RESUMO
Our understanding of how the brain assembles its circuits and how this goes awry in disease remains incomplete. There has been great progress in generating human neurons from stem cells in vitro and, more recently, in constructing circuits with human cells in vivo by transplantation. Here, I highlight approaches, promises, and challenges of growing human neurons in living animals to study human development and disease.
Assuntos
Encéfalo , Neurônios , Animais , Humanos , Células-TroncoRESUMO
The construction of the human nervous system is a distinctly complex although highly regulated process. Human tissue inaccessibility has impeded a molecular understanding of the developmental specializations from which our unique cognitive capacities arise. A confluence of recent technological advances in genomics and stem cell-based tissue modeling is laying the foundation for a new understanding of human neural development and dysfunction in neuropsychiatric disease. Here, we review recent progress on uncovering the cellular and molecular principles of human brain organogenesis in vivo as well as using organoids and assembloids in vitro to model features of human evolution and disease.
Assuntos
Transtorno do Espectro Autista/metabolismo , Encéfalo/embriologia , Encéfalo/crescimento & desenvolvimento , Epilepsia/metabolismo , Neurogênese/fisiologia , Esquizofrenia/metabolismo , Animais , Transtorno do Espectro Autista/genética , Encéfalo/metabolismo , Epilepsia/genética , Humanos , Mutação , Neurônios/citologia , Neurônios/metabolismo , Organoides/embriologia , Organoides/crescimento & desenvolvimento , Esquizofrenia/genéticaRESUMO
Genetic perturbations of cortical development can lead to neurodevelopmental disease, including autism spectrum disorder (ASD). To identify genomic regions crucial to corticogenesis, we mapped the activity of gene-regulatory elements generating a single-cell atlas of gene expression and chromatin accessibility both independently and jointly. This revealed waves of gene regulation by key transcription factors (TFs) across a nearly continuous differentiation trajectory, distinguished the expression programs of glial lineages, and identified lineage-determining TFs that exhibited strong correlation between linked gene-regulatory elements and expression levels. These highly connected genes adopted an active chromatin state in early differentiating cells, consistent with lineage commitment. Base-pair-resolution neural network models identified strong cell-type-specific enrichment of noncoding mutations predicted to be disruptive in a cohort of ASD individuals and identified frequently disrupted TF binding sites. This approach illustrates how cell-type-specific mapping can provide insights into the programs governing human development and disease.
Assuntos
Córtex Cerebral/embriologia , Cromatina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Análise de Célula Única , Astrócitos/citologia , Diferenciação Celular , Linhagem da Célula/genética , Análise por Conglomerados , Aprendizado Profundo , Epigênese Genética , Lógica Fuzzy , Glutamatos/metabolismo , Humanos , Mutação/genética , Neurônios/metabolismo , Sequências Reguladoras de Ácido Nucleico/genéticaRESUMO
Timothy syndrome (TS) is a severe, multisystem disorder characterized by autism, epilepsy, long-QT syndrome and other neuropsychiatric conditions1. TS type 1 (TS1) is caused by a gain-of-function variant in the alternatively spliced and developmentally enriched CACNA1C exon 8A, as opposed to its counterpart exon 8. We previously uncovered several phenotypes in neurons derived from patients with TS1, including delayed channel inactivation, prolonged depolarization-induced calcium rise, impaired interneuron migration, activity-dependent dendrite retraction and an unanticipated persistent expression of exon 8A2-6. We reasoned that switching CACNA1C exon utilization from 8A to 8 would represent a potential therapeutic strategy. Here we developed antisense oligonucleotides (ASOs) to effectively decrease the inclusion of exon 8A in human cells both in vitro and, following transplantation, in vivo. We discovered that the ASO-mediated switch from exon 8A to 8 robustly rescued defects in patient-derived cortical organoids and migration in forebrain assembloids. Leveraging a transplantation platform previously developed7, we found that a single intrathecal ASO administration rescued calcium changes and in vivo dendrite retraction of patient neurons, suggesting that suppression of CACNA1C exon 8A expression is a potential treatment for TS1. Broadly, these experiments illustrate how a multilevel, in vivo and in vitro stem cell model-based approach can identify strategies to reverse disease-relevant neural pathophysiology.
Assuntos
Transtorno Autístico , Síndrome do QT Longo , Oligonucleotídeos Antissenso , Sindactilia , Animais , Feminino , Humanos , Masculino , Camundongos , Processamento Alternativo/efeitos dos fármacos , Processamento Alternativo/genética , Transtorno Autístico/tratamento farmacológico , Transtorno Autístico/genética , Cálcio/metabolismo , Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio Tipo L/genética , Movimento Celular/efeitos dos fármacos , Dendritos/metabolismo , Éxons/genética , Síndrome do QT Longo/tratamento farmacológico , Síndrome do QT Longo/genética , Neurônios/metabolismo , Neurônios/efeitos dos fármacos , Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos Antissenso/uso terapêutico , Organoides/efeitos dos fármacos , Organoides/metabolismo , Prosencéfalo/metabolismo , Prosencéfalo/citologia , Sindactilia/tratamento farmacológico , Sindactilia/genética , Interneurônios/citologia , Interneurônios/efeitos dos fármacosRESUMO
The assembly of cortical circuits involves the generation and migration of interneurons from the ventral to the dorsal forebrain1-3, which has been challenging to study at inaccessible stages of late gestation and early postnatal human development4. Autism spectrum disorder and other neurodevelopmental disorders (NDDs) have been associated with abnormal cortical interneuron development5, but which of these NDD genes affect interneuron generation and migration, and how they mediate these effects remains unknown. We previously developed a platform to study interneuron development and migration in subpallial organoids and forebrain assembloids6. Here we integrate assembloids with CRISPR screening to investigate the involvement of 425 NDD genes in human interneuron development. The first screen aimed at interneuron generation revealed 13 candidate genes, including CSDE1 and SMAD4. We subsequently conducted an interneuron migration screen in more than 1,000 forebrain assembloids that identified 33 candidate genes, including cytoskeleton-related genes and the endoplasmic reticulum-related gene LNPK. We discovered that, during interneuron migration, the endoplasmic reticulum is displaced along the leading neuronal branch before nuclear translocation. LNPK deletion interfered with this endoplasmic reticulum displacement and resulted in abnormal migration. These results highlight the power of this CRISPR-assembloid platform to systematically map NDD genes onto human development and reveal disease mechanisms.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Transtornos do Neurodesenvolvimento , Feminino , Humanos , Recém-Nascido , Gravidez , Movimento Celular/genética , Sistemas CRISPR-Cas/genética , Interneurônios/citologia , Interneurônios/metabolismo , Interneurônios/patologia , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/patologia , Organoides/citologia , Organoides/embriologia , Organoides/crescimento & desenvolvimento , Organoides/metabolismo , Organoides/patologia , Retículo Endoplasmático/metabolismo , Prosencéfalo/citologia , Prosencéfalo/embriologia , Prosencéfalo/crescimento & desenvolvimento , Prosencéfalo/metabolismo , Prosencéfalo/patologia , Transporte Ativo do Núcleo CelularRESUMO
Self-organizing neural organoids represent a promising in vitro platform with which to model human development and disease1-5. However, organoids lack the connectivity that exists in vivo, which limits maturation and makes integration with other circuits that control behaviour impossible. Here we show that human stem cell-derived cortical organoids transplanted into the somatosensory cortex of newborn athymic rats develop mature cell types that integrate into sensory and motivation-related circuits. MRI reveals post-transplantation organoid growth across multiple stem cell lines and animals, whereas single-nucleus profiling shows progression of corticogenesis and the emergence of activity-dependent transcriptional programs. Indeed, transplanted cortical neurons display more complex morphological, synaptic and intrinsic membrane properties than their in vitro counterparts, which enables the discovery of defects in neurons derived from individuals with Timothy syndrome. Anatomical and functional tracings show that transplanted organoids receive thalamocortical and corticocortical inputs, and in vivo recordings of neural activity demonstrate that these inputs can produce sensory responses in human cells. Finally, cortical organoids extend axons throughout the rat brain and their optogenetic activation can drive reward-seeking behaviour. Thus, transplanted human cortical neurons mature and engage host circuits that control behaviour. We anticipate that this approach will be useful for detecting circuit-level phenotypes in patient-derived cells that cannot otherwise be uncovered.
Assuntos
Vias Neurais , Organoides , Animais , Animais Recém-Nascidos , Transtorno Autístico , Humanos , Síndrome do QT Longo , Motivação , Neurônios/fisiologia , Optogenética , Organoides/citologia , Organoides/inervação , Organoides/transplante , Ratos , Recompensa , Córtex Somatossensorial/citologia , Córtex Somatossensorial/fisiologia , Células-Tronco/citologia , SindactiliaRESUMO
Self-organizing three-dimensional cellular models derived from human pluripotent stem cells or primary tissue have great potential to provide insights into how the human nervous system develops, what makes it unique and how disorders of the nervous system arise, progress and could be treated. Here, to facilitate progress and improve communication with the scientific community and the public, we clarify and provide a basic framework for the nomenclature of human multicellular models of nervous system development and disease, including organoids, assembloids and transplants.
Assuntos
Consenso , Sistema Nervoso , Organoides , Terminologia como Assunto , Humanos , Modelos Biológicos , Sistema Nervoso/citologia , Sistema Nervoso/patologia , Organoides/citologia , Organoides/patologia , Células-Tronco Pluripotentes/citologiaRESUMO
Neurotransmitters play essential roles in regulating neural circuit dynamics both in the central nervous system as well as at the peripheral, including the gastrointestinal tract1-3. Their real-time monitoring will offer critical information for understanding neural function and diagnosing disease1-3. However, bioelectronic tools to monitor the dynamics of neurotransmitters in vivo, especially in the enteric nervous systems, are underdeveloped. This is mainly owing to the limited availability of biosensing tools that are capable of examining soft, complex and actively moving organs. Here we introduce a tissue-mimicking, stretchable, neurochemical biological interface termed NeuroString, which is prepared by laser patterning of a metal-complexed polyimide into an interconnected graphene/nanoparticle network embedded in an elastomer. NeuroString sensors allow chronic in vivo real-time, multichannel and multiplexed monoamine sensing in the brain of behaving mouse, as well as measuring serotonin dynamics in the gut without undesired stimulations and perturbing peristaltic movements. The described elastic and conformable biosensing interface has broad potential for studying the impact of neurotransmitters on gut microbes, brain-gut communication and may ultimately be extended to biomolecular sensing in other soft organs across the body.
Assuntos
Encéfalo , Sistema Nervoso Entérico , Trato Gastrointestinal , Neurotransmissores , Animais , Técnicas Biossensoriais , Encéfalo/metabolismo , Eixo Encéfalo-Intestino , Elastômeros , Sistema Nervoso Entérico/metabolismo , Trato Gastrointestinal/inervação , Trato Gastrointestinal/fisiologia , Grafite , Lasers , Camundongos , Nanopartículas , Neurotransmissores/análise , Serotonina/análiseRESUMO
Among primates, humans display a unique trajectory of development that is responsible for the many traits specific to our species. However, the inaccessibility of primary human and chimpanzee tissues has limited our ability to study human evolution. Comparative in vitro approaches using primate-derived induced pluripotent stem cells have begun to reveal species differences on the cellular and molecular levels1,2. In particular, brain organoids have emerged as a promising platform to study primate neural development in vitro3-5, although cross-species comparisons of organoids are complicated by differences in developmental timing and variability of differentiation6,7. Here we develop a new platform to address these limitations by fusing human and chimpanzee induced pluripotent stem cells to generate a panel of tetraploid hybrid stem cells. We applied this approach to study species divergence in cerebral cortical development by differentiating these cells into neural organoids. We found that hybrid organoids provide a controlled system for disentangling cis- and trans-acting gene-expression divergence across cell types and developmental stages, revealing a signature of selection on astrocyte-related genes. In addition, we identified an upregulation of the human somatostatin receptor 2 gene (SSTR2), which regulates neuronal calcium signalling and is associated with neuropsychiatric disorders8,9. We reveal a human-specific response to modulation of SSTR2 function in cortical neurons, underscoring the potential of this platform for elucidating the molecular basis of human evolution.
Assuntos
Fusão Celular , Regulação da Expressão Gênica no Desenvolvimento , Células Híbridas/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Neurogênese/genética , Alelos , Animais , Astrócitos/citologia , Sinalização do Cálcio , Córtex Cerebral/citologia , Feminino , Humanos , Masculino , Neurônios/citologia , Organoides/citologia , Pan troglodytes/genética , Receptores de Somatostatina/genética , Reprodutibilidade dos Testes , Transcrição GênicaRESUMO
Pluripotent stem cells show a remarkable ability to self-organize and differentiate in vitro in three-dimensional aggregates, known as organoids or organ spheroids, and to recapitulate aspects of human brain development and function. Region-specific 3D brain cultures can be derived from any individual and assembled to model complex cell-cell interactions and to generate circuits in human brain assembloids. Here I discuss how this approach can be used to understand unique features of the human brain and to gain insights into neuropsychiatric disorders. In addition, I consider the challenges faced by researchers in further improving and developing methods to probe and manipulate patient-derived 3D brain cultures.
Assuntos
Encéfalo/citologia , Técnicas de Cultura de Órgãos/métodos , Evolução Biológica , Encéfalo/embriologia , Encéfalo/crescimento & desenvolvimento , Diferenciação Celular , Humanos , Modelos Biológicos , Neurônios/citologia , Técnicas de Cultura de Órgãos/normas , Organogênese , Células-Tronco Pluripotentes/citologia , Controle de QualidadeRESUMO
The differentiation of pluripotent stem cells in three-dimensional cultures can recapitulate key aspects of brain development, but protocols are prone to variable results. Here we differentiated multiple human pluripotent stem cell lines for over 100 d using our previously developed approach to generate brain-region-specific organoids called cortical spheroids and, using several assays, found that spheroid generation was highly reliable and consistent. We anticipate the use of this approach for large-scale differentiation experiments and disease modeling.
Assuntos
Organoides/crescimento & desenvolvimento , Engenharia Tecidual , Linhagem Celular , Humanos , Células-Tronco Pluripotentes/citologia , Prosencéfalo/fisiologia , Reprodutibilidade dos Testes , Análise de Sequência de RNA , Análise de Célula Única/métodosAssuntos
Células-Tronco Embrionárias , Prosencéfalo , Animais , Blastocisto , Diferenciação Celular , Camundongos , OrganogêneseAssuntos
Encéfalo/fisiologia , Estado de Consciência/fisiologia , Experimentação Humana/ética , Modelos Biológicos , Neurociências/ética , Organoides/fisiologia , Animais , Encéfalo/citologia , Quimera , Morte , Embrião de Mamíferos/citologia , Xenoenxertos , Humanos , Técnicas In Vitro , Consentimento Livre e Esclarecido/ética , Camundongos , Organoides/citologia , Organoides/crescimento & desenvolvimento , Propriedade/ética , Ratos , SuínosRESUMO
Human neural organoids represent promising models for studying neural function; however, organoids grown in vitro lack certain microenvironments and sensory inputs that are thought to be essential for maturation. The transplantation of patient-derived neural organoids into animal hosts helps overcome some of these limitations and offers an approach for neural organoid maturation and circuit integration. Here, we describe a method for transplanting human stem cell-derived cortical organoids (hCOs) into the somatosensory cortex of newborn rats. The differentiation of human induced pluripotent stem cells into hCOs occurs over 30-60 days, and the transplantation procedure itself requires ~0.5-1 hours per animal. The use of neonatal hosts provides a developmentally appropriate stage for circuit integration and allows the generation and experimental manipulation of a unit of human neural tissue within the cortex of a living animal host. After transplantation, animals can be maintained for hundreds of days, and transplanted hCO growth can be monitored by using brain magnetic resonance imaging. We describe the assessment of human neural circuit function in vivo by monitoring genetically encoded calcium responses and extracellular activity. To demonstrate human neuron-host functional integration, we also describe a procedure for engaging host neural circuits and for modulating animal behavior by using an optogenetic behavioral training paradigm. The transplanted human neurons can then undergo ex vivo characterization across modalities including dendritic morphology reconstruction, single-nucleus transcriptomics, optogenetic manipulation and electrophysiology. This approach may enable the discovery of cellular phenotypes from patient-derived cells and uncover mechanisms that contribute to human brain evolution from previously inaccessible developmental stages.
RESUMO
Realizing the full potential of organoids and assembloids to model neural development and disease will require improved methods for long-term, minimally invasive recording of electrical activity. Current technologies, such as patch clamp, penetrating microelectrodes, planar electrode arrays and substrate-attached flexible electrodes, do not allow chronic recording of organoids in suspension, which is necessary to preserve architecture. Inspired by kirigami art, we developed flexible electronics that transition from a two-dimensional to a three-dimensional basket-like configuration with either spiral or honeycomb patterns to accommodate the long-term culture of organoids in suspension. Here we show that this platform, named kirigami electronics (KiriE), integrates with and enables chronic recording of cortical organoids for up to 120 days while preserving their morphology, cytoarchitecture and cell composition. We demonstrate integration of KiriE with optogenetic and pharmacological manipulation and modeling phenotypes related to a genetic disease. Moreover, KiriE can capture corticostriatal connectivity in assembloids following optogenetic stimulation. Thus, KiriE will enable investigation of disease and activity patterns underlying nervous system assembly.
RESUMO
BACKGROUND: CACNA1C encodes the voltage-gated L-type calcium channel CaV1.2. A specific gain-of-function pathogenic variant in CACNA1C causes Timothy syndrome type 1 (TS1) with cardiac long QT syndrome, syndactyly, and neuropsychiatric symptoms. Our previous work found that the TS1 mutation alters neuronal activity-dependent signaling and interneuron migration. Recent case series highlighted a broader spectrum of CACNA1C-related disorder (CRD) that includes isolated cardiac disease, isolated neurologic deficits, and TS, but it is unknown how the clinical presentation of other CRD variants relates to neural defects. We surveyed individuals with CRD to define the neuropsychiatric and developmental phenotype in an effort to guide future research into the role of calcium channels in neural development. METHODS: Caregivers of and individuals with CRD completed an online survey of pre- and perinatal events, cardiac events, developmental milestones, neuropsychiatric symptoms, and neuropsychiatric diagnoses. Multiple Mann-Whitney tests were used for comparison of categorical values and Fisher exact test for comparison of categorical variables between participants with and without cardiac arrhythmia. RESULTS: Twenty-four participants with germline CACNA1C variants including TS1 completed the survey. The most common neuropsychiatric symptoms and/or diagnoses were developmental delay in 92%, incoordination in 71%, hypotonia in 67%, autism spectrum disorder in 50% (autistic features in 92%), seizures in 37.5%, and attention-deficit/hyperactivity disorder in 21% of participants. There were no significant differences in symptoms between participants with and without arrhythmia. CONCLUSIONS: In our CRD cohort, there was an increased prevalence of multiple neuropsychiatric symptoms compared with the general population. These findings indicate the key role of CaV1.2 in brain development and the clinical importance of screening and therapeutically addressing neuropsychiatric symptoms in all individuals with CRD. Future directions include deep phenotyping of neuropsychiatric symptoms and efforts to relate these symptoms to cellular defects.
Assuntos
Transtorno do Espectro Autista , Síndrome do QT Longo , Gravidez , Feminino , Humanos , Estudos Transversais , Transtorno do Espectro Autista/genética , Síndrome do QT Longo/genética , Síndrome do QT Longo/diagnóstico , Mutação , Fenótipo , Canais de Cálcio Tipo L/genéticaRESUMO
The biofabrication of three-dimensional (3D) tissues that recapitulate organ-specific architecture and function would benefit from temporal and spatial control of cell-cell interactions. Bioprinting, while potentially capable of achieving such control, is poorly suited to organoids with conserved cytoarchitectures that are susceptible to plastic deformation. Here, we develop a platform, termed Spatially Patterned Organoid Transfer (SPOT), consisting of an iron-oxide nanoparticle laden hydrogel and magnetized 3D printer to enable the controlled lifting, transport, and deposition of organoids. We identify cellulose nanofibers as both an ideal biomaterial for encasing organoids with magnetic nanoparticles and a shear-thinning, self-healing support hydrogel for maintaining the spatial positioning of organoids to facilitate the generation of assembloids. We leverage SPOT to create precisely arranged assembloids composed of human pluripotent stem cell-derived neural organoids and patient-derived glioma organoids. In doing so, we demonstrate the potential for the SPOT platform to construct assembloids which recapitulate key developmental processes and disease etiologies.
Assuntos
Bioimpressão , Células-Tronco Pluripotentes , Humanos , Organoides , Bioimpressão/métodos , Hidrogéis , Materiais BiocompatíveisRESUMO
Understanding spinal cord assembly is essential to elucidate how motor behavior is controlled and how disorders arise. The human spinal cord is exquisitely organized, and this complex organization contributes to the diversity and intricacy of motor behavior and sensory processing. But how this complexity arises at the cellular level in the human spinal cord remains unknown. Here we transcriptomically profiled the midgestation human spinal cord with single-cell resolution and discovered remarkable heterogeneity across and within cell types. Glia displayed diversity related to positional identity along the dorso-ventral and rostro-caudal axes, while astrocytes with specialized transcriptional programs mapped into white and gray matter subtypes. Motor neurons clustered at this stage into groups suggestive of alpha and gamma neurons. We also integrated our data with multiple existing datasets of the developing human spinal cord spanning 22 weeks of gestation to investigate the cell diversity over time. Together with mapping of disease-related genes, this transcriptomic mapping of the developing human spinal cord opens new avenues for interrogating the cellular basis of motor control in humans and guides human stem cell-based models of disease.
Assuntos
Medula Espinal , Transcriptoma , Humanos , Neurônios Motores/metabolismo , Neuroglia , Substância CinzentaRESUMO
Assembloids generated from human pluripotent stem cells are self-organizing, multicellular in vitro models that recapitulate aspects of cell-cell interactions and circuit assembly during neural development. Here, we present protocols to functionally monitor, in forebrain assembloids, the migration of GABAergic interneurons from the ventral to the dorsal forebrain and the activity in early cortical networks. Specifically, we describe high-resolution imaging and analysis of neuronal migration as well as calcium imaging of network activity in forebrain assembloids. For complete details on the use and execution of this protocol, please refer to Birey et al. (2022).