ABSTRACT
The construction of the human nervous system is a distinctly complex although highly regulated process. Human tissue inaccessibility has impeded a molecular understanding of the developmental specializations from which our unique cognitive capacities arise. A confluence of recent technological advances in genomics and stem cell-based tissue modeling is laying the foundation for a new understanding of human neural development and dysfunction in neuropsychiatric disease. Here, we review recent progress on uncovering the cellular and molecular principles of human brain organogenesis in vivo as well as using organoids and assembloids in vitro to model features of human evolution and disease.
Subject(s)
Autism Spectrum Disorder/metabolism , Brain/embryology , Brain/growth & development , Epilepsy/metabolism , Neurogenesis/physiology , Schizophrenia/metabolism , Animals , Autism Spectrum Disorder/genetics , Brain/metabolism , Epilepsy/genetics , Humans , Mutation , Neurons/cytology , Neurons/metabolism , Organoids/embryology , Organoids/growth & development , Schizophrenia/geneticsABSTRACT
Organoids capable of forming tissue-like structures have transformed our ability to model human development and disease. With the notable exception of the human heart, lineage-specific self-organizing organoids have been reported for all major organs. Here, we established self-organizing cardioids from human pluripotent stem cells that intrinsically specify, pattern, and morph into chamber-like structures containing a cavity. Cardioid complexity can be controlled by signaling that instructs the separation of cardiomyocyte and endothelial layers and by directing epicardial spreading, inward migration, and differentiation. We find that cavity morphogenesis is governed by a mesodermal WNT-BMP signaling axis and requires its target HAND1, a transcription factor linked to developmental heart chamber defects. Upon cryoinjury, cardioids initiated a cell-type-dependent accumulation of extracellular matrix, an early hallmark of both regeneration and heart disease. Thus, human cardioids represent a powerful platform to mechanistically dissect self-organization, congenital heart defects and serve as a foundation for future translational research.
Subject(s)
Heart/embryology , Organogenesis , Organoids/embryology , Activins/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors/metabolism , Bone Morphogenetic Proteins/metabolism , Calcium/metabolism , Cell Line , Cell Lineage , Chickens , Endothelial Cells/cytology , Extracellular Matrix Proteins/metabolism , Female , Fibroblasts/cytology , Homeobox Protein Nkx-2.5/metabolism , Humans , Male , Mesoderm/embryology , Models, Biological , Myocardium/metabolism , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , Vascular Endothelial Growth Factor A/metabolism , Wnt Proteins/metabolismABSTRACT
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.
Subject(s)
Anatomy, Artistic , Atlases as Topic , Embryonic Development , Endoderm/embryology , Models, Biological , Organoids/embryology , CDX2 Transcription Factor/metabolism , Cell Line , Epidermal Growth Factor/pharmacology , Epithelial Cells/cytology , Female , Gastrulation , Gene Deletion , Gene Expression Regulation, Developmental/drug effects , Humans , Intestines/embryology , Male , Mesoderm/embryology , Middle Aged , Neuregulin-1/metabolism , Organ Specificity , Pluripotent Stem Cells/cytologyABSTRACT
The concomitant occurrence of tissue growth and organization is a hallmark of organismal development1-3. This often means that proliferating and differentiating cells are found at the same time in a continuously changing tissue environment. How cells adapt to architectural changes to prevent spatial interference remains unclear. Here, to understand how cell movements that are key for growth and organization are orchestrated, we study the emergence of photoreceptor neurons that occur during the peak of retinal growth, using zebrafish, human tissue and human organoids. Quantitative imaging reveals that successful retinal morphogenesis depends on the active bidirectional translocation of photoreceptors, leading to a transient transfer of the entire cell population away from the apical proliferative zone. This pattern of migration is driven by cytoskeletal machineries that differ depending on the direction: microtubules are exclusively required for basal translocation, whereas actomyosin is involved in apical movement. Blocking the basal translocation of photoreceptors induces apical congestion, which hampers the apical divisions of progenitor cells and leads to secondary defects in lamination. Thus, photoreceptor migration is crucial to prevent competition for space, and to allow concurrent tissue growth and lamination. This shows that neuronal migration, in addition to its canonical role in cell positioning4, can be involved in coordinating morphogenesis.
Subject(s)
Cell Movement , Morphogenesis , Photoreceptor Cells , Retina , Animals , Humans , Actomyosin/metabolism , Cell Competition , Cell Differentiation , Cell Movement/physiology , Cell Proliferation , Microtubules/metabolism , Morphogenesis/physiology , Organoids/cytology , Organoids/embryology , Photoreceptor Cells/cytology , Photoreceptor Cells/physiology , Retina/cytology , Retina/embryology , Zebrafish/embryologyABSTRACT
The assembly of cortical circuits involves the generation and migration of interneurons from the ventral to the dorsal forebrain1-3, which has been challenging to study at inaccessible stages of late gestation and early postnatal human development4. Autism spectrum disorder and other neurodevelopmental disorders (NDDs) have been associated with abnormal cortical interneuron development5, but which of these NDD genes affect interneuron generation and migration, and how they mediate these effects remains unknown. We previously developed a platform to study interneuron development and migration in subpallial organoids and forebrain assembloids6. Here we integrate assembloids with CRISPR screening to investigate the involvement of 425 NDD genes in human interneuron development. The first screen aimed at interneuron generation revealed 13 candidate genes, including CSDE1 and SMAD4. We subsequently conducted an interneuron migration screen in more than 1,000 forebrain assembloids that identified 33 candidate genes, including cytoskeleton-related genes and the endoplasmic reticulum-related gene LNPK. We discovered that, during interneuron migration, the endoplasmic reticulum is displaced along the leading neuronal branch before nuclear translocation. LNPK deletion interfered with this endoplasmic reticulum displacement and resulted in abnormal migration. These results highlight the power of this CRISPR-assembloid platform to systematically map NDD genes onto human development and reveal disease mechanisms.
Subject(s)
CRISPR-Cas Systems , Gene Editing , Neurodevelopmental Disorders , Female , Humans , Infant, Newborn , Pregnancy , Cell Movement/genetics , CRISPR-Cas Systems/genetics , Interneurons/cytology , Interneurons/metabolism , Interneurons/pathology , Neurodevelopmental Disorders/genetics , Neurodevelopmental Disorders/pathology , Organoids/cytology , Organoids/embryology , Organoids/growth & development , Organoids/metabolism , Organoids/pathology , Endoplasmic Reticulum/metabolism , Prosencephalon/cytology , Prosencephalon/embryology , Prosencephalon/growth & development , Prosencephalon/metabolism , Prosencephalon/pathology , Active Transport, Cell NucleusABSTRACT
The gastrointestinal (GI) tract is complex and consists of multiple organs with unique functions. Rare gene variants can cause congenital malformations of the human GI tract, although the molecular basis of these has been poorly studied. We identified a patient with compound-heterozygous variants in RFX6 presenting with duodenal malrotation and atresia, implicating RFX6 in development of the proximal intestine. To identify how mutations in RFX6 impact intestinal patterning and function, we derived induced pluripotent stem cells from this patient to generate human intestinal organoids (HIOs). We identified that the duodenal HIOs and human tissues had mixed regional identity, with gastric and ileal features. CRISPR-mediated correction of RFX6 restored duodenal identity. We then used gain- and loss-of-function and transcriptomic approaches in HIOs and Xenopus embryos to identify that PDX1 is a downstream transcriptional target of RFX6 required for duodenal development. However, RFX6 had additional PDX1-independent transcriptional targets involving multiple components of signaling pathways that are required for establishing early regional identity in the GI tract. In summary, we have identified RFX6 as a key regulator in intestinal patterning that acts by regulating transcriptional and signaling pathways.
Subject(s)
Gene Expression Regulation, Developmental , Homeodomain Proteins , Organoids , Regulatory Factor X Transcription Factors , Trans-Activators , Humans , Regulatory Factor X Transcription Factors/genetics , Regulatory Factor X Transcription Factors/metabolism , Animals , Homeodomain Proteins/metabolism , Homeodomain Proteins/genetics , Trans-Activators/metabolism , Trans-Activators/genetics , Organoids/metabolism , Organoids/embryology , Duodenum/metabolism , Duodenum/embryology , Intestines/embryology , Intestinal Atresia/genetics , Induced Pluripotent Stem Cells/metabolism , Body Patterning/genetics , Signal Transduction/genetics , Mutation/geneticsABSTRACT
Epithelial organoids, such as those derived from stem cells of the intestine, have great potential for modelling tissue and disease biology1-4. However, the approaches that are used at present to derive these organoids in three-dimensional matrices5,6 result in stochastically developing tissues with a closed, cystic architecture that restricts lifespan and size, limits experimental manipulation and prohibits homeostasis. Here, by using tissue engineering and the intrinsic self-organization properties of cells, we induce intestinal stem cells to form tube-shaped epithelia with an accessible lumen and a similar spatial arrangement of crypt- and villus-like domains to that in vivo. When connected to an external pumping system, the mini-gut tubes are perfusable; this allows the continuous removal of dead cells to prolong tissue lifespan by several weeks, and also enables the tubes to be colonized with microorganisms for modelling host-microorganism interactions. The mini-intestines include rare, specialized cell types that are seldom found in conventional organoids. They retain key physiological hallmarks of the intestine and have a notable capacity to regenerate. Our concept for extrinsically guiding the self-organization of stem cells into functional organoids-on-a-chip is broadly applicable and will enable the attainment of more physiologically relevant organoid shapes, sizes and functions.
Subject(s)
Homeostasis , Intestines/embryology , Morphogenesis , Organoids/embryology , Tissue Scaffolds , Animals , Body Patterning , Cell Differentiation , Cell Lineage , Cryptosporidium parvum/pathogenicity , Human Embryonic Stem Cells/cytology , Human Umbilical Vein Endothelial Cells , Humans , Intestines/cytology , Intestines/parasitology , Intestines/pathology , Mice , Models, Biological , Organoids/cytology , Organoids/parasitology , Organoids/pathology , Regeneration , Regenerative Medicine , Stem Cells , Tissue Culture Techniques/methods , Tissue EngineeringABSTRACT
The body plan of the mammalian embryo is shaped through the process of gastrulation, an early developmental event that transforms an isotropic group of cells into an ensemble of tissues that is ordered with reference to three orthogonal axes1. Although model organisms have provided much insight into this process, we know very little about gastrulation in humans, owing to the difficulty of obtaining embryos at such early stages of development and the ethical and technical restrictions that limit the feasibility of observing gastrulation ex vivo2. Here we show that human embryonic stem cells can be used to generate gastruloids-three-dimensional multicellular aggregates that differentiate to form derivatives of the three germ layers organized spatiotemporally, without additional extra-embryonic tissues. Human gastruloids undergo elongation along an anteroposterior axis, and we use spatial transcriptomics to show that they exhibit patterned gene expression. This includes a signature of somitogenesis that suggests that 72-h human gastruloids show some features of Carnegie-stage-9 embryos3. Our study represents an experimentally tractable model system to reveal and examine human-specific regulatory processes that occur during axial organization in early development.
Subject(s)
Body Patterning , Gastrula/cytology , Human Embryonic Stem Cells/cytology , Organoids/cytology , Organoids/embryology , Somites/cytology , Somites/embryology , Body Patterning/genetics , Gastrula/embryology , Gastrula/metabolism , Gene Expression Regulation, Developmental , Humans , In Vitro Techniques , Organoids/metabolism , Signal Transduction , Somites/metabolism , TranscriptomeABSTRACT
Gastruloids are three-dimensional aggregates of embryonic stem cells that display key features of mammalian development after implantation, including germ-layer specification and axial organization1-3. To date, the expression pattern of only a small number of genes in gastruloids has been explored with microscopy, and the extent to which genome-wide expression patterns in gastruloids mimic those in embryos is unclear. Here we compare mouse gastruloids with mouse embryos using single-cell RNA sequencing and spatial transcriptomics. We identify various embryonic cell types that were not previously known to be present in gastruloids, and show that key regulators of somitogenesis are expressed similarly between embryos and gastruloids. Using live imaging, we show that the somitogenesis clock is active in gastruloids and has dynamics that resemble those in vivo. Because gastruloids can be grown in large quantities, we performed a small screen that revealed how reduced FGF signalling induces a short-tail phenotype in embryos. Finally, we demonstrate that embedding in Matrigel induces gastruloids to generate somites with the correct rostral-caudal patterning, which appear sequentially in an anterior-to-posterior direction over time. This study thus shows the power of gastruloids as a model system for exploring development and somitogenesis in vitro in a high-throughput manner.
Subject(s)
Gastrula , Mouse Embryonic Stem Cells/cytology , Organoids/cytology , Organoids/embryology , Single-Cell Analysis , Somites/cytology , Somites/embryology , Transcriptome , Animals , Collagen , Drug Combinations , Embryo, Mammalian/cytology , Embryo, Mammalian/embryology , Embryo, Mammalian/metabolism , Embryonic Development , Female , Gastrula/cytology , Gastrula/embryology , Gastrula/metabolism , Gene Expression Regulation, Developmental , Laminin , Male , Mice , Mouse Embryonic Stem Cells/metabolism , Organoids/metabolism , Proteoglycans , RNA-Seq , Somites/metabolism , Time FactorsABSTRACT
Organogenesis is a complex and interconnected process that is orchestrated by multiple boundary tissue interactions1-7. However, it remains unclear how individual, neighbouring components coordinate to establish an integral multi-organ structure. Here we report the continuous patterning and dynamic morphogenesis of hepatic, biliary and pancreatic structures, invaginating from a three-dimensional culture of human pluripotent stem cells. The boundary interactions between anterior and posterior gut spheroids differentiated from human pluripotent stem cells enables retinoic acid-dependent emergence of hepato-biliary-pancreatic organ domains specified at the foregut-midgut boundary organoids in the absence of extrinsic factors. Whereas transplant-derived tissues are dominated by midgut derivatives, long-term-cultured microdissected hepato-biliary-pancreatic organoids develop into segregated multi-organ anlages, which then recapitulate early morphogenetic events including the invagination and branching of three different and interconnected organ structures, reminiscent of tissues derived from mouse explanted foregut-midgut culture. Mis-segregation of multi-organ domains caused by a genetic mutation in HES1 abolishes the biliary specification potential in culture, as seen in vivo8,9. In sum, we demonstrate that the experimental multi-organ integrated model can be established by the juxtapositioning of foregut and midgut tissues, and potentially serves as a tractable, manipulatable and easily accessible model for the study of complex human endoderm organogenesis.
Subject(s)
Biliary Tract/embryology , Intestines/embryology , Liver/embryology , Models, Biological , Morphogenesis , Pancreas/embryology , Animals , Biliary Tract/cytology , Biomarkers/analysis , Biomarkers/metabolism , Body Patterning , Endoderm/cytology , Endoderm/embryology , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Intestines/cytology , Liver/cytology , Male , Mice , Organoids/cytology , Organoids/embryology , Pancreas/cytology , Spheroids, Cellular/cytology , Spheroids, Cellular/metabolism , Spheroids, Cellular/transplantation , Transcription Factor HES-1/analysis , Transcription Factor HES-1/metabolismABSTRACT
The neocortex, the center for higher brain function, first emerged in mammals and has become massively expanded and folded in humans, constituting almost half the volume of the human brain. Primary microcephaly, a developmental disorder in which the brain is smaller than normal at birth, results mainly from there being fewer neurons in the neocortex because of defects in neural progenitor cells (NPCs). Outer radial glia (oRGs), NPCs that are abundant in gyrencephalic species but rare in lissencephalic species, are thought to play key roles in the expansion and folding of the neocortex. However, how oRGs expand, whether they are necessary for neocortical folding, and whether defects in oRGs cause microcephaly remain important questions in the study of brain development, evolution, and disease. Here, we show that oRG expansion in mice, ferrets, and human cerebral organoids requires cyclin-dependent kinase 6 (CDK6), the mutation of which causes primary microcephaly via an unknown mechanism. In a mouse model in which increased Hedgehog signaling expands oRGs and intermediate progenitor cells and induces neocortical folding, CDK6 loss selectively decreased oRGs and abolished neocortical folding. Remarkably, this function of CDK6 in oRG expansion did not require its kinase activity, was not shared by the highly similar CDK4 and CDK2, and was disrupted by the mutation causing microcephaly. Therefore, our results indicate that CDK6 is conserved to promote oRG expansion, that oRGs are necessary for neocortical folding, and that defects in oRG expansion may cause primary microcephaly.
Subject(s)
Cyclin-Dependent Kinase 6 , Ependymoglial Cells , Microcephaly , Neocortex , Animals , Cyclin-Dependent Kinase 6/genetics , Cyclin-Dependent Kinase 6/metabolism , Ependymoglial Cells/cytology , Ependymoglial Cells/enzymology , Ferrets , Hedgehog Proteins/metabolism , Humans , Mice , Microcephaly/genetics , Neocortex/abnormalities , Neocortex/enzymology , Neural Stem Cells/cytology , Neural Stem Cells/enzymology , Organoids/embryologyABSTRACT
Cardiac congenital disabilities are the most common organ malformations, but we still do not understand how they arise in the human embryo. Moreover, although cardiovascular disease is the most common cause of death globally, the development of new therapies is lagging compared with other fields. One major bottleneck hindering progress is the lack of self-organizing human cardiac models that recapitulate key aspects of human heart development, physiology and disease. Current in vitro cardiac three-dimensional systems are either engineered constructs or spherical aggregates of cardiomyocytes and other cell types. Although tissue engineering enables the modeling of some electro-mechanical properties, it falls short of mimicking heart development, morphogenetic defects and many clinically relevant aspects of cardiomyopathies. Here, we review different approaches and recent efforts to overcome these challenges in the field using a new generation of self-organizing embryonic and cardiac organoids.
Subject(s)
Heart/embryology , Models, Cardiovascular , Myocytes, Cardiac/metabolism , Organogenesis/physiology , Organoids/embryology , Tissue Engineering/methods , Animals , Cardiovascular Diseases , Coculture Techniques/methods , Humans , Induced Pluripotent Stem Cells/metabolismABSTRACT
The emergence of multiple axes is an essential element in the establishment of the mammalian body plan. This process takes place shortly after implantation of the embryo within the uterus and relies on the activity of gene regulatory networks that coordinate transcription in space and time. Whereas genetic approaches have revealed important aspects of these processes1, a mechanistic understanding is hampered by the poor experimental accessibility of early post-implantation stages. Here we show that small aggregates of mouse embryonic stem cells (ESCs), when stimulated to undergo gastrulation-like events and elongation in vitro, can organize a post-occipital pattern of neural, mesodermal and endodermal derivatives that mimic embryonic spatial and temporal gene expression. The establishment of the three major body axes in these 'gastruloids'2,3 suggests that the mechanisms involved are interdependent. Specifically, gastruloids display the hallmarks of axial gene regulatory systems as exemplified by the implementation of collinear Hox transcriptional patterns along an extending antero-posterior axis. These results reveal an unanticipated self-organizing capacity of aggregated ESCs and suggest that gastruloids could be used as a complementary system to study early developmental events in the mammalian embryo.
Subject(s)
Body Patterning , Gastrula/cytology , Gastrula/embryology , Mouse Embryonic Stem Cells/cytology , Organoids/cytology , Organoids/embryology , Animals , Body Patterning/genetics , Gastrula/metabolism , Gene Expression Profiling , Gene Expression Regulation, Developmental , Genes, Homeobox/genetics , In Vitro Techniques , Mice , Mouse Embryonic Stem Cells/metabolism , Organoids/metabolism , Time FactorsABSTRACT
Despite the global prevalence of gastric disease, there are few adequate models in which to study the fundus epithelium of the human stomach. We differentiated human pluripotent stem cells (hPSCs) into gastric organoids containing fundic epithelium by first identifying and then recapitulating key events in embryonic fundus development. We found that disruption of Wnt/ß-catenin signalling in mouse embryos led to conversion of fundic to antral epithelium, and that ß-catenin activation in hPSC-derived foregut progenitors promoted the development of human fundic-type gastric organoids (hFGOs). We then used hFGOs to identify temporally distinct roles for multiple signalling pathways in epithelial morphogenesis and differentiation of fundic cell types, including chief cells and functional parietal cells. hFGOs are a powerful model for studying the development of the human fundus and the molecular bases of human gastric physiology and pathophysiology, and also represent a new platform for drug discovery.
Subject(s)
Gastric Fundus/metabolism , Wnt Proteins/metabolism , Wnt Signaling Pathway , beta Catenin/metabolism , Animals , Body Patterning , Cell Differentiation , Cell Lineage , Drug Discovery/methods , Epithelial Cells/cytology , Epithelial Cells/metabolism , Epithelium/embryology , Epithelium/metabolism , Female , Gastric Fundus/cytology , Gastric Fundus/embryology , Homeodomain Proteins/metabolism , Humans , Male , Mice , Organoids/cytology , Organoids/embryology , Organoids/metabolism , Parietal Cells, Gastric/cytology , Parietal Cells, Gastric/metabolism , Pluripotent Stem Cells/cytology , SOXB1 Transcription Factors/metabolism , Spheroids, Cellular/cytology , Spheroids, Cellular/metabolism , Trans-Activators/metabolism , Wnt Signaling Pathway/genetics , beta Catenin/agonistsABSTRACT
Trachea-esophageal defects (TEDs), including esophageal atresia (EA), tracheoesophageal fistula (TEF), and laryngeal-tracheoesophageal clefts (LTEC), are a spectrum of life-threatening congenital anomalies in which the trachea and esophagus do not form properly. Up until recently, the developmental basis of these conditions and how the trachea and esophagus arise from a common fetal foregut was poorly understood. However, with significant advances in human genetics, organoids, and animal models, and integrating single cell genomics with high resolution imaging, we are revealing the molecular and cellular mechanisms that orchestrate tracheoesophageal morphogenesis and how disruption in these processes leads to birth defects. Here we review the current understanding of the genetic and developmental basis of TEDs. We suggest future opportunities for integrating developmental mechanisms elucidated from animals and organoids with human genetics and clinical data to gain insight into the genotype-phenotype basis of these heterogeneous birth defects. Finally, we envision how this will enhance diagnosis, improve treatment, and perhaps one day, lead to new tissue replacement therapy.
Subject(s)
Esophagus/abnormalities , Trachea/abnormalities , Animals , Digestive System Abnormalities/diagnosis , Digestive System Abnormalities/etiology , Digestive System Abnormalities/genetics , Disease Models, Animal , Esophagus/embryology , Humans , Organoids/embryology , Trachea/embryologyABSTRACT
Chronic kidney disease (CKD) and end stage renal disease (ESRD) are increasingly frequent and devastating conditions that have driven a surge in the need for kidney transplantation. A stark shortage of organs has fueled interest in generating viable replacement tissues ex vivo for transplantation. One promising approach has been self-organizing organoids, which mimic developmental processes and yield multicellular, organ-specific tissues. However, a recognized roadblock to this approach is that many organoid cell types fail to acquire full maturity and function. Here, we comprehensively assess the vasculature in two distinct kidney organoid models as well as in explanted embryonic kidneys. Using a variety of methods, we show that while organoids can develop a wide range of kidney cell types, as previously shown, endothelial cells (ECs) initially arise but then rapidly regress over time in culture. Vasculature of cultured embryonic kidneys exhibit similar regression. By contrast, engraftment of kidney organoids under the kidney capsule results in the formation of a stable, perfused vasculature that integrates into the organoid. This work demonstrates that kidney organoids offer a promising model system to define the complexities of vascular-nephron interactions, but the establishment and maintenance of a vascular network present unique challenges when grown ex vivo.
Subject(s)
Endothelium, Vascular/embryology , Kidney/blood supply , Kidney/embryology , Organogenesis , Organoids/embryology , Animals , Cells, Cultured , Endothelial Cells , Endothelium, Vascular/cytology , Female , Humans , Kidney/cytology , Male , Mice , Organoids/transplantation , RNA-Seq , Tissue Culture TechniquesABSTRACT
Balanced progenitor activities are crucial for the development and maintenance of high turn-over organs such as the esophagus. However, the molecular mechanisms regulating these progenitor activities in the esophagus remain to be elucidated. Here, we demonstrated that Yap is required for the proliferation of esophageal progenitor cells (EPCs) in the developing murine esophagus. We found that Yap deficiency reduces EPC proliferation and stratification whereas persistent Yap activation increases cell proliferation and causes aberrant stratification of the developing esophagus. We further demonstrated that the role of YAP signaling is conserved in the developing human esophagus by utilizing 3D human pluripotent stem cell (hPSC)-derived esophageal organoid culture. Taken together, our studies combining loss/gain-of-function murine models and hPSC differentiation support a key role for YAP in the self-renewal of EPCs and stratification of the esophageal epithelium.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Cell Cycle Proteins/metabolism , Esophagus/embryology , Models, Biological , Organoids/embryology , Pluripotent Stem Cells/metabolism , Transcription Factors/metabolism , Adaptor Proteins, Signal Transducing/genetics , Animals , Cell Cycle Proteins/genetics , Esophagus/cytology , Humans , Mice , Organoids/cytology , Pluripotent Stem Cells/cytology , Transcription Factors/genetics , YAP-Signaling ProteinsSubject(s)
Chimera/embryology , Embryo Research/ethics , Embryo, Mammalian/embryology , Macaca fascicularis/embryology , Animals , Cattle , Cell Communication , Embryo Research/economics , Embryo, Mammalian/cytology , Fertilization , Humans , Mice , National Institutes of Health (U.S.)/legislation & jurisprudence , Organoids/embryology , Pluripotent Stem Cells/cytology , Rats , Research Support as Topic/legislation & jurisprudence , Species Specificity , Swine/embryology , Time Factors , United StatesABSTRACT
The ability to reproduce early stages of human neurodevelopment in the laboratory is one of the most exciting fields in modern neuroscience. The inaccessibility of the healthy human brain developing in utero has delayed our understanding of the initial steps in the formation of one of the most complex tissues in the body. Animal models, postmortem human tissues and cellular systems have been instrumental in contributing to our understanding of the human brain. However, all model systems have intrinsic limitations. The emerging field of brain organoids, which are three-dimensional self-assembled multicellular structures derived from human pluripotent stem cells, offers a promising complementary cellular model for the study of the human brain. Here, we will discuss the initial experiments that were the foundation for this emerging field, highlight recent uses of the technology and offer our perspective on future directions that might guide further exploratory experimentation to improve the human brain organoid model system.
Subject(s)
Brain/embryology , Models, Biological , Nervous System Diseases/pathology , Organoids/embryology , Brain/drug effects , Brain/virology , Environmental Pollutants/toxicity , Humans , Nervous System Diseases/genetics , Organoids/drug effects , Viruses/metabolismABSTRACT
Loss of expression of the transcription regulator DC-SCRIPT (Zfp366) is a prominent prognostic event in estrogen receptor-positive breast cancer patients. Studying the inherent link between breast morphogenesis and tumorigenesis, we recently reported that DC-SCRIPT affects normal mammary branching morphogenesis and mammary epithelium homeostasis. Here we investigated the molecular mechanism involved in DC-SCRIPT mediated regulation of FGF2 induced mammary branching morphogenesis in a 3D organoid culture system. Our data show that the delayed mammary organoid branching observed in DC-SCRIPT-/- organoids cannot be compensated for by increasing FGF2 levels. Interestingly, FGFR1, the dominant FGF2 receptor, was expressed at a significantly lower level in basal epithelial cells of DC-SCRIPT deficient organoids relative to wildtype organoids. A potential link between DC-SCRIPT and FGFR1 was further supported by the predicted locations of the DC-SCRIPT DNA binding motif at the Fgfr1 gene. Moreover, ERK1/2 phosphorylation downstream of the FGFR1 pathway was decreased in basal epithelial cells of DC-SCRIPT deficient organoids. Altogether, this study shows a relationship between DC-SCRIPT and FGFR1 related pERK signaling in modulating the branching morphogenesis of mammary organoids in vitro.