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Primary cilia are finger-like sensory organelles that extend from the bodies of most cell types and have a distinct lipid and protein composition from the plasma membrane. This partitioning is maintained by a diffusion barrier that restricts the entry of non-ciliary proteins, and allows the selective entry of proteins harboring a ciliary targeting sequence (CTS). However, CTSs are not stereotyped and previously reported sequences are insufficient to drive efficient ciliary localisation across diverse cell types. Here, we describe a short peptide sequence that efficiently targets transmembrane proteins to primary cilia in all tested cell types, including human neurons. We generate human-induced pluripotent stem cell (hiPSC) lines stably expressing a transmembrane construct bearing an extracellular HaloTag and intracellular fluorescent protein, which enables the bright, specific labeling of primary cilia in neurons and other cell types to facilitate studies of cilia in health and disease. We demonstrate the utility of this resource by developing an image analysis pipeline for the automated measurement of primary cilia to detect changes in their length associated with altered signaling or disease state.
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Cílios , Células-Tronco Pluripotentes Induzidas , Proteínas de Membrana , Cílios/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Animais , Neurônios/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Transporte ProteicoRESUMO
Gracia-Diaz and colleagues analysed high-density DNA microarray and whole genome sequencing (WGS) data from the KOLF2.1J 'reference' human induced pluripotent stem cell (hiPSC) line1, and report the presence of five high-confidence heterozygous copy number variants (CNVs) at least 100kbp in length2. Since three of these CNVs span coding genes, some of which have been associated with neurodevelopmental disease, the authors raise the concern that these CNVs may compromise the utility of KOLF2.1J for neurological disease modelling. We appreciate their thorough analysis and thoughtful interpretation, and agree that potential users of this line should be made aware of all cases where KOLF2.1J differs from the reference genome. However, we believe that the benefits from the widespread use of KOLF2.1J outweigh the potential risks that might arise from the identified CNVs.
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Hypothalamic Adult Neurogenesis (hAN) has been implicated in regulating energy homeostasis. Adult-generated neurons and adult Neural Stem Cells (aNSCs) in the hypothalamus control food intake and body weight. Conversely, diet-induced obesity (DIO) by high fat diets (HFD) exerts adverse influence on hAN. However, the effects of anti-obesity compounds on hAN are not known. To address this, we administered a lipidized analogue of an anti-obesity neuropeptide, Prolactin Releasing Peptide (PrRP), so-called LiPR, to mice. In the HFD context, LiPR rescued the survival of adult-born hypothalamic neurons and increased the number of aNSCs by reducing their activation. LiPR also rescued the reduction of immature hippocampal neurons and modulated calcium dynamics in iPSC-derived human neurons. In addition, some of these neurogenic effects were exerted by another anti-obesity compound, Liraglutide. These results show for the first time that anti-obesity neuropeptides influence adult neurogenesis and suggest that the neurogenic process can serve as a target of anti-obesity pharmacotherapy.
Assuntos
Neuropeptídeos , Obesidade , Camundongos , Humanos , Animais , Hormônio Liberador de Prolactina/farmacologia , Hormônio Liberador de Prolactina/uso terapêutico , Obesidade/tratamento farmacológico , Peso Corporal , Neurogênese , HipotálamoRESUMO
Primary cilia protrude from most vertebrate cell bodies and act as specialized 'signalling antennae' that can substantially lengthen or retract in minutes to hours in response to specific stimuli. Here, we review the conditions and mechanisms responsible for regulating primary cilia length (PCL) in mammalian nonsensory neurons, and propose four models of how they could affect ciliary signalling and alter cell state and suggest experiments to distinguish between them. These models include (i) the passive indicator model, where changes in PCL have no consequence; (ii) the rheostat model, in which a longer cilium enhances signalling; (iii) the local concentration model, where ciliary shortening increases the local protein concentration to facilitate signalling; and (iv) the altered composition model where changes in PCL skew signalling.
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Cílios , Transdução de Sinais , Humanos , Animais , Cílios/metabolismo , Vertebrados , Neurônios , MamíferosRESUMO
Neurons in the hypothalamus orchestrate homeostatic physiological processes and behaviors essential for life. Human pluripotent stem cells (hPSCs) can be differentiated into many types of hypothalamic neurons, progenitors, and glia. This updated unit includes published studies and protocols with new advances in the differentiation, maturation, and interrogation by transcriptomic profiling and calcium imaging of human hypothalamic cell populations. Specifically, new methods to freeze and thaw hypothalamic progenitors after they have been patterned and before substantial neurogenesis has occurred are provided that will facilitate experimental flexibility and planning. Also included are updated recipes and protocols for neuronal maturation, with details on the equipment and methods for examining their transcriptomic response and cell-autonomous properties in culture in the presence of synaptic blockers. Together, these protocols facilitate the adoption and use of this model system for fundamental biological discovery and therapeutic translation to human diseases such as obesity, diabetes, sleep disorders, infertility, and chronic stress. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: hPSC maintenance Basic Protocol 2: Hypothalamic neuron differentiation Support Protocol 1: Cortical neuron (control) differentiation Basic Protocol 3: Neuronal maturation Support Protocol 2: Cryopreservation and thawing of neuronal progenitors Support Protocol 3: Quality control: Confirmation of hypothalamic patterning and neurogenesis Support Protocol 4: Bulk RNA sequencing of hypothalamic cultures Basic Protocol 4: Calcium imaging of hypothalamic neurons using Fura-2 AM Alternate Protocol: Calcium imaging of green fluorescent hypothalamic neurons using Rhod-3 AM.
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Neurônios , Transcriptoma , Humanos , Neurônios/fisiologia , Diferenciação Celular/fisiologia , Hipotálamo/diagnóstico por imagem , Neurogênese/genética , Cálcio da DietaRESUMO
Makorin ring finger protein 3 (MKRN3) was identified as an inhibitor of puberty initiation with the report of loss-of-function mutations in association with central precocious puberty. Consistent with this inhibitory role, a prepubertal decrease in Mkrn3 expression was observed in the mouse hypothalamus. Here, we investigated the mechanisms of action of MKRN3 in the central regulation of puberty onset. We showed that MKRN3 deletion in hypothalamic neurons derived from human induced pluripotent stem cells was associated with significant changes in expression of genes controlling hypothalamic development and plasticity. Mkrn3 deletion in a mouse model led to early puberty onset in female mice. We found that Mkrn3 deletion increased the number of dendritic spines in the arcuate nucleus but did not alter the morphology of GnRH neurons during postnatal development. In addition, we identified neurokinin B (NKB) as an Mkrn3 target. Using proteomics, we identified insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) as another target of MKRN3. Interactome analysis revealed that IGF2BP1 interacted with MKRN3, along with several members of the polyadenylate-binding protein family. Our data show that one of the mechanisms by which MKRN3 inhibits pubertal initiation is through regulation of prepubertal hypothalamic development and plasticity, as well as through effects on NKB and IGF2BP1.
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Células-Tronco Pluripotentes Induzidas , Puberdade Precoce , Humanos , Feminino , Camundongos , Animais , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Hipotálamo/metabolismo , Puberdade , Hormônio Liberador de Gonadotropina/metabolismo , Puberdade Precoce/genética , Puberdade Precoce/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
It is well established that human pluripotent stem cells (hPSCs) can acquire genetic and epigenetic changes during culture in vitro. Given the increasing use of hPSCs in research and therapy and the vast expansion in the number of hPSC lines available for researchers, the International Society for Stem Cell Research has recognized the need to reassess quality control standards for ensuring the genetic integrity of hPSCs. Here, we summarize current knowledge of the nature of recurrent genetic and epigenetic variants in hPSC culture, the methods for their detection, and what is known concerning their effects on cell behavior in vitro or in vivo. We argue that the potential consequences of low-level contamination of cell therapy products with cells bearing oncogenic variants are essentially unknown at present. We highlight the key challenges facing the field with particular reference to safety assessment of hPSC-derived cellular therapeutics.
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Epigenômica , Células-Tronco Pluripotentes , Humanos , Pesquisa com Células-Tronco , Oncogenes , Epigênese GenéticaRESUMO
Despite their widespread use in research, there has not yet been a systematic genomic analysis of human embryonic stem cell (hESC) lines at a single-nucleotide resolution. We therefore performed whole-genome sequencing (WGS) of 143 hESC lines and annotated their single-nucleotide and structural genetic variants. We found that while a substantial fraction of hESC lines contained large deleterious structural variants, finer-scale structural and single-nucleotide variants (SNVs) that are ascertainable only through WGS analyses were present in hESC genomes and human blood-derived genomes at similar frequencies. Moreover, WGS allowed us to identify SNVs associated with cancer and other diseases that could alter cellular phenotypes and compromise the safety of hESC-derived cellular products transplanted into humans. As a resource to enable reproducible hESC research and safer translation, we provide a user-friendly WGS data portal and a data-driven scheme for cell line maintenance and selection.
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Células-Tronco Embrionárias Humanas , Variação Genética , Genoma Humano/genética , Humanos , Nucleotídeos , Sequenciamento Completo do GenomaRESUMO
When mice are exposed to external warmth, nitric oxide synthase (NOS1) neurons in the median and medial preoptic (MnPO/MPO) hypothalamus induce sleep and concomitant body cooling. However, how these neurons regulate baseline sleep and body temperature is unknown. Using calcium photometry, we show that NOS1 neurons in MnPO/MPO are predominantly NREM and REM active, especially at the boundary of wake to NREM transitions, and in the later parts of REM bouts, with lower activity during wakefulness. In addition to releasing nitric oxide, NOS1 neurons in MnPO/MPO can release GABA, glutamate and peptides. We expressed tetanus-toxin light-chain in MnPO/MPO NOS1 cells to reduce vesicular release of transmitters. This induced changes in sleep structure: over 24 h, mice had less NREM sleep in their dark (active) phase, and more NREM sleep in their light (sleep) phase. REM sleep episodes in the dark phase were longer, and there were fewer REM transitions between other vigilance states. REM sleep had less theta power. Mice with synaptically blocked MnPO/MPO NOS1 neurons were also warmer than control mice at the dark-light transition (ZT0), as well as during the dark phase siesta (ZT16-20), where there is usually a body temperature dip. Also, at this siesta point of cooled body temperature, mice usually have more NREM, but mice with synaptically blocked MnPO/MPO NOS1 cells showed reduced NREM sleep at this time. Overall, MnPO/MPO NOS1 neurons promote both NREM and REM sleep and contribute to chronically lowering body temperature, particularly at transitions where the mice normally enter NREM sleep.
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DNA replication follows a strict spatiotemporal program that intersects with chromatin structure but has a poorly understood genetic basis. To systematically identify genetic regulators of replication timing, we exploited inter-individual variation in human pluripotent stem cells from 349 individuals. We show that the human genome's replication program is broadly encoded in DNA and identify 1,617 cis-acting replication timing quantitative trait loci (rtQTLs) - sequence determinants of replication initiation. rtQTLs function individually, or in combinations of proximal and distal regulators, and are enriched at sites of histone H3 trimethylation of lysines 4, 9, and 36 together with histone hyperacetylation. H3 trimethylation marks are individually repressive yet synergistically associate with early replication. We identify pluripotency-related transcription factors and boundary elements as positive and negative regulators of replication timing, respectively. Taken together, human replication timing is controlled by a multi-layered mechanism with dozens of effectors working combinatorially and following principles analogous to transcription regulation.
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Período de Replicação do DNA , Genoma Humano , Células-Tronco Pluripotentes/metabolismo , Acetilação , Variação Biológica da População/genética , Metilação de DNA , Conjuntos de Dados como Assunto , Feminino , Regulação da Expressão Gênica , Código das Histonas/genética , Histonas/metabolismo , Humanos , Masculino , Locos de Características Quantitativas , Fatores de Transcrição/metabolismo , Sequenciamento Completo do GenomaRESUMO
Hypothalamic pro-opiomelanocortin (POMC) neurons are known to trigger satiety. However, these neuronal cells encompass heterogeneous subpopulations that release γ-aminobutyric acid (GABA), glutamate, or both neurotransmitters, whose functions are poorly defined. Using conditional mutagenesis and chemogenetics, we show that blockade of the energy sensor mechanistic target of rapamycin complex 1 (mTORC1) in POMC neurons causes hyperphagia by mimicking a cellular negative energy state. This is associated with decreased POMC-derived anorexigenic α-melanocyte-stimulating hormone and recruitment of POMC/GABAergic neurotransmission, which is restrained by cannabinoid type 1 receptor signaling. Electrophysiology and optogenetic studies further reveal that pharmacological blockade of mTORC1 simultaneously activates POMC/GABAergic neurons and inhibits POMC/glutamatergic ones, implying that the functional specificity of these subpopulations relies on mTORC1 activity. Finally, POMC neurons with different neurotransmitter profiles possess specific molecular signatures and spatial distribution. Altogether, these findings suggest that mTORC1 orchestrates the activity of distinct POMC neurons subpopulations to regulate feeding behavior.
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Regulação do Apetite , Comportamento Alimentar , Neurônios GABAérgicos/metabolismo , Ácido Glutâmico/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Inibição Neural , Núcleo Hipotalâmico Paraventricular/metabolismo , Pró-Opiomelanocortina/metabolismo , Animais , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Pró-Opiomelanocortina/genética , Transdução de SinaisRESUMO
The third 'Symposium for the Next Generation of Stem Cell Research' (SY-Stem) was held virtually on 3-5 March 2021, having been cancelled in 2020 due to the COVID-19 pandemic. As in previous years, the meeting highlighted the work of early career researchers, ranging from postgraduate students to young group leaders working in developmental and stem cell biology. Here, we summarize the excellent work presented at the Symposium, which covered topics ranging from pluripotency, species-specific aspects of development and emerging technologies, through to organoids, single-cell technology and clinical applications.
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Congressos como Assunto/organização & administração , Invenções/tendências , Pesquisa com Células-Tronco , Animais , COVID-19/epidemiologia , Diferenciação Celular , Congressos como Assunto/história , Congressos como Assunto/tendências , História do Século XXI , Humanos , Internet , Invenções/história , Sistemas On-Line , Pandemias , Análise de Célula Única/métodos , Análise de Célula Única/tendências , Pesquisa com Células-Tronco/história , Células-Tronco/fisiologia , Técnicas de Cultura de Tecidos/métodos , Técnicas de Cultura de Tecidos/tendênciasRESUMO
Studying the function of common genetic variants in primary human tissues and during development is challenging. To address this, we use an efficient multiplexing strategy to differentiate 215 human induced pluripotent stem cell (iPSC) lines toward a midbrain neural fate, including dopaminergic neurons, and use single-cell RNA sequencing (scRNA-seq) to profile over 1 million cells across three differentiation time points. The proportion of neurons produced by each cell line is highly reproducible and is predictable by robust molecular markers expressed in pluripotent cells. Expression quantitative trait loci (eQTL) were characterized at different stages of neuronal development and in response to rotenone-induced oxidative stress. Of these, 1,284 eQTL colocalize with known neurological trait risk loci, and 46% are not found in the Genotype-Tissue Expression (GTEx) catalog. Our study illustrates how coupling scRNA-seq with long-term iPSC differentiation enables mechanistic studies of human trait-associated genetic variants in otherwise inaccessible cell states.
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Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/fisiologia , Células-Tronco Pluripotentes Induzidas/citologia , Locos de Características Quantitativas , Transcriptoma , Diferenciação Celular/genética , Predisposição Genética para Doença , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Neurogênese/genética , Estresse Oxidativo/efeitos dos fármacos , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Rotenona/toxicidade , Análise de Sequência de RNA , Análise de Célula ÚnicaRESUMO
Brain-derived neurotrophic factor (BDNF) signals through its high affinity receptor Tropomyosin receptor kinase-B (TrkB) to regulate neuronal development, synapse formation and plasticity. In rodents, genetic disruption of Bdnf and TrkB leads to weight gain and a spectrum of neurobehavioural phenotypes. Here, we functionally characterised a de novo missense variant in BDNF and seven rare variants in TrkB identified in a large cohort of people with severe, childhood-onset obesity. In cells, the E183K BDNF variant resulted in impaired processing and secretion of the mature peptide. Multiple variants in the kinase domain and one variant in the extracellular domain of TrkB led to a loss of function through multiple signalling pathways, impaired neurite outgrowth and dominantly inhibited glutamatergic synaptogenesis in hippocampal neurons. BDNF/TrkB variant carriers exhibited learning difficulties, impaired memory, hyperactivity, stereotyped and sometimes, maladaptive behaviours. In conclusion, human loss of function BDNF/TrkB variants that impair hippocampal synaptogenesis may contribute to a spectrum of neurobehavioural disorders.
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Fator Neurotrófico Derivado do Encéfalo/metabolismo , Neurogênese/efeitos dos fármacos , Receptor trkB/metabolismo , Adolescente , Criança , Pré-Escolar , Feminino , Hipocampo/metabolismo , Hipocampo/fisiologia , Humanos , Masculino , Neurogênese/fisiologia , Crescimento Neuronal/efeitos dos fármacos , Neurônios/metabolismo , Fosforilação , Proteínas Quinases , Transdução de Sinais/efeitos dos fármacosRESUMO
In outbred mice, susceptibility or resistance to diet-induced obesity is associated with rapid changes in hypothalamic proopiomelanocortin (POMC) levels. Here, we evaluated 3 hypotheses that potentially explain the development of the different obesity phenotypes in outbred Swiss mice. First, rapid and differential changes in the gut microbiota in obesity-prone (OP) and obesity-resistant (OR) mice fed on a high-fat diet (HFD) might cause differential efficiencies in fatty acid harvesting leading to changes in systemic fatty acid concentrations that in turn affect POMC expression and processing. Second, independently of the gut microbiota, OP mice might have increased blood fatty acid levels after the introduction of a HFD, which could affect POMC expression and processing. Third, fatty acids might act directly in the hypothalamus to differentially regulate POMC expression and/or processing in OP and OR mice. We evaluated OP and OR male Swiss mice using 16S rRNA sequencing for the determination of gut microbiota; gas chromatography for blood lipid determination; and immunoblot and real-time polymerase chain reaction for protein and transcript determination and indirect calorimetry. Some experiments were performed with human pluripotent stem cells differentiated into hypothalamic neurons. We did not find evidence supporting the first 2 hypotheses. However, we found that in OP but not in OR mice, palmitate induces a rapid increase in hypothalamic POMC, which is followed by increased expression of proprotein convertase subtilisin/kexin type 1 PC1/3. Lentiviral inhibition of hypothalamic PC1/3 increased caloric intake and body mass in both OP and OR mice. In human stem cell-derived hypothalamic cells, we found that palmitate potently suppressed the production of POMC-derived peptides. Palmitate directly regulates PC1/3 in OP mice and likely has a functional impact on POMC processing.
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Microbioma Gastrointestinal , Hipotálamo/metabolismo , Inflamação/metabolismo , Neurônios/metabolismo , Obesidade/metabolismo , Palmitatos/farmacologia , Pró-Opiomelanocortina/metabolismo , Animais , Dieta Hiperlipídica , Modelos Animais de Doenças , Humanos , Ácido Linoleico/farmacologia , Masculino , Camundongos , Obesidade/sangue , Obesidade/etiologia , Células-Tronco Pluripotentes , RNA Ribossômico 16SRESUMO
OBJECTIVE: The lack of pro-opiomelanocortin (POMC)-derived melanocortin peptides results in hypoadrenalism and severe obesity in both humans and rodents that is treatable with synthetic melanocortins. However, there are significant differences in POMC processing between humans and rodents, and little is known about the relative physiological importance of POMC products in the human brain. The aim of this study was to determine which POMC-derived peptides are present in the human brain, to establish their relative concentrations, and to test if their production is dynamically regulated. METHODS: We analysed both fresh post-mortem human hypothalamic tissue and hypothalamic neurons derived from human pluripotent stem cells (hPSCs) using liquid chromatography tandem mass spectrometry (LC-MS/MS) to determine the sequence and quantify the production of hypothalamic neuropeptides, including those derived from POMC. RESULTS: In both in vitro and in vivo hypothalamic cells, LC-MS/MS revealed the sequence of hundreds of neuropeptides as a resource for the field. Although the existence of ß-melanocyte stimulating hormone (MSH) is controversial, we found that both this peptide and desacetyl α-MSH (d-α-MSH) were produced in considerable excess of acetylated α-MSH. In hPSC-derived hypothalamic neurons, these POMC derivatives were appropriately trafficked, secreted, and their production was significantly (P < 0.0001) increased in response to the hormone leptin. CONCLUSIONS: Our findings challenge the assumed pre-eminence of α-MSH and suggest that in humans, d-α-MSH and ß-MSH are likely to be the predominant physiological products acting on melanocortin receptors.
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Melanocortinas/metabolismo , alfa-MSH/metabolismo , beta-MSH/metabolismo , Cromatografia Líquida , Feminino , Homeostase/fisiologia , Humanos , Hipotálamo , Leptina/metabolismo , Masculino , Espectrometria de Massas/métodos , Neurônios/metabolismo , Neuropeptídeos/metabolismo , Células-Tronco Pluripotentes/metabolismo , Pró-Opiomelanocortina/metabolismo , Receptores de Melanocortina/metabolismo , Espectrometria de Massas em TandemRESUMO
OBJECTIVE: Dietary proteins are sensed by hypothalamic neurons and strongly influence multiple aspects of metabolic health, including appetite, weight gain, and adiposity. However, little is known about the mechanisms by which hypothalamic neural circuits controlling behavior and metabolism sense protein availability. The aim of this study is to characterize how neurons from the mediobasal hypothalamus respond to a signal of protein availability: the amino acid l-leucine. METHODS: We used primary cultures of post-weaning murine mediobasal hypothalamic neurons, hypothalamic neurons derived from human induced pluripotent stem cells, and calcium imaging to characterize rapid neuronal responses to physiological changes in extracellular l-Leucine concentration. RESULTS: A neurochemically diverse subset of both mouse and human hypothalamic neurons responded rapidly to l-leucine. Consistent with l-leucine's anorexigenic role, we found that 25% of mouse MBH POMC neurons were activated by l-leucine. 10% of MBH NPY neurons were inhibited by l-leucine, and leucine rapidly reduced AGRP secretion, providing a mechanism for the rapid leucine-induced inhibition of foraging behavior in rodents. Surprisingly, none of the candidate mechanisms previously implicated in hypothalamic leucine sensing (KATP channels, mTORC1 signaling, amino-acid decarboxylation) were involved in the acute activity changes produced by l-leucine. Instead, our data indicate that leucine-induced neuronal activation involves a plasma membrane Ca2+ channel, whereas leucine-induced neuronal inhibition is mediated by inhibition of a store-operated Ca2+ current. CONCLUSIONS: A subset of neurons in the mediobasal hypothalamus rapidly respond to physiological changes in extracellular leucine concentration. Leucine can produce both increases and decreases in neuronal Ca2+ concentrations in a neurochemically-diverse group of neurons, including some POMC and NPY/AGRP neurons. Our data reveal that leucine can signal through novel mechanisms to rapidly affect neuronal activity.
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Hipotálamo/metabolismo , Leucina/farmacologia , Neurônios/metabolismo , Transdução de Sinais , Animais , Cálcio/metabolismo , Células Cultivadas , Humanos , Hipotálamo/citologia , Canais KATP/metabolismo , Leucina/metabolismo , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacosRESUMO
Neurons in the hypothalamus orchestrate homeostatic physiological processes and behaviors essential for life. Defects in the function of hypothalamic neurons cause a spectrum of human diseases, including obesity, infertility, growth defects, sleep disorders, social disorders, and stress disorders. These diseases have been studied in animal models such as mice, but the rarity and relative inaccessibility of mouse hypothalamic neurons and species-specific differences between mice and humans highlight the need for human cellular models of hypothalamic diseases. We and others have developed methods to differentiate human pluripotent stem cells (hPSCs) into hypothalamic neurons and related cell types, such as astrocytes. This protocol builds on published studies by providing detailed step-by-step instructions for neuronal differentiation, quality control, long-term neuronal maintenance, and the functional interrogation of hypothalamic cells by calcium imaging. Together, these protocols should enable any group with appropriate facilities to generate and study human hypothalamic cells. © 2017 by John Wiley & Sons, Inc.
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Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Hipotálamo/citologia , Neurogênese/fisiologia , Neurônios/fisiologia , Proteína Relacionada com Agouti/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Humanos , Hormônios Hipotalâmicos/metabolismo , Hipotálamo/embriologia , Neurogênese/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/fisiologia , Pró-Opiomelanocortina/metabolismo , Transdução de Sinais/fisiologia , Fatores de TempoRESUMO
Human pluripotent stem cells (hPS cells) can self-renew indefinitely, making them an attractive source for regenerative therapies. This expansion potential has been linked with the acquisition of large copy number variants that provide mutated cells with a growth advantage in culture. The nature, extent and functional effects of other acquired genome sequence mutations in cultured hPS cells are not known. Here we sequence the protein-coding genes (exomes) of 140 independent human embryonic stem cell (hES cell) lines, including 26 lines prepared for potential clinical use. We then apply computational strategies for identifying mutations present in a subset of cells in each hES cell line. Although such mosaic mutations were generally rare, we identified five unrelated hES cell lines that carried six mutations in the TP53 gene that encodes the tumour suppressor P53. The TP53 mutations we observed are dominant negative and are the mutations most commonly seen in human cancers. We found that the TP53 mutant allelic fraction increased with passage number under standard culture conditions, suggesting that the P53 mutations confer selective advantage. We then mined published RNA sequencing data from 117 hPS cell lines, and observed another nine TP53 mutations, all resulting in coding changes in the DNA-binding domain of P53. In three lines, the allelic fraction exceeded 50%, suggesting additional selective advantage resulting from the loss of heterozygosity at the TP53 locus. As the acquisition and expansion of cancer-associated mutations in hPS cells may go unnoticed during most applications, we suggest that careful genetic characterization of hPS cells and their differentiated derivatives be carried out before clinical use.