Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 3.762
Filtrar
1.
Cancer Sci ; 110(10): 3027-3037, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31348591

RESUMO

We previously established a method to generate myeloid cells with a proliferative capability from pluripotent stem cells and designated them iPS-ML. Human iPS-ML cells share features with physiological macrophages including the capability to infiltrate into cancer tissues. We observed therapeutic effects of human iPS-ML cells expressing interferon ß (iPS-ML/interferon (IFN)-ß) in xenograft cancer models. However, assessment of host immune system-mediated therapeutic and adverse effects of this therapy is impossible by xenograft models. We currently evaluated the therapeutic effects of a mouse equivalent of human iPS-ML/IFN, a mouse embryonic stem (ES) cell-derived myeloid cell line producing IFN (ES-ML/IFN). The ES-MLs producing IFN-ß (ß-ML) and IFN-γ (γ-ML) and originating from E14 ES cells derived from the 129 mouse strain (H-2b ) were generated, and the MHC (H-2Kb , Db , and I-Ab ) genes of the ES-ML/IFN were disrupted using the clustered regularly interspaced short palindromic repeats (CRISPR)/CAS9 method. We used the ES-ML/IFN to treat allogeneic BALB/c mice (H-2d ) transplanted with Colon26 cancer cells. Treatment with ß-ML but not with γ-ML cells repressed the growth of colon cancer in the peritoneal cavity and liver. The transferred ES-ML/IFN infiltrated into cancer tissues and enhanced infiltration of T cells into cancer tissues. ES-ML/IFN therapy increased the number of immune cells in the lymphoid organs. Sensitization of both cancer antigen-specific CD8+ T cells and natural killer (NK) cells were enhanced by the therapy, and CD8+ T cells were essential for the therapeutic effect, implying that donor MHC-deficient ß-ML exhibited a therapeutic effect through the activation of host immune cells derived from allogeneic recipient mice. The results suggested the usefulness of HLA-deficient human iPS-ML/IFN-ß cells for therapy of HLA-mismatched allogeneic cancer patients.


Assuntos
Neoplasias do Colo/terapia , Células-Tronco Embrionárias/citologia , Antígenos de Histocompatibilidade/genética , Interferon beta/metabolismo , Células Mieloides/transplante , Animais , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo/imunologia , Células-Tronco Embrionárias/metabolismo , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células Matadoras Naturais/metabolismo , Linfócitos do Interstício Tumoral/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Células Mieloides/citologia , Células Mieloides/metabolismo , Transplante Homólogo , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Biochemistry (Mosc) ; 84(3): 283-290, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31221066

RESUMO

Reprogramming of somatic cells is associated with overcoming the established epigenetic barrier. Key events in this process are changes in the DNA methylation landscape and histone modifications. Studying the factors affecting epigenetic plasticity will allow not only to reveal the principles underlying cell reprogramming but also to find possible ways to influence this process. Kaiso transcription factor is one of the protein interpreters of methylated DNA. By binding to methylated DNA, Kaiso attracts corepressor complexes affecting chromatin structure. In this work, we showed that the Kaiso gene knockout contributes to more efficient somatic reprogramming by affecting both cell proliferation and DNA methylation. The proposed mechanisms for the increase in the efficiency of somatic reprogramming associated with the Kaiso gene knockout is a decrease in the methylation level of the Oct4 promoter region in mouse embryonic fibroblasts before reprogramming.


Assuntos
Reprogramação Celular , Técnicas de Inativação de Genes , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de Transcrição/metabolismo
3.
Biochemistry (Mosc) ; 84(3): 291-298, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31221067

RESUMO

Generation of patient-specific induced pluripotent stem cells (iPSCs) and their subsequent differentiation into cardiomyocytes opened new opportunities for studying pathogenesis of inherited cardiovascular diseases. One of these diseases is hypertrophic cardiomyopathy (HCM) for which no efficient therapy methods have been developed so far. In this study, the approach based on patient-specific iPSCs was applied to create a model of the disease. Genetic analysis of a hypertrophic cardiomyopathy patient revealed R326Q mutation in the MYBPC3 gene. iPSCs of the patient were generated and characterized. The cells were differentiated into cardiomyocytes together with the control iPSCs from a healthy donor. The patient's iPSC-derived cardiomyocytes exhibited early HCM features, such as abnormal calcium handling and increased intracellular calcium concentration. Therefore, cardiomyocytes obtained by directed differentiation of iPSCs from the HCM patient can be used as a model system to study HCM pathogenesis.


Assuntos
Cardiomiopatia Hipertrófica/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Modelos Biológicos , Adulto , Cálcio/metabolismo , Cardiomiopatia Hipertrófica/diagnóstico , Cardiomiopatia Hipertrófica/genética , Diferenciação Celular , Células Cultivadas , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Masculino
4.
Biochemistry (Mosc) ; 84(3): 299-309, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31221068

RESUMO

Patient-specific induced pluripotent stem cells (iPSCs) capable of differentiation into required cell type are a promising model for studying various pathological processes and development of new therapeutic approaches. However, no conventional strategies for using iPSCs in disease research have been established yet. Genetically encoded biosensors can be used for monitoring messenger molecules, metabolites, and enzyme activity in real time with the following conversion of the registered signals in quantitative data, thus allowing evaluation of the impact of certain molecules on pathology development. In this article, we describe the development of a universal cell-based platform for studying pathological processes associated with amyotrophic lateral sclerosis. For this purpose, we have created a series of plasmid constructs for monitoring endoplasmic reticulum stress, oxidative stress, apoptosis, and Ca2+-dependent hyperexcitability and generated transgenic iPSC line carrying mutation in the superoxide dismutase 1 gene (SOD1) and healthy control cell line. Both cell lines have specific transactivator sequence required for doxycycline-controlled transcriptional activation and can be used for a single-step biosensor insertion.


Assuntos
Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/patologia , Técnicas Biossensoriais/métodos , Células-Tronco Pluripotentes Induzidas/metabolismo , Apoptose , Cálcio/metabolismo , Diferenciação Celular , Células Cultivadas , Estresse do Retículo Endoplasmático , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Mutação , Estresse Oxidativo , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo
5.
Biochemistry (Mosc) ; 84(3): 310-320, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31221069

RESUMO

Ionotropic glutamate and GABA receptors regulate the differentiation and determine the functional properties of mature neurons. Both insufficient and excessive activity of these neurotransmission systems are associated with various nervous system diseases. Our knowledge regarding the expression profiles of these receptors and the mechanisms of their regulation during the differentiation of specialized human neuron subtypes is limited. Here the expression profiles of the NMDA and GABAA receptor subunits were explored during in vitro differentiation of human induced pluripotent stem cells (iPSCs) into ventral mesencephalic neurons. The correlation between the neuronal maturation and the expression dynamics of these genes was investigated, and the functional activity of these receptors was assessed by calcium imaging. The role of NMDA and GABAA receptors in neurite outgrowth and the development of spontaneous activity was analyzed using the viral transduction of neural progenitors with the reporter genes TagGFP and TagRFP. The data indicate that agonists of the investigated receptors can be employed for optimization of existing protocols for neural differentiation of iPSCs, in particular for acceleration of neuronal maturation.


Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Mesencéfalo/metabolismo , Neurônios/metabolismo , Receptores de GABA-A/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Diferenciação Celular , Células Cultivadas , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Mesencéfalo/citologia , Neurônios/citologia , Receptores de GABA-A/genética , Receptores de N-Metil-D-Aspartato/genética
6.
Cell Mol Life Sci ; 76(20): 3953-3967, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31250034

RESUMO

The brain tissue has only a limited capacity for generating new neurons. Therefore, to treat neurological diseases, there is a need of other cell sources for brain repair. Different sources of cells have been subject of intense research over the years, including cells from primary tissue, stem cell-derived cells and reprogrammed cells. As an alternative, direct reprogramming of resident brain cells into neurons is a recent approach that could provide an attractive method for treating brain injuries or diseases as it uses the patient's own cells for generating novel neurons inside the brain. In vivo reprogramming is still in its early stages but holds great promise as an option for cell therapy. To date, both inhibitory and excitatory neurons have been obtained via in vivo reprogramming, but the precise phenotype or functionality of these cells has not been analysed in detail in most of the studies. Recent data shows that in vivo reprogrammed neurons are able to functionally mature and integrate into the existing brain circuitry, and compose interneuron phenotypes that seem to correlate to their endogenous counterparts. Interneurons are of particular importance as they are essential in physiological brain function and when disturbed lead to several neurological disorders. In this review, we describe a comprehensive overview of the existing studies involving brain repair, including in vivo reprogramming, with a focus on interneurons, along with an overview on current efforts to generate interneurons for cell therapy for a number of neurological diseases.


Assuntos
Lesões Encefálicas/terapia , Terapia Baseada em Transplante de Células e Tecidos/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Interneurônios/citologia , Doenças Neurodegenerativas/terapia , Regeneração/fisiologia , Animais , Biomarcadores/metabolismo , Encéfalo/citologia , Encéfalo/metabolismo , Lesões Encefálicas/genética , Lesões Encefálicas/metabolismo , Lesões Encefálicas/patologia , Transdiferenciação Celular , Reprogramação Celular , Fibroblastos/citologia , Fibroblastos/metabolismo , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Injeções Intraventriculares , Interneurônios/metabolismo , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Neurogênese/genética , Transplante de Células-Tronco/métodos
7.
Oxid Med Cell Longev ; 2019: 7623023, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31049138

RESUMO

Iron homeostasis in the cardiac tissue as well as the involvement of the hepcidin-ferroportin (HAMP-FPN) axis in this process and in cardiac functionality are not fully understood. Imbalance of iron homeostasis occurs in several cardiac diseases, including iron-overload cardiomyopathies such as Friedreich's ataxia (FRDA, OMIM no. 229300), a hereditary neurodegenerative disorder. Exploiting the induced pluripotent stem cells (iPSCs) technology and the iPSC capacity to differentiate into specific cell types, we derived cardiomyocytes of a FRDA patient and of a healthy control subject in order to study the cardiac iron homeostasis and the HAMP-FPN axis. Both CTR and FRDA iPSCs-derived cardiomyocytes express cardiac differentiation markers; in addition, FRDA cardiomyocytes maintain the FRDA-like phenotype. We found that FRDA cardiomyocytes show an increase in the protein expression of HAMP and FPN. Moreover, immunofluorescence analysis revealed for the first time an unexpected nuclear localization of FPN in both CTR and FRDA cardiomyocytes. However, the amount of the nuclear FPN was less in FRDA cardiomyocytes than in controls. These and other data suggest that iron handling and the HAMP-FPN axis regulation in FRDA cardiac cells are hampered and that FPN may have new, still not fully understood, functions. These findings underline the complexity of the cardiac iron homeostasis.


Assuntos
Cardiomiopatias/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Ataxia de Friedreich/metabolismo , Hepcidinas/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Ferro/metabolismo , Miócitos Cardíacos/metabolismo , Cardiomiopatias/patologia , Ataxia de Friedreich/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Miócitos Cardíacos/patologia
8.
Int J Mol Sci ; 20(7)2019 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-30987116

RESUMO

Stage-specific embryonic antigen 1 (SSEA-1) is an antigenic epitope (also called CD15 antigen) defined as a Lewis X carbohydrate structure and known to be expressed in murine embryonal carcinoma cells, mouse embryonic stem cells (ESCs), and murine and human germ cells, but not human ESCs/induced pluripotent stem cells (iPSCs). It is produced by α1,3-fucosyltransferase IX gene (FUT9), and F9 ECCs having a disrupted FUT9 locus by gene targeting are reported to exhibit loss of SSEA-1 expression on their cell surface. Mouse ESCs are pluripotent cells and therefore known as "naïve stem cells (NSCs)." In contrast, human ESCs/iPSCs are thought to be epiblast stem cells (EpiSCs) that are slightly more differentiated than NSCs. Recently, it has been demonstrated that treatment of EpiSCs with several reprograming-related drugs can convert EpiSCs to cells similar to NSCs, which led us to speculate that SSEA-1 may have been expressed in these NSC-like EpiSCs. Immunocytochemical staining of these cells with anti-SSEA-1 revealed increased expression of this epitope. RT-PCR analysis also confirmed increased expression of FUT9 transcripts as well as other stemness-related transcripts such as REX-1 (ZFP42). These results suggest that SSEA-1 can be an excellent marker for human NSCs.


Assuntos
Membrana Celular/metabolismo , Polpa Dentária/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Antígenos CD15/metabolismo , Dente Decíduo/citologia , Animais , Ensaio de Unidades Formadoras de Colônias , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Nus
9.
Int J Mol Sci ; 20(9)2019 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-31032819

RESUMO

Brugada syndrome is an inherited, rare cardiac arrhythmogenic disease, associated with sudden cardiac death. It accounts for up to 20% of sudden deaths in patients without structural cardiac abnormalities. The majority of mutations involve the cardiac sodium channel gene SCN5A and give rise to classical abnormal electrocardiogram with ST segment elevation in the right precordial leads V1 to V3 and a predisposition to ventricular fibrillation. The pathophysiological mechanisms of Brugada syndrome have been investigated using model systems including transgenic mice, canine heart preparations, and expression systems to study different SCN5A mutations. These models have a number of limitations. The recent development of pluripotent stem cell technology creates an opportunity to study cardiomyocytes derived from patients and healthy individuals. To date, only a few studies have been done using Brugada syndrome patient-specific iPS-CM, which have provided novel insights into the mechanisms and pathophysiology of Brugada syndrome. This review provides an evaluation of the strengths and limitations of each of these model systems and summarizes the key mechanisms that have been identified to date.


Assuntos
Síndrome de Brugada/etiologia , Síndrome de Brugada/fisiopatologia , Modelos Animais de Doenças , Animais , Animais Geneticamente Modificados , Biomarcadores , Síndrome de Brugada/diagnóstico , Síndrome de Brugada/terapia , Diferenciação Celular , Suscetibilidade a Doenças , Cães , Predisposição Genética para Doença , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Mutação , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/genética
10.
Int J Mol Sci ; 20(8)2019 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-31022972

RESUMO

Stem cells are undifferentiated cells that can give rise to several different cell types and can self-renew. Given their ability to differentiate into different lineages, stem cells retain huge therapeutic potential for regenerative medicine. Therefore, the understanding of the signaling pathways involved in stem cell pluripotency maintenance and differentiation has a paramount importance in order to understand these biological processes and to develop therapeutic strategies. In this review, we focus on phosphoinositide 3 kinase (PI3K) since its signaling pathway regulates many cellular processes, such as cell growth, proliferation, survival, and cellular transformation. Precisely, in human stem cells, the PI3K cascade is involved in different processes from pluripotency and induced pluripotent stem cell (iPSC) reprogramming to mesenchymal and oral mesenchymal differentiation, through different and interconnected mechanisms.


Assuntos
Diferenciação Celular , Reprogramação Celular , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Mesenquimais/citologia , Fosfatidilinositol 3-Quinase/metabolismo , Transdução de Sinais , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Mesenquimais/metabolismo
11.
Methods Mol Biol ; 1981: 373-382, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31016668

RESUMO

Cholangiocytes play a crucial role in the pathophysiology of cholestasis. However, research on human cholangiocytes has been restricted by challenges in long-term propagation and large-scale expansion of primary biliary epithelium. The advent of organoid technology has overcome this limitation allowing long-term culture of a variety of epithelia from multiple organs. Here, we describe two methods for growing human cholangiocytes in organoid format. The first applies to the generation of intrahepatic bile ducts using human induced pluripotent stem cells using a protocol of differentiation that recapitulates physiological bile duct development. The second method allows the propagation of primary biliary epithelium from the extrahepatic ducts or gallbladder. Both protocols result in large numbers of cholangiocyte organoids expressing biliary markers and maintaining key cholangiocyte functions.


Assuntos
Colestase/metabolismo , Colestase/patologia , Organoides/patologia , Ductos Biliares/metabolismo , Ductos Biliares/patologia , Vesícula Biliar/metabolismo , Vesícula Biliar/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Fígado/metabolismo , Fígado/patologia , Organoides/metabolismo
12.
PLoS Genet ; 15(4): e1008045, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-31002671

RESUMO

Quantification of gene expression levels at the single cell level has revealed that gene expression can vary substantially even across a population of homogeneous cells. However, it is currently unclear what genomic features control variation in gene expression levels, and whether common genetic variants may impact gene expression variation. Here, we take a genome-wide approach to identify expression variance quantitative trait loci (vQTLs). To this end, we generated single cell RNA-seq (scRNA-seq) data from induced pluripotent stem cells (iPSCs) derived from 53 Yoruba individuals. We collected data for a median of 95 cells per individual and a total of 5,447 single cells, and identified 235 mean expression QTLs (eQTLs) at 10% FDR, of which 79% replicate in bulk RNA-seq data from the same individuals. We further identified 5 vQTLs at 10% FDR, but demonstrate that these can also be explained as effects on mean expression. Our study suggests that dispersion QTLs (dQTLs) which could alter the variance of expression independently of the mean can have larger fold changes, but explain less phenotypic variance than eQTLs. We estimate 4,015 individuals as a lower bound to achieve 80% power to detect the strongest dQTLs in iPSCs. These results will guide the design of future studies on understanding the genetic control of gene expression variance.


Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Locos de Características Quantitativas , Grupo com Ancestrais do Continente Africano/genética , Linhagem Celular , Simulação por Computador , Perfilação da Expressão Gênica , Variação Genética , Estudo de Associação Genômica Ampla , Humanos , Modelos Genéticos , Nigéria , Fenótipo , Análise de Sequência de RNA , Análise de Célula Única
13.
Nat Commun ; 10(1): 1465, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30931944

RESUMO

Embryonic stem cells (ESCs) can propagate in an undifferentiated state indefinitely in culture and retain the potential to differentiate into any somatic lineage as well as germ cells. The catabolic process autophagy has been reported to be involved in ESC identity regulation, but the underlying mechanism is still largely unknown. Here we show that EPG5, a eukaryotic-specific autophagy regulator which mediates autophagosome/lysosome fusion, is highly expressed in ESCs and contributes to ESC identity maintenance. We identify that the deubiquitinating enzyme USP8 binds to the Coiled-coil domain of EPG5. Mechanistically, USP8 directly removes non-classical K63-linked ubiquitin chains from EPG5 at Lysine 252, leading to enhanced interaction between EPG5 and LC3. We propose that deubiquitination of EPG5 by USP8 guards the autophagic flux in ESCs to maintain their stemness. This work uncovers a novel crosstalk pathway between ubiquitination and autophagy through USP8-EPG5 interaction to regulate the stemness of ESCs.


Assuntos
Autofagia/genética , Endopeptidases/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas/metabolismo , Ubiquitina Tiolesterase/genética , Animais , Autofagossomos/metabolismo , Células-Tronco Embrionárias/metabolismo , Fibroblastos/metabolismo , Células HEK293 , Humanos , Lisina/metabolismo , Lisossomos/metabolismo , Fusão de Membrana/genética , Camundongos , Células-Tronco Neurais/metabolismo , Ubiquitinação
14.
Cell Mol Life Sci ; 76(14): 2739-2760, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31016348

RESUMO

Astrocytes are the most abundant cell type in the brain. They were long considered only as passive support for neuronal cells. However, recent data have revealed many active roles for these cells both in maintenance of the normal physiological homeostasis in the brain as well as in neurodegeneration and disease. Moreover, human astrocytes have been found to be much more complex than their rodent counterparts, and to date, astrocytes are known to actively participate in a multitude of processes such as neurotransmitter uptake and recycling, gliotransmitter release, neuroenergetics, inflammation, modulation of synaptic activity, ionic balance, maintenance of the blood-brain barrier, and many other crucial functions of the brain. This review focuses on the role of astrocytes in human neurodegenerative disease and the potential of the novel stem cell-based platforms in modeling astrocytic functions in health and in disease.


Assuntos
Astrócitos/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Doenças Neurodegenerativas/patologia , Neurônios/patologia , Astrócitos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Doenças Neurodegenerativas/metabolismo , Neurônios/metabolismo
15.
Methods Mol Biol ; 1961: 137-151, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30912045

RESUMO

The generation of targeted mutants is a crucial step toward studying the biomedical effect of genes of interest. The generation of such mutants in human induced pluripotent stem cells (iPSCs) is of an utmost importance as these cells carry the potential to be differentiated into any cell lineage. Using the CRISPR/Cas9 nuclease system for induction of targeted double-strand breaks, gene editing of target loci in iPSCs can be achieved with high efficiency. This chapter covers protocols for the preparation of reagents to target loci of interest, the transfection, and for the genotyping of single cell-derived iPSC clones. Furthermore, we provide a protocol for the convenient generation of plasmids enabling multiplex gene targeting.


Assuntos
Sistemas CRISPR-Cas/genética , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Edição de Genes/métodos , Genoma Humano/genética , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Plasmídeos/genética
16.
Methods Mol Biol ; 1961: 153-183, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30912046

RESUMO

Genome editing using the CRISPR/Cas9 system has rapidly established itself as an essential tool in the genetic manipulation of many organisms, including human cell lines. Its application to human induced pluripotent stem cells (hiPSCs) allows for the generation of isogenic cell pairs that differ in a single genetic lesion, and therefore the identification and characterization of causal genetic variants. We describe a simple, effective approach to perform delicate manipulations of the genome of hiPSCs through delivery of Cas9 RNPs along with ssDNA oligonucleotide repair templates that can generate mutations in up to 98% of single cell clones and introduce single nucleotide changes at an efficiency of up to 40%. We describe our use of a T7 endonuclease assay to identify active guide RNAs, and a high-throughput sequencing genotyping strategy that allows the identification of correctly edited clones. We also present our experiences of generating single nucleotide changes at 15 sites, which show considerable variability between both guides and target sites in the efficiency at which such changes can be introduced.


Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes , Genoma Humano/genética , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , RNA Guia/genética , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo
17.
Proc Natl Acad Sci U S A ; 116(14): 6969-6974, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30886088

RESUMO

Sudden unexpected death of an infant (SUDI) is a devastating occurrence for families. To investigate the genetic pathogenesis of SUDI, we sequenced >70 genes from 191 autopsy-negative SUDI victims. Ten infants sharing a previously unknown variant in troponin I (TnI) were identified. The mutation (TNNI1 R37C+/-) is in the fetal/neonatal paralog of TnI, a gene thought to be expressed in the heart up to the first 24 months of life. Using phylogenetic analysis and molecular dynamics simulations, it was determined that arginine at residue 37 in TNNI1 may play a critical functional role, suggesting that the variant may be pathogenic. We investigated the biophysical properties of the TNNI1 R37C mutation in human reconstituted thin filaments (RTFs) using fluorometry. RTFs reconstituted with the mutant R37C TnI exhibited reduced Ca2+-binding sensitivity due to an increased Ca2+ off-rate constant. Furthermore, we generated TNNI1 R37C+/- mutants in human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) using CRISPR-Cas9. In monolayers of hiPSC-CMs, we simultaneously monitored voltage and Ca2+ transients through optical mapping and compared them to their isogenic controls. We observed normal intrinsic beating patterns under control conditions in TNNI1 R37C+/- at stimulation frequencies of 55 beats/min (bpm), but these cells showed no restitution with increased stimulation frequency to 65 bpm and exhibited alternans at >75 bpm. The WT hiPSC-CMs did not exhibit any sign of arrhythmogenicity even at stimulation frequencies of 120 bpm. The approach used in this study provides critical physiological and mechanistic bases to investigate sarcomeric mutations in the pathogenesis of SUDI.


Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Simulação de Dinâmica Molecular , Mutação de Sentido Incorreto , Miócitos Cardíacos/metabolismo , Morte Súbita do Lactente/genética , Troponina I , Cálcio/química , Cálcio/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Recém-Nascido , Contração Miocárdica/genética , Miócitos Cardíacos/patologia , Sarcômeros/genética , Sarcômeros/metabolismo , Sarcômeros/patologia , Morte Súbita do Lactente/patologia , Troponina I/química , Troponina I/genética , Troponina I/metabolismo
18.
Neurosci Lett ; 703: 111-118, 2019 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-30904577

RESUMO

Alzheimer's disease (AD) is a progressive, degenerative disorder that mainly results in memory loss and a cognitive disorder. Although the cause of AD is still unknown, a minor percentage of AD cases are produced by genetic mutations in the presenilin-1 (PSEN1) gene. Differentiated neuronal cells derived from induced pluripotent stem cells (iPSCs) of patients can recapitulate key pathological features of AD in vitro; however, iPSCs studies focused on the p.E280 A mutation, which afflicts the largest family in the world with familial AD, have not been carried out yet. Although a link between the loss of the Y (LOY) chromosome in peripheral blood cells and risk for AD has been reported, LOY-associated phenotype has not been previously studied in PSEN1 E280 A carriers. Here, we report the reprogramming of fibroblast cells into iPSCs from a familial AD patient with the PSEN1 E280 A mutation, followed by neuronal differentiation into neural precursor cells (NPCs), and the differentiation of NPCs into differentiated neurons that lacked a Y chromosome. Although the PSEN1 E280 A iPSCs and NPCs were successfully obtained, after 8 days of differentiation, PSEN1 E280 A differentiated neurons massively died reflected by release and/ or activation of death markers, and failed to reach complete neural differentiation compared to PSEN 1 wild type cells.


Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Cromossomos Humanos Y , Células-Tronco Pluripotentes Induzidas/metabolismo , Fragmentos de Peptídeos/metabolismo , Presenilina-1/genética , Doença de Alzheimer/genética , Morte Celular , Diferenciação Celular , Reprogramação Celular , Espaço Extracelular/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Mutação , Células-Tronco Neurais/patologia , Neurônios/patologia
19.
Methods Mol Biol ; 1942: 61-69, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30900175

RESUMO

Human pluripotent stem cells (hPSCs) offer powerful platforms for studying mechanisms of human diseases and for evaluating potential treatments. Genome editing, particularly the CRISPR/Cas9-based method, is highly effective for generating cell and animal models to study genetic human diseases. However, the procedure for generating gene-edited hPSCs is laborious, time consuming and unintentional genetic changes may confound the consequent experiments and conclusions. Here we describe one-step knockin of the NanoLuc luciferase gene (Nluc) to the fragile X syndrome gene, FMR1, in a human embryonic stem cell line (hESC), H1, and a fragile X disease model human induced pluripotent stem cell line (hiPSC), FX-iPSC. The luciferase reporter cell lines provide new platforms for exploring potential treatments for fragile X syndrome. The shortened and scarless targeting method described here can be effectively applied to other genes.


Assuntos
Sistemas CRISPR-Cas , Proteína do X Frágil de Retardo Mental/genética , Edição de Genes/métodos , Técnicas de Introdução de Genes/métodos , Genoma Humano , Células-Tronco Pluripotentes Induzidas/metabolismo , Luciferases/metabolismo , Células Cultivadas , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Síndrome do Cromossomo X Frágil/genética , Humanos , Células-Tronco Pluripotentes Induzidas/citologia
20.
Mol Biol Cell ; 30(10): 1214-1229, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30840538

RESUMO

Correct wiring in the neocortex requires that responses to an individual guidance cue vary among neurons in the same location, and within the same neuron over time. Nestin is an atypical intermediate filament expressed strongly in neural progenitors and is thus used widely as a progenitor marker. Here we show a subpopulation of embryonic cortical neurons that transiently express nestin in their axons. Nestin expression is thus not restricted to neural progenitors, but persists for 2-3 d at lower levels in newborn neurons. We found that nestin-expressing neurons have smaller growth cones, suggesting that nestin affects cytoskeletal dynamics. Nestin, unlike other intermediate filament subtypes, regulates cdk5 kinase by binding the cdk5 activator p35. Cdk5 activity is induced by the repulsive guidance cue Semaphorin3a (Sema3a), leading to axonal growth cone collapse in vitro. Therefore, we tested whether nestin-expressing neurons showed altered responses to Sema3a. We find that nestin-expressing newborn neurons are more sensitive to Sema3a in a roscovitine-sensitive manner, whereas nestin knockdown results in lowered sensitivity to Sema3a. We propose that nestin functions in immature neurons to modulate cdk5 downstream of the Sema3a response. Thus, the transient expression of nestin could allow temporal and/or spatial modulation of a neuron's response to Sema3a, particularly during early axon guidance.


Assuntos
Cones de Crescimento/metabolismo , Nestina/metabolismo , Neurônios/metabolismo , Semaforina-3A/farmacologia , Animais , Axônios/metabolismo , Células-Tronco Embrionárias/metabolismo , Feminino , Gânglios Espinais/metabolismo , Cones de Crescimento/fisiologia , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Camundongos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurônios/citologia , Fosforilação , Cultura Primária de Células , Transdução de Sinais/fisiologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA