RESUMO
Three-dimensional (3D) genomics shows immense promise for studying X chromosome inactivation (XCI) by interrogating changes to the X chromosomes' 3D states. Here, we sought to characterize the 3D state of the X chromosome in naïve and primed human pluripotent stem cells (hPSCs). Using chromatin tracing, we analyzed X chromosome folding conformations in these cells with megabase genomic resolution. X chromosomes in female naïve hPSCs exhibit folding conformations similar to the active X chromosome (Xa) and the inactive X chromosome (Xi) in somatic cells. However, naïve X chromosomes do not exhibit the chromatin compaction typically associated with these somatic X chromosome states. In H7 naïve human embryonic stem cells, XIST accumulation observed on damaged X chromosomes demonstrates the potential for naïve hPSCs to activate XCI-related mechanisms. Overall, our findings provide insight into the X chromosome status of naïve hPSCs with a single-chromosome resolution and are critical in understanding the unique epigenetic regulation in early embryonic cells.
Assuntos
Células-Tronco Pluripotentes , RNA Longo não Codificante , Humanos , Feminino , Epigênese Genética , Cromossomos Humanos X/genética , RNA Longo não Codificante/genética , Cromatina/genéticaRESUMO
Rett syndrome (RTT), mainly caused by mutations in methyl-CpG binding protein 2 (MeCP2), is one of the most prevalent intellectual disorders without effective therapies. Here, we used 2D and 3D human brain cultures to investigate MeCP2 function. We found that MeCP2 mutations cause severe abnormalities in human interneurons (INs). Surprisingly, treatment with a BET inhibitor, JQ1, rescued the molecular and functional phenotypes of MeCP2 mutant INs. We uncovered that abnormal increases in chromatin binding of BRD4 and enhancer-promoter interactions underlie the abnormal transcription in MeCP2 mutant INs, which were recovered to normal levels by JQ1. We revealed cell-type-specific transcriptome impairment in MeCP2 mutant region-specific human brain organoids that were rescued by JQ1. Finally, JQ1 ameliorated RTT-like phenotypes in mice. These data demonstrate that BRD4 dysregulation is a critical driver for RTT etiology and suggest that targeting BRD4 could be a potential therapeutic opportunity for RTT.
Assuntos
Azepinas/farmacologia , Encéfalo/patologia , Proteínas de Ciclo Celular/metabolismo , Interneurônios/patologia , Proteína 2 de Ligação a Metil-CpG/fisiologia , Síndrome de Rett/patologia , Fatores de Transcrição/metabolismo , Transcriptoma/efeitos dos fármacos , Triazóis/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Proteínas de Ciclo Celular/genética , Feminino , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Embrionárias Humanas/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Interneurônios/efeitos dos fármacos , Interneurônios/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Fenótipo , Síndrome de Rett/tratamento farmacológico , Síndrome de Rett/genética , Síndrome de Rett/metabolismo , Fatores de Transcrição/genéticaRESUMO
Human cortical organoids (hCOs), derived from human embryonic stem cells (hESCs), provide a platform to study human brain development and diseases in complex three-dimensional tissue. However, current hCOs lack microvasculature, resulting in limited oxygen and nutrient delivery to the inner-most parts of hCOs. We engineered hESCs to ectopically express human ETS variant 2 (ETV2). ETV2-expressing cells in hCOs contributed to forming a complex vascular-like network in hCOs. Importantly, the presence of vasculature-like structures resulted in enhanced functional maturation of organoids. We found that vascularized hCOs (vhCOs) acquired several blood-brain barrier characteristics, including an increase in the expression of tight junctions, nutrient transporters and trans-endothelial electrical resistance. Finally, ETV2-induced endothelium supported the formation of perfused blood vessels in vivo. These vhCOs form vasculature-like structures that resemble the vasculature in early prenatal brain, and they present a robust model to study brain disease in vitro.
Assuntos
Encéfalo/irrigação sanguínea , Células-Tronco Embrionárias Humanas/citologia , Organoides/irrigação sanguínea , Engenharia Tecidual/métodos , Animais , Barreira Hematoencefálica , Células Cultivadas , Humanos , Camundongos , Análise de Célula Única , Fatores de Transcrição/fisiologiaRESUMO
A defining feature of embryonic stem cells (ESCs) is the ability to differentiate into all three germ layers. Pluripotency is maintained in part by a unique transcription network that maintains expression of pluripotency-specific transcription factors and represses developmental genes. While the mechanisms that establish this transcription network are well studied, little is known of the posttranscriptional surveillance pathways that degrade differentiation-related RNAs. We report that the surveillance pathway mediated by the RNA exosome nuclease complex represses ESC differentiation. Depletion of the exosome expedites differentiation of human ESCs into all three germ layers. LINE-1 retrotransposons and specific miRNAs, lncRNAs, and mRNAs that encode developmental regulators or affect their expression are all bound by the exosome and increase in level upon exosome depletion. The exosome restrains differentiation in part by degrading transcripts encoding FOXH1, a transcription factor crucial for mesendoderm formation. Our studies establish the exosome as a regulator of human ESC differentiation and reveal the importance of RNA decay in maintaining pluripotency.
Assuntos
Diferenciação Celular , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Reagentes de Ligações Cruzadas/química , Endoderma/embriologia , Endoderma/metabolismo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica , Células HeLa , Humanos , Elementos Nucleotídeos Longos e Dispersos/genética , Mesoderma/embriologia , Mesoderma/metabolismo , MicroRNAs/genética , Fenótipo , RNA/isolamento & purificação , RNA Longo não Codificante/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Transcrição Gênica , TransgenesRESUMO
Human brain organoid techniques have rapidly advanced to facilitate investigating human brain development and diseases. These efforts have largely focused on generating telencephalon due to its direct relevance in a variety of forebrain disorders. Despite its importance as a relay hub between cortex and peripheral tissues, the investigation of three-dimensional (3D) organoid models for the human thalamus has not been explored. Here, we describe a method to differentiate human embryonic stem cells (hESCs) to thalamic organoids (hThOs) that specifically recapitulate the development of thalamus. Single-cell RNA sequencing revealed a formation of distinct thalamic lineages, which diverge from telencephalic fate. Importantly, we developed a 3D system to create the reciprocal projections between thalamus and cortex by fusing the two distinct region-specific organoids representing the developing thalamus or cortex. Our study provides a platform for understanding human thalamic development and modeling circuit organizations and related disorders in the brain.
Assuntos
Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Organoides/citologia , Organoides/metabolismo , Tálamo/citologia , Humanos , Modelos BiológicosRESUMO
Embryonic stem cells (ESCs) maintain pluripotency through unique epigenetic states. When ESCs commit to a specific lineage, epigenetic changes in histones and DNA accompany the transition to specialized cell types. Investigating how epigenetic regulation controls lineage specification is critical in order to generate the required cell types for clinical applications. Uhrf1 is a widely known hemi-methylated DNA-binding protein, playing a role in DNA methylation through the recruitment of Dnmt1 and in heterochromatin formation alongside G9a, Trim28, and HDACs. Although Uhrf1 is not essential in ESC self-renewal, it remains elusive how Uhrf1 regulates cell specification. Here we report that Uhrf1 forms a complex with the active trithorax group, the Setd1a/COMPASS complex, to maintain bivalent histone marks, particularly those associated with neuroectoderm and mesoderm specification. Overall, our data demonstrate that Uhrf1 safeguards proper differentiation via bivalent histone modifications.
Assuntos
Reprogramação Celular/genética , Código das Histonas/genética , Histona-Lisina N-Metiltransferase/metabolismo , Proteínas Nucleares/metabolismo , Animais , Proteínas Estimuladoras de Ligação a CCAAT , Técnicas de Reprogramação Celular , Quimera , Metilação de DNA/fisiologia , Epigênese Genética , Feminino , Fibroblastos , Técnicas de Inativação de Genes , Células HEK293 , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/isolamento & purificação , Histonas/metabolismo , Humanos , Masculino , Mesoderma/citologia , Mesoderma/fisiologia , Camundongos , Células-Tronco Embrionárias Murinas , Placa Neural/citologia , Placa Neural/fisiologia , Proteínas Nucleares/genética , Cultura Primária de Células , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Ubiquitina-Proteína LigasesRESUMO
To ensure accurate glucose readings when dispensing glucose oxidase enzyme solution from a jetting dispenser onto glucose test strips fabricated from an immersion gold-plated printed circuit board, every drop of the enzyme solution needs to have nearly the same weight and to be dispensed on the reaction zone of the test strips. Experimental results in this study show that the filling pressure in the fluid reservoir containing the glucose enzyme solution to dispense onto the test strips significantly affect the glucose test results. A filling pressure of 12 psi produces test strips with lower coefficient of variation and standard deviation than 10 and 14 psi. Proper filling pressure for dispensing glucose enzyme onto glucose test strips needs to be determined for any enzyme compound formulation.
Assuntos
Análise Química do Sangue/instrumentação , Glicemia/análise , Equipamentos e Provisões Elétricas , Ouro , Pressão , Impressão , Fitas Reagentes , ImersãoRESUMO
Three-dimensional (3D) brain organoid culture has become an essential tool for investigating human brain development and modeling neurological disorders during the past few years. Given the specific regionalization during brain development, it is important to produce distinct brain organoids that reproduce different brain regions and their interaction. The authors' laboratory recently established the platform to generate brain organoids resembling the medial ganglionic eminence (MGE), a specific brain region responsible for interneurogenesis, and found when fusing with organoid resembling the cortex, the fused organoids enabled modeling of interneuron migration in the brain. This unit describes four basic protocols that have been successfully applied in the authors' laboratory, covering the generation of embryonic body (EB) with neuroectodermal fate, the production of MGE organoids (hMGEOs) and cortical organoids (hCOs), and the fusion of the two organoids.
Assuntos
Encéfalo , Técnicas de Cultura de Órgãos , Organoides , Humanos , Interneurônios/citologia , Eminência Mediana/citologia , Neurogênese , Células-Tronco PluripotentesRESUMO
Organoid techniques provide unique platforms to model brain development and neurological disorders. Whereas several methods for recapitulating corticogenesis have been described, a system modeling human medial ganglionic eminence (MGE) development, a critical ventral brain domain producing cortical interneurons and related lineages, has been lacking until recently. Here, we describe the generation of MGE and cortex-specific organoids from human pluripotent stem cells that recapitulate the development of MGE and cortex domains, respectively. Population and single-cell RNA sequencing (RNA-seq) profiling combined with bulk assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) analyses revealed transcriptional and chromatin accessibility dynamics and lineage relationships during MGE and cortical organoid development. Furthermore, MGE and cortical organoids generated physiologically functional neurons and neuronal networks. Finally, fusing region-specific organoids followed by live imaging enabled analysis of human interneuron migration and integration. Together, our study provides a platform for generating domain-specific brain organoids and modeling human interneuron migration and offers deeper insight into molecular dynamics during human brain development.
Assuntos
Encéfalo/embriologia , Movimento Celular , Interneurônios/citologia , Modelos Biológicos , Organoides/citologia , Células-Tronco Pluripotentes/citologia , Encéfalo/citologia , Diferenciação Celular , Linhagem da Célula , Córtex Cerebral/citologia , Cromatina/metabolismo , Humanos , Interneurônios/metabolismo , Eminência Mediana/citologia , Células-Tronco Pluripotentes/metabolismo , Análise de Sequência de RNA , Transcriptoma/genéticaRESUMO
Conventional DNA bisulfite sequencing has been extended to single cell level, but the coverage consistency is insufficient for parallel comparison. Here we report a novel method for genome-wide CpG island (CGI) methylation sequencing for single cells (scCGI-seq), combining methylation-sensitive restriction enzyme digestion and multiple displacement amplification for selective detection of methylated CGIs. We applied this method to analyzing single cells from two types of hematopoietic cells, K562 and GM12878 and small populations of fibroblasts and induced pluripotent stem cells. The method detected 21 798 CGIs (76% of all CGIs) per cell, and the number of CGIs consistently detected from all 16 profiled single cells was 20 864 (72.7%), with 12 961 promoters covered. This coverage represents a substantial improvement over results obtained using single cell reduced representation bisulfite sequencing, with a 66-fold increase in the fraction of consistently profiled CGIs across individual cells. Single cells of the same type were more similar to each other than to other types, but also displayed epigenetic heterogeneity. The method was further validated by comparing the CpG methylation pattern, methylation profile of CGIs/promoters and repeat regions and 41 classes of known regulatory markers to the ENCODE data. Although not every minor methylation differences between cells are detectable, scCGI-seq provides a solid tool for unsupervised stratification of a heterogeneous cell population.
Assuntos
Ilhas de CpG , Metilação de DNA , Epigênese Genética , Regiões Promotoras Genéticas , Análise de Célula Única/métodos , Linhagem Celular , Linhagem Celular Tumoral , Mapeamento Cromossômico , Enzimas de Restrição do DNA/química , Fibroblastos/citologia , Fibroblastos/metabolismo , Variação Genética , Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células K562 , Linfócitos/citologia , Linfócitos/metabolismoRESUMO
DNA methylation is an important epigenetic mark that regulates gene expression. Dnmt1 plays an important role in maintaining DNA methylation patterns on daughter DNA strands. Studies have shed light into the functional role of Dnmt1 regulation in the hematopoietic and epidermal systems. Here we show that Dnmt1 is required for myogenesis. Loss of Dnmt1 results in reduced expression of myogenic genes and defects in myogenic differentiation. We have utilized a conditional knockout mouse approach to examine the functional consequences of Dnmt1 depletion specifically in the developing muscle. These mice were born runted, with smaller body weights, and reduced ability to form myotubes in vitro. We show that expression of Id-1, a negative regulator of myogenesis, is enhanced in Dnmt1-deficient cultures, leading to enhanced transdifferentiation of myoblasts toward the osteogenic lineage. Thus, these studies demonstrate that Dnmt1 influences cellular identity and determines lineage fidelity.
Assuntos
Diferenciação Celular/genética , DNA (Citosina-5-)-Metiltransferase 1/genética , Proteína 1 Inibidora de Diferenciação/genética , Desenvolvimento Muscular/genética , Animais , Benzomorfanos , Linhagem da Célula/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Metilação de DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteína 1 Inibidora de Diferenciação/metabolismo , Camundongos , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
Reprogramming to pluripotency after overexpression of OCT4, SOX2, KLF4, and MYC is accompanied by global genomic and epigenomic changes. Histone modification and DNA methylation states in induced pluripotent stem cells (iPSCs) have been shown to be highly similar to embryonic stem cells (ESCs). However, epigenetic differences still exist between iPSCs and ESCs. In particular, aberrant DNA methylation states found in iPSCs are a major concern when using iPSCs in a clinical setting. Thus, it is critical to find factors that regulate DNA methylation states in reprogramming. Here, we found that the miR-29 family is an important epigenetic regulator during human somatic cell reprogramming. Our global DNA methylation and hydroxymethylation analysis shows that DNA demethylation is a major event mediated by miR-29a depletion during early reprogramming, and that iPSCs derived from miR-29a depletion are epigenetically closer to ESCs. Our findings uncover an important miRNA-based approach to generate clinically robust iPSCs.
Assuntos
Metilação de DNA/genética , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Pluripotentes Induzidas/citologia , MicroRNAs/genética , Reprogramação Celular/genética , Epigênese Genética/genética , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Fator 4 Semelhante a Kruppel , MicroRNAs/metabolismoRESUMO
Core pluripotency factors, such as Oct4, Sox2, and Nanog, play important roles in maintaining embryonic stem cell (ESC) identity by autoregulatory feedforward loops. Nevertheless, the mechanism that provides precise control of the levels of the ESC core factors without indefinite amplification has remained elusive. Here, we report the direct repression of core pluripotency factors by Tgif1, a previously known terminal repressor of TGFß/activin/nodal signaling. Overexpression of Tgif1 reduces the levels of ESC core factors, whereas its depletion leads to the induction of the pluripotency factors. We confirm the existence of physical associations between Tgif1 and Oct4, Nanog, and HDAC1/2 and further show the level of Tgif1 is not significantly altered by treatment with an activator/inhibitor of the TGFß/activin/nodal signaling. Collectively, our findings establish Tgif1 as an integral member of the core regulatory circuitry of mouse ESCs that counterbalances the levels of the core pluripotency factors in a TGFß/activin/nodal-independent manner.
Assuntos
Proteínas de Homeodomínio/genética , Células-Tronco Embrionárias Murinas/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Proteínas Repressoras/genética , Fatores de Transcrição SOXB1/genética , Ativinas/genética , Ativinas/metabolismo , Animais , Diferenciação Celular , Ectoderma/citologia , Ectoderma/metabolismo , Embrião de Mamíferos , Endoderma/citologia , Endoderma/metabolismo , Retroalimentação Fisiológica , Regulação da Expressão Gênica no Desenvolvimento , Histona Desacetilase 1/genética , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/genética , Histona Desacetilase 2/metabolismo , Proteínas de Homeodomínio/metabolismo , Mesoderma/citologia , Mesoderma/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
Chronic alcohol consumption may result in sustained gene expression alterations in the brain, leading to alcohol abuse or dependence. Because of ethical concerns of using live human brain cells in research, this hypothesis cannot be tested directly in live human brains. In the present study, we used human embryonic stem cell (hESC)-derived cortical neurons as in vitro cellular models to investigate alcohol-induced expression changes of genes involved in alcohol metabolism (ALDH2), anti-apoptosis (BCL2 and CCND2), neurotransmission (NMDA receptor subunit genes: GRIN1, GRIN2A, GRIN2B, and GRIN2D), calcium channel activity (ITPR2), or transcriptional repression (JARID2). hESCs were differentiated into cortical neurons, which were characterized by immunostaining using antibodies against cortical neuron-specific biomarkers. Ethanol-induced gene expression changes were determined by reverse-transcription quantitative polymerase chain reaction (RT-qPCR). After a 7-day ethanol (50 mM) exposure followed by a 24-hour ethanol withdrawal treatment, five of the above nine genes (including all four NMDA receptor subunit genes) were highly upregulated (GRIN1: 1.93-fold, P = 0.003; GRIN2A: 1.40-fold, P = 0.003; GRIN2B: 1.75-fold, P = 0.002; GRIN2D: 1.86-fold, P = 0.048; BCL2: 1.34-fold, P = 0.031), and the results of GRIN1, GRIN2A, and GRIN2B survived multiple comparison correction. Our findings suggest that alcohol responsive genes, particularly NMDA receptor genes, play an important role in regulating neuronal function and mediating chronic alcohol consumption-induced neuroadaptations.
Assuntos
Consumo de Bebidas Alcoólicas/genética , Etanol/administração & dosagem , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Neurônios/metabolismo , Consumo de Bebidas Alcoólicas/patologia , Diferenciação Celular/efeitos dos fármacos , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/patologia , Humanos , Neurônios/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacosRESUMO
Reprogramming of somatic cells produces induced pluripotent stem cells (iPSCs) that are invaluable resources for biomedical research. Here, we extended the previous transcriptome studies by performing RNA-seq on cells defined by a combination of multiple cellular surface markers. We found that transcriptome changes during early reprogramming occur independently from the opening of closed chromatin by OCT4, SOX2, KLF4, and MYC (OSKM). Furthermore, our data identify multiple spliced forms of genes uniquely expressed at each progressive stage of reprogramming. In particular, we found a pluripotency-specific spliced form of CCNE1 that is specific to human and significantly enhances reprogramming. In addition, single nucleotide polymorphism (SNP) expression analysis reveals that monoallelic gene expression is induced in the intermediate stages of reprogramming, while biallelic expression is recovered upon completion of reprogramming. Our transcriptome data provide unique opportunities in understanding human iPSC reprogramming.
Assuntos
Reprogramação Celular , Células-Tronco Pluripotentes Induzidas/metabolismo , Transcriptoma , Processamento Alternativo , Animais , Sequência de Bases , Ciclina E/genética , Ciclina E/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Dados de Sequência Molecular , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Polimorfismo de Nucleotídeo Único , Análise de Componente Principal , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA/química , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Análise de Sequência de RNARESUMO
Induced pluripotent stem cells (iPSCs) acquire embryonic stem cell (ESC)-like epigenetic states, including the X chromosome. Previous studies reported that human iPSCs retain the inactive X chromosome of parental cells, or acquire two active X chromosomes through reprogramming. Most studies investigated the X chromosome states in established human iPSC clones after completion of reprogramming. Thus, it is still not fully understood when and how the X chromosome reactivation occurs during reprogramming. Here, we report a dynamic change in the X chromosome state throughout reprogramming, with an initial robust reactivation of the inactive X chromosome followed by an inactivation upon generation of nascent iPSC clones. iPSCs with two active X chromosomes or an eroded X chromosome arise in passaging iPSCs. These data provide important insights into the plasticity of the X chromosome of human female iPSCs and will be crucial for the future application of such cells in cell therapy and X-linked disease modeling.
Assuntos
Cromossomos Humanos X/genética , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células Cultivadas , Reprogramação Celular/genética , Reprogramação Celular/fisiologia , Feminino , Humanos , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
Rett syndrome (RTT) is one of the most prevalent female mental disorders. De novo mutations in methyl CpG-binding protein 2 (MeCP2) are a major cause of RTT. MeCP2 regulates gene expression as a transcription regulator as well as through long-range chromatin interaction. Because MeCP2 is present on the X chromosome, RTT is manifested in an X-linked dominant manner. Investigation using murine MeCP2 null models and post-mortem human brain tissues has contributed to understanding the molecular and physiological function of MeCP2. In addition, RTT models using human induced pluripotent stem cells derived from RTT patients (RTT-iPSCs) provide novel resources to elucidate the regulatory mechanism of MeCP2. Previously, we obtained clones of female RTT-iPSCs that express either wild-type or mutant MECP2 due to the inactivation of one X chromosome. Reactivation of the X chromosome also allowed us to have RTT-iPSCs that express both wild-type and mutant MECP2. Using these unique pluripotent stem cells, we investigated the regulation of gene expression by MeCP2 in pluripotent stem cells by transcriptome analysis. We found that MeCP2 regulates genes encoding mitochondrial membrane proteins. In addition, loss of function in MeCP2 results in de-repression of genes on the inactive X chromosome. Furthermore, we showed that each mutation in MECP2 affects a partly different set of genes. These studies suggest that fundamental cellular physiology is affected by mutations in MECP2 from early development, and that a therapeutic approach targeting to unique forms of mutant MeCP2 is needed.
Assuntos
Regulação da Expressão Gênica , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteína 2 de Ligação a Metil-CpG/fisiologia , Transcrição Gênica , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Feminino , Ontologia Genética , Humanos , Mutação , Síndrome de Rett/genética , Síndrome de Rett/metabolismo , Síndrome de Rett/patologia , TranscriptomaRESUMO
Clinical, epidemiological, and genetic evidence suggest overlapping pathogenic mechanisms between autism spectrum disorder (ASD) and schizophrenia. We tested this hypothesis by asking if mutations in the ASD gene MECP2 which cause Rett syndrome affect the expression of genes encoding the schizophrenia risk factor dysbindin, a subunit of the biogenesis of lysosome-related organelles complex-1 (BLOC-1), and associated interacting proteins. We measured mRNA and protein levels of key components of a dysbindin interaction network by, quantitative real time PCR and quantitative immunohistochemistry in hippocampal samples of wild-type and Mecp2 mutant mice. In addition, we confirmed results by performing immunohistochemistry of normal human hippocampus and quantitative qRT-PCR of human inducible pluripotent stem cells (iPSCs)-derived human neurons from Rett syndrome patients. We defined the distribution of the BLOC-1 subunit pallidin in human and mouse hippocampus and contrasted this distribution with that of symptomatic Mecp2 mutant mice. Neurons from mutant mice and Rett syndrome patients displayed selectively reduced levels of pallidin transcript. Pallidin immunoreactivity decreased in the hippocampus of symptomatic Mecp2 mutant mice, a feature most prominent at asymmetric synapses as determined by immunoelectron microcopy. Pallidin immunoreactivity decreased concomitantly with reduced BDNF content in the hippocampus of Mecp2 mice. Similarly, BDNF content was reduced in the hippocampus of BLOC-1 deficient mice suggesting that genetic defects in BLOC-1 are upstream of the BDNF phenotype in Mecp2 deficient mice. Our results demonstrate that the ASD-related gene Mecp2 regulates the expression of components belonging to the dysbindin interactome and these molecular differences may contribute to synaptic phenotypes that characterize Mecp2 deficiencies and ASD.
Assuntos
Proteínas de Transporte/metabolismo , Regulação da Expressão Gênica , Hipocampo/citologia , Lectinas/metabolismo , Proteína 2 de Ligação a Metil-CpG/metabolismo , Neurônios/metabolismo , Sinapses/metabolismo , Animais , Proteínas de Transporte/genética , Biologia Computacional , Disbindina , Proteínas Associadas à Distrofina , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Lectinas/genética , Proteína 2 de Ligação a Metil-CpG/deficiência , Camundongos , Neurônios/citologia , Mapas de Interação de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
BACKGROUND: Supravalvular aortic stenosis (SVAS) is caused by mutations in the elastin (ELN) gene and is characterized by abnormal proliferation of vascular smooth muscle cells (SMCs) that can lead to narrowing or blockage of the ascending aorta and other arterial vessels. Having patient-specific SMCs available may facilitate the study of disease mechanisms and development of novel therapeutic interventions. METHODS AND RESULTS: Here, we report the development of a human induced pluripotent stem cell (iPSC) line from a patient with SVAS caused by the premature termination in exon 10 of the ELN gene resulting from an exon 9 four-nucleotide insertion. We showed that SVAS iPSC-derived SMCs (iPSC-SMCs) had significantly fewer organized networks of smooth muscle α-actin filament bundles, a hallmark of mature contractile SMCs, compared with control iPSC-SMCs. The addition of elastin recombinant protein or enhancement of small GTPase RhoA signaling was able to rescue the formation of smooth muscle α-actin filament bundles in SVAS iPSC-SMCs. Cell counts and BrdU analysis revealed a significantly higher proliferation rate in SVAS iPSC-SMCs than control iPSC-SMCs. Furthermore, SVAS iPSC-SMCs migrated at a markedly higher rate to the chemotactic agent platelet-derived growth factor compared with the control iPSC-SMCs. We also provided evidence that elevated activity of extracellular signal-regulated kinase 1/2 is required for hyperproliferation of SVAS iPSC-SMCs. The phenotype was confirmed in iPSC-SMCs generated from a patient with deletion of elastin owing to Williams-Beuren syndrome. CONCLUSIONS: SVAS iPSC-SMCs recapitulate key pathological features of patients with SVAS and may provide a promising strategy to study disease mechanisms and to develop novel therapies.
Assuntos
Estenose Aórtica Supravalvular/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Síndrome de Williams/patologia , Adulto , Animais , Células Cultivadas , Criança , Humanos , Masculino , CamundongosRESUMO
Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impairment in reciprocal social interaction and communication, as well as the manifestation of stereotyped behaviors. Despite much effort, ASDs are not yet fully understood. Advanced genetics and genomics technologies have recently identified novel ASD genes, and approaches using genetically engineered murine models or postmortem human brain have facilitated understanding ASD. Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) provides unprecedented opportunities in generating human disease models. Here, we present an overview of applying iPSCs in developing cellular models for understanding ASD. We also discuss future perspectives in the use of iPSCs as a source of cell therapy and as a screening platform for identifying small molecules with efficacy for alleviating ASD.