RESUMEN
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.
Asunto(s)
Encéfalo , Linaje de la Célula , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Organoides , Humanos , Encéfalo/citología , Encéfalo/metabolismo , Diferenciación Celular/genética , Linaje de la Célula/genética , Cromatina/genética , Organoides/citología , Organoides/metabolismo , Factores de Transcripción/metabolismo , TranscriptomaRESUMEN
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.
Asunto(s)
Corteza Cerebral/citología , Genes Reporteros , Células Madre Pluripotentes Inducidas/citología , Organoides/citología , Análisis de la Célula Individual/métodos , Sistemas CRISPR-Cas , Linaje de la Célula , Humanos , Microscopía/métodos , Mutación , Neuronas/citología , Neuronas/fisiología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ARN , Proteína 2 del Complejo de la Esclerosis Tuberosa/genéticaRESUMEN
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.
Asunto(s)
Encéfalo , Genómica , Organoides/citología , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/fisiología , Animales , Evolución Biológica , Encéfalo/citología , Encéfalo/embriología , Encéfalo/fisiología , Humanos , Macaca , Pan troglodytes , Análisis de la Célula Individual , Especificidad de la EspecieRESUMEN
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.
Asunto(s)
Encéfalo/metabolismo , Transcriptoma , Animales , Encéfalo/citología , Evolución Molecular , Humanos , Inmunohistoquímica , Macaca/genética , Neuronas/metabolismo , Pan paniscus/genética , Pan troglodytes/genética , RNA-Seq , Análisis de la Célula IndividualRESUMEN
Cell fate progression of pluripotent progenitors is strictly regulated, resulting in high human cell diversity. Epigenetic modifications also orchestrate cell fate restriction. Unveiling the epigenetic mechanisms underlying human cell diversity has been difficult. In this study, we use human brain and retina organoid models and present single-cell profiling of H3K27ac, H3K27me3 and H3K4me3 histone modifications from progenitor to differentiated neural fates to reconstruct the epigenomic trajectories regulating cell identity acquisition. We capture transitions from pluripotency through neuroepithelium to retinal and brain region and cell type specification. Switching of repressive and activating epigenetic modifications can precede and predict cell fate decisions at each stage, providing a temporal census of gene regulatory elements and transcription factors. Removing H3K27me3 at the neuroectoderm stage disrupts fate restriction, resulting in aberrant cell identity acquisition. Our single-cell epigenome-wide map of human neural organoid development serves as a blueprint to explore human cell fate determination.
Asunto(s)
Epigénesis Genética , Epigenómica , Organoides , Análisis de la Célula Individual , Humanos , Epigenómica/métodos , Encéfalo/citología , Células Madre Pluripotentes/fisiología , Diferenciación Celular/fisiología , Diferenciación Celular/genética , Retina/citología , Retina/crecimiento & desarrollo , Histonas/metabolismoRESUMEN
The liver has the remarkable capacity to regenerate. In the clinic, regeneration is induced by portal vein embolization, which redirects portal blood flow, resulting in liver hypertrophy in locations with increased blood supply, and atrophy of embolized segments. Here, we apply single-cell and single-nucleus transcriptomics on healthy, hypertrophied, and atrophied patient-derived liver samples to explore cell states in the regenerating liver. Our data unveils pervasive upregulation of genes associated with developmental processes, cellular adhesion, and inflammation in post-portal vein embolization liver, disrupted portal-central hepatocyte zonation, and altered cell subtype composition of endothelial and immune cells. Interlineage crosstalk analysis reveals mesenchymal cells as an interaction hub between immune and endothelial cells, and highlights the importance of extracellular matrix proteins in liver regeneration. Moreover, we establish tissue-scale iterative indirect immunofluorescence imaging for high-dimensional spatial analysis of perivascular microenvironments, uncovering changes to tissue architecture in regenerating liver lobules. Altogether, our data is a rich resource revealing cellular and histological changes in human liver regeneration.
Asunto(s)
Embolización Terapéutica , Regeneración Hepática , Hígado , Vena Porta , Humanos , Regeneración Hepática/fisiología , Embolización Terapéutica/métodos , Hepatocitos/metabolismo , Análisis de la Célula Individual , Transcriptoma , Masculino , Células Endoteliales/metabolismo , Femenino , Hipertrofia , Persona de Mediana EdadRESUMEN
In multicellular organisms, the specification, coordination, and compartmentalization of cell types enable the formation of complex body plans. However, some eukaryotic protists such as slime molds generate diverse and complex structures while remaining in a multinucleate syncytial state. It is unknown if different regions of these giant syncytial cells have distinct transcriptional responses to environmental encounters and if nuclei within the cell diversify into heterogeneous states. Here, we performed spatial transcriptome analysis of the slime mold Physarum polycephalum in the plasmodium state under different environmental conditions and used single-nucleus RNA-sequencing to dissect gene expression heterogeneity among nuclei. Our data identifies transcriptome regionality in the organism that associates with proliferation, syncytial substructures, and localized environmental conditions. Further, we find that nuclei are heterogenous in their transcriptional profile and may process local signals within the plasmodium to coordinate cell growth, metabolism, and reproduction. To understand how nuclei variation within the syncytium compares to heterogeneity in single-nucleus cells, we analyzed states in single Physarum amoebal cells. We observed amoebal cell states at different stages of mitosis and meiosis, and identified cytokinetic features that are specific to nuclei divisions within the syncytium. Notably, we do not find evidence for predefined transcriptomic states in the amoebae that are observed in the syncytium. Our data shows that a single-celled slime mold can control its gene expression in a region-specific manner while lacking cellular compartmentalization and suggests that nuclei are mobile processors facilitating local specialized functions. More broadly, slime molds offer the extraordinary opportunity to explore how organisms can evolve regulatory mechanisms to divide labor, specialize, balance competition with cooperation, and perform other foundational principles that govern the logic of life.
Asunto(s)
Células Gigantes/fisiología , Physarum polycephalum/metabolismo , Análisis de la Célula Individual , Transcriptoma , Regulación de la Expresión Génica , RNA-SeqRESUMEN
BACKGROUND: Skeletal muscle fiber type distribution has implications for human health, muscle function, and performance. This knowledge has been gathered using labor-intensive and costly methodology that limited these studies. Here, we present a method based on muscle tissue RNA sequencing data (totRNAseq) to estimate the distribution of skeletal muscle fiber types from frozen human samples, allowing for a larger number of individuals to be tested. METHODS: By using single-nuclei RNA sequencing (snRNAseq) data as a reference, cluster expression signatures were produced by averaging gene expression of cluster gene markers and then applying these to totRNAseq data and inferring muscle fiber nuclei type via linear matrix decomposition. This estimate was then compared with fiber type distribution measured by ATPase staining or myosin heavy chain protein isoform distribution of 62 muscle samples in two independent cohorts (n = 39 and 22). RESULTS: The correlation between the sequencing-based method and the other two were rATPas = 0.44 [0.13-0.67], [95% CI], and rmyosin = 0.83 [0.61-0.93], with p = 5.70 × 10-3 and 2.00 × 10-6, respectively. The deconvolution inference of fiber type composition was accurate even for very low totRNAseq sequencing depths, i.e., down to an average of ~ 10,000 paired-end reads. CONCLUSIONS: This new method ( https://github.com/OlaHanssonLab/PredictFiberType ) consequently allows for measurement of fiber type distribution of a larger number of samples using totRNAseq in a cost and labor-efficient way. It is now feasible to study the association between fiber type distribution and e.g. health outcomes in large well-powered studies.
Asunto(s)
Fibras Musculares Esqueléticas , ARN , Secuencia de Bases , Humanos , Análisis de Secuencia de ARN , Secuenciación del ExomaRESUMEN
Self-organizing tissues resembling brain structures generated from human stem cells offer exciting possibilities to study human brain development, disease, and evolution. These 3D models are complex and can contain cells at various stages of differentiation from different brain regions. Single-cell genomic methods provide powerful approaches to explore cell composition, differentiation trajectories, and genetic perturbations in brain organoid systems. However, it remains a major challenge to understand the heterogeneity observed within and between individual organoids. Here, we develop a set of computational tools (VoxHunt) to assess brain organoid patterning, developmental state, and cell identity through comparisons to spatial and single-cell transcriptome reference datasets. We use VoxHunt to characterize and visualize cell compositions using single-cell and bulk genomic data from multiple organoid protocols modeling different brain structures. VoxHunt will be useful to assess organoid engineering protocols and to annotate cell fates that emerge in organoids during genetic and environmental perturbation experiments.
Asunto(s)
Encéfalo , Organoides , Diferenciación Celular/genética , Genómica , Humanos , TranscriptomaRESUMEN
Human neurons engineered from induced pluripotent stem cells (iPSCs) through neurogenin 2 (NGN2) overexpression are widely used to study neuronal differentiation mechanisms and to model neurological diseases. However, the differentiation paths and heterogeneity of emerged neurons have not been fully explored. Here, we used single-cell transcriptomics to dissect the cell states that emerge during NGN2 overexpression across a time course from pluripotency to neuron functional maturation. We find a substantial molecular heterogeneity in the neuron types generated, with at least two populations that express genes associated with neurons of the peripheral nervous system. Neuron heterogeneity is observed across multiple iPSC clones and lines from different individuals. We find that neuron fate acquisition is sensitive to NGN2 expression level and the duration of NGN2-forced expression. Our data reveal that NGN2 dosage can regulate neuron fate acquisition, and that NGN2-iN heterogeneity can confound results that are sensitive to neuron type.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Diferenciación Celular/genética , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Proteínas del Tejido Nervioso/genética , Neurogénesis/genética , Neuronas/citología , Neuronas/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Línea Celular , Células Cultivadas , Biología Computacional/métodos , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Humanos , Ratones , Proteínas del Tejido Nervioso/metabolismo , RNA-Seq , TranscriptomaRESUMEN
Malformations of the human cortex represent a major cause of disability1. Mouse models with mutations in known causal genes only partially recapitulate the phenotypes and are therefore not unlimitedly suited for understanding the molecular and cellular mechanisms responsible for these conditions2. Here we study periventricular heterotopia (PH) by analyzing cerebral organoids derived from induced pluripotent stem cells (iPSCs) of patients with mutations in the cadherin receptor-ligand pair DCHS1 and FAT4 or from isogenic knockout (KO) lines1,3. Our results show that human cerebral organoids reproduce the cortical heterotopia associated with PH. Mutations in DCHS1 and FAT4 or knockdown of their expression causes changes in the morphology of neural progenitor cells and result in defective neuronal migration dynamics only in a subset of neurons. Single-cell RNA-sequencing (scRNA-seq) data reveal a subpopulation of mutant neurons with dysregulated genes involved in axon guidance, neuronal migration and patterning. We suggest that defective neural progenitor cell (NPC) morphology and an altered navigation system in a subset of neurons underlie this form of PH.
Asunto(s)
Movimiento Celular , Cerebro/patología , Neuronas/patología , Organoides/patología , Heterotopia Nodular Periventricular/patología , Proteínas Relacionadas con las Cadherinas , Cadherinas/genética , Línea Celular , Humanos , Recién Nacido , Mutación/genética , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Imagen de Lapso de Tiempo , Proteínas Supresoras de Tumor/genéticaRESUMEN
Amputation of the axolotl forelimb results in the formation of a blastema, a transient tissue where progenitor cells accumulate prior to limb regeneration. However, the molecular understanding of blastema formation had previously been hampered by the inability to identify and isolate blastema precursor cells in the adult tissue. We have used a combination of Cre-loxP reporter lineage tracking and single-cell messenger RNA sequencing (scRNA-seq) to molecularly track mature connective tissue (CT) cell heterogeneity and its transition to a limb blastema state. We have uncovered a multiphasic molecular program where CT cell types found in the uninjured adult limb revert to a relatively homogenous progenitor state that recapitulates an embryonic limb bud-like phenotype including multipotency within the CT lineage. Together, our data illuminate molecular and cellular reprogramming during complex organ regeneration in a vertebrate.
Asunto(s)
Reprogramación Celular/fisiología , Células del Tejido Conectivo/fisiología , Miembro Anterior/fisiología , Regeneración/fisiología , Ambystoma mexicanum , Animales , Linaje de la Célula , Rastreo Celular , Genes Reporteros , Integrasas , ARN Mensajero/genética , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual , Células Madre/fisiologíaRESUMEN
Ectopic expression of defined transcription factors can force direct cell-fate conversion from one lineage to another in the absence of cell division. Several transcription factor cocktails have enabled successful reprogramming of various somatic cell types into induced neurons (iNs) of distinct neurotransmitter phenotype. However, the nature of the intermediate states that drive the reprogramming trajectory toward distinct iN types is largely unknown. Here we show that successful direct reprogramming of adult human brain pericytes into functional iNs by Ascl1 and Sox2 encompasses transient activation of a neural stem cell-like gene expression program that precedes bifurcation into distinct neuronal lineages. During this transient state, key signaling components relevant for neural induction and neural stem cell maintenance are regulated by and functionally contribute to iN reprogramming and maturation. Thus, Ascl1- and Sox2-mediated reprogramming into a broad spectrum of iN types involves the unfolding of a developmental program via neural stem cell-like intermediates.