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1.
Cell ; 184(8): 2084-2102.e19, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33765444

RESUMO

The human brain has undergone rapid expansion since humans diverged from other great apes, but the mechanism of this human-specific enlargement is still unknown. Here, we use cerebral organoids derived from human, gorilla, and chimpanzee cells to study developmental mechanisms driving evolutionary brain expansion. We find that neuroepithelial differentiation is a protracted process in apes, involving a previously unrecognized transition state characterized by a change in cell shape. Furthermore, we show that human organoids are larger due to a delay in this transition, associated with differences in interkinetic nuclear migration and cell cycle length. Comparative RNA sequencing (RNA-seq) reveals differences in expression dynamics of cell morphogenesis factors, including ZEB2, a known epithelial-mesenchymal transition regulator. We show that ZEB2 promotes neuroepithelial transition, and its manipulation and downstream signaling leads to acquisition of nonhuman ape architecture in the human context and vice versa, establishing an important role for neuroepithelial cell shape in human brain expansion.


Assuntos
Evolução Biológica , Encéfalo/citologia , Forma Celular/fisiologia , Animais , Encéfalo/metabolismo , Diferenciação Celular , Linhagem Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Transição Epitelial-Mesenquimal/genética , Expressão Gênica , Gorilla gorilla , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurogênese , Neurônios/citologia , Neurônios/metabolismo , Organoides/citologia , Organoides/metabolismo , Pan troglodytes , Homeobox 2 de Ligação a E-box com Dedos de Zinco/genética , Homeobox 2 de Ligação a E-box com Dedos de Zinco/metabolismo
2.
Cell ; 182(6): 1623-1640.e34, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32946783

RESUMO

Human organoids recapitulating the cell-type diversity and function of their target organ are valuable for basic and translational research. We developed light-sensitive human retinal organoids with multiple nuclear and synaptic layers and functional synapses. We sequenced the RNA of 285,441 single cells from these organoids at seven developmental time points and from the periphery, fovea, pigment epithelium and choroid of light-responsive adult human retinas, and performed histochemistry. Cell types in organoids matured in vitro to a stable "developed" state at a rate similar to human retina development in vivo. Transcriptomes of organoid cell types converged toward the transcriptomes of adult peripheral retinal cell types. Expression of disease-associated genes was cell-type-specific in adult retina, and cell-type specificity was retained in organoids. We implicate unexpected cell types in diseases such as macular degeneration. This resource identifies cellular targets for studying disease mechanisms in organoids and for targeted repair in human retinas.


Assuntos
Diferenciação Celular/genética , Organoides/citologia , Organoides/metabolismo , Retina/citologia , Retina/metabolismo , Análise de Célula Única/métodos , Sinapses/fisiologia , Transcriptoma/genética , Técnicas de Cultura de Células/métodos , Linhagem Celular , Eletrofisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Predisposição Genética para Doença/genética , Humanos , Hibridização In Situ , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Microscopia Eletrônica , Família Multigênica , Naftoquinonas , Organoides/efeitos da radiação , Organoides/ultraestrutura , Retina/patologia , Retina/efeitos da radiação
3.
Cell ; 175(6): 1591-1606.e19, 2018 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-30500538

RESUMO

The mammalian liver possesses a remarkable regenerative ability. Two modes of damage response have been described: (1) The "oval cell" response emanates from the biliary tree when all hepatocytes are affected by chronic liver disease. (2) A massive, proliferative response of mature hepatocytes occurs upon acute liver damage such as partial hepatectomy (PHx). While the oval cell response has been captured in vitro by growing organoids from cholangiocytes, the hepatocyte proliferative response has not been recapitulated in culture. Here, we describe the establishment of a long-term 3D organoid culture system for mouse and human primary hepatocytes. Organoids can be established from single hepatocytes and grown for multiple months, while retaining key morphological, functional and gene expression features. Transcriptional profiles of the organoids resemble those of proliferating hepatocytes after PHx. Human hepatocyte organoids proliferate extensively after engraftment into mice and thus recapitulate the proliferative damage-response of hepatocytes.


Assuntos
Proliferação de Células , Hepatócitos/metabolismo , Organoides/metabolismo , Animais , Técnicas de Cultura de Células , Células Cultivadas , Hepatócitos/citologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Organoides/citologia , Células-Tronco/citologia , Células-Tronco/metabolismo , Fatores de Tempo
4.
Cell ; 173(2): 515-528.e17, 2018 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-29625057

RESUMO

Bladder cancer is the fifth most prevalent cancer in the U.S., yet is understudied, and few laboratory models exist that reflect the biology of the human disease. Here, we describe a biobank of patient-derived organoid lines that recapitulates the histopathological and molecular diversity of human bladder cancer. Organoid lines can be established efficiently from patient biopsies acquired before and after disease recurrence and are interconvertible with orthotopic xenografts. Notably, organoid lines often retain parental tumor heterogeneity and exhibit a spectrum of genomic changes that are consistent with tumor evolution in culture. Analyses of drug response using bladder tumor organoids show partial correlations with mutational profiles, as well as changes associated with treatment resistance, and specific responses can be validated using xenografts in vivo. Our studies indicate that patient-derived bladder tumor organoids represent a faithful model system for studying tumor evolution and treatment response in the context of precision cancer medicine.


Assuntos
Neoplasias da Bexiga Urinária/patologia , Idoso , Idoso de 80 Anos ou mais , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Sobrevivência Celular/efeitos dos fármacos , Variações do Número de Cópias de DNA , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Pessoa de Meia-Idade , Mutação , Organoides/citologia , Organoides/efeitos dos fármacos , Organoides/metabolismo , Medicina de Precisão , Transplante Heterólogo , Células Tumorais Cultivadas , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/metabolismo
5.
Cell ; 175(5): 1307-1320.e22, 2018 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-30392957

RESUMO

In the small intestine, a niche of accessory cell types supports the generation of mature epithelial cell types from intestinal stem cells (ISCs). It is unclear, however, if and how immune cells in the niche affect ISC fate or the balance between self-renewal and differentiation. Here, we use single-cell RNA sequencing (scRNA-seq) to identify MHC class II (MHCII) machinery enrichment in two subsets of Lgr5+ ISCs. We show that MHCII+ Lgr5+ ISCs are non-conventional antigen-presenting cells in co-cultures with CD4+ T helper (Th) cells. Stimulation of intestinal organoids with key Th cytokines affects Lgr5+ ISC renewal and differentiation in opposing ways: pro-inflammatory signals promote differentiation, while regulatory cells and cytokines reduce it. In vivo genetic perturbation of Th cells or MHCII expression on Lgr5+ ISCs impacts epithelial cell differentiation and IEC fate during infection. These interactions between Th cells and Lgr5+ ISCs, thus, orchestrate tissue-wide responses to external signals.


Assuntos
Diferenciação Celular , Autorrenovação Celular , Interleucina-10/metabolismo , Células-Tronco/citologia , Linfócitos T Auxiliares-Indutores/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Autorrenovação Celular/efeitos dos fármacos , Citocinas/farmacologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Antígenos de Histocompatibilidade Classe II/metabolismo , Sistema Imunitário/metabolismo , Intestinos/citologia , Intestinos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Organoides/citologia , Organoides/efeitos dos fármacos , Organoides/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Salmonella enterica/patogenicidade , Células-Tronco/metabolismo , Linfócitos T Auxiliares-Indutores/citologia
6.
Cell ; 170(6): 1149-1163.e12, 2017 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-28886383

RESUMO

The diversity of mesenchymal cell types in the lung that influence epithelial homeostasis and regeneration is poorly defined. We used genetic lineage tracing, single-cell RNA sequencing, and organoid culture approaches to show that Lgr5 and Lgr6, well-known markers of stem cells in epithelial tissues, are markers of mesenchymal cells in the adult lung. Lgr6+ cells comprise a subpopulation of smooth muscle cells surrounding airway epithelia and promote airway differentiation of epithelial progenitors via Wnt-Fgf10 cooperation. Genetic ablation of Lgr6+ cells impairs airway injury repair in vivo. Distinct Lgr5+ cells are located in alveolar compartments and are sufficient to promote alveolar differentiation of epithelial progenitors through Wnt activation. Modulating Wnt activity altered differentiation outcomes specified by mesenchymal cells. This identification of region- and lineage-specific crosstalk between epithelium and their neighboring mesenchymal partners provides new understanding of how different cell types are maintained in the adult lung.


Assuntos
Pulmão/citologia , Mesoderma/citologia , Animais , Homeostase , Pulmão/fisiologia , Camundongos , Organoides/citologia , Alvéolos Pulmonares/citologia , Receptores Acoplados a Proteínas G/análise , Análise de Sequência de RNA , Análise de Célula Única , Transcrição Gênica
7.
Cell ; 170(6): 1134-1148.e10, 2017 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-28886382

RESUMO

The lung is an architecturally complex organ comprising a heterogeneous mixture of various epithelial and mesenchymal lineages. We use single-cell RNA sequencing and signaling lineage reporters to generate a spatial and transcriptional map of the lung mesenchyme. We find that each mesenchymal lineage has a distinct spatial address and transcriptional profile leading to unique niche regulatory functions. The mesenchymal alveolar niche cell is Wnt responsive, expresses Pdgfrα, and is critical for alveolar epithelial cell growth and self-renewal. In contrast, the Axin2+ myofibrogenic progenitor cell preferentially generates pathologically deleterious myofibroblasts after injury. Analysis of the secretome and receptome of the alveolar niche reveals functional pathways that mediate growth and self-renewal of alveolar type 2 progenitor cells, including IL-6/Stat3, Bmp, and Fgf signaling. These studies define the cellular and molecular framework of lung mesenchymal niches and reveal the functional importance of developmental pathways in promoting self-renewal versus a pathological response to tissue injury.


Assuntos
Pulmão/citologia , Mesoderma/citologia , Algoritmos , Animais , Células Epiteliais/metabolismo , Fibrose/metabolismo , Perfilação da Expressão Gênica , Pulmão/patologia , Pulmão/fisiologia , Lesão Pulmonar/patologia , Camundongos , Organoides/citologia , Comunicação Parácrina , Regeneração , Transdução de Sinais , Análise de Célula Única , Células-Tronco/metabolismo
8.
Cell ; 165(7): 1586-1597, 2016 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-27315476

RESUMO

Recent advances in 3D culture technology allow embryonic and adult mammalian stem cells to exhibit their remarkable self-organizing properties, and the resulting organoids reflect key structural and functional properties of organs such as kidney, lung, gut, brain and retina. Organoid technology can therefore be used to model human organ development and various human pathologies 'in a dish." Additionally, patient-derived organoids hold promise to predict drug response in a personalized fashion. Organoids open up new avenues for regenerative medicine and, in combination with editing technology, for gene therapy. The many potential applications of this technology are only beginning to be explored.


Assuntos
Modelos Biológicos , Organoides/citologia , Animais , Humanos , Especificidade de Órgãos , Análise de Sequência de RNA , Análise de Célula Única , Células-Tronco/citologia
9.
Cell ; 160(1-2): 299-312, 2015 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-25533785

RESUMO

Despite the enormous replication potential of the human liver, there are currently no culture systems available that sustain hepatocyte replication and/or function in vitro. We have shown previously that single mouse Lgr5+ liver stem cells can be expanded as epithelial organoids in vitro and can be differentiated into functional hepatocytes in vitro and in vivo. We now describe conditions allowing long-term expansion of adult bile duct-derived bipotent progenitor cells from human liver. The expanded cells are highly stable at the chromosome and structural level, while single base changes occur at very low rates. The cells can readily be converted into functional hepatocytes in vitro and upon transplantation in vivo. Organoids from α1-antitrypsin deficiency and Alagille syndrome patients mirror the in vivo pathology. Clonal long-term expansion of primary adult liver stem cells opens up experimental avenues for disease modeling, toxicology studies, regenerative medicine, and gene therapy.


Assuntos
Fígado/citologia , Técnicas de Cultura de Órgãos , Animais , Instabilidade Genômica , Hepatócitos/citologia , Humanos , Camundongos , Organoides/citologia
10.
Nature ; 631(8019): 142-149, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38926573

RESUMO

Interindividual genetic variation affects the susceptibility to and progression of many diseases1,2. However, efforts to study how individual human brains differ in normal development and disease phenotypes are limited by the paucity of faithful cellular human models, and the difficulty of scaling current systems to represent multiple people. Here we present human brain Chimeroids, a highly reproducible, multidonor human brain cortical organoid model generated by the co-development of cells from a panel of individual donors in a single organoid. By reaggregating cells from multiple single-donor organoids at the neural stem cell or neural progenitor cell stage, we generate Chimeroids in which each donor produces all cell lineages of the cerebral cortex, even when using pluripotent stem cell lines with notable growth biases. We used Chimeroids to investigate interindividual variation in the susceptibility to neurotoxic triggers that exhibit high clinical phenotypic variability: ethanol and the antiepileptic drug valproic acid. Individual donors varied in both the penetrance of the effect on target cell types, and the molecular phenotype within each affected cell type. Our results suggest that human genetic background may be an important mediator of neurotoxin susceptibility and introduce Chimeroids as a scalable system for high-throughput investigation of interindividual variation in processes of brain development and disease.


Assuntos
Córtex Cerebral , Quimera , Predisposição Genética para Doença , Neurotoxinas , Organoides , Feminino , Humanos , Masculino , Linhagem da Célula/efeitos dos fármacos , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Quimera/genética , Etanol/efeitos adversos , Etanol/toxicidade , Variação Genética , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Neurotoxinas/toxicidade , Organoides/citologia , Organoides/efeitos dos fármacos , Organoides/metabolismo , Fenótipo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , Doadores de Tecidos , Ácido Valproico/efeitos adversos , Ácido Valproico/toxicidade , Predisposição Genética para Doença/genética
11.
Nature ; 621(7978): 365-372, 2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36198796

RESUMO

Self-organizing neural organoids grown from pluripotent stem cells1-3 combined with single-cell genomic technologies provide opportunities to examine gene regulatory networks underlying human brain development. Here we acquire single-cell transcriptome and accessible chromatin data over a dense time course in human organoids covering neuroepithelial formation, patterning, brain regionalization and neurogenesis, and identify temporally dynamic and brain-region-specific regulatory regions. We developed Pando-a flexible framework that incorporates multi-omic data and predictions of transcription-factor-binding sites to infer a global gene regulatory network describing organoid development. We use pooled genetic perturbation with single-cell transcriptome readout to assess transcription factor requirement for cell fate and state regulation in organoids. We find that certain factors regulate the abundance of cell fates, whereas other factors affect neuronal cell states after differentiation. We show that the transcription factor GLI3 is required for cortical fate establishment in humans, recapitulating previous research performed in mammalian model systems. We measure transcriptome and chromatin accessibility in normal or GLI3-perturbed cells and identify two distinct GLI3 regulomes that are central to telencephalic fate decisions: one regulating dorsoventral patterning with HES4/5 as direct GLI3 targets, and one controlling ganglionic eminence diversification later in development. Together, we provide a framework for how human model systems and single-cell technologies can be leveraged to reconstruct human developmental biology.


Assuntos
Encéfalo , Linhagem da Célula , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Organoides , Humanos , Encéfalo/citologia , Encéfalo/metabolismo , Diferenciação Celular/genética , Linhagem da Célula/genética , Cromatina/genética , Organoides/citologia , Organoides/metabolismo , Fatores de Transcrição/metabolismo , Transcriptoma
12.
Nature ; 623(7986): 397-405, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37914940

RESUMO

Microglia are specialized brain-resident macrophages that arise from primitive macrophages colonizing the embryonic brain1. Microglia contribute to multiple aspects of brain development, but their precise roles in the early human brain remain poorly understood owing to limited access to relevant tissues2-6. The generation of brain organoids from human induced pluripotent stem cells recapitulates some key features of human embryonic brain development7-10. However, current approaches do not incorporate microglia or address their role in organoid maturation11-21. Here we generated microglia-sufficient brain organoids by coculturing brain organoids with primitive-like macrophages generated from the same human induced pluripotent stem cells (iMac)22. In organoid cocultures, iMac differentiated into cells with microglia-like phenotypes and functions (iMicro) and modulated neuronal progenitor cell (NPC) differentiation, limiting NPC proliferation and promoting axonogenesis. Mechanistically, iMicro contained high levels of PLIN2+ lipid droplets that exported cholesterol and its esters, which were taken up by NPCs in the organoids. We also detected PLIN2+ lipid droplet-loaded microglia in mouse and human embryonic brains. Overall, our approach substantially advances current human brain organoid approaches by incorporating microglial cells, as illustrated by the discovery of a key pathway of lipid-mediated crosstalk between microglia and NPCs that leads to improved neurogenesis.


Assuntos
Encéfalo , Colesterol , Células-Tronco Pluripotentes Induzidas , Microglia , Células-Tronco Neurais , Neurogênese , Organoides , Animais , Humanos , Camundongos , Encéfalo/citologia , Encéfalo/metabolismo , Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Microglia/citologia , Microglia/metabolismo , Organoides/citologia , Organoides/metabolismo , Colesterol/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Axônios , Proliferação de Células , Ésteres/metabolismo , Gotículas Lipídicas/metabolismo
13.
Nature ; 620(7974): 615-624, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37558872

RESUMO

The concomitant occurrence of tissue growth and organization is a hallmark of organismal development1-3. This often means that proliferating and differentiating cells are found at the same time in a continuously changing tissue environment. How cells adapt to architectural changes to prevent spatial interference remains unclear. Here, to understand how cell movements that are key for growth and organization are orchestrated, we study the emergence of photoreceptor neurons that occur during the peak of retinal growth, using zebrafish, human tissue and human organoids. Quantitative imaging reveals that successful retinal morphogenesis depends on the active bidirectional translocation of photoreceptors, leading to a transient transfer of the entire cell population away from the apical proliferative zone. This pattern of migration is driven by cytoskeletal machineries that differ depending on the direction: microtubules are exclusively required for basal translocation, whereas actomyosin is involved in apical movement. Blocking the basal translocation of photoreceptors induces apical congestion, which hampers the apical divisions of progenitor cells and leads to secondary defects in lamination. Thus, photoreceptor migration is crucial to prevent competition for space, and to allow concurrent tissue growth and lamination. This shows that neuronal migration, in addition to its canonical role in cell positioning4, can be involved in coordinating morphogenesis.


Assuntos
Movimento Celular , Morfogênese , Células Fotorreceptoras , Retina , Animais , Humanos , Actomiosina/metabolismo , Competição entre as Células , Diferenciação Celular , Movimento Celular/fisiologia , Proliferação de Células , Microtúbulos/metabolismo , Morfogênese/fisiologia , Organoides/citologia , Organoides/embriologia , Células Fotorreceptoras/citologia , Células Fotorreceptoras/fisiologia , Retina/citologia , Retina/embriologia , Peixe-Zebra/embriologia
14.
Nature ; 616(7955): 143-151, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36991123

RESUMO

The relationship between the human placenta-the extraembryonic organ made by the fetus, and the decidua-the mucosal layer of the uterus, is essential to nurture and protect the fetus during pregnancy. Extravillous trophoblast cells (EVTs) derived from placental villi infiltrate the decidua, transforming the maternal arteries into high-conductance vessels1. Defects in trophoblast invasion and arterial transformation established during early pregnancy underlie common pregnancy disorders such as pre-eclampsia2. Here we have generated a spatially resolved multiomics single-cell atlas of the entire human maternal-fetal interface including the myometrium, which enables us to resolve the full trajectory of trophoblast differentiation. We have used this cellular map to infer the possible transcription factors mediating EVT invasion and show that they are preserved in in vitro models of EVT differentiation from primary trophoblast organoids3,4 and trophoblast stem cells5. We define the transcriptomes of the final cell states of trophoblast invasion: placental bed giant cells (fused multinucleated EVTs) and endovascular EVTs (which form plugs inside the maternal arteries). We predict the cell-cell communication events contributing to trophoblast invasion and placental bed giant cell formation, and model the dual role of interstitial EVTs and endovascular EVTs in mediating arterial transformation during early pregnancy. Together, our data provide a comprehensive analysis of postimplantation trophoblast differentiation that can be used to inform the design of experimental models of the human placenta in early pregnancy.


Assuntos
Multiômica , Primeiro Trimestre da Gravidez , Trofoblastos , Feminino , Humanos , Gravidez , Movimento Celular , Placenta/irrigação sanguínea , Placenta/citologia , Placenta/fisiologia , Primeiro Trimestre da Gravidez/fisiologia , Trofoblastos/citologia , Trofoblastos/metabolismo , Trofoblastos/fisiologia , Decídua/irrigação sanguínea , Decídua/citologia , Relações Materno-Fetais/fisiologia , Análise de Célula Única , Miométrio/citologia , Miométrio/fisiologia , Diferenciação Celular , Organoides/citologia , Organoides/fisiologia , Células-Tronco/citologia , Transcriptoma , Fatores de Transcrição/metabolismo , Comunicação Celular
15.
Nature ; 622(7982): 359-366, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37758944

RESUMO

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 Celular
16.
Nature ; 621(7978): 373-380, 2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37704762

RESUMO

The development of the human brain involves unique processes (not observed in many other species) that can contribute to neurodevelopmental disorders1-4. Cerebral organoids enable the study of neurodevelopmental disorders in a human context. We have developed the CRISPR-human organoids-single-cell RNA sequencing (CHOOSE) system, which uses verified pairs of guide RNAs, inducible CRISPR-Cas9-based genetic disruption and single-cell transcriptomics for pooled loss-of-function screening in mosaic organoids. Here we show that perturbation of 36 high-risk autism spectrum disorder genes related to transcriptional regulation uncovers their effects on cell fate determination. We find that dorsal intermediate progenitors, ventral progenitors and upper-layer excitatory neurons are among the most vulnerable cell types. We construct a developmental gene regulatory network of cerebral organoids from single-cell transcriptomes and chromatin modalities and identify autism spectrum disorder-associated and perturbation-enriched regulatory modules. Perturbing members of the BRG1/BRM-associated factor (BAF) chromatin remodelling complex leads to enrichment of ventral telencephalon progenitors. Specifically, mutating the BAF subunit ARID1B affects the fate transition of progenitors to oligodendrocyte and interneuron precursor cells, a phenotype that we confirmed in patient-specific induced pluripotent stem cell-derived organoids. Our study paves the way for high-throughput phenotypic characterization of disease susceptibility genes in organoid models with cell state, molecular pathway and gene regulatory network readouts.


Assuntos
Transtorno do Espectro Autista , Encéfalo , Deficiências do Desenvolvimento , Organoides , Análise da Expressão Gênica de Célula Única , Humanos , Transtorno do Espectro Autista/complicações , Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/patologia , Transtorno Autístico/complicações , Transtorno Autístico/genética , Transtorno Autístico/patologia , Encéfalo/citologia , Encéfalo/metabolismo , Linhagem da Célula/genética , Cromatina/genética , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Deficiências do Desenvolvimento/complicações , Deficiências do Desenvolvimento/genética , Deficiências do Desenvolvimento/patologia , Edição de Genes , Mutação com Perda de Função , Mosaicismo , Neurônios/metabolismo , Neurônios/patologia , Organoides/citologia , Organoides/metabolismo , RNA Guia de Sistemas CRISPR-Cas , Transcrição Gênica
17.
Annu Rev Neurosci ; 43: 375-389, 2020 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-32640930

RESUMO

Scientists have been fascinated by the human brain for centuries, yet knowledge of the cellular and molecular events that build the human brain during embryogenesis and of how abnormalities in this process lead to neurological disease remains very superficial. In particular, the lack of experimental models for a process that largely occurs during human in utero development, and is therefore poorly accessible for study, has hindered progress in mechanistic understanding. Advances in stem cell-derived models of human organogenesis, in the form of three-dimensional organoid cultures, and transformative new analytic technologies have opened new experimental pathways for investigation of aspects of development, evolution, and pathology of the human brain. Here, we consider the biology of brain organoids, compared and contrasted with the endogenous human brain, and highlight experimental strategies to use organoids to pioneer new understanding of human brain pathology.


Assuntos
Encéfalo/crescimento & desenvolvimento , Rede Nervosa/fisiologia , Organogênese/fisiologia , Organoides/citologia , Animais , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Doenças do Sistema Nervoso/patologia
18.
Cell ; 155(1): 19-20, 2013 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-24074857

RESUMO

A three-dimensional culture of cortical tissues derived from pluripotent stem cells offers an opportunity to model human brain development and disorders. In a recent issue of Nature, Lancaster et al. describe a new method for generating cerebral organoids in a dish and use it to model microcephaly.


Assuntos
Encéfalo/crescimento & desenvolvimento , Encéfalo/patologia , Microcefalia/patologia , Modelos Biológicos , Organoides/citologia , Organoides/crescimento & desenvolvimento , Técnicas de Cultura de Tecidos/métodos , Animais , Humanos
19.
Cell ; 155(2): 357-68, 2013 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-24120136

RESUMO

Proliferation of the self-renewing epithelium of the gastric corpus occurs almost exclusively in the isthmus of the glands, from where cells migrate bidirectionally toward pit and base. The isthmus is therefore generally viewed as the stem cell zone. We find that the stem cell marker Troy is expressed at the gland base by a small subpopulation of fully differentiated chief cells. By lineage tracing with a Troy-eGFP-ires-CreERT2 allele, single marked chief cells are shown to generate entirely labeled gastric units over periods of months. This phenomenon accelerates upon tissue damage. Troy(+) chief cells can be cultured to generate long-lived gastric organoids. Troy marks a specific subset of chief cells that display plasticity in that they are capable of replenishing entire gastric units, essentially serving as quiescent "reserve" stem cells. These observations challenge the notion that stem cell hierarchies represent a "one-way street."


Assuntos
Celulas Principais Gástricas/citologia , Células-Tronco/citologia , Estômago/citologia , Animais , Linhagem da Célula , Celulas Principais Gástricas/química , Mucosa Gástrica/citologia , Camundongos , Organoides/citologia , Receptores do Fator de Necrose Tumoral/análise , Via de Sinalização Wnt
20.
Nature ; 609(7929): 907-910, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36171373

RESUMO

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/citologia
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