RESUMO
Human induced pluripotent stem cells (iPSCs) have great potential in research, but pluripotency testing faces challenges due to non-standardized methods and ambiguous markers. Here, we use long-read nanopore transcriptome sequencing to discover 172 genes linked to cell states not covered by current guidelines. We validate 12 genes by qPCR as unique markers for specific cell fates: pluripotency (CNMD, NANOG, SPP1), endoderm (CER1, EOMES, GATA6), mesoderm (APLNR, HAND1, HOXB7), and ectoderm (HES5, PAMR1, PAX6). Using these genes, we develop a machine learning-based scoring system, "hiPSCore", trained on 15 iPSC lines and validated on 10 more. hiPSCore accurately classifies pluripotent and differentiated cells and predicts their potential to become specialized 2D cells and 3D organoids. Our re-evaluation of cell fate marker genes identifies key targets for future studies on cell fate assessment. hiPSCore improves iPSC testing by reducing time, subjectivity, and resource use, thus enhancing iPSC quality for scientific and medical applications.
Assuntos
Diferenciação Celular , Células-Tronco Pluripotentes Induzidas , Controle de Qualidade , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Humanos , Diferenciação Celular/genética , Biomarcadores/metabolismo , Aprendizado de Máquina , Endoderma/citologia , Endoderma/metabolismo , Transcriptoma , Mesoderma/metabolismo , Mesoderma/citologia , Linhagem Celular , Ectoderma/metabolismo , Ectoderma/citologia , Organoides/metabolismo , Perfilação da Expressão Gênica/métodos , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Proteínas com Domínio TRESUMO
Pluripotent mouse embryonic stem cells (ESCs) can differentiate to all germ layers and serve as an in vitro model of embryonic development. To better understand the differentiation paths traversed by ESCs committing to different lineages, we track individual differentiating ESCs by timelapse imaging followed by multiplexed high-dimensional Imaging Mass Cytometry (IMC) protein quantification. This links continuous live single-cell molecular NANOG and cellular dynamics quantification over 5-6 generations to protein expression of 37 different molecular regulators in the same single cells at the observation endpoints. Using this unique data set including kinship history and live lineage marker detection, we show that NANOG downregulation occurs generations prior to, but is not sufficient for neuroectoderm marker Sox1 upregulation. We identify a developmental cell type co-expressing both the canonical Sox1 neuroectoderm and FoxA2 endoderm markers in vitro and confirm the presence of such a population in the post-implantation embryo. RNASeq reveals cells co-expressing SOX1 and FOXA2 to have a unique cell state characterized by expression of both endoderm as well as neuroectoderm genes suggesting lineage potential towards both germ layers.
Assuntos
Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Fator 3-beta Nuclear de Hepatócito , Células-Tronco Embrionárias Murinas , Fatores de Transcrição SOXB1 , Animais , Camundongos , Fator 3-beta Nuclear de Hepatócito/metabolismo , Fator 3-beta Nuclear de Hepatócito/genética , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição SOXB1/genética , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Rastreamento de Células/métodos , Proteína Homeobox Nanog/metabolismo , Proteína Homeobox Nanog/genética , Linhagem da Célula , Endoderma/metabolismo , Endoderma/citologia , Análise de Célula Única/métodos , Desenvolvimento Embrionário/genética , Placa Neural/metabolismo , Placa Neural/embriologia , Placa Neural/citologia , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/citologiaRESUMO
Pharyngeal endoderm cells undergo convergence and extension (C&E), which is essential for endoderm pouch formation and craniofacial development. Our previous work implicates Gα13/RhoA-mediated signaling in regulating this process, but the underlying mechanisms remain unclear. Here, we have used endoderm-specific transgenic and Gα13 mutant zebrafish to demonstrate that Gα13 plays a crucial role in pharyngeal endoderm C&E by regulating RhoA activation and E-cadherin expression. We showed that during C&E, endodermal cells gradually establish stable cell-cell contacts, acquire apical-basal polarity and undergo actomyosin-driven apical constriction, which are processes that require Gα13. Additionally, we found that Gα13-deficient embryos exhibit reduced E-cadherin expression, partially contributing to endoderm C&E defects. Notably, interfering with RhoA function disrupts spatial actomyosin activation without affecting E-cadherin expression. Collectively, our findings identify crucial cellular processes for pharyngeal endoderm C&E and reveal that Gα13 controls this through two independent pathways - modulating RhoA activation and regulating E-cadherin expression - thus unveiling intricate mechanisms governing pharyngeal endoderm morphogenesis.
Assuntos
Caderinas , Endoderma , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP , Regulação da Expressão Gênica no Desenvolvimento , Faringe , Proteínas de Peixe-Zebra , Peixe-Zebra , Proteína rhoA de Ligação ao GTP , Animais , Endoderma/metabolismo , Endoderma/embriologia , Endoderma/citologia , Caderinas/metabolismo , Caderinas/genética , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Peixe-Zebra/genética , Proteína rhoA de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/genética , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/genética , Faringe/embriologia , Faringe/metabolismo , Actomiosina/metabolismo , Transdução de Sinais , Morfogênese/genética , Polaridade Celular , Animais Geneticamente Modificados , Embrião não Mamífero/metabolismoRESUMO
Vertebrate calcitonin-producing cells (C-cells) are neuroendocrine cells that secrete the small peptide hormone calcitonin in response to elevated blood calcium levels. Whereas mouse C-cells reside within the thyroid gland and derive from pharyngeal endoderm, avian C-cells are located within ultimobranchial glands and have been reported to derive from the neural crest. We use a comparative cell lineage tracing approach in a range of vertebrate model systems to resolve the ancestral embryonic origin of vertebrate C-cells. We find, contrary to previous studies, that chick C-cells derive from pharyngeal endoderm, with neural crest-derived cells instead contributing to connective tissue intimately associated with C-cells in the ultimobranchial gland. This endodermal origin of C-cells is conserved in a ray-finned bony fish (zebrafish) and a cartilaginous fish (the little skate, Leucoraja erinacea). Furthermore, we discover putative C-cell homologs within the endodermally-derived pharyngeal epithelium of the ascidian Ciona intestinalis and the amphioxus Branchiostoma lanceolatum, two invertebrate chordates that lack neural crest cells. Our findings point to a conserved endodermal origin of C-cells across vertebrates and to a pre-vertebrate origin of this cell type along the chordate stem.
Assuntos
Calcitonina , Linhagem da Célula , Ciona intestinalis , Endoderma , Crista Neural , Células Neuroendócrinas , Animais , Endoderma/metabolismo , Endoderma/citologia , Calcitonina/metabolismo , Células Neuroendócrinas/metabolismo , Células Neuroendócrinas/citologia , Ciona intestinalis/metabolismo , Ciona intestinalis/embriologia , Crista Neural/metabolismo , Crista Neural/citologia , Embrião de Galinha , Camundongos , Vertebrados/embriologia , Vertebrados/metabolismo , Peixe-Zebra/embriologia , Anfioxos/embriologia , Anfioxos/metabolismo , Anfioxos/genética , Corpo Ultimobranquial/metabolismoRESUMO
Cell size is a crucial physical property that significantly impacts cellular physiology and function. However, the influence of cell size on stem cell specification remains largely unknown. Here, we investigated the dynamic changes in cell size during the differentiation of human pluripotent stem cells into definitive endoderm (DE). Interestingly, cell size exhibited a gradual decrease as DE differentiation progressed with higher stiffness. Furthermore, the application of hypertonic pressure or chemical to accelerate the reduction in cell size significantly and specifically enhanced DE differentiation. By functionally intervening in mechanosensitive elements, we have identified actomyosin activity as a crucial mediator of both DE differentiation and cell size reduction. Mechanistically, the reduction in cell size induces actomyosin-dependent angiomotin (AMOT) nuclear translocation, which suppresses Yes-associated protein (YAP) activity and thus facilitates DE differentiation. Together, our study has established a novel connection between cell size diminution and DE differentiation, which is mediated by AMOT nuclear translocation. Additionally, our findings suggest that the application of osmotic pressure can effectively promote human endodermal lineage differentiation.
Assuntos
Actomiosina , Angiomotinas , Diferenciação Celular , Tamanho Celular , Endoderma , Transdução de Sinais , Fatores de Transcrição , Proteínas de Sinalização YAP , Humanos , Endoderma/citologia , Endoderma/metabolismo , Actomiosina/metabolismo , Proteínas de Sinalização YAP/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Pressão Osmótica , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/citologia , Núcleo Celular/metabolismoRESUMO
Mutations in GATA6 are associated with congenital heart disease, most notably conotruncal structural defects. However, how GATA6 regulates cardiac morphology during embryogenesis is undefined. We used knockout and conditional mutant zebrafish alleles to investigate the spatiotemporal role of gata6 during cardiogenesis. Loss of gata6 specifically impacts atrioventricular valve formation and recruitment of epicardium, with a prominent loss of arterial pole cardiac cells, including those of the ventricle and outflow tract. However, there are no obvious defects in cardiac progenitor cell specification, proliferation or death. Conditional loss of gata6 starting at 24â h is sufficient to disrupt the addition of late differentiating cardiomyocytes at the arterial pole, with decreased expression levels of anterior secondary heart field (SHF) markers spry4 and mef2cb. Conditional loss of gata6 in the endoderm is sufficient to phenocopy the straight knockout, resulting in a significant loss of ventricular and outflow tract tissue. Exposure to a Dusp6 inhibitor largely rescues the loss of ventricular cells in gata6-/- larvae. Thus, gata6 functions in endoderm are mediated by FGF signaling to regulate the addition of anterior SHF progenitor derivatives during heart formation.
Assuntos
Diferenciação Celular , Endoderma , Fator de Transcrição GATA6 , Coração , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Fator de Transcrição GATA6/metabolismo , Fator de Transcrição GATA6/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Endoderma/metabolismo , Endoderma/embriologia , Endoderma/citologia , Diferenciação Celular/genética , Coração/embriologia , Organogênese/genética , Regulação da Expressão Gênica no Desenvolvimento , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/citologia , Transdução de Sinais , Fatores de Crescimento de Fibroblastos/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Fosfatase 6 de Especificidade Dupla/metabolismo , Fosfatase 6 de Especificidade Dupla/genética , Fatores de Transcrição GATARESUMO
Effective lineage-specific differentiation is essential to fulfilling the great potentials of human pluripotent stem cells (hPSCs). In this report, we investigate how modulation of medium pH and associated metabolic changes influence mesendoderm differentiation from hPSCs. We show that daily medium pH fluctuations are critical for the heterogeneity of cell fates in the absence of exogenous inducers. Acidic environment alone leads to cardiomyocyte generation without other signaling modulators. In contrast, medium alkalinization is inhibitory to cardiac fate even in the presence of classic cardiac inducers. We then demonstrate that acidic environment suppresses glycolysis to facilitate cardiac differentiation, while alkaline condition promotes glycolysis and diverts the differentiation toward other cell types. We further show that glycolysis inhibition or AMPK activation can rescue cardiac differentiation under alkalinization, and glycolysis inhibition alone can drive cardiac cell fate. This study highlights that pH changes remodel metabolic patterns and modulate signaling pathways to control cell fate.
Assuntos
Diferenciação Celular , Glicólise , Miócitos Cardíacos , Células-Tronco Pluripotentes , Humanos , Diferenciação Celular/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Acidose/metabolismo , Endoderma/citologia , Endoderma/metabolismo , Linhagem da Célula/efeitos dos fármacos , Mesoderma/citologia , Mesoderma/metabolismo , Meios de Cultura/farmacologia , Meios de Cultura/química , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular , Proteínas Quinases Ativadas por AMP/metabolismoRESUMO
BACKGROUND: The Pharyngeal Endoderm (PE) is an extremely relevant developmental tissue, serving as the progenitor for the esophagus, parathyroids, thyroids, lungs, and thymus. While several studies have highlighted the importance of PE cells, a detailed transcriptional and epigenetic characterization of this important developmental stage is still missing, especially in humans, due to technical and ethical constraints pertaining to its early formation. RESULTS: Here we fill this knowledge gap by developing an in vitro protocol for the derivation of PE-like cells from human Embryonic Stem Cells (hESCs) and by providing an integrated multi-omics characterization. Our PE-like cells robustly express PE markers and are transcriptionally homogenous and similar to in vivo mouse PE cells. In addition, we define their epigenetic landscape and dynamic changes in response to Retinoic Acid by combining ATAC-Seq and ChIP-Seq of histone modifications. The integration of multiple high-throughput datasets leads to the identification of new putative regulatory regions and to the inference of a Retinoic Acid-centered transcription factor network orchestrating the development of PE-like cells. CONCLUSIONS: By combining hESCs differentiation with computational genomics, our work reveals the epigenetic dynamics that occur during human PE differentiation, providing a solid resource and foundation for research focused on the development of PE derivatives and the modeling of their developmental defects in genetic syndromes.
Assuntos
Diferenciação Celular , Endoderma , Epigênese Genética , Células-Tronco Embrionárias Humanas , Humanos , Endoderma/citologia , Endoderma/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Faringe/citologia , Faringe/metabolismo , Tretinoína/farmacologia , Tretinoína/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , CamundongosRESUMO
Organoids, three-dimensional, stem cell-based structures that mimic the cellular and functional architecture of tissues, have emerged as an innovative in vitro tool. They offer highly efficient models for studying both embryonic development and disease progression processes. Colon organoids can also be generated from biopsies obtained during a colonoscopy. However, the invasive nature of biopsy collection poses practical challenges and introduces biases when studying patients who are already afflicted. Therefore, the use of iPSC-derived colon organoids can be considered a more practical approach for researchers and patients alike. Numerous protocols have been published for generating colon organoids from iPSCs. While most of these protocols share a common developmental process, some are labor-intensive or require additional equipment. Taking these considerations into account, we present a cost-effective and straightforward yet functionally robust colon organoid protocol: (1) definitive endoderm differentiation, (2) hindgut endoderm differentiation, and (3) maturation of colon spheroids into mature organoids.
Assuntos
Diferenciação Celular , Colo , Células-Tronco Pluripotentes Induzidas , Organoides , Organoides/citologia , Colo/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Técnicas de Cultura de Células/métodos , Endoderma/citologiaRESUMO
Cell fate decisions remain poorly understood at the molecular level. Embryogenesis provides a unique opportunity to analyze molecular details associated with cell fate decisions. Works based on model organisms have provided a conceptual framework of genes that specify cell fate control, for example, transcription factors (TFs) controlling processes from pluripotency to immunity1. How TFs specify cell fate remains poorly understood. Here we report that SALL4 relies on NuRD (nucleosome-remodeling and deacetylase complex) to interpret BMP4 signal and decide cell fate in a well-controlled in vitro system. While NuRD complex cooperates with SALL4 to convert mouse embryonic fibroblasts or MEFs to pluripotency, BMP4 diverts the same process to an alternative fate, PrE (primitive endoderm). Mechanistically, BMP4 signals the dissociation of SALL4 from NuRD physically to establish a gene regulatory network for PrE. Our results provide a conceptual framework to explore the rich landscapes of cell fate choices intrinsic to development in higher organisms involving morphogen-TF-chromatin modifier pathways.
Assuntos
Proteína Morfogenética Óssea 4 , Diferenciação Celular , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase , Fatores de Transcrição , Animais , Camundongos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteína Morfogenética Óssea 4/metabolismo , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Cromatina/metabolismo , Redes Reguladoras de Genes , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Endoderma/metabolismo , Endoderma/citologia , Transdução de Sinais , Linhagem da Célula , Proteínas de Ligação a DNARESUMO
Dysfunction of pancreatic δ cells contributes to the etiology of diabetes. Despite their important role, human δ cells are scarce, limiting physiological studies and drug discovery targeting δ cells. To date, no directed δ-cell differentiation method has been established. Here, we demonstrate that fibroblast growth factor (FGF) 7 promotes pancreatic endoderm/progenitor differentiation, whereas FGF2 biases cells towards the pancreatic δ-cell lineage via FGF receptor 1. We develop a differentiation method to generate δ cells from human stem cells by combining FGF2 with FGF7, which synergistically directs pancreatic lineage differentiation and modulates the expression of transcription factors and SST activators during endoderm/endocrine precursor induction. These δ cells display mature RNA profiles and fine secretory granules, secrete somatostatin in response to various stimuli, and suppress insulin secretion from in vitro co-cultured ß cells and mouse ß cells upon transplantation. The generation of human pancreatic δ cells from stem cells in vitro would provide an unprecedented cell source for drug discovery and cell transplantation studies in diabetes.
Assuntos
Diferenciação Celular , Células-Tronco Pluripotentes , Humanos , Animais , Camundongos , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/citologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/citologia , Células Secretoras de Somatostatina/metabolismo , Células Secretoras de Somatostatina/citologia , Endoderma/citologia , Endoderma/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Pâncreas/citologia , Pâncreas/metabolismo , Somatostatina/metabolismo , Linhagem da Célula , Insulina/metabolismo , Secreção de InsulinaRESUMO
Embryonic development is highly robust. Morphogenetic variability between embryos (under ideal conditions) is largely quantitative. This robustness stands in contrast to in vitro embryo-like models, which, like most organoids, can display a high degree of tissue morphogenetic variability. The source of this difference is not fully understood. We use the mouse gastruloid model to study the morphogenetic progression of definitive endoderm (DE) and its divergence. We first catalog the different morphologies and characterize their statistics. We then learn predictive models for DE morphotype based on earlier expression and morphology measurements. Finally, we analyze these models to identify key drivers of morphotype variability and devise gastruloid-specific and global interventions that can lower this variability and steer morphotype choice. In the process, we identify two types of coordination lacking in the in vitro model but required for robust gut-tube formation. This approach can help improve the quality and usability of 3D embryo-like models.
Assuntos
Endoderma , Organoides , Animais , Endoderma/citologia , Camundongos , Organoides/citologia , Organoides/metabolismo , Morfogênese , Desenvolvimento Embrionário , Embrião de Mamíferos/citologia , Regulação da Expressão Gênica no Desenvolvimento , Gástrula/citologiaRESUMO
The anterior visceral endoderm (AVE) differs from the surrounding visceral endoderm (VE) in its migratory behavior and ability to restrict primitive streak formation to the opposite side of the mouse embryo. To characterize the molecular bases for the unique properties of the AVE, we combined single-cell RNA sequencing of the VE prior to and during AVE migration with phosphoproteomics, high-resolution live-imaging, and short-term lineage labeling and intervention. This identified the transient nature of the AVE with attenuation of "anteriorizing" gene expression as cells migrate and the emergence of heterogeneities in transcriptional states relative to the AVE's position. Using cell communication analysis, we identified the requirement of semaphorin signaling for normal AVE migration. Lattice light-sheet microscopy showed that Sema6D mutants have abnormalities in basal projections and migration speed. These findings point to a tight coupling between transcriptional state and position of the AVE and identify molecular controllers of AVE migration.
Assuntos
Movimento Celular , Endoderma , Regulação da Expressão Gênica no Desenvolvimento , Animais , Endoderma/metabolismo , Endoderma/citologia , Camundongos , Transdução de Sinais , Semaforinas/metabolismo , Semaforinas/genética , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/citologia , Vísceras/metabolismo , Vísceras/embriologia , Padronização Corporal/genéticaRESUMO
The anterior-posterior axis of the mammalian embryo is laid down by the anterior visceral endoderm (AVE), an extraembryonic signaling center that is specified within the visceral endoderm. Current models posit that AVE differentiation is promoted globally by epiblast-derived Nodal signals, and spatially restricted by a BMP gradient established by the extraembryonic ectoderm. Here, we report spatially restricted AVE differentiation in bilayered embryo-like aggregates made from mouse embryonic stem cells that lack an extraembryonic ectoderm. Notably, clusters of AVE cells also form in pure visceral endoderm cultures upon activation of Nodal signaling, indicating that tissue-intrinsic factors can restrict AVE differentiation. We identify ß-catenin activity as a tissue-intrinsic factor that antagonizes AVE-inducing Nodal signals. Together, our results show how an AVE-like population can arise through interactions between epiblast and visceral endoderm alone. This mechanism may be a flexible solution for axis patterning in a wide range of embryo geometries, and provide robustness to axis patterning when coupled with signal gradients.
Assuntos
Padronização Corporal , Diferenciação Celular , Endoderma , Proteína Nodal , Transdução de Sinais , beta Catenina , Animais , Endoderma/citologia , Endoderma/metabolismo , Endoderma/embriologia , beta Catenina/metabolismo , Camundongos , Proteína Nodal/metabolismo , Proteína Nodal/genética , Camadas Germinativas/metabolismo , Camadas Germinativas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Regulação da Expressão Gênica no Desenvolvimento , Embrião de Mamíferos/citologiaRESUMO
Despite a distinct developmental origin, extraembryonic cells in mice contribute to gut endoderm and converge to transcriptionally resemble their embryonic counterparts. Notably, all extraembryonic progenitors share a non-canonical epigenome, raising several pertinent questions, including whether this landscape is reset to match the embryonic regulation and if extraembryonic cells persist into later development. Here we developed a two-colour lineage-tracing strategy to track and isolate extraembryonic cells over time. We find that extraembryonic gut cells display substantial memory of their developmental origin including retention of the original DNA methylation landscape and resulting transcriptional signatures. Furthermore, we show that extraembryonic gut cells undergo programmed cell death and neighbouring embryonic cells clear their remnants via non-professional phagocytosis. By midgestation, we no longer detect extraembryonic cells in the wild-type gut, whereas they persist and differentiate further in p53-mutant embryos. Our study provides key insights into the molecular and developmental fate of extraembryonic cells inside the embryo.
Assuntos
Apoptose , Linhagem da Célula , Metilação de DNA , Endoderma , Regulação da Expressão Gênica no Desenvolvimento , Animais , Endoderma/citologia , Endoderma/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Fagocitose , Camundongos Endogâmicos C57BL , Camundongos , Diferenciação Celular , Feminino , Desenvolvimento Embrionário , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Camundongos Transgênicos , Trato Gastrointestinal/citologia , Trato Gastrointestinal/embriologia , Trato Gastrointestinal/metabolismoRESUMO
Single-cell RNA sequencing (scRNA-seq) has been widely used to characterize cell types based on their average gene expression profiles. However, most studies do not consider cell type-specific variation across donors. Modelling this cell type-specific inter-individual variation could help elucidate cell type-specific biology and inform genes and cell types underlying complex traits. We therefore develop a new model to detect and quantify cell type-specific variation across individuals called CTMM (Cell Type-specific linear Mixed Model). We use extensive simulations to show that CTMM is powerful and unbiased in realistic settings. We also derive calibrated tests for cell type-specific interindividual variation, which is challenging given the modest sample sizes in scRNA-seq. We apply CTMM to scRNA-seq data from human induced pluripotent stem cells to characterize the transcriptomic variation across donors as cells differentiate into endoderm. We find that almost 100% of transcriptome-wide variability between donors is differentiation stage-specific. CTMM also identifies individual genes with statistically significant stage-specific variability across samples, including 85 genes that do not have significant stage-specific mean expression. Finally, we extend CTMM to partition interindividual covariance between stages, which recapitulates the overall differentiation trajectory. Overall, CTMM is a powerful tool to illuminate cell type-specific biology in scRNA-seq.
Assuntos
Diferenciação Celular , Células-Tronco Pluripotentes Induzidas , Análise de Sequência de RNA , Análise de Célula Única , Transcriptoma , Humanos , Análise de Célula Única/métodos , Análise de Sequência de RNA/métodos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Diferenciação Celular/genética , Perfilação da Expressão Gênica/métodos , RNA-Seq/métodos , Endoderma/citologia , Endoderma/metabolismoRESUMO
Mammalian blastocyst formation involves the specification of the trophectoderm followed by the differentiation of the inner cell mass into embryonic epiblast and extra-embryonic primitive endoderm (PrE). During this time, the embryo maintains a window of plasticity and can redirect its cellular fate when challenged experimentally. In this context, we found that the PrE alone was sufficient to regenerate a complete blastocyst and continue post-implantation development. We identify an in vitro population similar to the early PrE in vivo that exhibits the same embryonic and extra-embryonic potency and can form complete stem cell-based embryo models, termed blastoids. Commitment in the PrE is suppressed by JAK/STAT signaling, collaborating with OCT4 and the sustained expression of a subset of pluripotency-related transcription factors that safeguard an enhancer landscape permissive for multi-lineage differentiation. Our observations support the notion that transcription factor persistence underlies plasticity in regulative development and highlight the importance of the PrE in perturbed development.
Assuntos
Blastocisto , Diferenciação Celular , Endoderma , Animais , Endoderma/metabolismo , Endoderma/citologia , Camundongos , Blastocisto/metabolismo , Blastocisto/citologia , Linhagem da Célula , Fator 3 de Transcrição de Octâmero/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Transdução de Sinais , Desenvolvimento Embrionário , Janus Quinases/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição STAT/metabolismo , Fatores de Transcrição/metabolismo , Feminino , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/citologiaRESUMO
Single-cell RNA sequencing allows us to model cellular state dynamics and fate decisions using expression similarity or RNA velocity to reconstruct state-change trajectories; however, trajectory inference does not incorporate valuable time point information or utilize additional modalities, whereas methods that address these different data views cannot be combined or do not scale. Here we present CellRank 2, a versatile and scalable framework to study cellular fate using multiview single-cell data of up to millions of cells in a unified fashion. CellRank 2 consistently recovers terminal states and fate probabilities across data modalities in human hematopoiesis and endodermal development. Our framework also allows combining transitions within and across experimental time points, a feature we use to recover genes promoting medullary thymic epithelial cell formation during pharyngeal endoderm development. Moreover, we enable estimating cell-specific transcription and degradation rates from metabolic-labeling data, which we apply to an intestinal organoid system to delineate differentiation trajectories and pinpoint regulatory strategies.
Assuntos
Diferenciação Celular , Análise de Célula Única , Análise de Célula Única/métodos , Humanos , Endoderma/citologia , Endoderma/metabolismo , Hematopoese , Linhagem da Célula , Análise de Sequência de RNA/métodos , Organoides/metabolismo , Organoides/citologiaRESUMO
Cell-fate decisions during mammalian gastrulation are poorly understood outside of rodent embryos. The embryonic disc of pig embryos mirrors humans, making them a useful proxy for studying gastrulation. Here we present a single-cell transcriptomic atlas of pig gastrulation, revealing cell-fate emergence dynamics, as well as conserved and divergent gene programs governing early porcine, primate, and murine development. We highlight heterochronicity in extraembryonic cell-types, despite the broad conservation of cell-type-specific transcriptional programs. We apply these findings in combination with functional investigations, to outline conserved spatial, molecular, and temporal events during definitive endoderm specification. We find early FOXA2 + /TBXT- embryonic disc cells directly form definitive endoderm, contrasting later-emerging FOXA2/TBXT+ node/notochord progenitors. Unlike mesoderm, none of these progenitors undergo epithelial-to-mesenchymal transition. Endoderm/Node fate hinges on balanced WNT and hypoblast-derived NODAL, which is extinguished upon endodermal differentiation. These findings emphasise the interplay between temporal and topological signalling in fate determination during gastrulation.
Assuntos
Embrião de Mamíferos , Endoderma , Gastrulação , Regulação da Expressão Gênica no Desenvolvimento , Análise de Célula Única , Animais , Endoderma/citologia , Endoderma/metabolismo , Endoderma/embriologia , Suínos , Camundongos , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Diferenciação Celular , Mesoderma/citologia , Mesoderma/embriologia , Mesoderma/metabolismo , Transcriptoma , Fator 3-beta Nuclear de Hepatócito/metabolismo , Fator 3-beta Nuclear de Hepatócito/genética , Linhagem da Célula , Proteínas com Domínio T/metabolismo , Proteínas com Domínio T/genética , Transição Epitelial-Mesenquimal/genéticaRESUMO
Genetic differences between pluripotent stem cell lines cause variable activity of extracellular signaling pathways, limiting reproducibility of directed differentiation protocols. Here we used human embryonic stem cells (hESCs) to interrogate how exogenous factors modulate endogenous signaling events during specification of foregut endoderm lineages. We find that transforming growth factor ß1 (TGF-ß1) activates a putative human OTX2/LHX1 gene regulatory network which promotes anterior fate by antagonizing endogenous Wnt signaling. In contrast to Porcupine inhibition, TGF-ß1 effects cannot be reversed by exogenous Wnt ligands, suggesting that induction of SHISA proteins and intracellular accumulation of Fzd receptors render TGF-ß1-treated cells refractory to Wnt signaling. Subsequently, TGF-ß1-mediated inhibition of BMP and Wnt signaling suppresses liver fate and promotes pancreas fate. Furthermore, combined TGF-ß1 treatment and Wnt inhibition during pancreatic specification reproducibly and robustly enhance INSULIN+ cell yield across hESC lines. This modification of widely used differentiation protocols will enhance pancreatic ß cell yield for cell-based therapeutic applications.