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BACKGROUND AND AIMS: Primary sclerosing cholangitis (PSC) is a chronic inflammatory liver disorder without effective medical treatment which is characterized by inflammation and fibrotic structures around the bile ducts. Biliary epithelial cells (cholangiocytes) are the target and potential disease drivers in PSC, yet little is known if cholangiocytes from PSC patients differ from non-PSC controls. To characterize cholangiocytes at early rather than end-stage disease, cholangiocyte organoids (COs) were derived from diseased bile ducts of PSC patients and compared to organoids generated from disease controls. METHODS: Cholangiocytes were obtained during endoscopic retrograde cholangiopancreatography (ERCP) brushing of diseased bile duct areas and expanded as organoids using previously established culture methods. Stable CO lines were analyzed for cell type identity, basic cholangiocyte function, and transcriptomic signature. RESULTS: We demonstrate that cholangiocytes, derived from the damaged area within the bile ducts of PSC patients, can be expanded in culture without displaying functional or genetic disease-related features. We further show that COs from patients who later were diagnosed with dysplasia exhibit higher expression of the cancer-associated genes PGC, FXYD2, MIR4435-2HG, and HES1. CONCLUSIONS: Our results demonstrate that PSC organoids are largely similar to control organoids after culture and highlight the significance of COs as a tool for regenerative medicine approaches as well as their potential for discovering new potential biomarkers for diagnosing cholangiocarcinoma.
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For many adult human organs, tissue regeneration during chronic disease remains a controversial subject. Regenerative processes are easily observed in animal models, and their underlying mechanisms are becoming well characterized1-4, but technical challenges and ethical aspects are limiting the validation of these results in humans. We decided to address this difficulty with respect to the liver. This organ displays the remarkable ability to regenerate after acute injury, although liver regeneration in the context of recurring injury remains to be fully demonstrated. Here we performed single-nucleus RNA sequencing (snRNA-seq) on 47 liver biopsies from patients with different stages of metabolic dysfunction-associated steatotic liver disease to establish a cellular map of the liver during disease progression. We then combined these single-cell-level data with advanced 3D imaging to reveal profound changes in the liver architecture. Hepatocytes lose their zonation and considerable reorganization of the biliary tree takes place. More importantly, our study uncovers transdifferentiation events that occur between hepatocytes and cholangiocytes without the presence of adult stem cells or developmental progenitor activation. Detailed analyses and functional validations using cholangiocyte organoids confirm the importance of the PI3K-AKT-mTOR pathway in this process, thereby connecting this acquisition of plasticity to insulin signalling. Together, our data indicate that chronic injury creates an environment that induces cellular plasticity in human organs, and understanding the underlying mechanisms of this process could open new therapeutic avenues in the management of chronic diseases.
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Transdiferenciación Celular , Hepatocitos , Hepatopatías , Hígado , Humanos , Sistema Biliar/citología , Sistema Biliar/metabolismo , Sistema Biliar/patología , Biopsia , Plasticidad de la Célula , Enfermedad Crónica , Progresión de la Enfermedad , Células Epiteliales/metabolismo , Células Epiteliales/citología , Células Epiteliales/patología , Hepatocitos/metabolismo , Hepatocitos/citología , Hepatocitos/patología , Insulina/metabolismo , Hígado/patología , Hígado/metabolismo , Hígado/citología , Hepatopatías/patología , Hepatopatías/metabolismo , Regeneración Hepática , Organoides/metabolismo , Organoides/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , RNA-Seq , Transducción de Señal , Análisis de la Célula Individual , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
The in vitro oxygen microenvironment profoundly affects the capacity of cell cultures to model physiological and pathophysiological states. Cell culture is often considered to be hyperoxic, but pericellular oxygen levels, which are affected by oxygen diffusivity and consumption, are rarely reported. Here, we provide evidence that several cell types in culture actually experience local hypoxia, with important implications for cell metabolism and function. We focused initially on adipocytes, as adipose tissue hypoxia is frequently observed in obesity and precedes diminished adipocyte function. Under standard conditions, cultured adipocytes are highly glycolytic and exhibit a transcriptional profile indicative of physiological hypoxia. Increasing pericellular oxygen diverted glucose flux toward mitochondria, lowered HIF1α activity, and resulted in widespread transcriptional rewiring. Functionally, adipocytes increased adipokine secretion and sensitivity to insulin and lipolytic stimuli, recapitulating a healthier adipocyte model. The functional benefits of increasing pericellular oxygen were also observed in macrophages, hPSC-derived hepatocytes and cardiac organoids. Our findings demonstrate that oxygen is limiting in many terminally-differentiated cell types, and that considering pericellular oxygen improves the quality, reproducibility and translatability of culture models.
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Adipocitos , Diferenciación Celular , Oxígeno , Oxígeno/metabolismo , Adipocitos/metabolismo , Adipocitos/citología , Humanos , Técnicas de Cultivo de Célula/métodos , Animales , Glucólisis , Hepatocitos/metabolismo , Hipoxia de la Célula , Mitocondrias/metabolismo , Ratones , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Células Cultivadas , Glucosa/metabolismo , Macrófagos/metabolismoRESUMEN
BACKGROUND: Pulmonary ionocytes have been identified in the airway epithelium as a small population of ion transporting cells expressing high levels of CFTR (cystic fibrosis transmembrane conductance regulator), the gene mutated in cystic fibrosis. By providing an infinite source of airway epithelial cells (AECs), the use of human induced pluripotent stem cells (hiPSCs) could overcome some challenges of studying ionocytes. However, the production of AEC epithelia containing ionocytes from hiPSCs has proven difficult. Here, we present a platform to produce hiPSC-derived AECs (hiPSC-AECs) including ionocytes and investigate their role in the airway epithelium. METHODS: hiPSCs were differentiated into lung progenitors, which were expanded as 3D organoids and matured by air-liquid interface culture as polarised hiPSC-AEC epithelia. Using CRISPR/Cas9 technology, we generated a hiPSCs knockout (KO) for FOXI1, a transcription factor that is essential for ionocyte specification. Differences between FOXI1 KO hiPSC-AECs and their wild-type (WT) isogenic controls were investigated by assessing gene and protein expression, epithelial composition, cilia coverage and motility, pH and transepithelial barrier properties. RESULTS: Mature hiPSC-AEC epithelia contained basal cells, secretory cells, ciliated cells with motile cilia, pulmonary neuroendocrine cells (PNECs) and ionocytes. There was no difference between FOXI1 WT and KO hiPSCs in terms of their capacity to differentiate into airway progenitors. However, FOXI1 KO led to mature hiPSC-AEC epithelia without ionocytes with reduced capacity to produce ciliated cells. CONCLUSION: Our results suggest that ionocytes could have role beyond transepithelial ion transport by regulating epithelial properties and homeostasis in the airway epithelium.
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Células Madre Pluripotentes Inducidas , Mucosa Respiratoria , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/citología , Diferenciación Celular/fisiología , Células Cultivadas , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Células Epiteliales/metabolismo , Organoides/metabolismoRESUMEN
Mechanisms specifying amniotic ectoderm and surface ectoderm are unresolved in humans due to their close similarities in expression patterns and signal requirements. This lack of knowledge hinders the development of protocols to accurately model human embryogenesis. Here, we developed a human pluripotent stem cell model to investigate the divergence between amniotic and surface ectoderms. In the established culture system, cells differentiated into functional amnioblast-like cells. Single-cell RNA sequencing analyses of amnioblast differentiation revealed an intermediate cell state with enhanced surface ectoderm gene expression. Furthermore, when the differentiation started at the confluent condition, cells retained the expression profile of surface ectoderm. Collectively, we propose that human amniotic ectoderm and surface ectoderm are specified along a common nonneural ectoderm trajectory based on cell density. Our culture system also generated extraembryonic mesoderm-like cells from the primed pluripotent state. Together, this study provides an integrative understanding of the human nonneural ectoderm development and a model for embryonic and extraembryonic human development around gastrulation.
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Ectodermo , Células Madre Pluripotentes , Humanos , Ectodermo/metabolismo , Diferenciación Celular/genética , MesodermoRESUMEN
Cilia density, distribution and beating frequency are important properties of airway epithelial tissues. These parameters are critical in diagnosing primary ciliary dyskinesia and examining in vitro models, including those derived from induced pluripotent stem cells. Video microscopy can be used to characterize these parameters, but most tools available at the moment are limited in the type of information they can provide, usually only describing the ciliary beat frequency of very small areas, while requiring human intervention and training for their use. We propose a novel and open-source method to fully characterize cilia beating frequency and motile cilia coverage in an automated fashion without user intervention. We demonstrate the ability to differentiate between different coverage densities, identifying even small patches of cilia in a larger field of view, and to fully characterize the cilia beating frequency of all moving areas. We also show that the method can be used to combine multiple fields of view to better describe a sample without relying on small pre-selected regions of interest. This is released with a simple graphical user interface for file handling, enabling a full analysis of individual fields of view in a few minutes on a typical personal computer.
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Nonalcoholic fatty liver disease (NAFLD) is a chronic liver disease affecting multiple cell types of the human liver. The high prevalence of NAFLD and the lack of approved therapies increase the demand for reliable models for the preclinical discovery of drug targets. In the last decade, multiple proof-of-principle studies have demonstrated human-specific NAFLD modeling in the dish. These systems have included technologies based on human induced pluripotent stem cell derivatives, liver tissue section cultures, intrahepatic cholangiocyte organoids, and liver-on-a-chip. These platforms differ in functional maturity, multicellularity, scalability, and spatial organization. Identifying an appropriate model for a specific NAFLD-related research question is challenging. Therefore, we review different platforms for their strengths and limitations in modeling NAFLD. To define the fidelity of the current human in vitro NAFLD models in depth, we define disease hallmarks within the NAFLD spectrum that range from steatosis to severe fibroinflammatory tissue injury. We discuss how the most common methods are efficacious in modeling genetic contributions and aspects of the early NAFLD-related tissue response. We also highlight the shortcoming of current models to recapitulate the complexity of inter-organ crosstalk and the chronic process of liver fibrosis-to-cirrhosis that usually takes decades in patients. Importantly, we provide methodological overviews and discuss implementation hurdles (eg, reproducibility or costs) to help choose the most appropriate NAFLD model for the individual research focus: hepatocyte injury, ductular reaction, cellular crosstalk, or other applications. In sum, we highlight current strategies and deficiencies to model NAFLD in the dish and propose a framework for the next generation of human-specific investigations.
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Células Madre Pluripotentes Inducidas , Enfermedad del Hígado Graso no Alcohólico , Humanos , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Reproducibilidad de los Resultados , Células Madre Pluripotentes Inducidas/metabolismo , Cirrosis Hepática , FibrosisRESUMEN
[This corrects the article DOI: 10.1038/s44161-022-00183-w.].
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Over 80% of patients with pancreatic ductal adenocarcinoma (PDAC) are diagnosed at a late stage and are locally advanced or with concurrent metastases. The aggressive phenotype and relative chemo- and radiotherapeutic resistance of PDAC is thought to be mediated largely by its prominent stroma, which is supported by an extracellular matrix (ECM). Therefore, we investigated the impact of tissue-matched human ECM in driving PDAC and the role of the ECM in promoting chemotherapy resistance. Decellularized human pancreata and livers were recellularized with PANC-1 and MIA PaCa-2 (PDAC cell lines), as well as PK-1 cells (liver-derived metastatic PDAC cell line). PANC-1 cells migrated into the pancreatic scaffolds, MIA PaCa-2 cells were able to migrate into both scaffolds, whereas PK-1 cells were able to migrate into the liver scaffolds only. These differences were supported by significant deregulations in gene and protein expression between the pancreas scaffolds, liver scaffolds, and 2D culture. Moreover, these cell lines were significantly more resistant to gemcitabine and doxorubicin chemotherapy treatments in the 3D models compared to 2D cultures, even after confirmed uptake by confocal microscopy. These results suggest that tissue-specific ECM provides the preserved native cues for primary and metastatic PDAC cells necessary for a more reliable in vitro cell culture.
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Adenocarcinoma , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Línea Celular Tumoral , Neoplasias Pancreáticas/patología , Carcinoma Ductal Pancreático/metabolismo , Páncreas/patología , Matriz Extracelular/metabolismo , Adenocarcinoma/metabolismo , Neoplasias PancreáticasRESUMEN
RB is a well-known cell cycle regulator controlling the G1 checkpoint. Previous reports have suggested that it can influence cell fate decisions not only by regulating cell proliferation and survival but also by interacting with transcription factors and epigenetic modifiers. However, the functional redundancy of RB family proteins (RB, RBL1 and RBL2) renders it difficult to investigate their roles during early development, especially in human. Here, we address this problem by generating human embryonic stem cells lacking RB family proteins. To achieve this goal, we first introduced frameshift mutations in RBL1 and RBL2 genes using the CRISPR/Cas9 technology, and then integrated the shRNA-expression cassette to knockdown RB upon tetracycline treatment. The resulting RBL1/2_dKO+RB_iKD cells remain pluripotent and efficiently differentiate into the primary germ layers in vitro even in the absence of the RB family proteins. In contrast, we observed that subsequent differentiation into foregut endoderm was impaired without the expression of RB, RBL1 and RBL2. Thus, it is suggested that RB proteins are dispensable for the maintenance and acquisition of cell identities during early development, but they are essential to generate advanced derivatives after the formation of primary germ layers. These results also indicate that our RBL1/2_dKO+RB_iKD cell lines are useful to depict the detailed molecular roles of RB family proteins in the maintenance and generation of various cell types accessible from human pluripotent stem cells.
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Células Madre Embrionarias Humanas , Células Madre Pluripotentes , Humanos , Diferenciación Celular/fisiología , Endodermo/metabolismo , Células Madre Embrionarias Humanas/metabolismo , Células Madre Pluripotentes/metabolismo , Proteína de Retinoblastoma/genética , Proteína p130 Similar a la del Retinoblastoma/genéticaRESUMEN
Microglia, the resident immune cells of the central nervous system (CNS), are derived from yolk-sac macrophages that populate the developing CNS during early embryonic development. Once established, the microglia population is self-maintained throughout life by local proliferation. As a scalable source of microglia-like cells (MGLs), we here present a forward programming protocol for their generation from human pluripotent stem cells (hPSCs). The transient overexpression of PU.1 and C/EBPß in hPSCs led to a homogenous population of mature microglia within 16 d. MGLs met microglia characteristics on a morphological, transcriptional, and functional level. MGLs facilitated the investigation of a human tauopathy model in cortical neuron-microglia cocultures, revealing a secondary dystrophic microglia phenotype. Single-cell RNA sequencing of microglia integrated into hPSC-derived cortical brain organoids demonstrated a shift of microglia signatures toward a more-developmental in vivo-like phenotype, inducing intercellular interactions promoting neurogenesis and arborization. Taken together, our microglia forward programming platform represents a tool for both reductionist studies in monocultures and complex coculture systems, including 3D brain organoids for the study of cellular interactions in healthy or diseased environments.
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Microglía , Células Madre Pluripotentes , Diferenciación Celular/genética , Sistema Nervioso Central , Humanos , Macrófagos , NeuronasRESUMEN
The liver has been studied extensively due to the broad number of diseases affecting its vital functions. However, therapeutic advances have been hampered by the lack of knowledge concerning human hepatic development. Here, we addressed this limitation by describing the developmental trajectories of different cell types that make up the human liver at single-cell resolution. These transcriptomic analyses revealed that sequential cell-to-cell interactions direct functional maturation of hepatocytes, with non-parenchymal cells playing essential roles during organogenesis. We utilized this information to derive bipotential hepatoblast organoids and then exploited this model system to validate the importance of signalling pathways in hepatocyte and cholangiocyte specification. Further insights into hepatic maturation also enabled the identification of stage-specific transcription factors to improve the functionality of hepatocyte-like cells generated from human pluripotent stem cells. Thus, our study establishes a platform to investigate the basic mechanisms directing human liver development and to produce cell types for clinical applications.
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Hepatocitos , Hígado , Humanos , Hígado/metabolismo , Hepatocitos/metabolismo , Diferenciación Celular , Organoides , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Production of large quantities of hepatocytes remains a major challenge for a number of clinical applications in the biomedical field. Directed differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells (HLCs) provides an advantageous solution and a number of protocols have been developed for this purpose. However, these methods usually follow different steps of liver development in vitro, which is time consuming and requires complex culture conditions. In addition, HLCs lack the full repertoire of functionalities characterising primary hepatocytes. Here, we explore the interest of forward programming to generate hepatocytes from hPSCs and to bypass these limitations. This approach relies on the overexpression of three hepatocyte nuclear factors (HNF1A, HNF6, and FOXA3) in combination with different nuclear receptors expressed in the adult liver using the OPTi-OX platform. Forward programming allows for the rapid production of hepatocytes (FoP-Heps) with functional characteristics using a simplified process. We also uncovered that the overexpression of nuclear receptors such as RORc can enhance specific functionalities of FoP-Heps thereby validating its role in lipid/glucose metabolism. Together, our results show that forward programming could offer a versatile alternative to direct differentiation for generating hepatocytes in vitro.
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Células Madre Pluripotentes Inducidas , Células Madre Pluripotentes , Diferenciación Celular , Hepatocitos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Hígado , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismoRESUMEN
Organoid-based models of murine and human innate lymphoid cell precursor (ILCP) maturation are presented. First, murine intestinal and pulmonary organoids are harnessed to demonstrate that the epithelial niche is sufficient to drive tissue-specific maturation of all innate lymphoid cell (ILC) groups in parallel, without requiring subset-specific cytokine supplementation. Then, more complex human induced pluripotent stem cell (hiPSC)-based gut and lung organoid models are used to demonstrate that human epithelial cells recapitulate maturation of ILC from a stringent systemic human ILCP population, but only when the organoid-associated stromal cells are depleted. These systems offer versatile and reductionist models to dissect the impact of environmental and mucosal niche cues on ILC maturation. In the future, these could provide insight into how ILC activity and development might become dysregulated in chronic inflammatory diseases.
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Células Madre Pluripotentes Inducidas , Organoides , Animales , Diferenciación Celular , Humanos , Inmunidad Innata , Inmunoterapia , Linfocitos , RatonesRESUMEN
Human cholangiocyte organoids show great promise for regenerative therapies and in vitro modeling of bile duct development and diseases. However, the cystic organoids lack the branching morphology of intrahepatic bile ducts (IHBDs). Here, we report establishing human branching cholangiocyte organoid (BRCO) cultures. BRCOs self-organize into complex tubular structures resembling the IHBD architecture. Single-cell transcriptomics and functional analysis showed high similarity to primary cholangiocytes, and importantly, the branching growth mimics aspects of tubular development and is dependent on JAG1/NOTCH2 signaling. When applied to cholangiocarcinoma tumor organoids, the morphology changes to an in vitro morphology like primary tumors. Moreover, these branching cholangiocarcinoma organoids (BRCCAOs) better match the transcriptomic profile of primary tumors and showed increased chemoresistance to gemcitabine and cisplatin. In conclusion, BRCOs recapitulate a complex process of branching morphogenesis in vitro. This provides an improved model to study tubular formation, bile duct functionality, and associated biliary diseases.
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Colangiocarcinoma , Organoides , Conductos Biliares , Células Epiteliales , Humanos , TranscriptomaRESUMEN
Long-term neuroepithelial-like stem cells (lt-NES) derived from human embryonic stem cells are a stable self-renewing progenitor population with high neurogenic potential and phenotypic plasticity. Lt-NES are amenable to regional patterning toward neurons and glia subtypes and thus represent a valuable source of cells for many biomedical applications. For use in regenerative medicine and cell therapy, lt-NES and their progeny require derivation with high-quality culture conditions suitable for clinical use. In this chapter, we describe a robust method to derive multipotent and expandable lt-NES based on good manufacturing practice and cell therapy-grade reagents. We further describe fully defined protocols to terminally differentiate lt-NES toward GABA-ergic, dopaminergic, and motor neurons.
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Células Madre Embrionarias Humanas , Células-Madre Neurales , Diferenciación Celular/fisiología , Tratamiento Basado en Trasplante de Células y Tejidos , Humanos , NeurogénesisRESUMEN
Re-activating quiescent adult epicardium represents a potential therapeutic approach for human cardiac regeneration. However, the exact molecular differences between inactive adult and active fetal epicardium are not known. In this study, we combined fetal and adult human hearts using single-cell and single-nuclei RNA sequencing and compared epicardial cells from both stages. We found that a migratory fibroblast-like epicardial population only in the fetal heart and fetal epicardium expressed angiogenic gene programs, whereas the adult epicardium was solely mesothelial and immune responsive. Furthermore, we predicted that adult hearts may still receive fetal epicardial paracrine communication, including WNT signaling with endocardium, reinforcing the validity of regenerative strategies that administer or reactivate epicardial cells in situ. Finally, we explained graft efficacy of our human embryonic stem-cell-derived epicardium model by noting its similarity to human fetal epicardium. Overall, our study defines epicardial programs of regenerative angiogenesis absent in adult hearts, contextualizes animal studies and defines epicardial states required for effective human heart regeneration.
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Gastrulation is the fundamental process in all multicellular animals through which the basic body plan is first laid down1-4. It is pivotal in generating cellular diversity coordinated with spatial patterning. In humans, gastrulation occurs in the third week after fertilization. Our understanding of this process in humans is relatively limited and based primarily on historical specimens5-8, experimental models9-12 or, more recently, in vitro cultured samples13-16. Here we characterize in a spatially resolved manner the single-cell transcriptional profile of an entire gastrulating human embryo, staged to be between 16 and 19 days after fertilization. We use these data to analyse the cell types present and to make comparisons with other model systems. In addition to pluripotent epiblast, we identified primordial germ cells, red blood cells and various mesodermal and endodermal cell types. This dataset offers a unique glimpse into a central but inaccessible stage of our development. This characterization provides new context for interpreting experiments in other model systems and represents a valuable resource for guiding directed differentiation of human cells in vitro.
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Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Gástrula/citología , Gastrulación/genética , Perfilación de la Expresión Génica , Análisis de la Célula Individual , Transcriptoma , Animales , Diferenciación Celular , Conjuntos de Datos como Asunto , Embrión de Mamíferos/embriología , Endodermo/citología , Eritrocitos/citología , Femenino , Gástrula/metabolismo , Células Germinativas/citología , Humanos , Masculino , Mesodermo/citología , RatonesRESUMEN
Heterozygous mutations in HNF1B in humans result in a multisystem disorder, including pancreatic hypoplasia and diabetes mellitus. Here we used a well-controlled human induced pluripotent stem cell pancreatic differentiation model to elucidate the molecular mechanisms underlying HNF1B-associated diabetes. Our results show that lack of HNF1B blocks specification of pancreatic fate from the foregut progenitor (FP) stage, but HNF1B haploinsufficiency allows differentiation of multipotent pancreatic progenitor cells (MPCs) and insulin-secreting ß-like cells. We show that HNF1B haploinsufficiency impairs cell proliferation in FPs and MPCs. This could be attributed to impaired induction of key pancreatic developmental genes, including SOX11, ROBO2, and additional TEAD1 target genes whose function is associated with MPC self-renewal. In this work we uncover an exhaustive list of potential HNF1B gene targets during human pancreas organogenesis whose downregulation might underlie HNF1B-associated diabetes onset in humans, thus providing an important resource to understand the pathogenesis of this disease.
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Diferenciación Celular/genética , Factor Nuclear 1-beta del Hepatocito/genética , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Modelos Biológicos , Organogénesis/genética , Páncreas/embriología , Páncreas/metabolismo , Biomarcadores , Sistemas CRISPR-Cas , Linaje de la Célula/genética , Diabetes Mellitus/etiología , Susceptibilidad a Enfermedades , Técnica del Anticuerpo Fluorescente , Edición Génica , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Haploinsuficiencia , Factor Nuclear 1-beta del Hepatocito/metabolismo , Humanos , Inmunofenotipificación , Células Secretoras de Insulina/metabolismo , Transducción de SeñalRESUMEN
The signalling pathways that maintain primed human pluripotent stem cells (hPSCs) have been well characterised, revealing a critical role for TGFß/Activin/Nodal signalling. In contrast, the signalling requirements of naive human pluripotency have not been fully established. Here, we demonstrate that TGFß signalling is required to maintain naive hPSCs. The downstream effector proteins - SMAD2/3 - bind common sites in naive and primed hPSCs, including shared pluripotency genes. In naive hPSCs, SMAD2/3 additionally bind to active regulatory regions near to naive pluripotency genes. Inhibiting TGFß signalling in naive hPSCs causes the downregulation of SMAD2/3-target genes and pluripotency exit. Single-cell analyses reveal that naive and primed hPSCs follow different transcriptional trajectories after inhibition of TGFß signalling. Primed hPSCs differentiate into neuroectoderm cells, whereas naive hPSCs transition into trophectoderm. These results establish that there is a continuum for TGFß pathway function in human pluripotency spanning a developmental window from naive to primed states.