RESUMEN
Human brain development involves an orchestrated, massive neural progenitor expansion while a multi-cellular tissue architecture is established. Continuously expanding organoids can be grown directly from multiple somatic tissues, yet to date, brain organoids can solely be established from pluripotent stem cells. Here, we show that healthy human fetal brain in vitro self-organizes into organoids (FeBOs), phenocopying aspects of in vivo cellular heterogeneity and complex organization. FeBOs can be expanded over long time periods. FeBO growth requires maintenance of tissue integrity, which ensures production of a tissue-like extracellular matrix (ECM) niche, ultimately endowing FeBO expansion. FeBO lines derived from different areas of the central nervous system (CNS), including dorsal and ventral forebrain, preserve their regional identity and allow to probe aspects of positional identity. Using CRISPR-Cas9, we showcase the generation of syngeneic mutant FeBO lines for the study of brain cancer. Taken together, FeBOs constitute a complementary CNS organoid platform.
Asunto(s)
Encéfalo , Organoides , Humanos , Encéfalo/citología , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Sistema Nervioso Central/metabolismo , Matriz Extracelular/metabolismo , Células Madre Pluripotentes/metabolismo , Prosencéfalo/citología , Técnicas de Cultivo de Tejidos , Células Madre/metabolismo , MorfogénesisRESUMEN
The Wnt pathway is central to a host of developmental and disease-related processes. The remarkable conservation of this intercellular signaling cascade throughout metazoan lineages indicates that it coevolved with multicellularity to regulate the generation and spatial arrangement of distinct cell types. By regulating cell fate specification, mitotic activity, and cell polarity, Wnt signaling orchestrates development and tissue homeostasis, and its dysregulation is implicated in developmental defects, cancer, and degenerative disorders. We review advances in our understanding of this key pathway, from Wnt protein production and secretion to relay of the signal in the cytoplasm of the receiving cell. We discuss the evolutionary history of this pathway as well as endogenous and synthetic modulators of its activity. Finally, we highlight remaining gaps in our knowledge of Wnt signal transduction and avenues for future research.
Asunto(s)
Neoplasias , Vía de Señalización Wnt , Animales , Diferenciación Celular , Neoplasias/genética , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Vía de Señalización Wnt/fisiología , beta Catenina/metabolismoRESUMEN
Few cancers can be targeted efficiently by engineered T cell strategies. Here, we show that γδ T cell antigen receptor (γδ TCR)-mediated cancer metabolome targeting can be combined with targeting of cancer-associated stress antigens (such as NKG2D ligands or CD277) through the addition of chimeric co-receptors. This strategy overcomes suboptimal γ9δ2 TCR engagement of αß T cells engineered to express a defined γδ TCR (TEGs) and improves serial killing, proliferation and persistence of TEGs. In vivo, the NKG2D-CD28WT chimera enabled control only of liquid tumors, whereas the NKG2D-4-1BBCD28TM chimera prolonged persistence of TEGs and improved control of liquid and solid tumors. The CD277-targeting chimera (103-4-1BB) was the most optimal co-stimulation format, eradicating both liquid and solid tumors. Single-cell transcriptomic analysis revealed that NKG2D-4-1BBCD28TM and 103-4-1BB chimeras reprogram TEGs through NF-κB. Owing to competition with naturally expressed NKG2D in CD8+ TEGs, the NKG2D-4-1BBCD28TM chimera mainly skewed CD4+ TEGs toward adhesion, proliferation, cytotoxicity and less exhausted signatures, whereas the 103-4-1BB chimera additionally shaped the CD8+ subset toward a proliferative state.
Asunto(s)
Neoplasias , Linfocitos T , Humanos , Subfamilia K de Receptores Similares a Lectina de Células NK/metabolismo , Neoplasias/genética , Neoplasias/terapia , Neoplasias/metabolismo , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Perfilación de la Expresión GénicaRESUMEN
It has generally proven challenging to produce functional ß cells in vitro. Here, we describe a previously unidentified protein C receptor positive (Procr+) cell population in adult mouse pancreas through single-cell RNA sequencing (scRNA-seq). The cells reside in islets, do not express differentiation markers, and feature epithelial-to-mesenchymal transition characteristics. By genetic lineage tracing, Procr+ islet cells undergo clonal expansion and generate all four endocrine cell types during adult homeostasis. Sorted Procr+ cells, representing â¼1% of islet cells, can robustly form islet-like organoids when cultured at clonal density. Exponential expansion can be maintained over long periods by serial passaging, while differentiation can be induced at any time point in culture. ß cells dominate in differentiated islet organoids, while α, δ, and PP cells occur at lower frequencies. The organoids are glucose-responsive and insulin-secreting. Upon transplantation in diabetic mice, these organoids reverse disease. These findings demonstrate that the adult mouse pancreatic islet contains a population of Procr+ endocrine progenitors.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Receptor de Proteína C Endotelial/metabolismo , Islotes Pancreáticos/citología , Animales , Diferenciación Celular/fisiología , Línea Celular , Células Cultivadas , Diabetes Mellitus Experimental/metabolismo , Transición Epitelial-Mesenquimal/fisiología , Femenino , Glucosa/metabolismo , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/citología , Islotes Pancreáticos/crecimiento & desarrollo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Desnudos , Organoides/crecimiento & desarrollo , Organoides/metabolismo , Páncreas/citología , Páncreas/metabolismo , Proteína C/metabolismo , Células Madre/citologíaRESUMEN
RNA viruses are among the most prevalent pathogens and are a major burden on society. Although RNA viruses have been studied extensively, little is known about the processes that occur during the first several hours of infection because of a lack of sensitive assays. Here we develop a single-molecule imaging assay, virus infection real-time imaging (VIRIM), to study translation and replication of individual RNA viruses in live cells. VIRIM uncovered a striking heterogeneity in replication dynamics between cells and revealed extensive coordination between translation and replication of single viral RNAs. Furthermore, using VIRIM, we identify the replication step of the incoming viral RNA as a major bottleneck of successful infection and identify host genes that are responsible for inhibition of early virus replication. Single-molecule imaging of virus infection is a powerful tool to study virus replication and virus-host interactions that may be broadly applicable to RNA viruses.
Asunto(s)
Biosíntesis de Proteínas , Virus ARN/fisiología , Replicación Viral/fisiología , Línea Celular Tumoral , Supervivencia Celular , Células HEK293 , Interacciones Huésped-Patógeno , Humanos , Interferones/metabolismo , Transporte de ARN , ARN Viral/genética , Reproducibilidad de los Resultados , Imagen Individual de Molécula , Factores de TiempoRESUMEN
Wnt dependency and Lgr5 expression define multiple mammalian epithelial stem cell types. Under defined growth factor conditions, such adult stem cells (ASCs) grow as 3D organoids that recapitulate essential features of the pertinent epithelium. Here, we establish long-term expanding venom gland organoids from several snake species. The newly assembled transcriptome of the Cape coral snake reveals that organoids express high levels of toxin transcripts. Single-cell RNA sequencing of both organoids and primary tissue identifies distinct venom-expressing cell types as well as proliferative cells expressing homologs of known mammalian stem cell markers. A hard-wired regional heterogeneity in the expression of individual venom components is maintained in organoid cultures. Harvested venom peptides reflect crude venom composition and display biological activity. This study extends organoid technology to reptilian tissues and describes an experimentally tractable model system representing the snake venom gland.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Organoides/crecimiento & desarrollo , Venenos de Serpiente/metabolismo , Células Madre Adultas/metabolismo , Animales , Serpientes de Coral/metabolismo , Perfilación de la Expresión Génica/métodos , Organoides/metabolismo , Glándulas Salivales/metabolismo , Venenos de Serpiente/genética , Serpientes/genética , Serpientes/crecimiento & desarrollo , Células Madre/metabolismo , Toxinas Biológicas/genética , Transcriptoma/genéticaRESUMEN
Enteroendocrine cells (EECs) sense intestinal content and release hormones to regulate gastrointestinal activity, systemic metabolism, and food intake. Little is known about the molecular make-up of human EEC subtypes and the regulated secretion of individual hormones. Here, we describe an organoid-based platform for functional studies of human EECs. EEC formation is induced in vitro by transient expression of NEUROG3. A set of gut organoids was engineered in which the major hormones are fluorescently tagged. A single-cell mRNA atlas was generated for the different EEC subtypes, and their secreted products were recorded by mass-spectrometry. We note key differences to murine EECs, including hormones, sensory receptors, and transcription factors. Notably, several hormone-like molecules were identified. Inter-EEC communication is exemplified by secretin-induced GLP-1 secretion. Indeed, individual EEC subtypes carry receptors for various EEC hormones. This study provides a rich resource to study human EEC development and function.
Asunto(s)
Células Enteroendocrinas/metabolismo , ARN Mensajero/genética , Células Cultivadas , Hormonas Gastrointestinales/genética , Tracto Gastrointestinal/metabolismo , Péptido 1 Similar al Glucagón/genética , Humanos , Organoides/metabolismo , Factores de Transcripción/genética , Transcriptoma/genéticaRESUMEN
Homeostatic regulation of the intestinal enteroendocrine lineage hierarchy is a poorly understood process. We resolved transcriptional changes during enteroendocrine differentiation in real time at single-cell level using a novel knockin allele of Neurog3, the master regulator gene briefly expressed at the onset of enteroendocrine specification. A bi-fluorescent reporter, Neurog3Chrono, measures time from the onset of enteroendocrine differentiation and enables precise positioning of single-cell transcriptomes along an absolute time axis. This approach yielded a definitive description of the enteroendocrine hierarchy and its sub-lineages, uncovered differential kinetics between sub-lineages, and revealed time-dependent hormonal plasticity in enterochromaffin and L cells. The time-resolved map of transcriptional changes predicted multiple novel molecular regulators. Nine of these were validated by conditional knockout in mice or CRISPR modification in intestinal organoids. Six novel candidate regulators (Sox4, Rfx6, Tox3, Myt1, Runx1t1, and Zcchc12) yielded specific enteroendocrine phenotypes. Our time-resolved single-cell transcriptional map presents a rich resource to unravel enteroendocrine differentiation.
Asunto(s)
Linaje de la Célula/genética , Células Enteroendocrinas/metabolismo , Perfilación de la Expresión Génica/métodos , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Diferenciación Celular/genética , Linaje de la Célula/fisiología , Células Enteroendocrinas/fisiología , Colorantes Fluorescentes , Proteínas de Homeodominio/genética , Mucosa Intestinal/citología , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Imagen Óptica/métodos , Organoides , Fenotipo , Análisis de la Célula Individual/métodos , Células Madre , Factores de Transcripción/genética , Transcriptoma/genéticaRESUMEN
Loss of BRCA1 p220 function often results in basal-like breast cancer (BLBC), but the underlying disease mechanism is largely opaque. In mammary epithelial cells (MECs), BRCA1 interacts with multiple proteins, including NUMB and HES1, to form complexes that participate in interstrand crosslink (ICL) DNA repair and MEC differentiation control. Unrepaired ICL damage results in aberrant transdifferentiation to a mesenchymal state of cultured, human basal-like MECs and to a basal/mesenchymal state in primary mouse luminal MECs. Loss of BRCA1, NUMB, or HES1 or chemically induced ICL damage in primary murine luminal MECs results in persistent DNA damage that triggers luminal to basal/mesenchymal transdifferentiation. In vivo single-cell analysis revealed a time-dependent evolution from normal luminal MECs to luminal progenitor-like tumor cells with basal/mesenchymal transdifferentiation during murine BRCA1 BLBC development. Growing DNA damage accompanied this malignant transformation.
Asunto(s)
Proteína BRCA1/genética , Neoplasias de la Mama/genética , Transdiferenciación Celular/genética , Daño del ADN/genética , Reparación del ADN/genética , Glándulas Mamarias Animales/patología , Animales , Proteína BRCA1/metabolismo , Neoplasias de la Mama/inducido químicamente , Neoplasias de la Mama/patología , Diferenciación Celular/genética , Transformación Celular Neoplásica , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Femenino , Células HEK293 , Humanos , Células MCF-7 , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/metabolismo , Factor de Transcripción HES-1/metabolismo , TransfecciónRESUMEN
In mammals, endogenous circadian clocks sense and respond to daily feeding and lighting cues, adjusting internal â¼24 h rhythms to resonate with, and anticipate, external cycles of day and night. The mechanism underlying circadian entrainment to feeding time is critical for understanding why mistimed feeding, as occurs during shift work, disrupts circadian physiology, a state that is associated with increased incidence of chronic diseases such as type 2 (T2) diabetes. We show that feeding-regulated hormones insulin and insulin-like growth factor 1 (IGF-1) reset circadian clocks in vivo and in vitro by induction of PERIOD proteins, and mistimed insulin signaling disrupts circadian organization of mouse behavior and clock gene expression. Insulin and IGF-1 receptor signaling is sufficient to determine essential circadian parameters, principally via increased PERIOD protein synthesis. This requires coincident mechanistic target of rapamycin (mTOR) activation, increased phosphoinositide signaling, and microRNA downregulation. Besides its well-known homeostatic functions, we propose insulin and IGF-1 are primary signals of feeding time to cellular clocks throughout the body.
Asunto(s)
Relojes Circadianos/fisiología , Conducta Alimentaria/fisiología , Proteínas Circadianas Period/metabolismo , Animales , Ritmo Circadiano/fisiología , Femenino , Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Masculino , Mamíferos/metabolismo , Ratones , Ratones Endogámicos C57BL , Receptor IGF Tipo 1/metabolismo , Transducción de SeñalRESUMEN
Central to the classical hematopoietic stem cell (HSC) paradigm is the concept that the maintenance of blood cell numbers is exclusively executed by a discrete physical entity: the transplantable HSC. The HSC paradigm has served as a stereotypic template in stem cell biology, yet the search for rare, hardwired professional stem cells has remained futile in most other tissues. In a more open approach, the focus on the search for stem cells as a physical entity may need to be replaced by the search for stem cell function, operationally defined as the ability of an organ to replace lost cells. The nature of such a cell may be different under steady state conditions and during tissue repair. We discuss emerging examples including the renewal strategies of the skin, gut epithelium, liver, lung, and mammary gland in comparison with those of the hematopoietic system. While certain key housekeeping and developmental signaling pathways are shared between different stem cell systems, there may be no general, deeper principles underlying the renewal mechanisms of the various individual tissues.
Asunto(s)
Células Madre Adultas/citología , Células Madre Adultas/fisiología , Animales , Diferenciación Celular , Linaje de la Célula , Proliferación Celular , Autorrenovación de las Células , Femenino , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/fisiología , Humanos , Masculino , Modelos Biológicos , Fenotipo , Transducción de SeñalRESUMEN
Intestinal stem cells at the bottom of crypts fuel the rapid renewal of the different cell types that constitute a multitasking tissue. The intestinal epithelium facilitates selective uptake of nutrients while acting as a barrier for hostile luminal contents. Recent discoveries have revealed that the lineage plasticity of committed cells - combined with redundant sources of niche signals - enables the epithelium to efficiently repair tissue damage. New approaches such as single-cell transcriptomics and the use of organoid models have led to the identification of the signals that guide fate specification of stem cell progeny into the six intestinal cell lineages. These cell types display context-dependent functionality and can adapt to different requirements over their lifetime, as dictated by their microenvironment. These new insights into stem cell regulation and fate specification could aid the development of therapies that exploit the regenerative capacity and functionality of the gut.
Asunto(s)
Diferenciación Celular , Linaje de la Célula , Mucosa Intestinal/citología , Regeneración , Células Madre/citología , Animales , Humanos , Transducción de SeñalRESUMEN
Cancer immunotherapies have shown substantial clinical activity for a subset of patients with epithelial cancers. Still, technological platforms to study cancer T-cell interactions for individual patients and understand determinants of responsiveness are presently lacking. Here, we establish and validate a platform to induce and analyze tumor-specific T cell responses to epithelial cancers in a personalized manner. We demonstrate that co-cultures of autologous tumor organoids and peripheral blood lymphocytes can be used to enrich tumor-reactive T cells from peripheral blood of patients with mismatch repair-deficient colorectal cancer and non-small-cell lung cancer. Furthermore, we demonstrate that these T cells can be used to assess the efficiency of killing of matched tumor organoids. This platform provides an unbiased strategy for the isolation of tumor-reactive T cells and provides a means by which to assess the sensitivity of tumor cells to T cell-mediated attack at the level of the individual patient.
Asunto(s)
Leucocitos Mononucleares/citología , Linfocitos T/inmunología , Anciano , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Técnicas de Cultivo de Célula , Técnicas de Cocultivo , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Femenino , Humanos , Técnicas In Vitro , Interferón gamma/farmacología , Leucocitos Mononucleares/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Activación de Linfocitos/efectos de los fármacos , Masculino , Persona de Mediana Edad , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
The mammalian liver possesses a remarkable regenerative ability. Two modes of damage response have been described: (1) The "oval cell" response emanates from the biliary tree when all hepatocytes are affected by chronic liver disease. (2) A massive, proliferative response of mature hepatocytes occurs upon acute liver damage such as partial hepatectomy (PHx). While the oval cell response has been captured in vitro by growing organoids from cholangiocytes, the hepatocyte proliferative response has not been recapitulated in culture. Here, we describe the establishment of a long-term 3D organoid culture system for mouse and human primary hepatocytes. Organoids can be established from single hepatocytes and grown for multiple months, while retaining key morphological, functional and gene expression features. Transcriptional profiles of the organoids resemble those of proliferating hepatocytes after PHx. Human hepatocyte organoids proliferate extensively after engraftment into mice and thus recapitulate the proliferative damage-response of hepatocytes.
Asunto(s)
Proliferación Celular , Hepatocitos/metabolismo , Organoides/metabolismo , Animales , Técnicas de Cultivo de Célula , Células Cultivadas , Hepatocitos/citología , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Organoides/citología , Células Madre/citología , Células Madre/metabolismo , Factores de TiempoRESUMEN
Breast cancer (BC) comprises multiple distinct subtypes that differ genetically, pathologically, and clinically. Here, we describe a robust protocol for long-term culturing of human mammary epithelial organoids. Using this protocol, >100 primary and metastatic BC organoid lines were generated, broadly recapitulating the diversity of the disease. BC organoid morphologies typically matched the histopathology, hormone receptor status, and HER2 status of the original tumor. DNA copy number variations as well as sequence changes were consistent within tumor-organoid pairs and largely retained even after extended passaging. BC organoids furthermore populated all major gene-expression-based classification groups and allowed in vitro drug screens that were consistent with in vivo xeno-transplantations and patient response. This study describes a representative collection of well-characterized BC organoids available for cancer research and drug development, as well as a strategy to assess in vitro drug response in a personalized fashion.
Asunto(s)
Neoplasias de la Mama/patología , Heterogeneidad Genética , Organoides/patología , Bancos de Tejidos , Animales , Antineoplásicos/farmacología , Neoplasias de la Mama/genética , Células Cultivadas , Ensayos de Selección de Medicamentos Antitumorales/métodos , Femenino , Humanos , Ratones , Ratones Desnudos , Organoides/efectos de los fármacos , Medicina de Precisión/métodosRESUMEN
Gut-brain signaling plays a central role in a range of homeostatic processes, yet details of this cross-talk remain enigmatic. In this issue of Cell, Bellono and colleagues identify a variety of luminal stimuli acting on serotonin-secreting enteroendocrine cells and, for the first time, demonstrate a functional synaptic interaction with neurons.
Asunto(s)
Células Enteroendocrinas , Serotonina , Transducción de Señal , OlfatoRESUMEN
The WNT signal transduction cascade is a main regulator of development throughout the animal kingdom. Wnts are also key drivers of most types of tissue stem cells in adult mammals. Unsurprisingly, mutated Wnt pathway components are causative to multiple growth-related pathologies and to cancer. Here, we describe the core Wnt/ß-catenin signaling pathway, how it controls stem cells, and contributes to disease. Finally, we discuss strategies for Wnt-based therapies.
Asunto(s)
Proteínas Wnt/metabolismo , Vía de Señalización Wnt , Animales , Anomalías Congénitas/metabolismo , Humanos , Terapia Molecular Dirigida , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Organoides/metabolismo , Células Madre/metabolismo , Proteínas Wnt/química , Proteínas Wnt/genética , Vía de Señalización Wnt/efectos de los fármacosRESUMEN
Recent advances in 3D culture technology allow embryonic and adult mammalian stem cells to exhibit their remarkable self-organizing properties, and the resulting organoids reflect key structural and functional properties of organs such as kidney, lung, gut, brain and retina. Organoid technology can therefore be used to model human organ development and various human pathologies 'in a dish." Additionally, patient-derived organoids hold promise to predict drug response in a personalized fashion. Organoids open up new avenues for regenerative medicine and, in combination with editing technology, for gene therapy. The many potential applications of this technology are only beginning to be explored.
Asunto(s)
Modelos Biológicos , Organoides/citología , Animales , Humanos , Especificidad de Órganos , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Células Madre/citologíaRESUMEN
Tissue stem cells require unique niche microenvironments. In the presence of specific combinations of niche factors, mouse and human epithelial tissues from stomach, small intestine, colon, pancreas duct, and liver bile duct efficiently form stereotypic organoids. The platform of epitheloid organoids can also be employed for in vitro generation of digestive tissue from human pluripotent stem cells. Organoids hold great promise for basic and translational research.
Asunto(s)
Organoides , Células Madre/citología , Técnicas de Cultivo de Tejidos , Animales , Sistema Digestivo/citología , Células Epiteliales/citología , Humanos , Células Madre Pluripotentes/citología , Investigación Biomédica TraslacionalRESUMEN
The adenomatous polyposis coli (APC) tumor suppressor is mutated in the vast majority of human colorectal cancers (CRC) and leads to deregulated Wnt signaling. To determine whether Apc disruption is required for tumor maintenance, we developed a mouse model of CRC whereby Apc can be conditionally suppressed using a doxycycline-regulated shRNA. Apc suppression produces adenomas in both the small intestine and colon that, in the presence of Kras and p53 mutations, can progress to invasive carcinoma. In established tumors, Apc restoration drives rapid and widespread tumor-cell differentiation and sustained regression without relapse. Tumor regression is accompanied by the re-establishment of normal crypt-villus homeostasis, such that once aberrantly proliferating cells reacquire self-renewal and multi-lineage differentiation capability. Our study reveals that CRC cells can revert to functioning normal cells given appropriate signals and provide compelling in vivo validation of the Wnt pathway as a therapeutic target for treatment of CRC.