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1.
Transl Psychiatry ; 11(1): 504, 2021 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-34601489

RESUMO

Transcriptomic changes in specific brain regions can influence the risk of alcohol use disorder (AUD), but the underlying mechanism is not fully understood. We investigated AUD-associated miRNA-mRNA regulatory networks in multiple brain regions by analyzing transcriptomic changes in two sets of postmortem brain tissue samples and ethanol-exposed human embryonic stem cell (hESC)-derived cortical interneurons. miRNA and mRNA transcriptomes were profiled in 192 tissue samples (Set 1) from eight brain regions (amygdala, caudate nucleus, cerebellum, hippocampus, nucleus accumbens, prefrontal cortex, putamen, and ventral tegmental area) of 12 AUD and 12 control European Australians. Nineteen differentially expressed miRNAs (fold-change>2.0 & P < 0.05) and 97 differentially expressed mRNAs (fold-change>2.0 & P < 0.001) were identified in one or multiple brain regions of AUD subjects. AUD-associated miRNA-mRNA regulatory networks in each brain region were constructed using differentially expressed and negatively correlated miRNA-mRNA pairs. AUD-relevant pathways (including CREB Signaling, IL-8 Signaling, and Axonal Guidance Signaling) were potentially regulated by AUD-associated brain miRNA-mRNA pairs. Moreover, miRNA and mRNA transcriptomes were mapped in additional 96 tissue samples (Set 2) from six of the above eight brain regions of eight AUD and eight control European Australians. Some of the AUD-associated miRNA-mRNA regulatory networks were confirmed. In addition, miRNA and mRNA transcriptomes were analyzed in hESC-derived cortical interneurons with or without ethanol exposure, and ethanol-influenced miRNA-mRNA regulatory networks were constructed. This study provided evidence that alcohol could induce concerted miRNA and mRNA expression changes in reward-related or alcohol-responsive brain regions. We concluded that altered brain miRNA-mRNA regulatory networks might contribute to AUD development.


Assuntos
Alcoolismo , MicroRNAs , Austrália , Encéfalo , Redes Reguladoras de Genes , Humanos , MicroRNAs/genética , RNA Mensageiro
2.
Nat Biomed Eng ; 5(8): 880-896, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34426676

RESUMO

Fibroblasts can be directly reprogrammed into cardiomyocytes, endothelial cells or smooth muscle cells. Here we report the reprogramming of mouse tail-tip fibroblasts simultaneously into cells resembling these three cell types using the microRNA mimic miR-208b-3p, ascorbic acid and bone morphogenetic protein 4, as well as the formation of tissue-like structures formed by the directly reprogrammed cells. Implantation of the formed cardiovascular tissue into the infarcted hearts of mice led to the migration of reprogrammed cells to the injured tissue, reducing regional cardiac strain and improving cardiac function. The migrated endothelial cells and smooth muscle cells contributed to vessel formation, and the migrated cardiomyocytes, which initially displayed immature characteristics, became mature over time and formed gap junctions with host cardiomyocytes. Direct reprogramming of somatic cells to make cardiac tissue may aid the development of applications in cell therapy, disease modelling and drug discovery for cardiovascular diseases.


Assuntos
Células Endoteliais/transplante , Coração/fisiologia , Infarto do Miocárdio/terapia , Miócitos de Músculo Liso/transplante , Regeneração , Animais , Ácido Ascórbico/farmacologia , Proteína Morfogenética Óssea 4/farmacologia , Reprogramação Celular/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Junções Comunicantes/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Miocárdio/citologia , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Neovascularização Fisiológica , Transcriptoma
3.
Exp Neurol ; 342: 113724, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33915166

RESUMO

Temporal lobe epilepsy (TLE) is characterized by recurrent spontaneous seizures and behavioral comorbidities. Reduced hippocampal theta oscillations and hyperexcitability that contribute to cognitive deficits and spontaneous seizures are present beyond the sclerotic hippocampus in TLE. However, the mechanisms underlying compromised network oscillations and hyperexcitability observed in circuits remote from the sclerotic hippocampus are largely unknown. Cholecystokinin (CCK)-expressing basket cells (CCKBCs) critically participate in hippocampal theta rhythmogenesis, and regulate neuronal excitability. Thus, we examined whether CCKBCs were vulnerable in nonsclerotic regions of the ventral hippocampus remote from dorsal sclerotic hippocampus using the intrahippocampal kainate (IHK) mouse model of TLE, targeting unilateral dorsal hippocampus. We found a decrease in the number of CCK+ interneurons in ipsilateral ventral CA1 regions from epileptic mice compared to those from sham controls. We also found that the number of boutons from CCK+ interneurons was reduced in the stratum pyramidale, but not in other CA1 layers, of ipsilateral hippocampus in epileptic mice, suggesting that CCKBCs are vulnerable. Electrical recordings showed that synaptic connectivity and strength from surviving CCKBCs to CA1 pyramidal cells (PCs) were similar between epileptic mice and sham controls. In agreement with reduced CCKBC number in TLE, electrical recordings revealed a significant reduction in amplitude and frequency of IPSCs in CA1 PCs evoked by carbachol (commonly used to excite CCK+ interneurons) in ventral CA1 regions from epileptic mice versus sham controls. These findings suggest that loss of CCKBCs beyond the hippocampal lesion may contribute to hyperexcitability and compromised network oscillations in TLE.


Assuntos
Região CA1 Hipocampal/metabolismo , Colecistocinina/biossíntese , Epilepsia do Lobo Temporal/metabolismo , Neurônios GABAérgicos/metabolismo , Interneurônios/metabolismo , Ácido Caínico/toxicidade , Animais , Região CA1 Hipocampal/efeitos dos fármacos , Colecistocinina/genética , Modelos Animais de Doenças , Epilepsia do Lobo Temporal/induzido quimicamente , Epilepsia do Lobo Temporal/genética , Feminino , Neurônios GABAérgicos/efeitos dos fármacos , Expressão Gênica , Interneurônios/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL
4.
J Mol Med (Berl) ; 99(4): 489-500, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33651139

RESUMO

Along with emergence of the organoids, their application in biomedical research has been currently one of the most fascinating themes. For the past few years, scientists have made significant contributions to deriving organoids representing the whole brain and specific brain regions. Coupled with somatic cell reprogramming and CRISPR/Cas9 editing, the organoid technologies were applied for disease modeling and drug screening. The methods to develop organoids further improved for rapid and efficient generation of cerebral organoids. Additionally, refining the methods to develop the regionally specified brain organoids enabled the investigation of development and interaction of the specific brain regions. Recent studies started resolving the issue in the lack of non-neuroectodermal cells in brain organoids, including vascular endothelial cells and microglia, which play fundamental roles in neurodevelopment and are involved in the pathophysiology of acute and chronic neural disorders. In this review, we highlight recent advances of neuronal organoid technologies, focusing on the region-specific brain organoids and complementation with endothelial cells and microglia, and discuss their potential applications to neuronal diseases.

5.
iScience ; 24(2): 102063, 2021 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-33554067

RESUMO

Brain organoids closely recapitulate many features and characteristics of in vivo brain tissue. This technology in turn allows unprecedented possibilities to investigate brain development and function in the dish. Several brain organoid protocols have been established, and the studies have focused on validating the architecture, cellular composition, and function of the organoids. In future, the improved and advanced organoid models will enable us to understand cellular and molecular features of the developing brain. However, several obstacles, such as the quality of the organoids, 3D structural analysis, and measurement of the neural connectivity need to be improved. In this perspective, we will provide an overview of the current state of the art of the brain organoid field, with a focus on protocols and organoid characterization. Additionally, we will address the current limitations of this evolving field and provide an understanding of the current brain organoid landscape and insight toward the next steps.

6.
Semin Cell Dev Biol ; 111: 40-51, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32553582

RESUMO

Brain organoids, three-dimensional neural cultures recapitulating the spatiotemporal organization and function of the brain in a dish, offer unique opportunities for investigating the human brain development and diseases. To model distinct parts of the brain, various region-specific human brain organoids have been developed. In this article, we review current approaches to produce human region-specific brain organoids, developed through the endeavor of many researchers. We highlight the applications of human region-specific brain organoids, especially in reconstructing regional interactions in the brain through organoid fusion. We also outline the existing challenges to drive forward further the brain organoid technology and its applications for future studies.

7.
Exp Neurol ; 335: 113523, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33157092

RESUMO

Congenital hydrocephalus (CH) is caused by genetic mutations, but whether factors impacting human genetic mutations are disease-specific remains elusive. Given two factors associated with high mutation rates, we reviewed how many disease-susceptible genes match with (i) proximity to telomeres or (ii) high adenine and thymine (A + T) content in human CH as compared to other disorders of the central nervous system (CNS). We extracted genomic information using a genome data viewer. Importantly, 98 of 108 genes causing CH satisfied (i) or (ii), resulting in >90% matching rate. However, such a high accordance no longer sustained as we checked two factors in Alzheimer's disease (AD) and/or familial Parkinson's disease (fPD), resulting in 84% and 59% matching, respectively. A disease-specific matching of telomere proximity or high A + T content predicts causative genes of CH much better than neurodegenerative diseases and other CNS conditions, likely due to sufficient number of known causative genes (n = 108) and precise determination and classification of the genotype and phenotype. Our analysis suggests a need for identifying genetic basis of both factors before human clinical studies, to prioritize putative genes found in preclinical models into the likely (meeting at least one) and more likely candidate (meeting both), which predisposes human genes to mutations.


Assuntos
Cromossomos/genética , DNA/química , DNA/genética , Hidrocefalia/genética , Telômero/ultraestrutura , Adenina , Doença de Alzheimer/genética , Animais , Proteínas de Transporte/genética , Mapeamento Cromossômico , Bases de Dados Genéticas , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Mutação/genética , Defeitos do Tubo Neural/genética , Proteínas Nucleares/genética , Doença de Parkinson/genética , Timina
8.
Stem Cells ; 39(1): 43-54, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33075202

RESUMO

There is wide variability in the propensity of somatic cells to reprogram into pluripotency in response to the Yamanaka factors. How to segregate these variabilities to enrich for cells of specific traits that reprogram efficiently remains challenging. Here we report that the variability in reprogramming propensity is associated with the activity of the MKL1/SRF transcription factor and concurs with small cell size as well as rapid cell cycle. Reprogramming progressive cells can be prospectively identified by their low activity of a widely used synthetic promoter, CAG. CAGlow cells arise and expand during cell cycle acceleration in the early reprogramming culture of both mouse and human fibroblasts. Our work illustrates a molecular scenario underlying the distinct reprogramming propensities and demonstrates a convenient practical approach for their enrichment.

9.
Front Cell Neurosci ; 14: 546659, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33100972

RESUMO

In subacute and chronic phases of the stroke, there are no therapeutics available at present to promote functional recovery. Human umbilical cord-derived mesenchymal stromal cells (hUC-MSCs) are one of the candidate cell types for treating subacute-phase stroke. The benefits of cell-based therapy largely depend on the migratory capacity of products administered, as well as their potential for engraftment in targeted tissues and paracrine activities. Timing and delivery modes may also influence the outcomes of stem-cell therapy. Still, the functional recuperative effects of differing hUC-MSC delivery modes, about cell replacement and cell-to-cell paracrine activity levels, have yet to be clarified in subacute phases of stroke.This study was conducted to compare the therapeutic effects of various delivery routes when administering Good Manufacturing Practice (GMP)-grade hUC-MSCs in a rodent model of subacute-phase stroke. Cell aliquots (1 × 106) were given to rats as intravenous (IV) injections or intracerebral (IC) transplants 1 week after middle cerebral artery occlusion (MCAo). Transplanted rats were examined up to 7 weeks later using various behavioral tests and immunohistochemical analyses. Most IC-transplanted cells survived for short periods (i.e., <4 weeks after receipt) and gradually disappeared, whereas IV-injected cells were undetectable in the brain at the same time points (i.e., 3 days, 4 weeks, or 7 weeks after injection). Although short-lived, IC-transplanted cells effectively improved behavioral deficits, serving to reduce infarct volumes and glial scar formation, increase subventricular counts of proliferating neuroblasts, and promote cerebrovascular ingrowth in ischemic penumbra regions. IV injection, however, failed to improve behavioral function or histologic parameters during the same 7-week time frame. These findings overall suggest that IC transplantation is preferable to IV injection for delivery of hUC-MSCs during subacute phases of stroke.

10.
STAR Protoc ; 1(1)2020 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-33103124

RESUMO

Thalamus is a critical information relay hub in the cortex; its malfunction causes multiple neurological and psychiatric disorders. However, there are no model systems to study the development and function of human thalamus. Here, we present a protocol to generate regionally specified human brain organoids that recapitulate the development of the thalamus using human pluripotent stem cells (hPSCs). Thalamic organoids can be used to study human thalamus development, to model related diseases, and to discover potential therapeutics. For complete information on human thalamic organoids and their application, please refer to the paper by Xiang et al. (2019).

11.
Arterioscler Thromb Vasc Biol ; 40(9): 2171-2186, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32640906

RESUMO

OBJECTIVE: Cerebral cavernous malformations (CCM), consisting of dilated capillary channels formed by a single layer of endothelial cells lacking surrounding mural cells. It is unclear why CCM lesions are primarily confined to brain vasculature, although the 3 CCM-associated genes (CCM1, CCM2, and CCM3) are ubiquitously expressed in all tissues. We aimed to determine the role of CCM gene in brain mural cell in CCM pathogenesis. Approach and Results: SM22α-Cre was used to drive a specific deletion of Ccm3 in mural cells, including pericytes and smooth muscle cells (Ccm3smKO). Ccm3smKO mice developed CCM lesions in the brain with onset at neonatal stages. One-third of Ccm3smKO mice survived upto 6 weeks of age, exhibiting seizures, and severe brain hemorrhage. The early CCM lesions in Ccm3smKO neonates were loosely wrapped by mural cells, and adult Ccm3smKO mice had clustered and enlarged capillary channels (caverns) formed by a single layer of endothelium lacking mural cell coverage. Importantly, CCM lesions throughout the entire brain in Ccm3smKO mice, which more accurately mimicked human disease than the current endothelial cell-specific CCM3 deletion models. Mechanistically, CCM3 loss in brain pericytes dramatically increased paxillin stability and focal adhesion formation, enhancing ITG-ß1 (integrin ß1) activity and extracellular matrix adhesion but reducing cell migration and endothelial cell-pericyte associations. Moreover, CCM3-wild type, but not a paxillin-binding defective mutant, rescued the phenotypes in CCM3-deficient pericytes. CONCLUSIONS: Our data demonstrate for the first time that deletion of a CCM gene in the brain mural cell induces CCM pathogenesis.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Encéfalo/irrigação sanguínea , Células Endoteliais/metabolismo , Deleção de Genes , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Microvasos/metabolismo , Miócitos de Músculo Liso/metabolismo , Pericitos/metabolismo , Animais , Proteínas Reguladoras de Apoptose/deficiência , Proteínas Reguladoras de Apoptose/metabolismo , Comunicação Celular , Movimento Celular , Células Cultivadas , Técnicas de Cocultura , Células Endoteliais/patologia , Feminino , Adesões Focais/genética , Adesões Focais/metabolismo , Adesões Focais/patologia , Predisposição Genética para Doença , Hemangioma Cavernoso do Sistema Nervoso Central/metabolismo , Hemangioma Cavernoso do Sistema Nervoso Central/patologia , Humanos , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Knockout , Microvasos/anormalidades , Miócitos de Músculo Liso/patologia , Paxilina/metabolismo , Pericitos/patologia , Fenótipo , Estabilidade Proteica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais
12.
Mol Cell ; 79(1): 84-98.e9, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32526163

RESUMO

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/genética
13.
Stem Cells ; 38(8): 936-947, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32374064

RESUMO

Huntington's disease (HD) is a devastating, autosomal-dominant neurodegenerative disease, for which there are currently no disease-modifying therapies. Clinical trials to replace the damaged striatal medium spiny neurons (MSNs) have been attempted in the past two decades but have met with only limited success. In this study, we investigated whether a clonal, conditionally immortalized neural stem cell line (CTX0E03), which has already shown safety and signals of efficacy in chronic ischemic stroke patients, could rescue deficits seen in an animal model of HD. After CTX0E03 transplantation into the quinolinic acid-lesioned rat model of HD, behavioral changes were measured using the rotarod, stepping, and staircase tests. In vivo differentiation and neuronal connections of the transplanted CTX0E03 cells were evaluated with immunohistochemical staining and retrograde tracing with Fluoro-Gold. We found that transplantation of CTX0E03 gave rise to a significant behavioral improvement compared with the sham- or fibroblast-transplanted group. Transplanted CTX0E03 formed MSNs (DARPP-32) and GABAergic neurons (GABA, GAD65/67) with BDNF expression in the striatum, while cortically transplanted cells formed Tbr1-positive neurons. Using a retrograde label, we also found stable engraftment and connection of the transplanted cells with host brain tissues. CTX0E03 transplantation also reduced glial scar formation and inflammation, as well as increasing endogenous neurogenesis and angiogenesis. Overall, our results demonstrate that CTX0E03, a clinical-grade neural stem cell line, is effective for preclinical test in HD, and, therefore, will be useful for clinical development in the treatment of HD patients.

14.
Cell Rep ; 30(6): 1682-1689.e3, 2020 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-32049002

RESUMO

Human brain organoid systems offer unprecedented opportunities to investigate both neurodevelopmental and neurological disease. Single-cell-based transcriptomics or epigenomics have dissected the cellular and molecular heterogeneity in the brain organoids, revealing a complex organization. Similar but distinct protocols from different labs have been applied to generate brain organoids, providing a large resource to perform a comparative analysis of brain developmental processes. Here, we take a systematic approach to compare the single-cell transcriptomes of various human cortical brain organoids together with fetal brain to define the identity of specific cell types and differentiation routes in each method. Importantly, we identify unique developmental programs in each protocol compared to fetal brain, which will be a critical benchmark for the utility of human brain organoids in the future.


Assuntos
Encéfalo/crescimento & desenvolvimento , Organoides/crescimento & desenvolvimento , Transcriptoma/genética , Feminino , Feto , Humanos , Masculino
15.
Neuropharmacology ; 162: 107787, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31550457

RESUMO

Gamma network oscillations in the brain are fast rhythmic network oscillations in the gamma frequency range (~30-100 Hz), playing key roles in the hippocampus for learning, memory, and spatial processing. There is evidence indicating that GABAergic interneurons, including parvalbumin-expressing basket cells (PVBCs), contribute to cortical gamma oscillations through synaptic interactions with excitatory cells. However, the molecular, cellular, and circuit underpinnings underlying generation and maintenance of cortical gamma oscillations are largely elusive. Recent studies demonstrated that intrinsic and synaptic properties of GABAergic interneurons and excitatory cells are regulated by a slowly inactivating or non-inactivating sodium current (i.e., persistent sodium current, INaP), suggesting that INaP is involved in gamma oscillations. Here, we tested whether INaP plays a role in hippocampal gamma oscillations using pharmacological, optogenetic, and electrophysiological approaches. We found that INaP blockers, phenytoin (40 µM and 100 µM) and riluzole (10 µM), reduced gamma oscillations induced by optogenetic stimulation of CaMKII-expressing cells in CA1 networks. Whole-cell patch-clamp recordings further demonstrated that phenytoin (100 µM) reduced INaP and firing frequencies in both PVBCs and pyramidal cells without altering threshold and amplitude of action potentials, but increased rheobase in both cell types. These results suggest that INaP in pyramidal cells and PVBCs is required for hippocampal gamma oscillations, supporting a pyramidal-interneuron network gamma model. Phenytoin-mediated modulation of hippocampal gamma oscillations may be a mechanism underlying its anticonvulsant efficacy, as well as its contribution to cognitive impairments in epilepsy patients.


Assuntos
Região CA1 Hipocampal/fisiologia , Neurônios GABAérgicos/fisiologia , Ritmo Gama/fisiologia , Interneurônios/fisiologia , Células Piramidais/fisiologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Animais , Região CA1 Hipocampal/citologia , Região CA1 Hipocampal/efeitos dos fármacos , Região CA1 Hipocampal/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Antagonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Neurônios GABAérgicos/efeitos dos fármacos , Neurônios GABAérgicos/metabolismo , Ritmo Gama/efeitos dos fármacos , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/fisiologia , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Potenciais Pós-Sinápticos Inibidores/fisiologia , Interneurônios/efeitos dos fármacos , Interneurônios/metabolismo , Camundongos , Optogenética , Parvalbuminas/metabolismo , Técnicas de Patch-Clamp , Fenitoína/farmacologia , Células Piramidais/efeitos dos fármacos , Riluzol/farmacologia , Sódio/metabolismo
16.
Nat Methods ; 16(11): 1169-1175, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31591580

RESUMO

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/fisiologia
17.
J Cell Biol ; 218(8): 2564-2582, 2019 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-31308215

RESUMO

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 para 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 Genética , Transgenes
18.
Cell Stem Cell ; 24(3): 487-497.e7, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30799279

RESUMO

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 Biológicos
19.
Nat Commun ; 9(1): 2583, 2018 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-29968706

RESUMO

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 Ligases
20.
Tissue Eng Regen Med ; 15(1): 125, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-30603540

RESUMO

[This corrects the article DOI: 10.1007/s13770-017-0096-4.].

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