RESUMO
Recent studies demonstrate that metabolic disturbance, such as augmented glycolysis, contributes to fibrosis. The molecular regulation of this metabolic perturbation in fibrosis, however, has been elusive. COUP-TFII (also known as NR2F2) is an important regulator of glucose and lipid metabolism. Its contribution to organ fibrosis is undefined. Here, we found increased COUP-TFII expression in myofibroblasts in human fibrotic kidneys, lungs, kidney organoids, and mouse kidneys after injury. Genetic ablation of COUP-TFII in mice resulted in attenuation of injury-induced kidney fibrosis. A non-biased proteomic study revealed the suppression of fatty acid oxidation and the enhancement of glycolysis pathways in COUP-TFII overexpressing fibroblasts. Overexpression of COUP-TFII in fibroblasts also induced production of alpha-smooth muscle actin (αSMA) and collagen 1. Knockout of COUP-TFII decreased glycolysis and collagen 1 levels in fibroblasts. Chip-qPCR revealed the binding of COUP-TFII on the promoter of PGC1α. Overexpression of COUP-TFII reduced the cellular level of PGC1α. Targeting COUP-TFII serves as a novel treatment approach for mitigating fibrosis in chronic kidney disease and potentially fibrosis in other organs.
Assuntos
Fator II de Transcrição COUP , Receptores Nucleares Órfãos , Animais , Fator II de Transcrição COUP/genética , Fator II de Transcrição COUP/metabolismo , Fibrose , Glicólise/genética , Rim , Camundongos , Camundongos Knockout , Miofibroblastos , Receptores Nucleares Órfãos/metabolismo , ProteômicaRESUMO
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.
Assuntos
Fundo Gástrico/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo , Animais , Padronização Corporal , Diferenciação Celular , Linhagem da Célula , Descoberta de Drogas/métodos , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Epitélio/embriologia , Epitélio/metabolismo , Feminino , Fundo Gástrico/citologia , Fundo Gástrico/embriologia , Proteínas de Homeodomínio/metabolismo , Humanos , Masculino , Camundongos , Organoides/citologia , Organoides/embriologia , Organoides/metabolismo , Células Parietais Gástricas/citologia , Células Parietais Gástricas/metabolismo , Células-Tronco Pluripotentes/citologia , Fatores de Transcrição SOXB1/metabolismo , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo , Transativadores/metabolismo , Via de Sinalização Wnt/genética , beta Catenina/agonistasRESUMO
Gastric diseases, including peptic ulcer disease and gastric cancer, affect 10% of the world's population and are largely due to chronic Helicobacter pylori infection. Species differences in embryonic development and architecture of the adult stomach make animal models suboptimal for studying human stomach organogenesis and pathogenesis, and there is no experimental model of normal human gastric mucosa. Here we report the de novo generation of three-dimensional human gastric tissue in vitro through the directed differentiation of human pluripotent stem cells. We show that temporal manipulation of the FGF, WNT, BMP, retinoic acid and EGF signalling pathways and three-dimensional growth are sufficient to generate human gastric organoids (hGOs). Developing hGOs progressed through molecular and morphogenetic stages that were nearly identical to the developing antrum of the mouse stomach. Organoids formed primitive gastric gland- and pit-like domains, proliferative zones containing LGR5-expressing cells, surface and antral mucous cells, and a diversity of gastric endocrine cells. We used hGO cultures to identify novel signalling mechanisms that regulate early endoderm patterning and gastric endocrine cell differentiation upstream of the transcription factor NEUROG3. Using hGOs to model pathogenesis of human disease, we found that H. pylori infection resulted in rapid association of the virulence factor CagA with the c-Met receptor, activation of signalling and induction of epithelial proliferation. Together, these studies describe a new and robust in vitro system for elucidating the mechanisms underlying human stomach development and disease.
Assuntos
Infecções por Helicobacter/fisiopatologia , Modelos Biológicos , Organogênese , Organoides/citologia , Células-Tronco Pluripotentes/citologia , Estômago/citologia , Diferenciação Celular , Helicobacter pylori , Humanos , Organoides/microbiologia , Transdução de SinaisRESUMO
The stomach is a digestive organ that has important roles in human physiology and pathophysiology. The developmental origin of the stomach is the embryonic foregut, which also gives rise a number of other structures. There are several signaling pathways and transcription factors that are known to regulate stomach development at different stages, including foregut patterning, stomach specification, and gastric regionalization. These developmental events have important implications in later homeostasis and disease in the adult stomach. Here we will review the literature that has shaped our current understanding of the molecular mechanisms that coordinate gastric organogenesis. Further we will discuss how developmental paradigms have guided recent efforts to differentiate stomach tissue from pluripotent stem cells.
Assuntos
Desenvolvimento Embrionário/genética , Endoderma/embriologia , Estômago/embriologia , Fatores de Transcrição/metabolismo , HumanosRESUMO
A small number of signaling pathways are used repeatedly during organogenesis, and they can have drastically different effects on the same population of cells depending on the embryonic stage. How cellular competence changes over developmental time is not well understood. Here we used Xenopus, mouse, and human pluripotent stem cells to investigate how the temporal sequence of Wnt, BMP, and retinoic acid (RA) signals regulates endoderm developmental competence and organ induction, focusing on respiratory fate. While Nkx2-1+ lung fate is not induced until late somitogenesis stages, here we show that lung competence is restricted by the gastrula stage as a result of Wnt and BMP-dependent anterior-posterior (A-P) patterning. These early Wnt and BMP signals make posterior endoderm refractory to subsequent RA/Wnt/BMP-dependent lung induction. We further mapped how RA modulates the response to Wnt and BMP in a temporal specific manner. In the gastrula RA promotes posterior identity, however in early somite stages of development RA regulates respiratory versus pharyngeal potential in anterior endoderm and midgut versus hindgut potential in posterior endoderm. Together our data suggest a dynamic and conserved response of vertebrate endoderm during organogenesis, wherein early Wnt/BMP/RA impacts how cells respond to later Wnt/BMP/RA signals, illustrating how reiterative combinatorial signaling can regulate both developmental competence and subsequent fate specification.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Endoderma/embriologia , Organogênese/efeitos dos fármacos , Tretinoína/farmacologia , Proteínas Wnt/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Endoderma/citologia , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Organogênese/fisiologia , Somitos/citologia , Somitos/embriologia , Especificidade da Espécie , Xenopus laevisRESUMO
Recent advances in generating pancreatic cell types from human pluripotent stem cells has depended on our knowledge of the developmental processes that regulate pancreas development in vivo. The developmental events between gastrulation and formation of the embryonic pancreatic primordia are both rapid and dynamic and studies in frog, fish, chick, and mouse have identified the molecular basis of how the pancreas develops from multipotent endoderm progenitors. Here, we review the current status of our understanding of molecular mechanisms that control endoderm formation, endoderm patterning, and pancreas specification and highlight how these discoveries have allowed for the development of robust methods to generate pancreatic cells from human pluripotent stem cells.
Assuntos
Organogênese , Pâncreas/embriologia , Células-Tronco Pluripotentes/metabolismo , Transdução de Sinais , Animais , Endoderma/embriologia , Endoderma/metabolismo , Humanos , Pâncreas/metabolismoRESUMO
To investigate the co-development of vasculature, mesenchyme, and epithelium crucial for organogenesis and the acquisition of organ-specific characteristics, we constructed a human pluripotent stem cell-derived organoid system comprising lung or intestinal epithelium surrounded by organotypic mesenchyme and vasculature. We demonstrated the pivotal role of co-differentiating mesoderm and endoderm via precise BMP regulation in generating multilineage organoids and gut tube patterning. Single-cell RNA-seq analysis revealed organ specificity in endothelium and mesenchyme, and uncovered key ligands driving endothelial specification in the lung (e.g., WNT2B and Semaphorins) or intestine (e.g., GDF15). Upon transplantation under the kidney capsule in mice, these organoids further matured and developed perfusable human-specific sub-epithelial capillaries. Additionally, our model recapitulated the abnormal endothelial-epithelial crosstalk in patients with FOXF1 deletion or mutations. Multilineage organoids provide a unique platform to study developmental cues guiding endothelial and mesenchymal cell fate determination, and investigate intricate cell-cell communications in human organogenesis and disease. Highlights: BMP signaling fine-tunes the co-differentiation of mesoderm and endoderm.The cellular composition in multilineage organoids resembles that of human fetal organs.Mesenchyme and endothelium co-developed within the organoids adopt organ-specific characteristics.Multilineage organoids recapitulate abnormal endothelial-epithelial crosstalk in FOXF1-associated disorders.
RESUMO
Enteroendocrine cells of the gastrointestinal (GI) tract play a central role in metabolism, digestion, satiety and lipid absorption, yet their development remains poorly understood. Here we show that Arx, a homeodomain-containing transcription factor, is required for the normal development of mouse and human enteroendocrine cells. Arx expression is detected in a subset of Neurogenin3 (Ngn3)-positive endocrine progenitors and is also found in a subset of hormone-producing cells. In mice, removal of Arx from the developing endoderm results in a decrease of enteroendocrine cell types including gastrin-, glucagon/GLP-1-, CCK-, secretin-producing cell populations and an increase of somatostatin-expressing cells. This phenotype is also observed in mice with endocrine-progenitor-specific Arx ablation suggesting that Arx is required in the progenitor for enteroendocrine cell development. In addition, depletion of human ARX in developing human intestinal tissue results in a profound deficit in expression of the enteroendocrine cell markers CCK, secretin and glucagon while expression of a pan-intestinal epithelial marker, CDX2, and other non-endocrine markers remained unchanged. Taken together, our findings uncover a novel and conserved role of Arx in mammalian endocrine cell development and provide a potential cause for the chronic diarrhea seen in both humans and mice carrying Arx mutations.
Assuntos
Endoderma/embriologia , Células Enteroendócrinas/citologia , Proteínas de Homeodomínio/fisiologia , Fatores de Transcrição/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular , Linhagem da Célula , Endoderma/citologia , Humanos , Camundongos , Proteínas do Tecido Nervoso/metabolismoRESUMO
Developing models of human kidney tissue in vitro is an important challenge in regenerative nephrology research, given the paucity of novel and effective therapies in kidney disease. However, the de novo generation of kidney tissues from human pluripotent stem cells (hPSCs) is challenging owing to the structural and functional complexity of the organ, as well its developmental origin from two distinct embryologic populations: the metanephric mesenchyme and the ureteric bud (UB). Directed differentiation strategies have been developed to generate kidney organoids containing nephron-like structures; we recently reported an efficient and practical method to generate UB tissues. Here, we describe a detailed step-by-step protocol for differentiation of hPSCs into three-dimensonal UB organoids that exhibit complex morphological development and the capacity to differentiate into functional collecting duct tissues. Over 3 d, hPSCs are induced into PAX2+GATA3+ pronephric (anterior) intermediate mesoderm fates in monolayer cultures at high efficiency. The cells are aggregated into three-dimensional spheroids, which then assemble and organize into nephric duct-like tissue over 4 d. When embedded into an extracellular matrix, the spheroids grow into UB organoids that recapitulate fetal branching morphogenesis for 1 week of culture. When switched to permissive conditions, the UB organoids spontaneously differentiate to form collecting duct principal cells. This approach provides robust and reproducible methods that can be readily adopted by users with basic experience in hPSC and organoid differentiation to generate UB tissues, which may be used to investigate human kidney development, model disease processes and catalyze further efforts in engineering functional kidney tissue.
Assuntos
Rim , Células-Tronco Pluripotentes , Humanos , Organoides , Diferenciação Celular , Engenharia Tecidual/métodosRESUMO
Directed differentiation of human pluripotent stem cells (hPSCs) into functional ureteric and collecting duct (CD) epithelia is essential to kidney regenerative medicine. Here we describe highly efficient, serum-free differentiation of hPSCs into ureteric bud (UB) organoids and functional CD cells. The hPSCs are first induced into pronephric progenitor cells at 90% efficiency and then aggregated into spheres with a molecular signature similar to the nephric duct. In a three-dimensional matrix, the spheres form UB organoids that exhibit branching morphogenesis similar to the fetal UB and correct distal tip localization of RET expression. Organoid-derived cells incorporate into the UB tips of the progenitor niche in chimeric fetal kidney explant culture. At later stages, the UB organoids differentiate into CD organoids, which contain >95% CD cell types as estimated by single-cell RNA sequencing. The CD epithelia demonstrate renal electrophysiologic functions, with ENaC-mediated vectorial sodium transport by principal cells and V-type ATPase proton pump activity by FOXI1-induced intercalated cells.
Assuntos
Células-Tronco Pluripotentes , Ureter , Humanos , Rim , Ureter/metabolismo , Diferenciação Celular , Organoides , Morfogênese , Fatores de Transcrição Forkhead/metabolismoRESUMO
Autosomal Recessive Renal Tubular Dysgenesis (AR-RTD) is a fatal genetic disorder characterized by complete absence or severe depletion of proximal tubules (PT) in patients harboring pathogenic variants in genes involved in the Renin-Angiotensin-Aldosterone System. To uncover the pathomechanism of AR-RTD, differentiation of ACE-/- and AGTR1-/- induced pluripotent stem cells (iPSCs) and AR-RTD patient-derived iPSCs into kidney organoids is leveraged. Comprehensive marker analyses show that both mutant and control organoids generate indistinguishable PT in vitro under normoxic (21% O2) or hypoxic (2% O2) conditions. Fully differentiated (d24) AGTR1-/- and control organoids transplanted under the kidney capsule of immunodeficient mice engraft and mature well, as do renal vesicle stage (d14) control organoids. By contrast, d14 AGTR1-/- organoids fail to engraft due to insufficient pro-angiogenic VEGF-A expression. Notably, growth under hypoxic conditions induces VEGF-A expression and rescues engraftment of AGTR1-/- organoids at d14, as does ectopic expression of VEGF-A. We propose that PT dysgenesis in AR-RTD is primarily a non-autonomous consequence of delayed angiogenesis, starving PT at a critical time in their development.
Assuntos
Angiogênese , Sistema Renina-Angiotensina , Humanos , Animais , Camundongos , Sistema Renina-Angiotensina/genética , Fator A de Crescimento do Endotélio Vascular , Túbulos Renais Proximais/patologia , OrganoidesRESUMO
The human stomach contains two primary domains: the corpus, which contains the fundic epithelium, and the antrum. Each of these domains has distinct cell types and functions, and therefore each presents with unique disease pathologies. Here, we detail two protocols to differentiate human pluripotent stem cells (hPSCs) into human gastric organoids (hGOs) that recapitulate both domains. Both protocols begin with the differentiation of hPSCs into definitive endoderm (DE) using activin A, followed by the generation of free-floating 3D posterior foregut spheroids using FGF4, Wnt pathway agonist CHIR99021 (CHIR), BMP pathway antagonist Noggin, and retinoic acid. Embedding spheroids in Matrigel and continuing 3D growth in epidermal growth factor (EGF)-containing medium for 4 weeks results in antral hGOs (hAGOs). To obtain fundic hGOs (hFGOs), spheroids are additionally treated with CHIR and FGF10. Induced differentiation of acid-secreting parietal cells in hFGOs requires temporal treatment of BMP4 and the MEK inhibitor PD0325901 for 48 h on protocol day 30. In total, it takes ~34 d to generate hGOs from hPSCs. To date, this is the only approach that generates functional human differentiated gastric cells de novo from hPSCs.
Assuntos
Técnicas de Cultura de Células , Endoderma/citologia , Células Epiteliais/citologia , Fundo Gástrico/citologia , Organoides/citologia , Células-Tronco Pluripotentes/citologia , Antro Pilórico/citologia , Ativinas/farmacologia , Benzamidas/farmacologia , Proteínas de Transporte/farmacologia , Diferenciação Celular , Colágeno/química , Meios de Cultura/química , Meios de Cultura/farmacologia , Difenilamina/análogos & derivados , Difenilamina/farmacologia , Combinação de Medicamentos , Endoderma/efeitos dos fármacos , Endoderma/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Fator 10 de Crescimento de Fibroblastos/farmacologia , Fator 4 de Crescimento de Fibroblastos/farmacologia , Fundo Gástrico/metabolismo , Humanos , Laminina/química , Especificidade de Órgãos , Organoides/efeitos dos fármacos , Organoides/metabolismo , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , Proteoglicanas/química , Antro Pilórico/metabolismo , Piridinas/farmacologia , Pirimidinas/farmacologia , Tretinoína/farmacologia , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
Tracheal and esophageal disorders are prevalent in humans and difficult to accurately model in mice. We therefore established a three-dimensional organoid model of esophageal development through directed differentiation of human pluripotent stem cells. Sequential manipulation of bone morphogenic protein (BMP), Wnt, and RA signaling pathways was required to pattern definitive endoderm into foregut, anterior foregut (AFG), and dorsal AFG spheroids. Dorsal AFG spheroids grown in a 3D matrix formed human esophageal organoids (HEOs), and HEO cells could be transitioned into two-dimensional cultures and grown as esophageal organotypic rafts. In both configurations, esophageal tissues had proliferative basal progenitors and a differentiated stratified squamous epithelium. Using HEO cultures to model human esophageal birth defects, we identified that Sox2 promotes esophageal specification in part through repressing Wnt signaling in dorsal AFG and promoting survival. Consistently, Sox2 ablation in mice causes esophageal agenesis. Thus, HEOs present a powerful platform for modeling human pathologies and tissue engineering.
Assuntos
Doenças do Esôfago/metabolismo , Doenças do Esôfago/patologia , Esôfago/citologia , Esôfago/metabolismo , Organoides/metabolismo , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Adolescente , Animais , Células Cultivadas , Criança , Pré-Escolar , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NODRESUMO
Organogenesis of the trachea and lungs requires a complex series of mesoderm-endoderm interactions mediated by WNT, BMP, retinoic acid (RA), and hedgehog (Hh), but how these pathways interact in a gene regulatory network is less clear. Using Xenopus embryology, mouse genetics, and human ES cell cultures, we identified a conserved signaling cascade that initiates respiratory lineage specification. We show that RA has multiple roles; first RA pre-patterns the lateral plate mesoderm and then it promotes Hh ligand expression in the foregut endoderm. Hh subsequently signals back to the pre-patterned mesoderm to promote expression of the lung-inducing ligands Wnt2/2b and Bmp4. Finally, RA regulates the competence of the endoderm to activate the Nkx2-1+ respiratory program in response to these mesodermal WNT and BMP signals. These data provide insights into early lung development and a paradigm for how mesenchymal signals are coordinated with epithelial competence during organogenesis.
Assuntos
Padronização Corporal , Endoderma/embriologia , Proteínas Hedgehog/metabolismo , Pulmão/embriologia , Mesoderma/embriologia , Transdução de Sinais , Tretinoína/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Embrião de Mamíferos/metabolismo , Embrião não Mamífero/metabolismo , Endoderma/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Intestinos/embriologia , Ligantes , Pulmão/metabolismo , Mesoderma/metabolismo , Camundongos , Respiração , Células-Tronco/metabolismo , Proteínas Wnt/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMO
Here we describe a protocol for generating 3D human intestinal tissues (called organoids) in vitro from human pluripotent stem cells (hPSCs). To generate intestinal organoids, pluripotent stem cells are first differentiated into FOXA2(+)SOX17(+) endoderm by treating the cells with activin A for 3 d. After endoderm induction, the pluripotent stem cells are patterned into CDX2(+) mid- and hindgut tissue using FGF4 and WNT3a. During this patterning step, 3D mid- or hindgut spheroids bud from the monolayer epithelium attached to the tissue culture dish. The 3D spheroids are further cultured in Matrigel along with prointestinal growth factors, and they proliferate and expand over 1-3 months to give rise to intestinal tissue, complete with intestinal mesenchyme and epithelium comprising all of the major intestinal cell types. To date, this is the only method for efficiently directing the differentiation of hPSCs into 3D human intestinal tissue in vitro.