RESUMO
The mammalian liver possesses a remarkable regenerative ability. Two modes of damage response have been described: (1) The "oval cell" response emanates from the biliary tree when all hepatocytes are affected by chronic liver disease. (2) A massive, proliferative response of mature hepatocytes occurs upon acute liver damage such as partial hepatectomy (PHx). While the oval cell response has been captured in vitro by growing organoids from cholangiocytes, the hepatocyte proliferative response has not been recapitulated in culture. Here, we describe the establishment of a long-term 3D organoid culture system for mouse and human primary hepatocytes. Organoids can be established from single hepatocytes and grown for multiple months, while retaining key morphological, functional and gene expression features. Transcriptional profiles of the organoids resemble those of proliferating hepatocytes after PHx. Human hepatocyte organoids proliferate extensively after engraftment into mice and thus recapitulate the proliferative damage-response of hepatocytes.
Assuntos
Proliferação de Células , Hepatócitos/metabolismo , Organoides/metabolismo , Animais , Técnicas de Cultura de Células , Células Cultivadas , Hepatócitos/citologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Organoides/citologia , Células-Tronco/citologia , Células-Tronco/metabolismo , Fatores de TempoRESUMO
Tissue-resident memory T cells (TRM cells) in the airways mediate protection against respiratory infection. We characterized TRM cells expressing integrin αE (CD103) that reside within the epithelial barrier of human lungs. These cells had specialized profiles of chemokine receptors and adhesion molecules, consistent with their unique localization. Lung TRM cells were poised for rapid responsiveness by constitutive expression of deployment-ready mRNA encoding effector molecules, but they also expressed many inhibitory regulators, suggestive of programmed restraint. A distinct set of transcription factors was active in CD103+ TRM cells, including Notch. Genetic and pharmacological experiments with mice revealed that Notch activity was required for the maintenance of CD103+ TRM cells. We have thus identified specialized programs underlying the residence, persistence, vigilance and tight control of human lung TRM cells.
Assuntos
Linfócitos T CD8-Positivos/fisiologia , Memória Imunológica , Vírus da Influenza A Subtipo H3N2/imunologia , Pulmão/imunologia , Infecções por Orthomyxoviridae/imunologia , Receptor Notch1/metabolismo , Receptor Notch2/metabolismo , Infecções Respiratórias/imunologia , Animais , Antígenos CD/metabolismo , Células Cultivadas , Feminino , Humanos , Cadeias alfa de Integrinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pessoa de Meia-Idade , Receptor Notch1/genética , Receptor Notch2/genéticaRESUMO
Proliferation of the self-renewing epithelium of the gastric corpus occurs almost exclusively in the isthmus of the glands, from where cells migrate bidirectionally toward pit and base. The isthmus is therefore generally viewed as the stem cell zone. We find that the stem cell marker Troy is expressed at the gland base by a small subpopulation of fully differentiated chief cells. By lineage tracing with a Troy-eGFP-ires-CreERT2 allele, single marked chief cells are shown to generate entirely labeled gastric units over periods of months. This phenomenon accelerates upon tissue damage. Troy(+) chief cells can be cultured to generate long-lived gastric organoids. Troy marks a specific subset of chief cells that display plasticity in that they are capable of replenishing entire gastric units, essentially serving as quiescent "reserve" stem cells. These observations challenge the notion that stem cell hierarchies represent a "one-way street."
Assuntos
Celulas Principais Gástricas/citologia , Células-Tronco/citologia , Estômago/citologia , Animais , Linhagem da Célula , Celulas Principais Gástricas/química , Mucosa Gástrica/citologia , Camundongos , Organoides/citologia , Receptores do Fator de Necrose Tumoral/análise , Via de Sinalização WntRESUMO
Astrocytes are highly abundant in the mammalian brain, and their functions are of vital importance for all aspects of development, adaption, and aging of the central nervous system (CNS). Mounting evidence indicates the important contributions of astrocytes to a wide range of neuropathies. Still, our understanding of astrocyte development significantly lags behind that of other CNS cells. We here combine immunohistochemical approaches with genetic fate-mapping, behavioural paradigms, single-cell transcriptomics, and in vivo two-photon imaging, to comprehensively assess the generation and the proliferation of astrocytes in the dentate gyrus (DG) across the life span of a mouse. Astrogenesis in the DG is initiated by radial glia-like neural stem cells giving rise to locally dividing astrocytes that enlarge the astrocyte compartment in an outside-in-pattern. Also in the adult DG, the vast majority of astrogenesis is mediated through the proliferation of local astrocytes. Interestingly, locally dividing astrocytes were able to adapt their proliferation to environmental and behavioral stimuli revealing an unexpected plasticity. Our study establishes astrocytes as enduring plastic elements in DG circuits, implicating a vital contribution of astrocyte dynamics to hippocampal plasticity.
Assuntos
Células-Tronco Neurais , Neurogênese , Animais , Astrócitos/fisiologia , Giro Denteado , Hipocampo/fisiologia , Mamíferos , Camundongos , Células-Tronco Neurais/fisiologia , Neurogênese/fisiologiaRESUMO
The complex structure and function of the human central nervous system that develops from the neural tube made in vitro modeling quite challenging until the discovery of brain organoids. Human-induced pluripotent stem cells-derived brain organoids offer recapitulation of the features of early human neurodevelopment in vitro, including the generation, proliferation, and differentiation into mature neurons and micro-macroglial cells, as well as the complex interactions among these diverse cell types of the developing brain. Recent advancements in brain organoids, microfluidic systems, real-time sensing technologies, and their cutting-edge integrated use provide excellent models and tools for emulation of fundamental neurodevelopmental processes, the pathology of neurological disorders, personalized transplantation therapy, and high-throughput neurotoxicity testing by bridging the gap between two-dimensional models and the complex three-dimensional environment in vivo. In this review, we summarize how bioengineering approaches are applied to mitigate the limitations of brain organoids for biomedical and clinical research. We further provide an extensive overview and future perspectives of the humanized brain organoids-on-chip platforms with integrated sensors toward brain organoid intelligence and biocomputing studies. Such approaches might pave the way for increasing approvable clinical applications by solving their current limitations.
Assuntos
Células-Tronco Pluripotentes Induzidas , Doenças do Sistema Nervoso , Humanos , Encéfalo , Neurônios , Células-Tronco Pluripotentes Induzidas/metabolismo , OrganoidesRESUMO
Mammalian Wnt proteins are believed to act as short-range signals, yet have not been previously visualized in vivo. Self-renewal, proliferation and differentiation are coordinated along a putative Wnt gradient in the intestinal crypt. Wnt3 is produced specifically by Paneth cells. Here we have generated an epitope-tagged, functional Wnt3 knock-in allele. Wnt3 covers basolateral membranes of neighbouring stem cells. In intestinal organoids, Wnt3-transfer involves direct contact between Paneth cells and stem cells. Plasma membrane localization requires surface expression of Frizzled receptors, which in turn is regulated by the transmembrane E3 ligases Rnf43/Znrf3 and their antagonists Lgr4-5/R-spondin. By manipulating Wnt3 secretion and by arresting stem-cell proliferation, we demonstrate that Wnt3 mainly travels away from its source in a cell-bound manner through cell division, and not through diffusion. We conclude that stem-cell membranes constitute a reservoir for Wnt proteins, while Frizzled receptor turnover and 'plasma membrane dilution' through cell division shape the epithelial Wnt3 gradient.
Assuntos
Membrana Celular/metabolismo , Mucosa Intestinal/citologia , Nicho de Células-Tronco , Células-Tronco/citologia , Células-Tronco/metabolismo , Via de Sinalização Wnt , Proteína Wnt3/metabolismo , Alelos , Animais , Adesão Celular , Divisão Celular , Difusão , Feminino , Receptores Frizzled/metabolismo , Técnicas de Introdução de Genes , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Camundongos , Organoides/citologia , Organoides/metabolismo , Celulas de Paneth/citologia , Celulas de Paneth/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteína Wnt3/genéticaRESUMO
Mammalian epidermal stem cells maintain homeostasis of the skin epidermis and contribute to its regeneration throughout adult life. While 2D mouse epidermal stem cell cultures have been established decades ago, a long-term, feeder cell- and serum-free culture system recapitulating murine epidermal architecture has not been available. Here we describe an epidermal organoid culture system that allows long-term, genetically stable expansion of adult epidermal stem cells. Our epidermal expansion media combines atypically high calcium concentrations, activation of cAMP, FGF, and R-spondin signaling with inhibition of bone morphogenetic protein (BMP) signaling. Organoids are established robustly from adult mouse skin and expand over at least 6 mo, while maintaining the basal-apical organization of the mouse interfollicular epidermis. The system represents a powerful tool to study epidermal homeostasis and disease in vitro.
Assuntos
Células-Tronco Adultas/fisiologia , Técnicas de Cultura de Células/métodos , Epiderme/fisiologia , Queratinócitos/fisiologia , Organoides/fisiologia , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Técnicas de Introdução de Genes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores Acoplados a Proteínas G/genética , Fatores de TempoRESUMO
Understanding the development and function of an organ requires the characterization of all of its cell types. Traditional methods for visualizing and isolating subpopulations of cells are based on messenger RNA or protein expression of only a few known marker genes. The unequivocal identification of a specific marker gene, however, poses a major challenge, particularly if this cell type is rare. Identifying rare cell types, such as stem cells, short-lived progenitors, cancer stem cells, or circulating tumour cells, is crucial to acquire a better understanding of normal or diseased tissue biology. To address this challenge we first sequenced the transcriptome of hundreds of randomly selected cells from mouse intestinal organoids, cultured self-organizing epithelial structures that contain all cell lineages of the mammalian intestine. Organoid buds, like intestinal crypts, harbour stem cells that continuously differentiate into a variety of cell types, occurring at widely different abundances. Since available computational methods can only resolve more abundant cell types, we developed RaceID, an algorithm for rare cell type identification in complex populations of single cells. We demonstrate that this algorithm can resolve cell types represented by only a single cell in a population of randomly sampled organoid cells. We use this algorithm to identify Reg4 as a novel marker for enteroendocrine cells, a rare population of hormone-producing intestinal cells. Next, we use Reg4 expression to enrich for these rare cells and investigate the heterogeneity within this population. RaceID confirmed the existence of known enteroendocrine lineages, and moreover discovered novel subtypes, which we subsequently validated in vivo. Having validated RaceID we then applied the algorithm to ex vivo-isolated Lgr5-positive stem cells and their direct progeny. We find that Lgr5-positive cells represent a homogenous abundant population of stem cells mixed with a rare population of Lgr5-positive secretory cells. We envision broad applicability of our method for discovering rare cell types and the corresponding marker genes in healthy and diseased organs.
Assuntos
Separação Celular/métodos , Intestino Delgado/citologia , RNA Mensageiro/genética , Análise de Sequência de RNA , Análise de Célula Única , Algoritmos , Animais , Biomarcadores/análise , Diferenciação Celular/genética , Linhagem da Célula , Hibridização in Situ Fluorescente , Camundongos , Proteínas de Neoplasias/genética , Organoides/citologia , Proteínas Associadas a Pancreatite , Celulas de Paneth/citologia , Celulas de Paneth/metabolismo , Receptores Acoplados a Proteínas G/genética , Reprodutibilidade dos Testes , Células-Tronco/citologia , Células-Tronco/metabolismo , Transcriptoma/genéticaRESUMO
The adult mouse subependymal zone provides a niche for mammalian neural stem cells (NSCs). However, the molecular signature, self-renewal potential, and fate behavior of NSCs remain poorly defined. Here we propose a model in which the fate of active NSCs is coupled to the total number of neighboring NSCs in a shared niche. Using knock-in reporter alleles and single-cell RNA sequencing, we show that the Wnt target Tnfrsf19/Troy identifies both active and quiescent NSCs. Quantitative analysis of genetic lineage tracing of individual NSCs under homeostasis or in response to injury reveals rapid expansion of stem-cell number before some return to quiescence. This behavior is best explained by stochastic fate decisions, where stem-cell number within a shared niche fluctuates over time. Fate mapping proliferating cells using a Ki67iresCreER allele confirms that active NSCs reversibly return to quiescence, achieving long-term self-renewal. Our findings suggest a niche-based mechanism for the regulation of NSC fate and number.
Assuntos
Ventrículos Laterais/citologia , Células-Tronco Neurais/fisiologia , Nicho de Células-Tronco , Animais , Linhagem da Célula , Proliferação de Células , Camundongos , Neurogênese , Receptores do Fator de Necrose Tumoral/metabolismo , Análise de Célula Única , TranscriptomaRESUMO
The significance of cardiac stem cell (CSC) populations for cardiac regeneration remains disputed. Here, we apply the most direct definition of stem cell function (the ability to replace lost tissue through cell division) to interrogate the existence of CSCs. By single-cell mRNA sequencing and genetic lineage tracing using two Ki67 knockin mouse models, we map all proliferating cells and their progeny in homoeostatic and regenerating murine hearts. Cycling cardiomyocytes were only robustly observed in the early postnatal growth phase, while cycling cells in homoeostatic and damaged adult myocardium represented various noncardiomyocyte cell types. Proliferative postdamage fibroblasts expressing follistatin-like protein 1 (FSTL1) closely resemble neonatal cardiac fibroblasts and form the fibrotic scar. Genetic deletion of Fstl1 in cardiac fibroblasts results in postdamage cardiac rupture. We find no evidence for the existence of a quiescent CSC population, for transdifferentiation of other cell types toward cardiomyocytes, or for proliferation of significant numbers of cardiomyocytes in response to cardiac injury.
Assuntos
Proliferação de Células , Traumatismos Cardíacos/fisiopatologia , Animais , Células Cultivadas , Modelos Animais de Doenças , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Proteínas Relacionadas à Folistatina/genética , Proteínas Relacionadas à Folistatina/metabolismo , Traumatismos Cardíacos/genética , Traumatismos Cardíacos/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocárdio/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Gravidez , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
Cycling Lgr5+ stem cells fuel the rapid turnover of the adult intestinal epithelium. The existence of quiescent Lgr5+ cells has been reported, while an alternative quiescent stem cell population is believed to reside at crypt position +4. Here, we generated a novel Ki67RFP knock-in allele that identifies dividing cells. Using Lgr5-GFP;Ki67RFP mice, we isolated crypt stem and progenitor cells with distinct Wnt signaling levels and cell cycle features and generated their molecular signature using microarrays. Stem cell potential of these populations was further characterized using the intestinal organoid culture. We found that Lgr5high stem cells are continuously in cell cycle, while a fraction of Lgr5low progenitors that reside predominantly at +4 position exit the cell cycle. Unlike fast dividing CBCs, Lgr5low Ki67- cells have lost their ability to initiate organoid cultures, are enriched in secretory differentiation factors, and resemble the Dll1 secretory precursors and the label-retaining cells of Winton and colleagues. Our findings support the cycling stem cell hypothesis and highlight the cell cycle heterogeneity of early progenitors during lineage commitment.
Assuntos
Diferenciação Celular , Perfilação da Expressão Gênica , Genes Reporter , Receptores Acoplados a Proteínas G/análise , Células-Tronco/fisiologia , Animais , Divisão Celular , Técnicas de Introdução de Genes , Mucosa Intestinal/citologia , Mucosa Intestinal/fisiologia , Antígeno Ki-67/biossíntese , Antígeno Ki-67/genética , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Camundongos , Análise em Microsséries , Células-Tronco/química , Via de Sinalização WntRESUMO
BACKGROUND: Perturbations of epidermal and follicular homeostasis have been attributed to a variety of skin diseases affecting dogs. The availability of an in vitro system to investigate these diseases is important to understand underlying pathomechanisms. OBJECTIVES: To establish an accurate and reliable in vitro 3D system of canine keratinocyte organoids to lay the basis for studying functional defects in interfollicular epidermis (IFE) and hair follicle (HF) morphogenesis, reconstitution and differentiation that lead to alopecic and epidermal diseases. ANIMALS: Skin biopsies were obtained from freshly euthanized dogs of different breeds with no skin abnormalities. METHODS: Cells derived from microdissected IFE and HFs were seeded in Matrigel and keratinocyte organoids were grown and characterized using immunohistochemistry, RT-qPCR and RNA sequencing. RESULTS: Both organoid lines develop into a basal IFE-like cell type. Gene and protein expression analysis revealed high mRNA and protein levels of keratins 5 and 14, IFE differentiation markers and intercellular molecules. Key markers of HF stem cells were lacking. Withdrawal of growth factors resulted in upregulation of markers such as KRT16, Involucrin, KRT17 and SOX9, showing the potential of the organoids to develop towards more differentiated tissue. CONCLUSION AND CLINICAL IMPORTANCE: Our 3D in vitro culture system provides the basis to explore epidermal function, to investigate the culture conditions necessary for the development of organoids with a HF signature and to address cutaneous disorders in dogs. However, for induction of HF signatures or hair growth, addition of different growth factors or co-culture with dermal papilla will be required.
Assuntos
Cães/anatomia & histologia , Queratinócitos/citologia , Técnicas de Cultura de Órgãos/veterinária , Animais , Biópsia/veterinária , Células Cultivadas/citologia , Células Epidérmicas , Queratinócitos/patologia , Técnicas de Cultura de Órgãos/métodos , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Análise de Sequência de RNA/veterinária , Pele/citologia , Pele/patologia , Dermatopatias/patologia , Dermatopatias/veterináriaRESUMO
Neural stem cells (NSCs) in the ventricular domain of the subventricular zone (V-SVZ) of rodents produce neurons throughout life while those in humans become largely inactive or may be lost during infancy. Most adult NSCs are quiescent, express glial markers, and depend on Notch signaling for their self-renewal and the generation of neurons. Using genetic markers and lineage tracing, we identified subpopulations of adult V-SVZ NSCs (type 1, 2, and 3) indicating a striking heterogeneity including activated, brain lipid binding protein (BLBP, FABP7) expressing stem cells. BLBP(+) NSCs are mitotically active components of pinwheel structures in the lateral ventricle walls and persistently generate neurons in adulthood. BLBP(+) NSCs express epidermal growth factor (EGF) receptor, proliferate in response to EGF, and are a major clonogenic population in the SVZ. We also find BLBP expressed by proliferative ventricular and subventricular progenitors in the fetal and postnatal human brain. Loss of BLBP(+) stem/progenitor cells correlates with reduced neurogenesis in aging rodents and postnatal humans. These findings of molecular heterogeneity and proliferative differences subdivide the NSC population and have implications for neurogenesis in the forebrain of mammals during aging.
Assuntos
Células-Tronco Neurais/citologia , Neurônios/citologia , Prosencéfalo/citologia , Animais , Processos de Crescimento Celular/fisiologia , Humanos , Camundongos , Camundongos Transgênicos , Células-Tronco Neurais/metabolismo , Neurogênese , Neurônios/metabolismo , Prosencéfalo/metabolismo , Transdução de SinaisRESUMO
Glial fibrillary acidic protein (GFAP) is an intermediate filament (IF) protein expressed in specific types of glial cells in the nervous system. The expression of GFAP is highly regulated during brain development and in neurological diseases. The presence of distinct GFAP-isoforms in various cell types, developmental stages, and diseases indicates that GFAP (post-)transcriptional regulation has a role in glial cell physiology and pathology. GFAP-isoforms differ in sub-cellular localisation, IF-network assembly properties, and IF-dynamics which results in distinct molecular interactions and mechanical properties of the IF-network. Therefore, GFAP (post-)transcriptional regulation is likely a mechanism by which radial glia, astrocytes, and glioma cells can modulate cellular function.
Assuntos
Astrócitos , Filamentos Intermediários , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Filamentos Intermediários/metabolismo , Astrócitos/metabolismo , Astrócitos/patologia , Isoformas de Proteínas/genética , Regulação da Expressão GênicaRESUMO
The adult mammalian forebrain contains neural stem/progenitor cells (NSCs) that generate neurons throughout life. As in other somatic stem cell systems, NSCs are proposed to be predominantly quiescent and proliferate only sporadically to produce more committed progeny. However, quiescence has recently been shown not to be an essential criterion for stem cells. It is not known whether NSCs show differences in molecular dependence based on their proliferation state. The subventricular zone (SVZ) of the adult mouse brain has a remarkable capacity for repair by activation of NSCs. The molecular interplay controlling adult NSCs during neurogenesis or regeneration is not clear but resolving these interactions is critical in order to understand brain homeostasis and repair. Using conditional genetics and fate mapping, we show that Notch signaling is essential for neurogenesis in the SVZ. By mosaic analysis, we uncovered a surprising difference in Notch dependence between active neurogenic and regenerative NSCs. While both active and regenerative NSCs depend upon canonical Notch signaling, Notch1-deletion results in a selective loss of active NSCs (aNSCs). In sharp contrast, quiescent NSCs (qNSCs) remain after Notch1 ablation until induced during regeneration or aging, whereupon they become Notch1-dependent and fail to fully reinstate neurogenesis. Our results suggest that Notch1 is a key component of the adult SVZ niche, promoting maintenance of aNSCs, and that this function is compensated in qNSCs. Therefore, we confirm the importance of Notch signaling for maintaining NSCs and neurogenesis in the adult SVZ and reveal that NSCs display a selective reliance on Notch1 that may be dictated by mitotic state.
Assuntos
Células-Tronco Adultas/fisiologia , Ventrículos Laterais/citologia , Neurogênese/fisiologia , Receptor Notch1/metabolismo , Nicho de Células-Tronco/fisiologia , Células-Tronco Adultas/efeitos dos fármacos , Animais , Proteínas de Transporte/genética , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Diferenciação Celular/genética , Citarabina/farmacologia , Antagonistas de Estrogênios/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Imunossupressores/farmacologia , Proteínas de Filamentos Intermediários/genética , Ventrículos Laterais/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Biológicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Nestina , Neurogênese/efeitos dos fármacos , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas/genética , Proteínas/metabolismo , RNA não Traduzido , Receptor Notch1/deficiência , Receptores de Estrogênio/genética , Nicho de Células-Tronco/genética , Tamoxifeno/farmacologia , Fatores de TempoRESUMO
Following the decline of neurogenesis at birth, progenitors of the subventricular zone (SVZ) remain mostly in a quiescent state in the adult human brain. The mechanisms that regulate this quiescent state are still unclear. Here, we isolate CD271+ progenitors from the aged human SVZ for single-cell RNA sequencing analysis. Our transcriptome data reveal the identity of progenitors of the aged human SVZ as late oligodendrocyte progenitor cells. We identify the Wnt pathway antagonist SFRP1 as a possible signal that promotes quiescence of progenitors from the aged human SVZ. Administration of WAY-316606, a small molecule that inhibits SFRP1 function, stimulates activation of neural stem cells both in vitro and in vivo under homeostatic conditions. Our data unravel a possible mechanism through which progenitors of the adult human SVZ are maintained in a quiescent state and a potential target for stimulating progenitors to re-activate.
Assuntos
Ventrículos Laterais , Células-Tronco Neurais , Idoso , Encéfalo/metabolismo , Diferenciação Celular/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Ventrículos Laterais/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Células-Tronco Neurais/metabolismo , Neurogênese/genética , TranscriptomaRESUMO
Spinal cord ependymal cells display neural stem cell properties in vitro and generate scar-forming astrocytes and remyelinating oligodendrocytes after injury. We report that ependymal cells are functionally heterogeneous and identify a small subpopulation (8% of ependymal cells and 0.1% of all cells in a spinal cord segment), which we denote ependymal A (EpA) cells, that accounts for the in vitro stem cell potential in the adult spinal cord. After spinal cord injury, EpA cells undergo self-renewing cell division as they give rise to differentiated progeny. Single-cell transcriptome analysis revealed a loss of ependymal cell gene expression programs as EpA cells gained signaling entropy and dedifferentiated to a stem-cell-like transcriptional state after an injury. We conclude that EpA cells are highly differentiated cells that can revert to a stem cell state and constitute a therapeutic target for spinal cord repair.
Assuntos
Células-Tronco Neurais , Traumatismos da Medula Espinal , Diferenciação Celular/fisiologia , Humanos , Células-Tronco Neurais/metabolismo , Neuroglia , Medula Espinal/metabolismo , Traumatismos da Medula Espinal/metabolismoRESUMO
The habenula plays a key role in various motivated and pathological behaviors and is composed of molecularly distinct neuron subtypes. Despite progress in identifying mature habenula neuron subtypes, how these subtypes develop and organize into functional brain circuits remains largely unknown. Here, we performed single-cell transcriptional profiling of mouse habenular neurons at critical developmental stages, instructed by detailed three-dimensional anatomical data. Our data reveal cellular and molecular trajectories during embryonic and postnatal development, leading to different habenular subtypes. Further, based on this analysis, our work establishes the distinctive functional properties and projection target of a subtype of Cartpt+ habenula neurons. Finally, we show how comparison of single-cell transcriptional profiles and GWAS data links specific developing habenular subtypes to psychiatric disease. Together, our study begins to dissect the mechanisms underlying habenula neuron subtype-specific development and creates a framework for further interrogation of habenular development in normal and disease states.
Assuntos
Habenula , Animais , Habenula/fisiologia , Camundongos , Neurogênese/genética , NeurôniosRESUMO
The development of the hippocampal dentate gyrus is a complex process in which several signaling pathways are involved and likely interact with each other. The extracellular matrix molecule Reelin is necessary both for normal development of the dentate gyrus radial glia and neuronal migration. In Reelin-deficient Reeler mice, the hippocampal radial glial scaffold fails to form, and granule cells are dispersed throughout the dentate gyrus. Here, we show that both formation of the radial glia scaffold and lamination of the dentate gyrus depend on intact Notch signaling. Inhibition of Notch signaling in organotypic hippocampal slice cultures induced a phenotype reminiscent of the Reelin-deficient hippocampus, i.e., a reduced density of radial glia fibers and granule cell dispersion. Moreover, a Reelin-dependent rescue of the Reeler phenotype was blocked by inhibition of Notch activation. In the Reeler dentate gyrus, we found reduced Notch1 signaling; the activated Notch intracellular domain as well as the transcriptional targets, brain lipid-binding protein, and Hes5 are decreased. Disabled1, a component of the Reelin-signaling pathway colocalizes with Notch1, thus indicating a direct interaction between the Reelin- and Notch1-signaling pathways. These results suggest that Reelin enhances Notch1 signaling, thereby contributing to the formation of the radial glial scaffold and the normal development of the dentate gyrus.