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1.
Am J Hum Genet ; 108(9): 1647-1668, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34416157

RESUMO

Interpretation of the function of non-coding risk loci for neuropsychiatric disorders and brain-relevant traits via gene expression and alternative splicing quantitative trait locus (e/sQTL) analyses is generally performed in bulk post-mortem adult tissue. However, genetic risk loci are enriched in regulatory elements active during neocortical differentiation, and regulatory effects of risk variants may be masked by heterogeneity in bulk tissue. Here, we map e/sQTLs, and allele-specific expression in cultured cells representing two major developmental stages, primary human neural progenitors (n = 85) and their sorted neuronal progeny (n = 74), identifying numerous loci not detected in either bulk developing cortical wall or adult cortex. Using colocalization and genetic imputation via transcriptome-wide association, we uncover cell-type-specific regulatory mechanisms underlying risk for brain-relevant traits that are active during neocortical differentiation. Specifically, we identified a progenitor-specific eQTL for CENPW co-localized with common variant associations for cortical surface area and educational attainment.


Assuntos
Proteínas Cromossômicas não Histona/genética , Regulação da Expressão Gênica no Desenvolvimento , Neocórtex/metabolismo , Neurogênese/genética , Neurônios/metabolismo , Locos de Características Quantitativas , Alelos , Doença de Alzheimer/diagnóstico , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Diferenciação Celular , Cromatina/química , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Mapeamento Cromossômico , Escolaridade , Feminino , Feto , Predisposição Genética para Doença , Genoma Humano , Estudo de Associação Genômica Ampla , Humanos , Masculino , Neocórtex/citologia , Neocórtex/crescimento & desenvolvimento , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurônios/citologia , Neuroticismo , Doença de Parkinson/diagnóstico , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Cultura Primária de Células , Prognóstico , Esquizofrenia/diagnóstico , Esquizofrenia/genética , Esquizofrenia/metabolismo , Transcriptoma
2.
Science ; 373(6558)2021 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-34446581

RESUMO

Organ development is orchestrated by cell- and time-specific gene regulatory networks. In this study, we investigated the regulatory basis of mouse cerebellum development from early neurogenesis to adulthood. By acquiring snATAC-seq (single-nucleus assay for transposase accessible chromatin using sequencing) profiles for ~90,000 cells spanning 11 stages, we mapped cerebellar cell types and identified candidate cis-regulatory elements (CREs). We detected extensive spatiotemporal heterogeneity among progenitor cells and a gradual divergence in the regulatory programs of cerebellar neurons during differentiation. Comparisons to vertebrate genomes and snATAC-seq profiles for ∼20,000 cerebellar cells from the marsupial opossum revealed a shared decrease in CRE conservation during development and differentiation as well as differences in constraint between cell types. Our work delineates the developmental and evolutionary dynamics of gene regulation in cerebellar cells and provides insights into mammalian organ development.


Assuntos
Evolução Biológica , Cerebelo/citologia , Cerebelo/crescimento & desenvolvimento , Neurônios/fisiologia , Elementos Reguladores de Transcrição , Animais , Cerebelo/embriologia , Cromatina/genética , Cromatina/metabolismo , DNA Intergênico , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Masculino , Camundongos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/fisiologia , Neurogênese , Gambás/genética
3.
Int J Mol Sci ; 22(15)2021 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-34361088

RESUMO

Mouse embryonic stem cells (ESCs) are useful tools for studying early embryonic development and tissue formation in mammals. Since neural lineage differentiation is a major subject of organogenesis, the development of efficient techniques to induce neural stem cells (NSCs) from pluripotent stem cells must be preceded. However, the currently available NSC differentiation methods are complicated and time consuming. This study aimed to propose an efficient method for the derivation of NSCs from mouse ESCs; early neural lineage commitment was achieved using a three-dimensional (3D) culture system, followed by a two-dimensional (2D) NSC derivation. To select early neural lineage cell types during differentiation, Sox1-GFP transgenic ESCs were used. They were differentiated into early neural lineage, forming spherical aggregates, which were subsequently picked for the establishment of 2D NSCs. The latter showed a morphology similar to that of brain-derived NSCs and expressed NSC markers, Musashi, Nestin, N-cadherin, and Sox2. Moreover, the NSCs could differentiate into three subtypes of neural lineages, neurons, astrocytes, and oligodendrocytes. The results together suggested that ESCs could efficiently differentiate into tripotent NSCs through specification in 3D culture (for approximately 10 days) followed by 2D culture (for seven days).


Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Neurais/citologia , Neurônios/citologia , Células-Tronco Pluripotentes/citologia , Animais , Diferenciação Celular , Células Cultivadas , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Nestina/metabolismo , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição SOXB1/metabolismo
4.
Nat Commun ; 12(1): 4857, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34381039

RESUMO

Physical exercise stimulates adult neurogenesis, yet the underlying mechanisms remain poorly understood. A fundamental component of the innate neuroregenerative capacity of zebrafish is the proliferative and neurogenic ability of the neural stem/progenitor cells. Here, we show that in the intact spinal cord, this plasticity response can be activated by physical exercise by demonstrating that the cholinergic neurotransmission from spinal locomotor neurons activates spinal neural stem/progenitor cells, leading to neurogenesis in the adult zebrafish. We also show that GABA acts in a non-synaptic fashion to maintain neural stem/progenitor cell quiescence in the spinal cord and that training-induced activation of neurogenesis requires a reduction of GABAA receptors. Furthermore, both pharmacological stimulation of cholinergic receptors, as well as interference with GABAergic signaling, promote functional recovery after spinal cord injury. Our findings provide a model for locomotor networks' activity-dependent neurogenesis during homeostasis and regeneration in the adult zebrafish spinal cord.


Assuntos
Locomoção , Neuroglia/metabolismo , Neurônios/metabolismo , Medula Espinal/crescimento & desenvolvimento , Animais , Interneurônios/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurogênese , Condicionamento Físico Animal , Receptores Colinérgicos/metabolismo , Receptores de GABA-A/metabolismo , Recuperação de Função Fisiológica , Medula Espinal/citologia , Medula Espinal/fisiologia , Transmissão Sináptica , Peixe-Zebra , Ácido gama-Aminobutírico/metabolismo
5.
Int J Mol Sci ; 22(14)2021 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-34299053

RESUMO

Chronic spinal cord injury (SCI) is a catastrophic condition associated with significant neurological deficit and social and financial burdens. It is currently being managed symptomatically with no real therapeutic strategies available. In recent years, a number of innovative regenerative strategies have emerged and have been continuously investigated in clinical trials. In addition, several more are coming down the translational pipeline. Among ongoing and completed trials are those reporting the use of mesenchymal stem cells, neural stem/progenitor cells, induced pluripotent stem cells, olfactory ensheathing cells, and Schwann cells. The advancements in stem cell technology, combined with the powerful neuroimaging modalities, can now accelerate the pathway of promising novel therapeutic strategies from bench to bedside. Various combinations of different molecular therapies have been combined with supportive scaffolds to facilitate favorable cell-material interactions. In this review, we summarized some of the most recent insights into the preclinical and clinical studies using stem cells and other supportive drugs to unlock the microenvironment in chronic SCI to treat patients with this condition. Successful future therapies will require these stem cells and other synergistic approaches to address the persistent barriers to regeneration, including glial scarring, loss of structural framework, and immunorejection.


Assuntos
Células-Tronco Neurais/citologia , Traumatismos da Medula Espinal/terapia , Medula Espinal/citologia , Transplante de Células-Tronco/métodos , Animais , Humanos
6.
Int J Mol Sci ; 22(14)2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-34299185

RESUMO

Nervous system development involves proliferation and cell specification of progenitor cells into neurons and glial cells. Unveiling how this complex process is orchestrated under physiological conditions and deciphering the molecular and cellular changes leading to neurological diseases is mandatory. To date, great efforts have been aimed at identifying gene mutations associated with many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Mutations in the RNA/DNA binding protein Fused in Sarcoma/Translocated in Liposarcoma (FUS/TLS) have been associated with motor neuron degeneration in rodents and humans. Furthermore, increased levels of the wild-type protein can promote neuronal cell death. Despite the well-established causal link between FUS mutations and ALS, its role in neural cells remains elusive. In order to shed new light on FUS functions we studied its role in the control of neural stem progenitor cell (NSPC) properties. Here, we report that human wild-type Fused in Sarcoma (WT FUS), exogenously expressed in mouse embryonic spinal cord-derived NSPCs, was localized in the nucleus, caused cell cycle arrest in G1 phase by affecting cell cycle regulator expression, and strongly reduced neuronal differentiation. Furthermore, the expression of the human mutant form of FUS (P525L-FUS), associated with early-onset ALS, drives the cells preferentially towards a glial lineage, strongly reducing the number of developing neurons. These results provide insight into the involvement of FUS in NSPC proliferation and differentiation into neurons and glia.


Assuntos
Mutação , Células-Tronco Neurais/citologia , Neuroglia/citologia , Neurônios/patologia , Proteína FUS de Ligação a RNA/metabolismo , Medula Espinal/citologia , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Camundongos , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Neuroglia/metabolismo , Neuroglia/patologia , Neurônios/metabolismo , Proteína FUS de Ligação a RNA/genética , Medula Espinal/embriologia , Medula Espinal/metabolismo , Medula Espinal/patologia
7.
Int J Mol Sci ; 22(14)2021 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-34299091

RESUMO

The differentiation of human pluripotent stem cells (hPSCs) to neural stem cells (NSCs) is the key initial event in neurogenesis and is thought to be dependent on the family of Wnt growth factors, their receptors and signaling proteins. The delineation of the transcriptional pathways that mediate Wnt-induced hPSCs to NSCs differentiation is vital for understanding the global genomic mechanisms of the development of NSCs and, potentially, the creation of new protocols in regenerative medicine. To understand the genomic mechanism of Wnt signaling during NSCs development, we treated hPSCs with Wnt activator (CHIR-99021) and leukemia inhibitory factor (LIF) in a chemically defined medium (N2B27) to induce NSCs, referred to as CLNSCs. The CLNSCs were subcultured for more than 40 passages in vitro; were positive for AP staining; expressed neural progenitor markers such as NESTIN, PAX6, SOX2, and SOX1; and were able to differentiate into three neural lineage cells: neurons, astrocytes, and oligodendrocytes in vitro. Our transcriptome analyses revealed that the Wnt and Hedgehog signaling pathways regulate hPSCs cell fate decisions for neural lineages and maintain the self-renewal of CLNSCs. One interesting network could be the deregulation of the Wnt/ß-catenin signaling pathway in CLNSCs via the downregulation of c-MYC, which may promote exit from pluripotency and neural differentiation. The Wnt-induced spinal markers HOXA1-4, HOXA7, HOXB1-4, and HOXC4 were increased, however, the brain markers FOXG1 and OTX2, were absent in the CLNSCs, indicating that CLNSCs have partial spinal cord properties. Finally, a CLNSC simple culture condition, when applied to hPSCs, supports the generation of NSCs, and provides a new and efficient cell model with which to untangle the mechanisms during neurogenesis.


Assuntos
Biomarcadores/análise , Células-Tronco Neurais/citologia , Neurogênese , Neurônios/citologia , Células-Tronco Pluripotentes/citologia , Transcriptoma , Via de Sinalização Wnt , Diferenciação Celular , Células Cultivadas , Humanos , Fator Inibidor de Leucemia/administração & dosagem , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo
8.
Int J Mol Sci ; 22(13)2021 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-34202161

RESUMO

The autonomic nervous system derives from the neural crest (NC) and supplies motor innervation to the smooth muscle of visceral organs, including the lower urinary tract (LUT). During fetal development, sacral NC cells colonize the urogenital sinus to form pelvic ganglia (PG) flanking the bladder neck. The coordinated activity of PG neurons is required for normal urination; however, little is known about the development of PG neuronal diversity. To discover candidate genes involved in PG neurogenesis, the transcriptome profiling of sacral NC and developing PG was performed, and we identified the enrichment of the type 3 serotonin receptor (5-HT3, encoded by Htr3a and Htr3b). We determined that Htr3a is one of the first serotonin receptor genes that is up-regulated in sacral NC progenitors and is maintained in differentiating PG neurons. In vitro cultures showed that the disruption of 5-HT3 signaling alters the differentiation outcomes of sacral NC cells, while the stimulation of 5-HT3 in explanted fetal pelvic ganglia severely diminished neurite arbor outgrowth. Overall, this study provides a valuable resource for the analysis of signaling pathways in PG development, identifies 5-HT3 as a novel regulator of NC lineage diversification and neuronal maturation in the peripheral nervous system, and indicates that the perturbation of 5-HT3 signaling in gestation has the potential to alter bladder function later in life.


Assuntos
Crista Neural/metabolismo , Receptores 5-HT3 de Serotonina/metabolismo , Transdução de Sinais , Sistema Urinário/inervação , Sistema Urinário/metabolismo , Animais , Sistema Nervoso Autônomo , Diferenciação Celular , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Camundongos , Crista Neural/embriologia , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neuritos/metabolismo , Neurogênese , Crescimento Neuronal , Neurônios/metabolismo , Receptores de Serotonina/metabolismo , Receptores 5-HT3 de Serotonina/genética , Transcriptoma , Sistema Urinário/embriologia
9.
Aging (Albany NY) ; 13(12): 16062-16071, 2021 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-34120891

RESUMO

Neural stem cells play pivotal roles during prenatal development and throughout life. Here, we report that Paired immunoglobulin-like receptor B (PirB) functions as a suppressor during brain neurogenesis in the adult mouse. PirB expression increased with age during development, and its deficiency promoted neural stem cell proliferation and differentiation in vivo and in vitro. Furthermore, we detected an increase in Type 1 neural stem cells in PirB-deficient mice compared to their wild-type littermates. PirB deficiency promoted stemness marker gene expression of Sox2 and KLF4 by activating Akt1 phosphorylation. These findings suggest that PirB inhibits the self-renewal and differentiation capacities of neural stem cells. Thus, PirB may have the potential to serve as a therapeutic target for treatment of reduced neurogenesis in adults due to aging or other pathological conditions.


Assuntos
Hipocampo/citologia , Células-Tronco Neurais/metabolismo , Receptores Imunológicos/metabolismo , Envelhecimento/metabolismo , Animais , Diferenciação Celular , Autorrenovação Celular , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/citologia , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais
10.
Nat Biomed Eng ; 5(8): 830-846, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34127820

RESUMO

The neurovascular unit, which consists of vascular cells surrounded by astrocytic end-feet and neurons, controls cerebral blood flow and the permeability of the blood-brain barrier (BBB) to maintain homeostasis in the neuronal milieu. Studying how some pathogens and drugs can penetrate the human BBB and disrupt neuronal homeostasis requires in vitro microphysiological models of the neurovascular unit. Here we show that the neurotropism of Cryptococcus neoformans-the most common pathogen causing fungal meningitis-and its ability to penetrate the BBB can be modelled by the co-culture of human neural stem cells, brain microvascular endothelial cells and brain vascular pericytes in a human-neurovascular-unit-on-a-chip maintained by a stepwise gravity-driven unidirectional flow and recapitulating the structural and functional features of the BBB. We found that the pathogen forms clusters of cells that penetrate the BBB without altering tight junctions, suggesting a transcytosis-mediated mechanism. The neurovascular-unit-on-a-chip may facilitate the study of the mechanisms of brain infection by pathogens, and the development of drugs for a range of brain diseases.


Assuntos
Barreira Hematoencefálica/metabolismo , Cryptococcus neoformans/fisiologia , Dispositivos Lab-On-A-Chip , Modelos Biológicos , Barreira Hematoencefálica/química , Barreira Hematoencefálica/microbiologia , Técnicas de Cocultura , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Matriz Extracelular/química , Humanos , Hidrogéis/química , Meningite/microbiologia , Meningite/patologia , Microvasos/citologia , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Pericitos/citologia , Pericitos/metabolismo , Transcitose
11.
Cell Prolif ; 54(8): e13082, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34152047

RESUMO

OBJECTIVES: To investigate whether human HLA-homozygous induced pluripotent stem cell (iPSC)-derived neural precursor cells (iPSC-NPCs) can provide functional benefits in Huntington's disease (HD), we transplanted them into the YAC128 transgenic HD mouse model. MATERIALS AND METHODS: CHAi001-A, an HLA-homozygous iPSC line (A*33:03-B*44:03-DRB1*13:02), was differentiated into neural precursor cells, and then, they were transplanted into 6 months-old YAC128 mice. Various behavioural and histological analyses were performed for five months after transplantation. RESULTS: Motor and cognitive functions were significantly improved in transplanted animals. Cells transplanted in the striatum showed multipotential differentiation. Five months after transplantation, the donor cells had differentiated into neurons, oligodendrocytes and astrocytes. Transplantation restored DARPP-32 expression, synaptophysin density, myelin basic protein expression in the corpus callosum and astrocyte function. CONCLUSION: Altogether, these results strongly suggest that iPSC-NPCs transplantation induces neuroprotection and functional recovery in a mouse model of HD and should be taken forward for clinical trials in HD patients.


Assuntos
Diferenciação Celular , Doença de Huntington/patologia , Células-Tronco Neurais/transplante , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Comportamento Animal , Linhagem Celular , Corpo Caloso/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Fosfoproteína 32 Regulada por cAMP e Dopamina/metabolismo , Humanos , Doença de Huntington/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Aprendizagem em Labirinto , Camundongos , Camundongos Transgênicos , Proteína Básica da Mielina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo
12.
Cell Prolif ; 54(7): e13027, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33988263

RESUMO

OBJECTIVES: The area of the subventricular zone (SVZ) in the adult brain exhibits the highest number of proliferative cells, which, together with the olfactory bulb (OB), maintains constant brain plasticity through the generation, migration and integration of newly born neurons. Despite Tau and its malfunction is increasingly related to deficits of adult hippocampal neurogenesis and brain plasticity under pathological conditions [e.g. in Alzheimer's disease (AD)], it remains unknown whether Tau plays a role in the neurogenic process of the SVZ and OB system under conditions of chronic stress, a well-known sculptor of brain and risk factor for AD. MATERIALS AND METHODS: Different types of newly born cells in SVZ and OB were analysed in animals that lack Tau gene (Tau-KO) and their wild-type littermates (WT) under control or chronic stress conditions. RESULTS: We demonstrate that chronic stress reduced the number of proliferating cells and neuroblasts in the SVZ leading to decreased number of newborn neurons in the OB of adult WT, but not Tau-KO, mice. Interestingly, while stress-evoked changes were not detected in OB granular cell layer, Tau-KO exhibited increased number of mature neurons in this layer indicating altered neuronal migration due to Tau loss. CONCLUSIONS: Our findings suggest the critical involvement of Tau in the neurogenesis suppression of SVZ and OB neurogenic niche under stressful conditions highlighting the role of Tau protein as an essential regulator of stress-driven plasticity deficits.


Assuntos
Ventrículos Laterais/metabolismo , Bulbo Olfatório/metabolismo , Estresse Fisiológico , Proteínas tau/metabolismo , Animais , Comportamento Animal , Proliferação de Células , Sobrevivência Celular , Ventrículos Laterais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurogênese , Bulbo Olfatório/patologia , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Proteínas tau/genética
13.
Biochem Biophys Res Commun ; 560: 152-158, 2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-33989907

RESUMO

Dp71 and Dp40 are the main products of the DMD gene in the central nervous system, and they are developmentally regulated from the early stages of embryonic development to adulthood. To further study the roles of Dp71 and Dp40 during cell proliferation and neural differentiation, we analyzed Dp71/Dp40 isoform expression at the mRNA level by RT-PCR assays to identify alternative splicing (AS) in the isoforms expressed in rat neural stem/progenitor cells (NSPCs) and in differentiated cells (neurons and glia). We found that proliferating NSPCs expressed Dp71d, Dp71dΔ71, Dp71f, Dp71fΔ71, Dp71dΔ74 and Dp40, as well as two Dp40 isoforms: Dp40Δ63,64 and Dp40Δ64-67. In differentiated cells we also found the expression of Dp71d, Dp71dΔ71, Dp71f, Dp71fΔ71 and Dp40. However, the expression frequencies were different in both stages. In addition, in differentiated cells, we found Dp71fΔ71-74, and interestingly, we did not find the expression of Dp71dΔ74 or the newly identified Dp40 isoforms. In this work we show that NSPC differentiation is accompanied by changes in Dp71/Dp40 isoform expression, suggesting different roles for these isoforms in NSPCs proliferation and neuronal differentiation, and we describe, for the first time, alternative splicing of Dp40.


Assuntos
Processamento Alternativo , Distrofina/genética , Células-Tronco Neurais/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Distrofina/metabolismo , Células-Tronco Neurais/citologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de RNA/metabolismo , Ratos Wistar
14.
Life Sci ; 282: 119393, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34004249

RESUMO

AIMS: During vertebrate development, the posterior end of the embryo progressively elongates in a head-to-tail direction to form the body plan. Recent lineage tracing experiments revealed that bi-potent progenitors, called neuromesodermal progenitors (NMPs), produce caudal neural and mesodermal tissues during axial elongation. However, their precise location and contribution to spinal cord development remain elusive. MAIN METHODS: Here we used NMP-specific markers (Sox2 and BraT) and a genetic lineage tracing system to localize NMP progeny in vivo. KEY FINDINGS: Sox2 and BraT double positive cells were initially located at the tail tip, but were later found in the caudal neural tube, which is a unique feature of mouse development. In the neural tube, they produced neural progenitors (NPCs) and contributed to the spinal cord gradually along the AP axis during axial elongation. Interestingly, NMP-derived NPCs preferentially contributed to the ventral side first and later to the dorsal side at the lumbar spinal cord level, which may be associated with atypical junctional neurulation in mice. SIGNIFICANCE: Our current observations detail the contribution of NMP progeny to spinal cord elongation and provide insights into how different species uniquely execute caudal morphogenesis.


Assuntos
Mesoderma/embriologia , Camundongos/embriologia , Células-Tronco Neurais/citologia , Medula Espinal/embriologia , Animais , Embrião de Mamíferos/embriologia , Feminino , Camundongos Endogâmicos C57BL
15.
Nat Commun ; 12(1): 2614, 2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-33972525

RESUMO

The differentiation of neural stem cells (NSCs) into neurons is proposed to be critical in devising potential cell-based therapeutic strategies for central nervous system (CNS) diseases, however, the determination and prediction of differentiation is complex and not yet clearly established, especially at the early stage. We hypothesize that deep learning could extract minutiae from large-scale datasets, and present a deep neural network model for predictable reliable identification of NSCs fate. Remarkably, using only bright field images without artificial labelling, our model is surprisingly effective at identifying the differentiated cell types, even as early as 1 day of culture. Moreover, our approach showcases superior precision and robustness in designed independent test scenarios involving various inducers, including neurotrophins, hormones, small molecule compounds and even nanoparticles, suggesting excellent generalizability and applicability. We anticipate that our accurate and robust deep learning-based platform for NSCs differentiation identification will accelerate the progress of NSCs applications.


Assuntos
Aprendizado Profundo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurogênese/genética , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Células Cultivadas , Simulação por Computador , Imunofluorescência , Hormônios/farmacologia , Microscopia Eletrônica de Transmissão , Nanopartículas/química , Nanopartículas/ultraestrutura , Fatores de Crescimento Neural/farmacologia , Redes Neurais de Computação , Células-Tronco Neurais/efeitos dos fármacos , Neurônios/citologia , Neurônios/metabolismo , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Ratos
16.
Nat Commun ; 12(1): 2594, 2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-33972529

RESUMO

Adult neural stem cells (NSCs) must tightly regulate quiescence and proliferation. Single-cell analysis has suggested a continuum of cell states as NSCs exit quiescence. Here we capture and characterize in vitro primed quiescent NSCs and identify LRIG1 as an important regulator. We show that BMP-4 signaling induces a dormant non-cycling quiescent state (d-qNSCs), whereas combined BMP-4/FGF-2 signaling induces a distinct primed quiescent state poised for cell cycle re-entry. Primed quiescent NSCs (p-qNSCs) are defined by high levels of LRIG1 and CD9, as well as an interferon response signature, and can efficiently engraft into the adult subventricular zone (SVZ) niche. Genetic disruption of Lrig1 in vivo within the SVZ NSCs leads an enhanced proliferation. Mechanistically, LRIG1 primes quiescent NSCs for cell cycle re-entry and EGFR responsiveness by enabling EGFR protein levels to increase but limiting signaling activation. LRIG1 is therefore an important functional regulator of NSC exit from quiescence.


Assuntos
Células-Tronco Adultas/metabolismo , Ventrículos Laterais/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/metabolismo , Neurogênese/genética , Células-Tronco Adultas/citologia , Células-Tronco Adultas/efeitos dos fármacos , Animais , Proteína Morfogenética Óssea 4/farmacologia , Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células/genética , Proteínas de Ligação a DNA/metabolismo , Receptores ErbB/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Ontologia Genética , Imuno-Histoquímica , Interferons/farmacologia , Ventrículos Laterais/citologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Glicoproteínas de Membrana/genética , Camundongos , Proteínas do Tecido Nervoso/genética , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Proteômica , RNA-Seq , Regeneração/efeitos dos fármacos , Tetraspanina 29/metabolismo , Regulação para Cima
17.
J Vis Exp ; (170)2021 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-33938879

RESUMO

Physiological electric fields (EF) play vital roles in cell migration, differentiation, division, and death. This paper describes a microfluidic cell culture system that was used for a long-term cell differentiation study using microscopy. The microfluidic system consists of the following major components: an optically transparent electrotactic chip, a transparent indium-tin-oxide (ITO) heater, a culture media-filling pump, an electrical power supply, a high-frequency power amplifier, an EF multiplexer, a programmable X-Y-Z motorized stage, and an inverted phase-contrast microscope equipped with a digital camera. The microfluidic system is beneficial in simplifying the overall experimental setup and, in turn, the reagent and sample consumption. This work involves the differentiation of neural stem and progenitor cells (NPCs) induced by direct current (DC) pulse stimulation. In the stem cell maintenance medium, the mouse NPCs (mNPCs) differentiated into neurons, astrocytes, and oligodendrocytes after the DC pulse stimulation. The results suggest that simple DC pulse treatment could control the fate of mNPCs and could be used to develop therapeutic strategies for nervous system disorders. The system can be used for cell culture in multiple channels, for long-term EF stimulation, for cell morphological observation, and for automatic time-lapse image acquisition. This microfluidic system not only shortens the required experimental time, but also increases the accuracy of control on the microenvironment.


Assuntos
Diferenciação Celular , Animais , Astrócitos/citologia , Técnicas de Cultura de Células , Eletricidade , Dispositivos Lab-On-A-Chip , Camundongos , Células-Tronco Neurais/citologia , Neurônios/citologia , Oligodendroglia/citologia
18.
J Med Chem ; 64(9): 6070-6084, 2021 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-33945688

RESUMO

A small library of phorbol 12,13-diesters bearing low lipophilicity ester chains was prepared as potential neurogenic agents in the adult brain. They were also used in a targeted UHPLC-HRMS screening of the latex of Euphorbia resinifera. Two new 12-deoxy-16-hydroxyphorbol 13,16-diesters were isolated, and their structures were deduced using two-dimensional NMR spectroscopy and NOE experiments. The ability of natural and synthetic compounds to stimulate transforming growth factor alpha (TFGα) release, to increase neural progenitor cell proliferation, and to stimulate neurogenesis was evaluated. All compounds that facilitated TGFα release promoted neural progenitor cell proliferation. The presence of two acyloxy moieties on the tigliane skeleton led to higher levels of activity, which decreased when a free hydroxyl group was at C-12. Remarkably, the compound bearing isobutyryloxy groups was the most potent on the TGFα assay and at inducing neural progenitor cell proliferation in vitro, also leading to enhanced neurogenesis in vivo when administered intranasally to mice.


Assuntos
Neurogênese/efeitos dos fármacos , Ésteres de Forbol/química , Ésteres de Forbol/farmacologia , Fator de Crescimento Transformador alfa/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Camundongos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos
19.
Cell Prolif ; 54(6): e13042, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33955094

RESUMO

OBJECTIVES: The effects of general anaesthetics on fetal brain development remain elusive. Radial glial progenitors (RGPs) generate the majority of neurons in developing brains. Here, we evaluated the acute alterations in RGPs after maternal sevoflurane exposure. METHODS: Pregnant mice were exposed to 2.5% sevoflurane for 6 hours on gestational day 14.5. Interkinetic nuclear migration (INM) of RGPs in the ventricular zone (VZ) of the fetal brain was evaluated by thymidine analogues labelling. Cell fate of RGP progeny was determined by immunostaining using various neural markers. The Morris water maze (MWM) was used to assess the neurocognitive behaviours of the offspring. RNA sequencing (RNA-Seq) was performed for the potential mechanism, and the potential mechanism validated by quantitative real-time PCR (qPCR), Western blot and rescue experiments. Furthermore, INM was examined in human embryonic stem cell (hESC)-derived 3D cerebral organoids. RESULTS: Maternal sevoflurane exposure induced temporary abnormities in INM, and disturbed the cell cycle progression of RGPs in both rodents and cerebral organoids without cell fate alternation. RNA-Seq analysis, qPCR and Western blot showed that the Notch signalling pathway was a potential downstream target. Reactivation of Notch by Jag1 and NICD overexpression rescued the defects in INM. Young adult offspring showed no obvious cognitive impairments in MWM. CONCLUSIONS: Maternal sevoflurane exposure during neurogenic period temporarily induced abnormal INM of RGPs by targeting the Notch signalling pathway without inducing long-term effects on RGP progeny cell fate or offspring cognitive behaviours. More importantly, the defects of INM in hESC-derived cerebral organoids provide a novel insight into the effects of general anaesthesia on human brain development.


Assuntos
Anestésicos Inalatórios/efeitos adversos , Córtex Cerebral/efeitos dos fármacos , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Receptores Notch/metabolismo , Sevoflurano/efeitos adversos , Animais , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Córtex Cerebral/patologia , Feminino , Feto/efeitos dos fármacos , Feto/metabolismo , Feto/patologia , Humanos , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/patologia , Neurogênese/efeitos dos fármacos , Neuroglia/citologia , Neuroglia/efeitos dos fármacos , Neuroglia/patologia , Gravidez , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Efeitos Tardios da Exposição Pré-Natal/patologia , Transdução de Sinais/efeitos dos fármacos
20.
Int J Mol Sci ; 22(9)2021 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-33946340

RESUMO

During brain development, the genome must be repeatedly reconfigured in order to facilitate neuronal and glial differentiation. A host of chromatin remodeling complexes facilitates this process. At the genetic level, the non-redundancy of these complexes suggests that neurodevelopment may require a lexicon of remodelers with different specificities and activities. Here, we focus on the nucleosome remodeling and deacetylase (NuRD) complex. We review NuRD biochemistry, genetics, and functions in neural progenitors and neurons.


Assuntos
Encéfalo/crescimento & desenvolvimento , Montagem e Desmontagem da Cromatina , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Animais , Encéfalo/embriologia , Encéfalo/metabolismo , Humanos , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurogênese , Neurônios/citologia , Neurônios/metabolismo
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