocytes as a physiologically relevant human platform. The methods presented here highlight the use of commercially available iPSC-derived cardiomyocytes, iCell cardiomyocytes, for contractility evaluation recorded by the motion vector system.


Assuntos
Bioensaio/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Arritmias Cardíacas/terapia , Células Cultivadas , Humanos , Síndrome do QT Longo/terapia , Torsades de Pointes/terapia
17.
Methods Mol Biol ; 2320: 161-170, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302657

RESUMO

Recent advances in stem cell technologies and tissue engineering are enabling the fabrication of dynamically beating cardiac tissues from human induced pluripotent stem cells. These engineered human cardiac tissues are expected to be used for cardiac regenerative therapies, in vitro drug testing, and pathological investigations. Here we describe the method to fabricate engineered cardiac tissues from human induced pluripotent stem cell-derived cardiomyocytes and to measure the contractile force.


Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Contração Muscular/fisiologia , Miócitos Cardíacos/citologia , Células Cultivadas , Humanos , Engenharia Tecidual/métodos
18.
Methods Mol Biol ; 2320: 171-180, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302658

RESUMO

Engineered cardiac tissue (ECT) derived from human induced pluripotent stem cells (iPSCs) can replicate human heart in vitro and be applied to drug discovery and heart disease models. The contraction force of ECT is an important indicator of its function and of the disease phenotype. Here we describe a construction method of ECT using the Flexcell® Tissue Train® culture system and a contraction force measurement method based on the Frank-Starling law.


Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Contração Miocárdica/fisiologia , Miócitos Cardíacos/citologia , Engenharia Tecidual/métodos , Células Cultivadas , Humanos
19.
Methods Mol Biol ; 2352: 45-55, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34324179

RESUMO

Astrocytes play important roles in neurodevelopment and diseases. Previous studies described ways to derive astrocytes from somatic cells by going through iPSC or iNSC/iNPC intermediates. Here we describe a method to directly convert mouse fibroblasts into functional astrocytes using small molecules without transgenes or viral transduction. The direct chemical reprogramming method described in this study provides a more rapid way to derive astrocytes from fibroblasts.


Assuntos
Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Técnicas de Reprogramação Celular , Reprogramação Celular/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Animais , Astrócitos/metabolismo , Biomarcadores , Diferenciação Celular/genética , Separação Celular , Células Cultivadas , Reprogramação Celular/genética , Fibroblastos/metabolismo , Expressão Gênica , Humanos , Imuno-Histoquímica , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Fatores de Transcrição/genética , Transgenes
20.
Methods Mol Biol ; 2352: 127-132, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34324184

RESUMO

Human motor neurons are important materials for the research of the pathogenesis and drug discovery of motor neuron diseases. Various methods to generate motor neurons (MNs) from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) by the addition of signaling molecules have been reported. However, they require multiple steps and complicated processes. Here we describe an approach for generating human MNs from ESCs/iPSCs using a single Sendai virus vector encoding three transcription factors-Lhx3, Ngn2, and Isl1. This approach enabled us to generate MNs in one step, adding Sendai virus vector in culture medium. This simple method significantly reduces the efforts to generate MNs, and it provides a useful tool for motor neuron disease research.


Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/citologia , Vetores Genéticos , Células-Tronco Pluripotentes Induzidas/citologia , Neurônios Motores/citologia , Vírus Sendai , Diferenciação Celular/genética , Linhagem Celular , Células-Tronco Embrionárias/metabolismo , Expressão Gênica , Vetores Genéticos/genética , Humanos , Imuno-Histoquímica , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurônios Motores/metabolismo , Vírus Sendai/genética , Fatores de Transcrição/genética , Transgenes
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...