RESUMEN
Over the years, techniques have been developed to culture and assemble neurons, which brought us closer to creating neuronal circuits that functionally and structurally mimic parts of the brain. Starting with primary culture of neurons, preparations of neuronal culture have advanced substantially. Development of stem cell research and brain organoids has opened a new path for generating three-dimensional human neural circuits. Along with the progress in biology, engineering technologies advanced and paved the way for construction of neural circuit structures. In this article, we overview research progress and discuss perspective of in vitro neural circuits and their ability and potential to acquire functions. Construction of in vitro neural circuits with complex higher-order functions would be achieved by converging development in diverse major disciplines including neuroscience, stem cell biology, tissue engineering, electrical engineering and computer science.
Asunto(s)
Neuronas , Neurociencias , Encéfalo/fisiología , Humanos , Neuronas/fisiología , Células MadreRESUMEN
Current gene silencing tools based on RNA interference (RNAi) or, more recently, clustered regularly interspaced short palindromic repeats (CRISPR)âCas13 systems have critical drawbacks, such as off-target effects (RNAi) or collateral mRNA cleavage (CRISPRâCas13). Thus, a more specific method of gene knockdown is needed. Here, we develop CRISPRδ, an approach for translational silencing, harnessing catalytically inactive Cas13 proteins (dCas13). Owing to its tight association with mRNA, dCas13 serves as a physical roadblock for scanning ribosomes during translation initiation and does not affect mRNA stability. Guide RNAs covering the start codon lead to the highest efficacy regardless of the translation initiation mechanism: cap-dependent, internal ribosome entry site (IRES)-dependent, or repeat-associated non-AUG (RAN) translation. Strikingly, genome-wide ribosome profiling reveals the ultrahigh gene silencing specificity of CRISPRδ. Moreover, the fusion of a translational repressor to dCas13 further improves the performance. Our method provides a framework for translational repression-based gene silencing in eukaryotes.
Asunto(s)
ARN Guía de Sistemas CRISPR-Cas , Ribosomas , Animales , Codón Iniciador/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Silenciador del Gen , Biosíntesis de Proteínas/genética , Iniciación de la Cadena Peptídica Traduccional , Mamíferos/genéticaRESUMEN
An inter-regional cortical tract is one of the most fundamental architectural motifs that integrates neural circuits to orchestrate and generate complex functions of the human brain. To understand the mechanistic significance of inter-regional projections on development of neural circuits, we investigated an in vitro neural tissue model for inter-regional connections, in which two cerebral organoids are connected with a bundle of reciprocally extended axons. The connected organoids produced more complex and intense oscillatory activity than conventional or directly fused cerebral organoids, suggesting the inter-organoid axonal connections enhance and support the complex network activity. In addition, optogenetic stimulation of the inter-organoid axon bundles could entrain the activity of the organoids and induce robust short-term plasticity of the macroscopic circuit. These results demonstrated that the projection axons could serve as a structural hub that boosts functionality of the organoid-circuits. This model could contribute to further investigation on development and functions of macroscopic neuronal circuits in vitro.
Asunto(s)
Axones , Neuronas , Humanos , Axones/fisiología , Neuronas/fisiología , Organoides/fisiología , EncéfaloRESUMEN
Ribosome profiling, which is based on deep sequencing of ribosome footprints, has served as a powerful tool for elucidating the regulatory mechanism of protein synthesis. However, the current method has substantial issues: contamination by rRNAs and the lack of appropriate methods to measure ribosome numbers in transcripts. Here, we overcome these hurdles through the development of "Ribo-FilterOut", which is based on the separation of footprints from ribosome subunits by ultrafiltration, and "Ribo-Calibration", which relies on external spike-ins of stoichiometrically defined mRNA-ribosome complexes. A combination of these approaches estimates the number of ribosomes on a transcript, the translation initiation rate, and the overall number of translation events before its decay, all in a genome-wide manner. Moreover, our method reveals the allocation of ribosomes under heat shock stress, during aging, and across cell types. Our strategy of modified ribosome profiling measures kinetic and stoichiometric parameters of cellular translation across the transcriptome.
Asunto(s)
Biosíntesis de Proteínas , ARN Mensajero , Ribosomas , Ribosomas/metabolismo , ARN Mensajero/metabolismo , ARN Mensajero/genética , Humanos , Transcriptoma , ARN Ribosómico/metabolismo , ARN Ribosómico/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Calibración , Respuesta al Choque Térmico/genética , Perfilado de RibosomasRESUMEN
Mutations in ARID1B, a member of the mSWI/SNF complex, cause severe neurodevelopmental phenotypes with elusive mechanisms in humans. The most common structural abnormality in the brain of ARID1B patients is agenesis of the corpus callosum (ACC), characterized by the absence of an interhemispheric white matter tract that connects distant cortical regions. Here, we find that neurons expressing SATB2, a determinant of callosal projection neuron (CPN) identity, show impaired maturation in ARID1B+/- neural organoids. Molecularly, a reduction in chromatin accessibility of genomic regions targeted by TCF-like, NFI-like, and ARID-like transcription factors drives the differential expression of genes required for corpus callosum (CC) development. Through an in vitro model of the CC tract, we demonstrate that this transcriptional dysregulation impairs the formation of long-range axonal projections, causing structural underconnectivity. Our study uncovers new functions of the mSWI/SNF during human corticogenesis, identifying cell-autonomous axonogenesis defects in SATB2+ neurons as a cause of ACC in ARID1B patients.
Asunto(s)
Axones , Cuerpo Calloso , Proteínas de Unión al ADN , Organoides , Factores de Transcripción , Humanos , Cuerpo Calloso/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Organoides/metabolismo , Axones/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión a la Región de Fijación a la Matriz/metabolismo , Proteínas de Unión a la Región de Fijación a la Matriz/genética , Transcripción Genética , Neuronas/metabolismoRESUMEN
Degeneration of axons is characteristic of many devastating diseases including amyotrophic lateral sclerosis (ALS). However, lack of an in vitro neuronal culture system that mimics damages on nerves and axonal tracts hampered development of effective treatments. Here, we describe a method to model degeneration of motor neuron axons using motor nerve organoids that are formed with human induced pluripotent stem cells. In this protocol, motor neuron axon degeneration can be rapidly induced with chemical damages. Neuroprotective effects of compounds can be examined using the degenerated axons. This motor neuron axon bundle degeneration model should facilitate future screening for drugs against diseases affecting axon fascicles.
Asunto(s)
Esclerosis Amiotrófica Lateral , Células Madre Pluripotentes Inducidas , Tejido Nervioso , Humanos , Neuronas Motoras , OrganoidesRESUMEN
Melanocytes are surrounded by diverse cells, including sensory neurons in our skin, but their interaction and functional importance have been poorly investigated. In this study, we find that melanocytes and nociceptive neurons contact more in human skin color patch tissue than control. Co-culture with human iPSC-derived sensory neurons significantly induces morphogenesis and pigmentation of human melanocytes. To reveal melanocyte-stimulating factors secreted from neurons, we perform proteomic analyses and identify RGMB in the sensory neuron-conditioned medium. RGMB protein induces morphogenesis and melanin production of melanocytes, demonstrating that RGMB is a melanocyte-stimulating factor released from sensory neurons. Transcriptome analysis suggests that the melanosome transport machinery can be controlled by RGMB, leading us to identify the vesicle production response of melanocytes upon RGMB treatment. This study discovers a role of sensory neurons in modulating multiple aspects of human melanocytes through secretion of a key factor: RGMB.
Asunto(s)
Melaninas , Proteómica , Medios de Cultivo Condicionados/farmacología , Humanos , Melaninas/metabolismo , Melanocitos/metabolismo , Células Receptoras Sensoriales/metabolismoRESUMEN
Light-based therapy is an emerging treatment for skin cancer, which has received increased attention due to its drug-free and non-invasive approach. However, the limitation of current light therapy methods is the inability for light to penetrate the skin and reach deep lesions. As such, we have developed a polylactic acid (PLA) microneedles array as a novel light transmission platform to perform in vitro evaluation regarding the effect of light therapy on skin cancer. For the first time, we designed and fabricated a microneedle array system with a height fixation device that can be installed in a cell culture dish and an LED array for blue light irradiation. The effect of the blue light combined with the microneedles on cell apoptosis was evaluated using B16F10 melanoma cells and analyzed by Hoechst staining. Our results demonstrate that blue light can be transmitted by microneedles to skin cells and effectively affect cell viability.
RESUMEN
A fascicle of axons is one of the major structural motifs observed in the nervous system. Disruption of axon fascicles could cause developmental and neurodegenerative diseases. Although numerous studies of axons have been conducted, our understanding of formation and dysfunction of axon fascicles is still limited due to the lack of robust three-dimensional in vitro models. Here, we describe a step-by-step protocol for the rapid generation of a motor nerve organoid (MNO) from human induced pluripotent stem (iPS) cells in a microfluidic-based tissue culture chip. First, fabrication of chips used for the method is described. From human iPS cells, a motor neuron spheroid (MNS) is formed. Next, the differentiated MNS is transferred into the chip. Thereafter, axons spontaneously grow out of the spheroid and assemble into a fascicle within a microchannel equipped in the chip, which generates an MNO tissue carrying a bundle of axons extended from the spheroid. For the downstream analysis, MNOs can be taken out of the chip to be fixed for morphological analyses or dissected for biochemical analyses, as well as calcium imaging and multi-electrode array recordings. MNOs generated with this protocol can facilitate drug testing and screening and can contribute to understanding of mechanisms underlying development and diseases of axon fascicles.
Asunto(s)
Neuronas Motoras/fisiología , Organoides/fisiología , Animales , Calcio/metabolismo , Diferenciación Celular , Dimetilpolisiloxanos/química , Electrodos , Compuestos Epoxi/química , Humanos , Células Madre Pluripotentes Inducidas/citología , Microfluídica , Polímeros/química , Técnicas de Cultivo de TejidosRESUMEN
Cerebral tracts connect separated regions within a brain and serve as fundamental structures that support integrative brain functions. However, understanding the mechanisms of cerebral tract development, macro-circuit formation, and related disorders has been hampered by the lack of an in vitro model. Here, we developed a human stem cell-derived model of cerebral tracts, which is composed of two spheroids of cortical neurons and a robust fascicle of axons linking these spheroids reciprocally. In a microdevice, two spheroids of cerebral neurons extended axons into a microchannel between the spheroids and spontaneously formed an axon fascicle, mimicking a cerebral tract. We found that the formation of axon fascicle was significantly promoted when two spheroids extended axons toward each other compared with axons extended from only one spheroid. The two spheroids were able to communicate electrically through the axon fascicle. This model tissue could facilitate studies of cerebral tract development and diseases.