RESUMEN
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.
Asunto(s)
Anatomía Artística , Atlas como Asunto , Desarrollo Embrionario , Endodermo/embriología , Modelos Biológicos , Organoides/embriología , Factor de Transcripción CDX2/metabolismo , Línea Celular , Factor de Crecimiento Epidérmico/farmacología , Células Epiteliales/citología , Femenino , Gastrulación , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Humanos , Intestinos/embriología , Masculino , Mesodermo/embriología , Persona de Mediana Edad , Neurregulina-1/metabolismo , Especificidad de Órganos , Células Madre Pluripotentes/citologíaRESUMEN
The human nervous system is a highly complex but organized organ. The foundation of its complexity and organization is laid down during regional patterning of the neural tube, the embryonic precursor to the human nervous system. Historically, studies of neural tube patterning have relied on animal models to uncover underlying principles. Recently, models of neurodevelopment based on human pluripotent stem cells, including neural organoids1-5 and bioengineered neural tube development models6-10, have emerged. However, such models fail to recapitulate neural patterning along both rostral-caudal and dorsal-ventral axes in a three-dimensional tubular geometry, a hallmark of neural tube development. Here we report a human pluripotent stem cell-based, microfluidic neural tube-like structure, the development of which recapitulates several crucial aspects of neural patterning in brain and spinal cord regions and along rostral-caudal and dorsal-ventral axes. This structure was utilized for studying neuronal lineage development, which revealed pre-patterning of axial identities of neural crest progenitors and functional roles of neuromesodermal progenitors and the caudal gene CDX2 in spinal cord and trunk neural crest development. We further developed dorsal-ventral patterned microfluidic forebrain-like structures with spatially segregated dorsal and ventral regions and layered apicobasal cellular organizations that mimic development of the human forebrain pallium and subpallium, respectively. Together, these microfluidics-based neurodevelopment models provide three-dimensional lumenal tissue architectures with in vivo-like spatiotemporal cell differentiation and organization, which will facilitate the study of human neurodevelopment and disease.
Asunto(s)
Tipificación del Cuerpo , Microfluídica , Tubo Neural , Humanos , Técnicas de Cultivo Tridimensional de Células , Diferenciación Celular , Cresta Neural/citología , Cresta Neural/embriología , Tubo Neural/citología , Tubo Neural/embriología , Células Madre Pluripotentes/citología , Prosencéfalo/citología , Prosencéfalo/embriología , Médula Espinal/citología , Médula Espinal/embriologíaRESUMEN
Using scRNA-seq and microscopy, we describe a cell that is enriched in the lower airways of the developing human lung and identified by the unique coexpression of SCGB3A2/SFTPB/CFTR. To functionally interrogate these cells, we apply a single-cell barcode-based lineage tracing method, called CellTagging, to track the fate of SCGB3A2/SFTPB/CFTR cells during airway organoid differentiation in vitro. Lineage tracing reveals that these cells have a distinct differentiation potential from basal cells, giving rise predominantly to pulmonary neuroendocrine cells and a subset of multiciliated cells distinguished by high C6 and low MUC16 expression. Lineage tracing results are supported by studies using organoids and isolated cells from the lower noncartilaginous airway. We conclude that SCGB3A2/SFTPB/CFTR cells are enriched in the lower airways of the developing human lung and contribute to the epithelial diversity and heterogeneity in this region.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística , Pulmón , Humanos , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Células Madre/metabolismo , Diferenciación Celular , Linaje de la Célula , Organoides , Células Epiteliales/metabolismoRESUMEN
Bud tip progenitors (BTPs) in the developing lung give rise to all epithelial cell types found in the airways and alveoli. This work aimed to develop an iPSC organoid model enriched with NKX2-1+ BTP-like cells. Building on previous studies, we optimized a directed differentiation paradigm to generate spheroids with more robust NKX2-1 expression. Spheroids were expanded into organoids that possessed NKX2-1+/CPM+ BTP-like cells, which increased in number over time. Single cell RNA-sequencing analysis revealed a high degree of transcriptional similarity between induced BTPs (iBTPs) and in vivo BTPs. Using FACS, iBTPs were purified and expanded as induced bud tip progenitor organoids (iBTOs), which maintained an enriched population of bud tip progenitors. When iBTOs were directed to differentiate into airway or alveolar cell types using well-established methods, they gave rise to organoids composed of organized airway or alveolar epithelium, respectively. Collectively, iBTOs are transcriptionally and functionally similar to in vivo BTPs, providing an important model for studying human lung development and differentiation.
Asunto(s)
Células Madre Pluripotentes Inducidas , Células Madre Pluripotentes , Factor Nuclear Tiroideo 1/metabolismo , Células Epiteliales Alveolares , Diferenciación Celular , Humanos , Pulmón , OrganoidesRESUMEN
Many esophageal diseases can arise during development or throughout life. Therefore, well-characterized in vitro models and detailed methods are essential for studying human esophageal development, homeostasis and disease. Here, we (1) create an atlas of the cell types observed in the normal adult human esophagus; (2) establish an ancestrally diverse biobank of in vitro esophagus tissue to interrogate homeostasis and injury; and (3) benchmark in vitro models using the adult human esophagus atlas. We created a single-cell RNA sequencing reference atlas using fresh adult esophagus biopsies and a continuously expanding biobank of patient-derived in vitro cultures (n=55 lines). We identify and validate several transcriptionally distinct cell classes in the native human adult esophagus, with four populations belonging to the epithelial layer, including basal, epibasal, early differentiating and terminally differentiated luminal cells. Benchmarking in vitro esophagus cultures to the in vivo reference using single-cell RNA sequencing shows that the basal stem cells are robustly maintained in vitro, and the diversity of epithelial cell types in culture is dependent on cell density. We also demonstrate that cultures can be grown in 2D or as 3D organoids, and these methods can be employed for modeling the complete epithelial layers, thereby enabling in vitro modeling of the human adult esophagus.
Asunto(s)
Esófago , Organoides , Adulto , Humanos , Células Madre , Células Epiteliales/metabolismo , Diferenciación CelularRESUMEN
The physiological basis and mechanistic requirements for a large number of functional immunoreceptor tyrosine-based activation motifs (ITAMs; high ITAM multiplicity) in the complex of the T cell antigen receptor (TCR) and the invariant signaling protein CD3 remain obscure. Here we found that whereas a low multiplicity of TCR-CD3 ITAMs was sufficient to engage canonical TCR-induced signaling events that led to cytokine secretion, a high multiplicity of TCR-CD3 ITAMs was required for TCR-driven proliferation. This was dependent on the formation of compact immunological synapses, interaction of the adaptor Vav1 with phosphorylated CD3 ITAMs to mediate the recruitment and activation of the oncogenic transcription factor Notch1 and, ultimately, proliferation induced by the cell-cycle regulator c-Myc. Analogous mechanistic events were also needed to drive proliferation in response to weak peptide agonists. Thus, the TCR-driven pathways that initiate cytokine secretion and proliferation are separable and are coordinated by the multiplicity of phosphorylated ITAMs in TCR-CD3.
Asunto(s)
Complejo CD3/inmunología , Citocinas/biosíntesis , Motivo de Activación del Inmunorreceptor Basado en Tirosina/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Animales , Complejo CD3/metabolismo , Línea Celular , Proliferación Celular , Células HEK293 , Humanos , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas c-vav/metabolismo , Receptor Notch1/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Linfocitos T/metabolismoRESUMEN
Between embryonic days 10.5 and 14.5, active proliferation drives rapid elongation of the murine midgut epithelial tube. Within this pseudostratified epithelium, nuclei synthesize DNA near the basal surface and move apically to divide. After mitosis, the majority of daughter cells extend a long, basally oriented filopodial protrusion, building a de novo path along which their nuclei can return to the basal side. WNT5A, which is secreted by surrounding mesenchymal cells, acts as a guidance cue to orchestrate this epithelial pathfinding behavior, but how this signal is received by epithelial cells is unknown. Here, we have investigated two known WNT5A receptors: ROR2 and RYK. We found that epithelial ROR2 is dispensable for midgut elongation. However, loss of Ryk phenocopies the Wnt5a-/- phenotype, perturbing post-mitotic pathfinding and leading to apoptosis. These studies reveal that the ligand-receptor pair WNT5A-RYK acts as a navigation system to instruct filopodial pathfinding, a process that is crucial for continuous cell cycling to fuel rapid midgut elongation.
Asunto(s)
Sistema Digestivo/crecimiento & desarrollo , Sistema Digestivo/metabolismo , Seudópodos/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Animales , Apoptosis , Núcleo Celular/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Epitelio/metabolismo , Femenino , Masculino , Mesodermo/metabolismo , Ratones Endogámicos C57BL , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/metabolismoRESUMEN
As embryos mature, cells undergo remarkable transitions that are accompanied by shifts in transcription factor regulatory networks. Mechanisms driving developmental transitions are incompletely understood. The embryonic intestine transitions from a rapidly proliferating tube with pseudostratified epithelium prior to murine embryonic day (E) 14.5 to an exquisitely folded columnar epithelium in fetal stages. We sought to identify factors driving mouse fetal intestinal maturation by mining chromatin accessibility data for transcription factor motifs. ATAC-seq accessible regions shift during tissue maturation, with CDX2 transcription factor motifs abundant at chromatin-accessible regions of the embryo. Hepatocyte nuclear factor 4 (HNF4) transcription factor motifs are the most abundant in the fetal stages (>E16.5). Genetic inactivation of Hnf4a and its paralog Hnf4g revealed that HNF4 factors are redundantly required for fetal maturation. CDX2 binds to and activates Hnf4 gene loci to elevate HNF4 expression at fetal stages. HNF4 and CDX2 transcription factors then occupy shared genomic regulatory sites to promote chromatin accessibility and gene expression in the maturing intestine. Thus, HNF4 paralogs are key components of an intestinal transcription factor network shift during the embryonic to fetal transition.
Asunto(s)
Cromatina/metabolismo , Feto/embriología , Factor Nuclear 4 del Hepatocito/metabolismo , Intestinos/embriología , Animales , Factor de Transcripción CDX2/metabolismo , Línea Celular , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Elementos de Facilitación Genéticos/genética , Regulación del Desarrollo de la Expresión Génica , Humanos , Ratones , Modelos Biológicos , MorfogénesisRESUMEN
Lineage-restricted transcription factors, such as the intestine-specifying factor CDX2, often have dual requirements across developmental time. Embryonic loss of CDX2 triggers homeotic transformation of intestinal fate, whereas adult-onset loss compromises crucial physiological functions but preserves intestinal identity. It is unclear how such diverse requirements are executed across the developmental continuum. Using primary and engineered human tissues, mouse genetics, and a multi-omics approach, we demonstrate that divergent CDX2 loss-of-function phenotypes in embryonic versus adult intestines correspond to divergent CDX2 chromatin-binding profiles in embryonic versus adult stages. CDX2 binds and activates distinct target genes in developing versus adult mouse and human intestinal cells. We find that temporal shifts in chromatin accessibility correspond to these context-specific CDX2 activities. Thus, CDX2 is not sufficient to activate a mature intestinal program; rather, CDX2 responds to its environment, targeting stage-specific genes to contribute to either intestinal patterning or mature intestinal function. This study provides insights into the mechanisms through which lineage-specific regulatory factors achieve divergent functions over developmental time.
Asunto(s)
Factor de Transcripción CDX2/metabolismo , Cromatina/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Intestinos/embriología , Animales , Factor de Transcripción CDX2/genética , Sistemas CRISPR-Cas , Diferenciación Celular , Linaje de la Célula , Femenino , Humanos , Mucosa Intestinal/metabolismo , Ratones , Ratones Noqueados , Mutación , Células Madre Pluripotentes/citología , Unión Proteica , Dominios Proteicos , Transactivadores/metabolismoRESUMEN
The intestine is maintained by stem cells located at the base of crypts and distinguished by the expression of LGR5. Genetically engineered mouse models have provided a wealth of information about intestinal stem cells, whereas less is known about human intestinal stem cells owing to difficulty detecting and isolating these cells. We established an organoid repository from patient-derived adenomas, adenocarcinomas and normal colon, which we analyzed for variants in 71 colorectal cancer (CRC)-associated genes. Normal and neoplastic colon tissue organoids were analyzed by immunohistochemistry and fluorescent-activated cell sorting for LGR5. LGR5-positive cells were isolated from four adenoma organoid lines and were subjected to RNA sequencing. We found that LGR5 expression in the epithelium and stroma was associated with tumor stage, and by integrating functional experiments with LGR5-sorted cell RNA sequencing data from adenoma and normal organoids, we found correlations between LGR5 and CRC-specific genes, including dickkopf WNT signaling pathway inhibitor 4 (DKK4) and SPARC-related modular calcium binding 2 (SMOC2). Collectively, this work provides resources, methods and new markers to isolate and study stem cells in human tissue homeostasis and carcinogenesis.
Asunto(s)
Adenoma/metabolismo , Colon/metabolismo , Neoplasias del Colon/metabolismo , Mucosa Intestinal/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Adenoma/genética , Línea Celular Tumoral , Colon/patología , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Citometría de Flujo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inmunohistoquímica , Mucosa Intestinal/citología , Organoides/metabolismo , Transducción de SeñalRESUMEN
The intestine plays a central role in digestion, nutrient absorption and metabolism, with individual regions of the intestine having distinct functional roles. Many examples of region-specific gene expression in the adult intestine are known, but how intestinal regional identity is established during development is a largely unresolved issue. Here, we have identified several genes that are expressed in a region-specific manner in the developing human intestine. Using human embryonic stem cell-derived intestinal organoids, we demonstrate that the duration of exposure to active FGF and WNT signaling controls regional identity. Short-term exposure to FGF4 and CHIR99021 (a GSK3ß inhibitor that stabilizes ß-catenin) resulted in organoids with gene expression patterns similar to developing human duodenum, whereas longer exposure resulted in organoids similar to ileum. When region-specific organoids were transplanted into immunocompromised mice, duodenum-like organoids and ileum-like organoids retained their regional identity, demonstrating that regional identity of organoids is stable after initial patterning occurs. This work provides insights into the mechanisms that control regional specification of the developing human intestine and provides new tools for basic and translational research.
Asunto(s)
Tipificación del Cuerpo , Desarrollo Embrionario , Feto/embriología , Intestinos/embriología , Células Madre Pluripotentes/citología , Animales , Biomarcadores/metabolismo , Tipificación del Cuerpo/genética , Diferenciación Celular/genética , Biología Computacional , Desarrollo Embrionario/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Humanos , Ratones , Organoides/metabolismo , Organoides/trasplante , Células Madre Pluripotentes/metabolismo , Reproducibilidad de los Resultados , Análisis de Secuencia de ARN , Transducción de Señal/genética , Proteínas Wnt/metabolismoRESUMEN
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.
Asunto(s)
Infecciones por Helicobacter/fisiopatología , Modelos Biológicos , Organogénesis , Organoides/citología , Células Madre Pluripotentes/citología , Estómago/citología , Diferenciación Celular , Helicobacter pylori , Humanos , Organoides/microbiología , Transducción de SeñalRESUMEN
BACKGROUND & AIMS: A disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) is a cell surface sheddase that regulates physiologic processes, including Notch signaling. ADAM10 is expressed in all intestinal epithelial cell types, but the requirement for ADAM10 signaling in crypt homeostasis is not well defined. METHODS: We analyzed intestinal tissues from mice with constitutive (Vil-Cre;Adam10(f/f) mice) and conditional (Vil-CreER;Adam10(f/f) and Leucine-rich repeat-containing GPCR5 [Lgr5]-CreER;Adam10(f/f) mice) deletion of ADAM10. We performed cell lineage-tracing experiments in mice that expressed a gain-of-function allele of Notch in the intestine (Rosa26(NICD)), or mice with intestine-specific disruption of Notch (Rosa26(DN-MAML)), to examine the effects of ADAM10 deletion on cell fate specification and intestinal stem cell maintenance. RESULTS: Loss of ADAM10 from developing and adult intestine caused lethality associated with altered intestinal morphology, reduced progenitor cell proliferation, and increased secretory cell differentiation. ADAM10 deletion led to the replacement of intestinal cell progenitors with 2 distinct, post-mitotic, secretory cell lineages: intermediate-like (Paneth/goblet) and enteroendocrine cells. Based on analysis of Rosa26(NICD) and Rosa26(DN-MAML) mice, we determined that ADAM10 controls these cell fate decisions by regulating Notch signaling. Cell lineage-tracing experiments showed that ADAM10 is required for survival of Lgr5(+) crypt-based columnar cells. Our findings indicate that Notch-activated stem cells have a competitive advantage for occupation of the stem cell niche. CONCLUSIONS: ADAM10 acts in a cell autonomous manner within the intestinal crypt compartment to regulate Notch signaling. This process is required for progenitor cell lineage specification and crypt-based columnar cell maintenance.
Asunto(s)
Proteínas ADAM/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Intestinos/enzimología , Proteínas de la Membrana/metabolismo , Receptores Notch/metabolismo , Nicho de Células Madre , Células Madre/enzimología , Proteínas ADAM/deficiencia , Proteínas ADAM/genética , Proteína ADAM10 , Secretasas de la Proteína Precursora del Amiloide/deficiencia , Secretasas de la Proteína Precursora del Amiloide/genética , Animales , Diferenciación Celular , Línea Celular , Linaje de la Célula , Proliferación Celular , Supervivencia Celular , Células Enteroendocrinas/enzimología , Células Caliciformes/enzimología , Intestinos/citología , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Organoides , Células de Paneth/enzimología , Fenotipo , Transducción de Señal , Factores de TiempoRESUMEN
Notch signaling is known to regulate the proliferation and differentiation of intestinal stem and progenitor cells; however, direct cellular targets and specific functions of Notch signals had not been identified. We show here in mice that Notch directly targets the crypt base columnar (CBC) cell to maintain stem cell activity. Notch inhibition induced rapid CBC cell loss, with reduced proliferation, apoptotic cell death and reduced efficiency of organoid initiation. Furthermore, expression of the CBC stem cell-specific marker Olfm4 was directly dependent on Notch signaling, with transcription activated through RBP-Jκ binding sites in the promoter. Notch inhibition also led to precocious differentiation of epithelial progenitors into secretory cell types, including large numbers of cells that expressed both Paneth and goblet cell markers. Analysis of Notch function in Atoh1-deficient intestine demonstrated that the cellular changes were dependent on Atoh1, whereas Notch regulation of Olfm4 gene expression was Atoh1 independent. Our findings suggest that Notch targets distinct progenitor cell populations to maintain adult intestinal stem cells and to regulate cell fate choice to control epithelial cell homeostasis.
Asunto(s)
Diferenciación Celular , Proliferación Celular , Regulación de la Expresión Génica , Intestino Delgado/citología , Receptor Notch1/metabolismo , Receptor Notch2/metabolismo , Animales , Apoptosis/efectos de los fármacos , Secuencia de Bases , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Células Caliciformes/metabolismo , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Intestino Delgado/efectos de los fármacos , Intestino Delgado/metabolismo , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Técnicas de Cultivo de Órganos , Células de Paneth/metabolismo , Regiones Promotoras Genéticas , Receptor Notch1/antagonistas & inhibidores , Receptor Notch2/antagonistas & inhibidores , Transducción de Señal , Células Madre/citología , Células Madre/fisiologíaRESUMEN
p40, a Lactobacillus rhamnosus GG (LGG)-derived soluble protein, ameliorates intestinal injury and colitis, reduces apoptosis, and preserves barrier function by transactivation of the EGF receptor (EGFR) in intestinal epithelial cells. The aim of this study is to determine the mechanisms by which p40 transactivates the EGFR in intestinal epithelial cells. Here we show that p40-conditioned medium activates EGFR in young adult mouse colon epithelial cells and human colonic epithelial cell line, T84 cells. p40 up-regulates a disintegrin and metalloproteinase domain-containing protein 17 (ADAM17) catalytic activity, and broad spectrum metalloproteinase inhibitors block EGFR transactivation by p40 in these two cell lines. In ADAM17-deficient mouse colonic epithelial (ADAM17(-/-) MCE) cells, p40 transactivation of EGFR is blocked, but can be rescued by re-expression with WT ADAM17. Furthermore, p40 stimulates release of heparin binding (HB)-EGF, but not transforming growth factor (TGF)α or amphiregulin, in young adult mouse colon cells and ADAM17(-/-) MCE cells overexpressing WT ADAM17. Knockdown of HB-EGF expression by siRNA suppresses p40 effects on transactivating EGFR and Akt, preventing apoptosis, and preserving tight junction function. The effects of p40 on HB-EGF release and ADAM17 activation in vivo are examined after administration of p40-containing pectin/zein hydrogel beads to mice. p40 stimulates ADAM17 activity and EGFR activation in colonic epithelial cells and increases HB-EGF levels in blood from WT mice, but not from mice with intestinal epithelial cell-specific ADAM17 deletion. Thus, these data define a mechanism of a probiotic-derived soluble protein in modulating intestinal epithelial cell homeostasis through ADAM17-mediated HB-EGF release, leading to transactivation of EGFR.
Asunto(s)
Proteínas Bacterianas/metabolismo , Células Epiteliales/metabolismo , Receptores ErbB/biosíntesis , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Mucosa Intestinal/metabolismo , Lacticaseibacillus rhamnosus/metabolismo , Probióticos/metabolismo , Activación Transcripcional , Proteínas ADAM/biosíntesis , Proteínas ADAM/genética , Proteína ADAM17 , Animales , Línea Celular Tumoral , Activación Enzimática/genética , Células Epiteliales/citología , Células Epiteliales/microbiología , Receptores ErbB/genética , Regulación Enzimológica de la Expresión Génica/genética , Técnicas de Silenciamiento del Gen , Factor de Crecimiento Similar a EGF de Unión a Heparina , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Mucosa Intestinal/citología , Mucosa Intestinal/microbiología , Ratones , Ratones Noqueados , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factor de Crecimiento Transformador alfa/genética , Factor de Crecimiento Transformador alfa/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
Epithelial organoids derived from intestinal tissue, called enteroids, recapitulate many aspects of the organ in vitro and can be used for biological discovery, personalized medicine, and drug development. Here, we interrogated the cell signaling environment within the developing human intestine to identify niche cues that may be important for epithelial development and homeostasis. We identified an EGF family member, EPIREGULIN (EREG), which is robustly expressed in the developing human crypt. Enteroids generated from the developing human intestine grown in standard culture conditions, which contain EGF, are dominated by stem and progenitor cells and feature little differentiation and no spatial organization. Our results demonstrate that EREG can replace EGF in vitro, and EREG leads to spatially resolved enteroids that feature budded and proliferative crypt domains and a differentiated villus-like central lumen. Multiomic (transcriptome plus epigenome) profiling of native crypts, EGF-grown enteroids, and EREG-grown enteroids showed that EGF enteroids have an altered chromatin landscape that is dependent on EGF concentration, downregulate the master intestinal transcription factor CDX2, and ectopically express stomach genes, a phenomenon that is reversible. This is in contrast to EREG-grown enteroids, which remain intestine like in culture. Thus, EREG creates a homeostatic intestinal niche in vitro, enabling interrogation of stem cell function, cellular differentiation, and disease modeling.
Asunto(s)
Factor de Crecimiento Epidérmico , Intestinos , Humanos , Epirregulina , Mucosa Intestinal , Diferenciación CelularRESUMEN
NOTCH signaling is a key regulator involved in maintaining intestinal stem cell (ISC) homeostasis and for balancing differentiation. Using single-cell transcriptomics, we observed that OLFM4, a NOTCH target gene present in ISCs, is first expressed at 13 weeks post-conception in the developing human intestine and increases over time. This led us to hypothesize that the requirement for NOTCH signaling is acquired across human development. To test this, we established a series of epithelium-only organoids (enteroids) from different developmental stages and used γ-secretase inhibitors (dibenzazepine [DBZ] or DAPT) to functionally block NOTCH signaling. Using quantitative enteroid-forming assays, we observed a decrease in enteroid forming efficiency in response to γ-secretase inhibition as development progress. When DBZ was added to cultures and maintained during routine passaging, enteroids isolated from tissue before 20 weeks had higher recovery rates following single-cell serial passaging. Finally, bulk RNA sequencing (RNA-seq) analysis 1 day and 3 days after DBZ treatment showed major differences in the transcriptional changes between developing or adult enteroids. Collectively, these data suggest that ISC dependence on NOTCH signaling increases as the human intestine matures.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide , Receptores Notch , Células Madre , Secretasas de la Proteína Precursora del Amiloide/genética , Diferenciación Celular , Humanos , Mucosa Intestinal , Intestinos , Organoides , Receptores Notch/genéticaRESUMEN
Pluripotent-stem-cell-derived human intestinal organoids (HIOs) model some aspects of intestinal development and disease, but current culture methods do not fully recapitulate the diverse cell types and complex organization of the human intestine and are reliant on 3D extracellular matrix or hydrogel systems, which limit experimental control and translational potential for regenerative medicine. We describe suspension culture as a simple, low-maintenance method for culturing HIOs and for promoting in vitro differentiation of an organized serosal mesothelial layer that is similar to primary human intestinal serosal mesothelium based on single-cell RNA sequencing and histological analysis. Functionally, HIO serosal mesothelium has the capacity to differentiate into smooth-muscle-like cells and exhibits fibrinolytic activity. An inhibitor screen identifies Hedgehog and WNT signaling as regulators of human serosal mesothelial differentiation. Collectively, suspension HIOs represent a three-dimensional model to study the human serosal mesothelium.
Asunto(s)
Epitelio/crecimiento & desarrollo , Intestinos/crecimiento & desarrollo , Organoides/crecimiento & desarrollo , Membrana Serosa/crecimiento & desarrollo , Técnicas de Cultivo de Tejidos , Alginatos/farmacología , Adhesión Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular , Colágeno/farmacología , Combinación de Medicamentos , Epitelio/efectos de los fármacos , Proteínas Hedgehog/metabolismo , Humanos , Intestinos/ultraestructura , Laminina/farmacología , Músculo Liso/citología , Organoides/efectos de los fármacos , Organoides/ultraestructura , Proteoglicanos/farmacología , Membrana Serosa/efectos de los fármacos , Membrana Serosa/ultraestructura , Transducción de Señal/efectos de los fármacos , Suspensiones , Proteínas Wnt/metabolismoRESUMEN
The human respiratory epithelium is derived from a progenitor cell in the distal buds of the developing lung. These "bud tip progenitors" are regulated by reciprocal signaling with surrounding mesenchyme; however, mesenchymal heterogeneity and function in the developing human lung are poorly understood. We interrogated single-cell RNA sequencing data from multiple human lung specimens and identified a mesenchymal cell population present during development that is highly enriched for expression of the WNT agonist RSPO2, and we found that the adjacent bud tip progenitors are enriched for the RSPO2 receptor LGR5. Functional experiments using organoid models, explant cultures, and FACS-isolated RSPO2+ mesenchyme show that RSPO2 is a critical niche cue that potentiates WNT signaling in bud tip progenitors to support their maintenance and multipotency.
Asunto(s)
Células Madre Mesenquimatosas , Organogénesis , Humanos , Pulmón , Organoides , Vía de Señalización WntRESUMEN
The human intestinal stem cell niche supports self-renewal and epithelial function, but little is known about its development. We used single-cell mRNA sequencing with in situ validation approaches to interrogate human intestinal development from 7-21 weeks post conception, assigning molecular identities and spatial locations to cells and factors that comprise the niche. Smooth muscle cells of the muscularis mucosa, in close proximity to proliferative crypts, are a source of WNT and RSPONDIN ligands, whereas EGF is expressed far from crypts in the villus epithelium. Instead, an PDGFRAHI/F3HI/DLL1HI mesenchymal population lines the crypt-villus axis and is the source of the epidermal growth factor (EGF) family member NEUREGULIN1 (NRG1). In developing intestine enteroid cultures, NRG1, but not EGF, permitted increased cellular diversity via differentiation of secretory lineages. This work highlights the complexities of intestinal EGF/ERBB signaling and delineates key niche cells and signals of the developing intestine.