RESUMEN
The transfer of somatic cell nuclei into oocytes can give rise to pluripotent stem cells that are consistently equivalent to embryonic stem cells, holding promise for autologous cell replacement therapy. Although methods to induce pluripotent stem cells from somatic cells by transcription factors are widely used in basic research, numerous differences between induced pluripotent stem cells and embryonic stem cells have been reported, potentially affecting their clinical use. Because of the therapeutic potential of diploid embryonic stem-cell lines derived from adult cells of diseased human subjects, we have systematically investigated the parameters affecting efficiency of blastocyst development and stem-cell derivation. Here we show that improvements to the oocyte activation protocol, including the use of both kinase and translation inhibitors, and cell culture in the presence of histone deacetylase inhibitors, promote development to the blastocyst stage. Developmental efficiency varied between oocyte donors, and was inversely related to the number of days of hormonal stimulation required for oocyte maturation, whereas the daily dose of gonadotropin or the total number of metaphase II oocytes retrieved did not affect developmental outcome. Because the use of concentrated Sendai virus for cell fusion induced an increase in intracellular calcium concentration, causing premature oocyte activation, we used diluted Sendai virus in calcium-free medium. Using this modified nuclear transfer protocol, we derived diploid pluripotent stem-cell lines from somatic cells of a newborn and, for the first time, an adult, a female with type 1 diabetes.
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Núcleo Celular/genética , Reprogramación Celular , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/patología , Diploidia , Oocitos/citología , Células Madre Pluripotentes/citología , Adulto , Blastocisto/efectos de los fármacos , Fusión Celular , Cromosomas de los Mamíferos/metabolismo , Femenino , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Recién Nacido , Metafase , Oocitos/metabolismo , Oogénesis , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/patología , Virus Sendai , Huso Acromático/metabolismoRESUMEN
Cortical interneurons (INs) are a diverse group of neurons that project locally and shape the function of neural networks throughout the brain. Multiple lines of evidence suggest that a proper balance of glutamate and GABA signaling is essential for both the proper function and development of the brain. Dysregulation of this system may lead to neurodevelopmental disorders, including autism spectrum condition (ASC). We evaluate the development and function of INs in rodent and human models and examine how neurodevelopmental dysfunction may produce core symptoms of ASC. Finding common physiological mechanisms that underlie neurodevelopmental disorders may lead to novel pharmacological targets and candidates that could improve the cognitive and emotional symptoms associated with ASC.
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Trastorno del Espectro Autista/fisiopatología , Corteza Cerebral/patología , Interneuronas/patología , Animales , Trastorno del Espectro Autista/metabolismo , Corteza Cerebral/metabolismo , Electroencefalografía , Ácido Glutámico/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/patología , Interneuronas/metabolismo , Ácido gamma-Aminobutírico/metabolismoRESUMEN
BACKGROUND: Intracellular calcium is an important ion involved in the regulation and modulation of many neuronal functions. From regulating cell cycle and proliferation to initiating signaling cascades and regulating presynaptic neurotransmitter release, the concentration and timing of calcium activity governs the function and fate of neurons. Changes in calcium transients can be used in high-throughput screening applications as a basic measure of neuronal maturity, especially in developing or immature neuronal cultures derived from stem cells. RESULTS: Using human induced pluripotent stem cell derived neurons and dissociated mouse cortical neurons combined with the calcium indicator Fluo-4, we demonstrate that PeakCaller reduces type I and type II error in automated peak calling when compared to the oft-used PeakFinder algorithm under both basal and pharmacologically induced conditions. CONCLUSION: Here we describe PeakCaller, a novel MATLAB script and graphical user interface for the quantification of intracellular calcium transients in neuronal cultures. PeakCaller allows the user to set peak parameters and smoothing algorithms to best fit their data set. This new analysis script will allow for automation of calcium measurements and is a powerful software tool for researchers interested in high-throughput measurements of intracellular calcium.
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Algoritmos , Señalización del Calcio/fisiología , Calcio/metabolismo , Neurogénesis/fisiología , Animales , Automatización/instrumentación , Señalización del Calcio/efectos de los fármacos , Modelos Animales de Enfermedad , Humanos , Células Madre Pluripotentes Inducidas/citología , Ratones , Neuronas/metabolismo , Programas Informáticos/estadística & datos numéricosRESUMEN
Production and isolation of forebrain interneuron progenitors are essential for understanding cortical development and developing cell-based therapies for developmental and neurodegenerative disorders. We demonstrate production of a population of putative calretinin-positive bipolar interneurons that express markers consistent with caudal ganglionic eminence identities. Using serum-free embryoid bodies (SFEBs) generated from human inducible pluripotent stem cells (iPSCs), we demonstrate that these interneuron progenitors exhibit morphological, immunocytochemical, and electrophysiological hallmarks of developing cortical interneurons. Finally, we develop a fluorescence-activated cell-sorting strategy to isolate interneuron progenitors from SFEBs to allow development of a purified population of these cells. Identification of this critical neuronal cell type within iPSC-derived SFEBs is an important and novel step in describing cortical development in this iPSC preparation.
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Corteza Cerebral/citología , Corteza Cerebral/fisiología , Cuerpos Embrioides/fisiología , Células Madre Pluripotentes Inducidas/fisiología , Interneuronas/fisiología , Animales , Células Cultivadas , Fibroblastos/fisiología , Humanos , Ratones , Ratones NoqueadosRESUMEN
The heterogeneous expression of voltage-gated channels in dendrites suggests that neurons perform local microdomain computations at different regions. It has been shown that A-type K(+) channels have a nonuniform distribution along the primary apical dendrite in CA1 pyramidal neurons, increasing with distance from the soma. Kv4.2 channels, which are responsible for the somatodendritic A-type K(+) current in CA1 pyramidal neurons, shape local synaptic input, and regulate the back-propagation of APs into dendrites. Experiments were performed to test the hypothesis that Kv4.2 channels are differentially trafficked at different regions along the apical dendrite during basal activity and upon stimulation in CA1 neurons. Proximal (50-150 µm from the soma, primary and oblique) and distal (>200 µm) apical dendrites were selected. The fluorescence recovery after photobleaching (FRAP) technique was used to measure basal cycling rates of EGFP-tagged Kv4.2 (Kv4.2g). We found that the cycling rate of Kv4.2 channels was one order of magnitude slower at both primary and oblique dendrites between 50 and 150 µm from the soma. Kv4.2 channel cycling increased significantly at 200 to 250 µm from the soma. Expression of a Kv4.2 mutant lacking a phosphorylation site for protein kinase-A (Kv4.2gS552A) abolished this distance-dependent change in channel cycling; demonstrating that phosphorylation by PKA underlies the increased mobility in distal dendrites. Neuronal stimulation by α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) treatment increased cycling of Kv4.2 channels significantly at distal sites only. This activity-dependent increase in Kv4.2 cycling at distal dendrites was blocked by expression of Kv4.2gS552A. These results indicate that distance-dependent Kv4.2 mobility is regulated by activity-dependent phosphorylation of Kv4.2 by PKA.
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Región CA1 Hipocampal/citología , Región CA1 Hipocampal/metabolismo , Dendritas/metabolismo , Transporte de Proteínas/fisiología , Células Piramidales/metabolismo , Canales de Potasio Shal/metabolismo , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Endocitosis/fisiología , Recuperación de Fluorescencia tras Fotoblanqueo , Fosforilación , Bloqueadores de los Canales de Potasio/farmacología , Transporte de Proteínas/efectos de los fármacos , Células Piramidales/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacologíaRESUMEN
At present, the neuronal mechanisms underlying the diagnosis of autism spectrum disorder (ASD) have not been established. However, studies from human postmortem ASD brains have consistently revealed disruptions in cerebellar circuitry, specifically reductions in Purkinje cell (PC) number and size. Alterations in cerebellar circuitry would have important implications for information processing within the cerebellum and affect a wide range of human motor and non-motor behaviors. Laser capture microdissection was performed to obtain pure PC populations from a cohort of postmortem control and ASD cases and transcriptional profiles were compared. The 427 differentially expressed genes were enriched for gene ontology biological processes related to developmental organization/connectivity, extracellular matrix organization, calcium ion response, immune function and PC signaling alterations. Given the complexity of PCs and their far-ranging roles in response to sensory stimuli and motor function regulation, understanding transcriptional differences in this subset of cerebellar cells in ASD may inform on convergent pathways that impact neuronal function.
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Genetic studies have linked FAT1 (FAT atypical cadherin 1) with autism spectrum disorder (ASD); however, the role that FAT1 plays in ASD remains unknown. In mice, the function of Fat1 has been primarily implicated in embryonic nervous system development with less known about its role in postnatal development. We show for the first time that FAT1 protein is expressed in mouse postnatal brains and is enriched in the cerebellum, where it localizes to granule neurons and Golgi cells in the granule layer, as well as inhibitory neurons in the molecular layer. Furthermore, subcellular characterization revealed FAT1 localization in neurites and soma of granule neurons, as well as being present in the synaptic plasma membrane and postsynaptic densities. Interestingly, FAT1 expression was decreased in induced pluripotent stem cell (iPSC)-derived neural precursor cells (NPCs) from individuals with ASD. These findings suggest a novel role for FAT1 in postnatal development and may be particularly important for cerebellum function. As the cerebellum is one of the vulnerable brain regions in ASD, our study warrants further investigation of FAT1 in the disease etiology.
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Trastorno Autístico/etiología , Cadherinas/genética , Susceptibilidad a Enfermedades , Animales , Trastorno Autístico/metabolismo , Biomarcadores , Cadherinas/metabolismo , Cerebelo/metabolismo , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Hipocampo/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Interneuronas/metabolismo , Ratones , Neuritas/metabolismo , Transporte de Proteínas , TranscriptomaRESUMEN
Autism spectrum disorder (ASD) is a neurologic condition characterized by alterations in social interaction and communication, and restricted and/or repetitive behaviors. The classical Type II cadherins cadherin-8 (Cdh8, CDH8) and cadherin-11 (Cdh11, CDH11) have been implicated as autism risk gene candidates. To explore the role of cadherins in the etiology of autism, we investigated their expression patterns during mouse brain development and in autism-specific human tissue. In mice, expression of cadherin-8 and cadherin-11 was developmentally regulated and enriched in the cortex, hippocampus, and thalamus/striatum during the peak of dendrite formation and synaptogenesis. Both cadherins were expressed in synaptic compartments but only cadherin-8 associated with the excitatory synaptic marker neuroligin-1. Induced pluripotent stem cell (iPSC)-derived cortical neural precursor cells (NPCs) and cortical organoids generated from individuals with autism showed upregulated CDH8 expression levels, but downregulated CDH11. We used Cdh11 knock-out (KO) mice of both sexes to analyze the function of cadherin-11, which could help explain phenotypes observed in autism. Cdh11-/- hippocampal neurons exhibited increased dendritic complexity along with altered neuronal and synaptic activity. Similar to the expression profiles in human tissue, levels of cadherin-8 were significantly elevated in Cdh11 KO brains. Additionally, excitatory synaptic markers neuroligin-1 and postsynaptic density (PSD)-95 were both increased. Together, these results strongly suggest that cadherin-11 is involved in regulating the development of neuronal circuitry and that alterations in the expression levels of cadherin-11 may contribute to the etiology of autism.
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Trastorno del Espectro Autista , Trastorno Autístico , Cadherinas , Células-Madre Neurales , Animales , Trastorno del Espectro Autista/genética , Cadherinas/genética , Femenino , Humanos , Masculino , Ratones , Ratones NoqueadosRESUMEN
Clustered regularly interspaced short palindromic repeats (CRISPR) genome editing has already reinvented the direction of genetic and stem cell research. For more complex diseases it allows scientists to simultaneously create multiple genetic changes to a single cell. Technologies for correcting multiple mutations in an in vivo system are already in development. On the surface, the advent and use of gene editing technologies is a powerful tool to reduce human suffering by eradicating complex disease that has a genetic etiology. Gene drives are CRISPR mediated alterations to genes that allow them to be passed on to subsequent populations at rates that approach one hundred per cent transmission. Therefore, from an anticipatory biomedical ethics perspective, it is possible to conceive gene drive being used with CRISPR to permanently ameliorate aberrant genes from wild-type populations containing mutations. However, there are also a number of possible side effects that could develop as the result of combining gene editing and gene drive technologies in an effort to eradicate complex diseases. In this paper, we critically analyse the hypothesis that the combination of CRISPR and gene drive will have a deleterious effect on human populations from an ethical perspective by developing an anticipatory ethical analysis of the implications for the use of CRISPR together with gene drive in humans.
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Bioética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Análisis Ético , Tecnología de Genética Dirigida/ética , Edición Génica/ética , Humanos , Formulación de PolíticasRESUMEN
Autism spectrum condition (ASC) is a complex set of behavioral and neurological responses reflecting a likely interaction between autism susceptibility genes and the environment. Autism represents a spectrum in which heterogeneous genetic backgrounds are expressed with similar heterogeneity in the affected domains of communication, social interaction, and behavior. The impact of gene-environment interactions may also account for differences in underlying neurology and wide variation in observed behaviors. For these reasons, it has been difficult for geneticists and neuroscientists to build adequate systems to model the complex neurobiology causes of autism. In addition, the development of therapeutics for individuals with autism has been painstakingly slow, with most treatment options reduced to repurposed medications developed for other neurological diseases. Adequately developing therapeutics that are sensitive to the genetic and neurobiological diversity of individuals with autism necessitates personalized models of ASC that can capture some common pathways that reflect the neurophysiological and genetic backgrounds of varying individuals. Testing cohorts of individuals with and without autism for these potentially convergent pathways on a scalable platform for therapeutic development requires large numbers of samples from a diverse population. To date, human induced pluripotent stem cells (iPSCs) represent one of the best systems for conducting these types of assays in a clinically relevant and scalable way. The discovery of the four Yamanaka transcription factors (OCT3/4, SOX2, c-Myc, and KLF4) [1] allows for the induction of iPSCs from fibroblasts [2], peripheral blood mononuclear cells (PBMCs, i.e. lymphocytes and monocytes) [3, 4], or dental pulp cells [5] that retain the original genetics of the individual from which they were derived [6], making iPSCs a powerful tool to model neurophysiological conditions. iPSCs are a readily renewable cell type that can be developed on a small scale for boutique-style proof-of-principle phenotypic studies and scaled to an industrial level for drug screening and other high-content assays. This flexibility, along with the ability to represent the true genetic diversity of autism, underscores the importance of using iPSCs to model neurophysiological aspects of ASC.
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Trastorno del Espectro Autista , Trastorno Autístico , Células Madre Pluripotentes Inducidas , Trastorno del Espectro Autista/genética , Humanos , Factor 4 Similar a Kruppel , Leucocitos Mononucleares , OrganoidesRESUMEN
BACKGROUND: The need for scalable high-throughput screening (HTS) approaches for 3D human stem cell platforms remains a central challenge for disease modeling and drug discovery. We have developed a workflow to screen cortical organoids across platforms. NEW METHOD: We used serum-free embryoid bodies (SFEBs) derived from human induced pluripotent stem cells (hiPSCs) and employed high-content imaging (HCI) to assess neurite outgrowth and cellular composition within SFEBs. We multiplexed this screening assay with both multi-electrode arrays (MEAs) and single-cell calcium imaging. RESULTS: HCI was used to assess the number of excitatory neurons (VGlut+) in experimental replicates of hiPSC-derived SFEBs, demonstrating experiment-to-experiment consistency. Neurite detection using HCI was applied to assess neurite morphology. MEA analysis showed that firing and burst rates in SFEBs decreased with blockade of NMDARs and AMPARs and increased with GABAR blockade. We also demonstrate effective combination of both MEA and HCI to analyze VGlut+ populations surrounding electrodes within MEAs. HCI-based (Ca2+) transient analysis revealed firing in individual cells surrounding active MEA electrodes. COMPARISON WITH EXISTING METHODS: Current methods to generate neural organoids show high degrees of variability, and often require sectioning or special handling for analysis. The protocol outlined in this manuscript generates SFEBs with high degree of consistency making them amenable to complex assays combining HTS and electrophysiology allowing for an in-depth, unbiased analysis. CONCLUSIONS: SFEBs can be used in combination with HTS to compensate for experimental variability common in 3D cultures, while significantly decreasing processing speed, making this an efficient starting point for phenotypic drug screening.
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Células Madre Pluripotentes Inducidas , Encéfalo , Ensayos Analíticos de Alto Rendimiento , Humanos , Neuronas , OrganoidesRESUMEN
Ephrin (Eph) signaling via Eph receptors affects neuronal structure and function. We report here that exogenous ephrinAs (EphAs) induce outgrowth of filopodial processes from astrocytes within minutes in rat hippocampal slice cultures. Identical effects were induced by release of endogenous ephrinAs by cleavage of their glycosylphosphatidylinositol anchor. Reverse transcription-PCR and immunocytochemistry revealed the expression of multiple EphA receptors (EphARs) in astrocytes. Exogenous and endogenous ephrins did not induce process outgrowth from astrocytes transfected with a kinase-dead EphAR construct, indicating that the critical EphARs were located on glia. Concomitant with these morphological changes, ephrinA reduced the frequency of (S)-3,5-dihydroxyphenylglycine-evoked NMDA receptor-mediated inward currents in CA1 pyramidal cells, elicited by release of glutamate from glial cells. The sensitivity of CA1 cell synaptic or extrasynaptic NMDA receptors was unaffected by ephrinA, indicating that this effect was mediated by inhibition of glutamate release from glial cells. Finally, ephrinA application decreased the frequency and increased the duration of spontaneous oscillations of the intracellular [Ca2+] in astrocytes. We conclude that ephrinA-EphA signaling is a pluripotent regulator of neuron-astrocyte interactions mediating rapid structural and functional plasticity.
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Astrocitos/fisiología , Comunicación Celular/fisiología , Neuroglía/fisiología , Plasticidad Neuronal/fisiología , Neuronas/fisiología , Receptores de la Familia Eph/fisiología , Animales , Animales Recién Nacidos , Astrocitos/citología , Células Cultivadas , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuroglía/citología , Neuronas/citología , RatasRESUMEN
The primary cilium is a microtubule-enriched cell-communication organelle that participates in mechanisms controlling tissue development and maintenance, including cerebellar architecture. Centrosomal protein of 290 kDa (CEP290) is a protein important for centrosomal function and ciliogenesis. Mutations in CEP290 have been linked to a group of multi-organ disorders - termed ciliopathies. The neurophysiological deficits observed in ciliopathies are sometimes associated with the progression of autistic traits. Here, the cellular function of two rare variants of CEP290 identified from recent exome sequencing of autistic individuals are investigated. Cells expressing Cep290 carrying the missense mutation R1747Q in mouse exhibited a defective Sonic hedgehog (Shh) signalling response, mislocalisation of the Shh receptor Smoothened (Smo), and dysregulation of ciliary protein mobility, which ultimately disrupted the proliferation of cerebellar granule progenitors (CGPs). This data was furthermore corroborated in an autism patient-derived iPSC line harbouring the R1746Q rare CEP290 variant. Evidence from this study suggests that the R1746Q mutation interferes with the function of CEP290 to maintain the ciliary diffusion barrier and disrupts the integrity of the molecular composition in the primary cilium, which may contribute to alterations in neuroarchitecture.
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Antígenos de Neoplasias/genética , Proteínas de Ciclo Celular/genética , Cilios/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas Hedgehog/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Mutación Missense , Animales , Antígenos de Neoplasias/metabolismo , Trastorno Autístico/genética , Proteínas de Ciclo Celular/metabolismo , Proliferación Celular , Cilios/patología , Proteínas del Citoesqueleto/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células 3T3 NIH , Receptor Patched-1/metabolismo , Transducción de Señal/genética , Receptor Smoothened/metabolismoRESUMEN
Potentially pathogenic alterations have been identified in individuals with autism spectrum disorders (ASDs) within a variety of key neurodevelopment genes. While this hints at a common ASD molecular etiology, gaps persist in our understanding of the neurodevelopmental mechanisms impacted by genetic variants enriched in ASD patients. Induced pluripotent stem cells (iPSCs) can model neurodevelopment in vitro, permitting the characterization of pathogenic mechanisms that manifest during corticogenesis. Taking this approach, we examined the transcriptional differences between iPSC-derived cortical neurons from patients with idiopathic ASD and unaffected controls over a 135-day course of neuronal differentiation. Our data show ASD-specific misregulation of genes involved in neuronal differentiation, axon guidance, cell migration, DNA and RNA metabolism, and neural region patterning. Furthermore, functional analysis revealed defects in neuronal migration and electrophysiological activity, providing compelling support for the transcriptome analysis data. This study reveals important and functionally validated insights into common processes altered in early neuronal development and corticogenesis and may contribute to ASD pathogenesis.
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Trastorno Autístico/genética , Trastorno Autístico/patología , Perfilación de la Expresión Génica , Neuronas/metabolismo , Adolescente , Señalización del Calcio , Diferenciación Celular , Movimiento Celular , Niño , Preescolar , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Neuronas/patología , Sinapsis/patología , Adulto JovenRESUMEN
Although a number of in vitro disease models have been developed using hiPSCs, one limitation is that these two-dimensional (2-D) systems may not represent the underlying cytoarchitectural and functional complexity of the affected individuals carrying suspected disease variants. Conventional 2-D models remain incomplete representations of in vivo-like structures and do not adequately capture the complexity of the brain. Thus, there is an emerging need for more 3-D hiPSC-based models that can better recapitulate the cellular interactions and functions seen in an in vivo system. Here we report a protocol to develop a 3-D system from undifferentiated hiPSCs based on the serum free embryoid body (SFEB). This 3-D model mirrors aspects of a developing ventralized neocortex and allows for studies into functions integral to living neural cells and intact tissue such as migration, connectivity, communication, and maturation. Specifically, we demonstrate that the SFEBs using our protocol can be interrogated using physiologically relevant and high-content cell based assays such as calcium imaging, and multi-electrode array (MEA) recordings without cryosectioning. In the case of MEA recordings, we demonstrate that SFEBs increase both spike activity and network-level bursting activity during long-term culturing. This SFEB protocol provides a robust and scalable system for the study of developing network formation in a 3-D model that captures aspects of early cortical development.
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Técnicas de Cultivo de Célula/métodos , Técnicas de Cocultivo/métodos , Cuerpos Embrioides/fisiología , Células Madre Pluripotentes Inducidas/fisiología , Animales , Diferenciación Celular , Humanos , Ratones , Neuronas/fisiologíaRESUMEN
Variants in CNTNAP2, a member of the neurexin family of genes that function as cell adhesion molecules, have been associated with multiple neuropsychiatric conditions such as schizophrenia, autism spectrum disorder and intellectual disability; animal studies indicate a role for CNTNAP2 in axon guidance, dendritic arborization and synaptogenesis. We previously reprogrammed fibroblasts from a family trio consisting of two carriers of heterozygous intragenic CNTNAP2 deletions into human induced pluripotent stem cells (hiPSCs) and described decreased migration in the neural progenitor cells (NPCs) differentiated from the affected CNTNAP2 carrier in this trio. Here, we report the effect of this heterozygous intragenic deletion in CNTNAP2 on global gene expression and neuronal activity in the same cohort. Our findings suggest that heterozygous CNTNAP2 deletions affect genes involved in neuronal development and neuronal activity; however, these data reflect only one family trio and therefore more deletion carriers, with a variety of genetic backgrounds, will be needed to understand the molecular mechanisms underlying CNTNAP2 deletions.
RESUMEN
Autism Spectrum Disorder (ASD) is a behaviorally defined neurodevelopmental condition. Symptoms of ASD cover the spectrum from mild qualitative differences in social interaction to severe communication and social and behavioral challenges that require lifelong support. Attempts at understanding the pathophysiology of ASD have been hampered by a multifactorial etiology that stretches the limits of current behavioral and cell based models. Recent progress has implicated numerous autism-risk genes but efforts to gain a better understanding of the underlying biological mechanisms have seen slow progress. This is in part due to lack of appropriate models for complete molecular and pharmacological studies. The advent of induced pluripotent stem cells (iPSC) has reinvigorated efforts to establish more complete model systems that more reliably identify molecular pathways and predict effective drug targets and candidates in ASD. iPSCs are particularly appealing because they can be derived from human patients and controls for research purposes and provide a technology for the development of a personalized treatment regimen for ASD patients. The pluripotency of iPSCs allow them to be reprogrammed into a number of CNS cell types and phenotypically screened across many patients. This quality is already being exploited in protocols to generate 2-dimensional (2-D) and three-dimensional (3-D) models of neurons and developing brain structures. iPSC models make powerful platforms that can be interrogated using electrophysiology, gene expression studies, and other cell-based quantitative assays. iPSC technology has limitations but when combined with other model systems has great potential for helping define the underlying pathophysiology of ASD. Autism Res 2016, 9: 513-535. © 2015 International Society for Autism Research, Wiley Periodicals, Inc.
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Trastorno del Espectro Autista/genética , Trastorno del Espectro Autista/fisiopatología , Genómica/métodos , Células Madre Pluripotentes Inducidas , Animales , Trastorno del Espectro Autista/terapia , Humanos , Modelos BiológicosRESUMEN
Many protocols have been designed to differentiate human embryonic stem cells (ESCs) and human induced pluripotent stem cells (iPSCs) into neurons. Despite the relevance of electrophysiological properties for proper neuronal function, little is known about the evolution over time of important neuronal electrophysiological parameters in iPSC-derived neurons. Yet, understanding the development of basic electrophysiological characteristics of iPSC-derived neurons is critical for evaluating their usefulness in basic and translational research. Therefore, we analyzed the basic electrophysiological parameters of forebrain neurons differentiated from human iPSCs, from day 31 to day 55 after the initiation of neuronal differentiation. We assayed the developmental progression of various properties, including resting membrane potential, action potential, sodium and potassium channel currents, somatic calcium transients and synaptic activity. During the maturation of iPSC-derived neurons, the resting membrane potential became more negative, the expression of voltage-gated sodium channels increased, the membrane became capable of generating action potentials following adequate depolarization and, at day 48-55, 50% of the cells were capable of firing action potentials in response to a prolonged depolarizing current step, of which 30% produced multiple action potentials. The percentage of cells exhibiting miniature excitatory post-synaptic currents increased over time with a significant increase in their frequency and amplitude. These changes were associated with an increase of Ca2+ transient frequency. Co-culturing iPSC-derived neurons with mouse glial cells enhanced the development of electrophysiological parameters as compared to pure iPSC-derived neuronal cultures. This study demonstrates the importance of properly evaluating the electrophysiological status of the newly generated neurons when using stem cell technology, as electrophysiological properties of iPSC-derived neurons mature over time.
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Diferenciación Celular , Células Madre Pluripotentes Inducidas/citología , Neuronas/citología , Neuronas/fisiología , Animales , Calcio/metabolismo , Técnicas de Cocultivo , Fenómenos Electrofisiológicos , Humanos , Inmunofenotipificación , Ratones , Neuroglía , Técnicas de Placa-Clamp , Potenciales Sinápticos , Transmisión Sináptica , Factores de TiempoRESUMEN
Presenilin 1 (PSEN1) encodes the catalytic subunit of γ-secretase, and PSEN1 mutations are the most common cause of early onset familial Alzheimer's disease (FAD). In order to elucidate pathways downstream of PSEN1, we characterized neural progenitor cells (NPCs) derived from FAD mutant PSEN1 subjects. Thus, we generated induced pluripotent stem cells (iPSCs) from affected and unaffected individuals from two families carrying PSEN1 mutations. PSEN1 mutant fibroblasts, and NPCs produced greater ratios of Aß42 to Aß40 relative to their control counterparts, with the elevated ratio even more apparent in PSEN1 NPCs than in fibroblasts. Molecular profiling identified 14 genes differentially-regulated in PSEN1 NPCs relative to control NPCs. Five of these targets showed differential expression in late onset AD/Intermediate AD pathology brains. Therefore, in our PSEN1 iPSC model, we have reconstituted an essential feature in the molecular pathogenesis of FAD, increased generation of Aß42/40, and have characterized novel expression changes.