RESUMO
Organs are composed of diverse cell types that traverse transient states during organogenesis. To interrogate this diversity during human development, we generate a single-cell transcriptome atlas from multiple developing endodermal organs of the respiratory and gastrointestinal tract. We illuminate cell states, transcription factors, and organ-specific epithelial stem cell and mesenchyme interactions across lineages. We implement the atlas as a high-dimensional search space to benchmark human pluripotent stem cell (hPSC)-derived intestinal organoids (HIOs) under multiple culture conditions. We show that HIOs recapitulate reference cell states and use HIOs to reconstruct the molecular dynamics of intestinal epithelium and mesenchyme emergence. We show that the mesenchyme-derived niche cue NRG1 enhances intestinal stem cell maturation in vitro and that the homeobox transcription factor CDX2 is required for regionalization of intestinal epithelium and mesenchyme in humans. This work combines cell atlases and organoid technologies to understand how human organ development is orchestrated.
Assuntos
Anatomia Artística , Atlas como Assunto , Desenvolvimento Embrionário , Endoderma/embriologia , Modelos Biológicos , Organoides/embriologia , Fator de Transcrição CDX2/metabolismo , Linhagem Celular , Fator de Crescimento Epidérmico/farmacologia , Células Epiteliais/citologia , Feminino , Gastrulação , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Intestinos/embriologia , Masculino , Mesoderma/embriologia , Pessoa de Meia-Idade , Neuregulina-1/metabolismo , Especificidade de Órgãos , Células-Tronco Pluripotentes/citologiaRESUMO
Intestinal Th17 cells are induced and accumulate in response to colonization with a subgroup of intestinal microbes such as segmented filamentous bacteria (SFB) and certain extracellular pathogens. Here, we show that adhesion of microbes to intestinal epithelial cells (ECs) is a critical cue for Th17 induction. Upon monocolonization of germ-free mice or rats with SFB indigenous to mice (M-SFB) or rats (R-SFB), M-SFB and R-SFB showed host-specific adhesion to small intestinal ECs, accompanied by host-specific induction of Th17 cells. Citrobacter rodentium and Escherichia coli O157 triggered similar Th17 responses, whereas adhesion-defective mutants of these microbes failed to do so. Moreover, a mixture of 20 bacterial strains, which were selected and isolated from fecal samples of a patient with ulcerative colitis on the basis of their ability to cause a robust induction of Th17 cells in the mouse colon, also exhibited EC-adhesive characteristics.
Assuntos
Aderência Bacteriana , Citrobacter rodentium/fisiologia , Infecções por Enterobacteriaceae/imunologia , Infecções por Escherichia coli/imunologia , Escherichia coli O157/fisiologia , Mucosa Intestinal/imunologia , Células Th17/imunologia , Animais , Infecções Bacterianas/imunologia , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Células Epiteliais/ultraestrutura , Fezes/microbiologia , Humanos , Imunoglobulina A/imunologia , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Microscopia Eletrônica de Varredura , Ratos , Ratos Endogâmicos F344 , Especificidade da EspécieRESUMO
The intimate relationship between the epithelium and immune system is crucial for maintaining tissue homeostasis, with perturbations therein linked to autoimmune disease and cancer1-3. Whereas stem cell-derived organoids are powerful models of epithelial function4, they lack tissue-resident immune cells that are essential for capturing organ-level processes. We describe human intestinal immuno-organoids (IIOs), formed through self-organization of epithelial organoids and autologous tissue-resident memory T (TRM) cells, a portion of which integrate within the epithelium and continuously survey the barrier. TRM cell migration and interaction with epithelial cells was orchestrated by TRM cell-enriched transcriptomic programs governing cell motility and adhesion. We combined IIOs and single-cell transcriptomics to investigate intestinal inflammation triggered by cancer-targeting biologics in patients. Inflammation was associated with the emergence of an activated population of CD8+ T cells that progressively acquired intraepithelial and cytotoxic features. The appearance of this effector population was preceded and potentiated by a T helper-1-like CD4+ population, which initially produced cytokines and subsequently became cytotoxic itself. As a system amenable to direct perturbation, IIOs allowed us to identify the Rho pathway as a new target for mitigation of immunotherapy-associated intestinal inflammation. Given that they recapitulate both the phenotypic outcomes and underlying interlineage immune interactions, IIOs can be used to study tissue-resident immune responses in the context of tumorigenesis and infectious and autoimmune diseases.
RESUMO
Our ancestors acquired morphological, cognitive and metabolic modifications that enabled humans to colonize diverse habitats, develop extraordinary technologies and reshape the biosphere. Understanding the genetic, developmental and molecular bases for these changes will provide insights into how we became human. Connecting human-specific genetic changes to species differences has been challenging owing to an abundance of low-effect size genetic changes, limited descriptions of phenotypic differences across development at the level of cell types and lack of experimental models. Emerging approaches for single-cell sequencing, genetic manipulation and stem cell culture now support descriptive and functional studies in defined cell types with a human or ape genetic background. In this Review, we describe how the sequencing of genomes from modern and archaic hominins, great apes and other primates is revealing human-specific genetic changes and how new molecular and cellular approaches - including cell atlases and organoids - are enabling exploration of the candidate causal factors that underlie human-specific traits.
Assuntos
Hominidae , Animais , Humanos , Hominidae/genética , Organoides , Evolução Biológica , Evolução MolecularRESUMO
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 , TranscriptomaRESUMO
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
The Human Developmental Cell Atlas (HDCA) initiative, which is part of the Human Cell Atlas, aims to create a comprehensive reference map of cells during development. This will be critical to understanding normal organogenesis, the effect of mutations, environmental factors and infectious agents on human development, congenital and childhood disorders, and the cellular basis of ageing, cancer and regenerative medicine. Here we outline the HDCA initiative and the challenges of mapping and modelling human development using state-of-the-art technologies to create a reference atlas across gestation. Similar to the Human Genome Project, the HDCA will integrate the output from a growing community of scientists who are mapping human development into a unified atlas. We describe the early milestones that have been achieved and the use of human stem-cell-derived cultures, organoids and animal models to inform the HDCA, especially for prenatal tissues that are hard to acquire. Finally, we provide a roadmap towards a complete atlas of human development.
Assuntos
Movimento Celular , Rastreamento de Células , Células/citologia , Biologia do Desenvolvimento/métodos , Embrião de Mamíferos/citologia , Feto/citologia , Disseminação de Informação , Organogênese , Adulto , Animais , Atlas como Assunto , Técnicas de Cultura de Células , Sobrevivência Celular , Visualização de Dados , Feminino , Humanos , Imageamento Tridimensional , Masculino , Modelos Animais , Organogênese/genética , Organoides/citologia , Células-Tronco/citologiaRESUMO
Induced pluripotent stem cell (iPSC)-derived organoids provide models to study human organ development. Single-cell transcriptomics enable highly resolved descriptions of cell states within these systems; however, approaches are needed to directly measure lineage relationships. Here we establish iTracer, a lineage recorder that combines reporter barcodes with inducible CRISPR-Cas9 scarring and is compatible with single-cell and spatial transcriptomics. We apply iTracer to explore clonality and lineage dynamics during cerebral organoid development and identify a time window of fate restriction as well as variation in neurogenic dynamics between progenitor neuron families. We also establish long-term four-dimensional light-sheet microscopy for spatial lineage recording in cerebral organoids and confirm regional clonality in the developing neuroepithelium. We incorporate gene perturbation (iTracer-perturb) and assess the effect of mosaic TSC2 mutations on cerebral organoid development. Our data shed light on how lineages and fates are established during cerebral organoid formation. More broadly, our techniques can be adapted in any iPSC-derived culture system to dissect lineage alterations during normal or perturbed development.
Assuntos
Córtex Cerebral/citologia , Genes Reporter , Células-Tronco Pluripotentes Induzidas/citologia , Organoides/citologia , Análise de Célula Única/métodos , Sistemas CRISPR-Cas , Linhagem da Célula , Humanos , Microscopia/métodos , Mutação , Neurônios/citologia , Neurônios/fisiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise de Sequência de RNA , Proteína 2 do Complexo Esclerose Tuberosa/genéticaRESUMO
The human brain has undergone substantial change since humans diverged from chimpanzees and the other great apes1,2. However, the genetic and developmental programs that underlie this divergence are not fully understood. Here we have analysed stem cell-derived cerebral organoids using single-cell transcriptomics and accessible chromatin profiling to investigate gene-regulatory changes that are specific to humans. We first analysed cell composition and reconstructed differentiation trajectories over the entire course of human cerebral organoid development from pluripotency, through neuroectoderm and neuroepithelial stages, followed by divergence into neuronal fates within the dorsal and ventral forebrain, midbrain and hindbrain regions. Brain-region composition varied in organoids from different iPSC lines, but regional gene-expression patterns remained largely reproducible across individuals. We analysed chimpanzee and macaque cerebral organoids and found that human neuronal development occurs at a slower pace relative to the other two primates. Using pseudotemporal alignment of differentiation paths, we found that human-specific gene expression resolved to distinct cell states along progenitor-to-neuron lineages in the cortex. Chromatin accessibility was dynamic during cortex development, and we identified divergence in accessibility between human and chimpanzee that correlated with human-specific gene expression and genetic change. Finally, we mapped human-specific expression in adult prefrontal cortex using single-nucleus RNA sequencing analysis and identified developmental differences that persist into adulthood, as well as cell-state-specific changes that occur exclusively in the adult brain. Our data provide a temporal cell atlas of great ape forebrain development, and illuminate dynamic gene-regulatory features that are unique to humans.
Assuntos
Encéfalo , Genômica , Organoides/citologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/fisiologia , Animais , Evolução Biológica , Encéfalo/citologia , Encéfalo/embriologia , Encéfalo/fisiologia , Humanos , Macaca , Pan troglodytes , Análise de Célula Única , Especificidade da EspécieRESUMO
Identification of gene expression traits unique to the human brain sheds light on the molecular mechanisms underlying human evolution. Here, we searched for uniquely human gene expression traits by analyzing 422 brain samples from humans, chimpanzees, bonobos, and macaques representing 33 anatomical regions, as well as 88,047 cell nuclei composing three of these regions. Among 33 regions, cerebral cortex areas, hypothalamus, and cerebellar gray and white matter evolved rapidly in humans. At the cellular level, astrocytes and oligodendrocyte progenitors displayed more differences in the human evolutionary lineage than the neurons. Comparison of the bulk tissue and single-nuclei sequencing revealed that conventional RNA sequencing did not detect up to two-thirds of cell-type-specific evolutionary differences.
Assuntos
Encéfalo/metabolismo , Transcriptoma , Animais , Encéfalo/citologia , Evolução Molecular , Humanos , Imuno-Histoquímica , Macaca/genética , Neurônios/metabolismo , Pan paniscus/genética , Pan troglodytes/genética , RNA-Seq , Análise de Célula ÚnicaRESUMO
Conventional two-dimensional differentiation from pluripotency fails to recapitulate cell interactions occurring during organogenesis. Three-dimensional organoids generate complex organ-like tissues; however, it is unclear how heterotypic interactions affect lineage identity. Here we use single-cell RNA sequencing to reconstruct hepatocyte-like lineage progression from pluripotency in two-dimensional culture. We then derive three-dimensional liver bud organoids by reconstituting hepatic, stromal, and endothelial interactions, and deconstruct heterogeneity during liver bud development. We find that liver bud hepatoblasts diverge from the two-dimensional lineage, and express epithelial migration signatures characteristic of organ budding. We benchmark three-dimensional liver buds against fetal and adult human liver single-cell RNA sequencing data, and find a striking correspondence between the three-dimensional liver bud and fetal liver cells. We use a receptor-ligand pairing analysis and a high-throughput inhibitor assay to interrogate signalling in liver buds, and show that vascular endothelial growth factor (VEGF) crosstalk potentiates endothelial network formation and hepatoblast differentiation. Our molecular dissection reveals interlineage communication regulating organoid development, and illuminates previously inaccessible aspects of human liver development.
Assuntos
Comunicação Celular , Diferenciação Celular , Linhagem da Célula , Fígado/citologia , Fígado/embriologia , Organogênese , Técnicas de Cultura de Tecidos/métodos , Idoso , Hipóxia Celular , Movimento Celular , Endotélio/citologia , Células Epiteliais/citologia , Matriz Extracelular/metabolismo , Feminino , Feto/citologia , Hepatócitos/citologia , Humanos , Masculino , Pessoa de Meia-Idade , Organoides/citologia , Células-Tronco Pluripotentes/citologia , Análise de Sequência de RNA , Transdução de Sinais , Análise de Célula Única , Fator A de Crescimento do Endotélio Vascular/metabolismo , Adulto JovemRESUMO
The establishment of the small intestinal (SI) lineage during human embryogenesis ensures functional integrity of the intestine after birth. The chromatin dynamics that drive SI lineage formation and regional patterning in humans are essentially unknown. To fill this knowledge void, we apply a cutting-edge genomic technology to a state-of-the-art human model of early SI development. Specifically, we leverage chromatin run-on sequencing (ChRO-seq) to define the landscape of active promoters, enhancers and gene bodies across distinct stages of directed differentiation of human pluripotent stem cells into SI spheroids with regional specification. Through comprehensive ChRO-seq analysis we identify candidate stage-specific chromatin activity states, novel markers and enhancer hotspots during the directed differentiation. Moreover, we propose a detailed transcriptional network associated with SI lineage formation or regional patterning. Our ChRO-seq analyses uncover a previously undescribed pattern of enhancer activity and transcription at HOX gene loci underlying SI regional patterning. We also validated this unique HOX dynamics by the analysis of single cell RNA-seq data from human fetal SI. Overall, the results lead to a new proposed working model for the regulatory underpinnings of human SI development, thereby adding a novel dimension to the literature that has relied almost exclusively on non-human models.
Assuntos
Montagem e Desmontagem da Cromatina , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Embrionárias Humanas/metabolismo , Intestino Delgado/embriologia , Modelos Biológicos , Animais , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Elementos Facilitadores Genéticos , Genes Homeobox , Células-Tronco Embrionárias Humanas/citologia , Humanos , Intestino Delgado/metabolismo , Camundongos , Organoides , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Análise de Sequência de RNA/métodos , Análise de Célula Única , Transcrição GênicaRESUMO
Direct lineage reprogramming represents a remarkable conversion of cellular and transcriptome states. However, the intermediate stages through which individual cells progress during reprogramming are largely undefined. Here we use single-cell RNA sequencing at multiple time points to dissect direct reprogramming from mouse embryonic fibroblasts to induced neuronal cells. By deconstructing heterogeneity at each time point and ordering cells by transcriptome similarity, we find that the molecular reprogramming path is remarkably continuous. Overexpression of the proneural pioneer factor Ascl1 results in a well-defined initialization, causing cells to exit the cell cycle and re-focus gene expression through distinct neural transcription factors. The initial transcriptional response is relatively homogeneous among fibroblasts, suggesting that the early steps are not limiting for productive reprogramming. Instead, the later emergence of a competing myogenic program and variable transgene dynamics over time appear to be the major efficiency limits of direct reprogramming. Moreover, a transcriptional state, distinct from donor and target cell programs, is transiently induced in cells undergoing productive reprogramming. Our data provide a high-resolution approach for understanding transcriptome states during lineage differentiation.
Assuntos
Reprogramação Celular/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Análise de Sequência de RNA , Análise de Célula Única , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Ciclo Celular/genética , Linhagem da Célula/genética , Transdiferenciação Celular/genética , Embrião de Mamíferos/citologia , Perfilação da Expressão Gênica , Inativação Gênica , Proteínas de Homeodomínio/metabolismo , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Fatores do Domínio POU/metabolismo , Fatores de Tempo , Fatores de Transcrição/metabolismo , Transcriptoma/genética , Transgenes/genéticaRESUMO
A specific subpopulation of neural progenitor cells, the basal radial glial cells (bRGCs) of the outer subventricular zone (OSVZ), are thought to have a key role in the evolutionary expansion of the mammalian neocortex. In the developing lissencephalic mouse neocortex, bRGCs exist at low abundance and show significant molecular differences from bRGCs in developing gyrencephalic species. Here, we demonstrate that the developing mouse medial neocortex (medNcx), in contrast to the canonically studied lateral neocortex (latNcx), exhibits an OSVZ and an abundance of bRGCs similar to that in developing gyrencephalic neocortex. Unlike bRGCs in developing mouse latNcx, the bRGCs in medNcx exhibit human bRGC-like gene expression, including expression of Hopx, a human bRGC marker. Disruption of Hopx expression in mouse embryonic medNcx and forced Hopx expression in mouse embryonic latNcx demonstrate that Hopx is required and sufficient, respectively, for bRGC abundance as found in the developing gyrencephalic neocortex. Taken together, our data identify a novel bRGC subpopulation in developing mouse medNcx that is highly related to bRGCs of developing gyrencephalic neocortex.
Assuntos
Células Ependimogliais/metabolismo , Proteínas de Homeodomínio/metabolismo , Neocórtex/citologia , Neocórtex/embriologia , Animais , Sistemas CRISPR-Cas/genética , Proliferação de Células , Embrião de Mamíferos/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Ventrículos Laterais/embriologia , Camundongos Endogâmicos C57BL , Neocórtex/metabolismo , Fator de Transcrição PAX6/metabolismo , Células-Tronco/citologiaRESUMO
To understand human development and disease, as well as to regenerate damaged tissues, scientists are working to engineer certain cell types in vitro and to create 3D microenvironments in which cells behave physiologically. Single-cell genomics (SCG) technologies are being applied to primary human organs and to engineered cells and tissues to generate atlases of cell diversity in these systems at unparalleled resolution. Moving beyond atlases, SCG methods are powerful tools for gaining insight into the engineering and disease process. Here we discuss how scientists can use single-cell sequencing to optimize human cell and tissue engineering by measuring precision, detecting inefficiencies, and assessing accuracy. We also provide a perspective on how emerging SCG methods can be used to reverse-engineer human cells and tissues and unravel disease mechanisms.
Assuntos
Genômica , Análise de Célula Única , Células-Tronco/citologia , Engenharia Tecidual , Epigênese Genética , Humanos , Modelos BiológicosRESUMO
Innovative methods designed to recapitulate human organogenesis from pluripotent stem cells provide a means to explore human developmental biology. New technologies to sequence and analyze single-cell transcriptomes can deconstruct these 'organoids' into constituent parts, and reconstruct lineage trajectories during cell differentiation. In this Spotlight article we summarize the different approaches to performing single-cell transcriptomics on organoids, and discuss the opportunities and challenges of applying these techniques to generate organ-level, mechanistic models of human development and disease. Together, these technologies will move past characterization to the prediction of human developmental and disease-related phenomena.
Assuntos
Perfilação da Expressão Gênica/métodos , Crescimento e Desenvolvimento , Organoides/metabolismo , Análise de Célula Única/métodos , Linhagem da Célula , Humanos , Organoides/citologiaRESUMO
The intestinal epithelium serves critical physiologic functions that are shared among all vertebrates. However, it is unknown how the transcriptional regulatory mechanisms underlying these functions have changed over the course of vertebrate evolution. We generated genome-wide mRNA and accessible chromatin data from adult intestinal epithelial cells (IECs) in zebrafish, stickleback, mouse, and human species to determine if conserved IEC functions are achieved through common transcriptional regulation. We found evidence for substantial common regulation and conservation of gene expression regionally along the length of the intestine from fish to mammals and identified a core set of genes comprising a vertebrate IEC signature. We also identified transcriptional start sites and other putative regulatory regions that are differentially accessible in IECs in all 4 species. Although these sites rarely showed sequence conservation from fish to mammals, surprisingly, they drove highly conserved IEC expression in a zebrafish reporter assay. Common putative transcription factor binding sites (TFBS) found at these sites in multiple species indicate that sequence conservation alone is insufficient to identify much of the functionally conserved IEC regulatory information. Among the rare, highly sequence-conserved, IEC-specific regulatory regions, we discovered an ancient enhancer upstream from her6/HES1 that is active in a distinct population of Notch-positive cells in the intestinal epithelium. Together, these results show how combining accessible chromatin and mRNA datasets with TFBS prediction and in vivo reporter assays can reveal tissue-specific regulatory information conserved across 420 million years of vertebrate evolution. We define an IEC transcriptional regulatory network that is shared between fish and mammals and establish an experimental platform for studying how evolutionarily distilled regulatory information commonly controls IEC development and physiology.