RESUMEN
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
Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal human malignancies, owing in part to its propensity for metastasis. Here, we used an organoid culture system to investigate how transcription and the enhancer landscape become altered during discrete stages of disease progression in a PDA mouse model. This approach revealed that the metastatic transition is accompanied by massive and recurrent alterations in enhancer activity. We implicate the pioneer factor FOXA1 as a driver of enhancer activation in this system, a mechanism that renders PDA cells more invasive and less anchorage-dependent for growth in vitro, as well as more metastatic in vivo. In this context, FOXA1-dependent enhancer reprogramming activates a transcriptional program of embryonic foregut endoderm. Collectively, our study implicates enhancer reprogramming, FOXA1 upregulation, and a retrograde developmental transition in PDA metastasis.
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Adenocarcinoma/genética , Carcinoma Ductal Pancreático/genética , Elementos de Facilitación Genéticos , Regulación Neoplásica de la Expresión Génica , Factor Nuclear 3-alfa del Hepatocito/genética , Neoplasias Pancreáticas/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Animales , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Epigenómica , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Metástasis de la Neoplasia , Organoides/metabolismo , Páncreas/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologíaRESUMEN
Stem-cell-based therapies can potentially reverse organ dysfunction and diseases, but the removal of impaired tissue and activation of a program leading to organ regeneration pose major challenges. In mice, a 4-day fasting mimicking diet (FMD) induces a stepwise expression of Sox17 and Pdx-1, followed by Ngn3-driven generation of insulin-producing ß cells, resembling that observed during pancreatic development. FMD cycles restore insulin secretion and glucose homeostasis in both type 2 and type 1 diabetes mouse models. In human type 1 diabetes pancreatic islets, fasting conditions reduce PKA and mTOR activity and induce Sox2 and Ngn3 expression and insulin production. The effects of the FMD are reversed by IGF-1 treatment and recapitulated by PKA and mTOR inhibition. These results indicate that a FMD promotes the reprogramming of pancreatic cells to restore insulin generation in islets from T1D patients and reverse both T1D and T2D phenotypes in mouse models. PAPERCLIP.
Asunto(s)
Diabetes Mellitus Tipo 1/dietoterapia , Diabetes Mellitus Tipo 2/dietoterapia , Ayuno , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Dieta , Prueba de Tolerancia a la Glucosa , Humanos , Técnicas In Vitro , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos , Ratones , Proteínas del Tejido Nervioso/genética , Páncreas/citología , Páncreas/metabolismo , Transducción de Señal , TranscriptomaRESUMEN
Stellate cells are resident lipid-storing cells of the pancreas and liver that transdifferentiate to a myofibroblastic state in the context of tissue injury. Beyond having roles in tissue homeostasis, stellate cells are increasingly implicated in pathological fibrogenic and inflammatory programs that contribute to tissue fibrosis and that constitute a growth-permissive tumor microenvironment. Although the capacity of stellate cells for extracellular matrix production and remodeling has long been appreciated, recent research efforts have demonstrated diverse roles for stellate cells in regulation of epithelial cell fate, immune modulation, and tissue health. Our present understanding of stellate cell biology in health and disease is discussed here, as are emerging means to target these multifaceted cells for therapeutic benefit.
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Células Estrelladas Hepáticas/metabolismo , Inflamación/genética , Neoplasias/genética , Células Estrelladas Pancreáticas/metabolismo , Transdiferenciación Celular/genética , Células Estrelladas Hepáticas/patología , Humanos , Inflamación/patología , Hígado/metabolismo , Hígado/patología , Miofibroblastos/metabolismo , Miofibroblastos/patología , Neoplasias/patología , Páncreas/lesiones , Páncreas/metabolismo , Páncreas/patología , Células Estrelladas Pancreáticas/patología , Microambiente Tumoral/genética , Cicatrización de HeridasRESUMEN
Aging is the major risk factor for many human diseases. In vitro studies have demonstrated that cellular reprogramming to pluripotency reverses cellular age, but alteration of the aging process through reprogramming has not been directly demonstrated in vivo. Here, we report that partial reprogramming by short-term cyclic expression of Oct4, Sox2, Klf4, and c-Myc (OSKM) ameliorates cellular and physiological hallmarks of aging and prolongs lifespan in a mouse model of premature aging. Similarly, expression of OSKM in vivo improves recovery from metabolic disease and muscle injury in older wild-type mice. The amelioration of age-associated phenotypes by epigenetic remodeling during cellular reprogramming highlights the role of epigenetic dysregulation as a driver of mammalian aging. Establishing in vivo platforms to modulate age-associated epigenetic marks may provide further insights into the biology of aging.
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Envejecimiento/genética , Reprogramación Celular , Epigénesis Genética , Enfermedades Metabólicas/genética , Factores de Transcripción/metabolismo , Envejecimiento Prematuro/genética , Envejecimiento Prematuro/metabolismo , Animales , Diabetes Mellitus Tipo 2/inducido químicamente , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Factor 4 Similar a Kruppel , Lamina Tipo A/genética , Enfermedades Metabólicas/metabolismo , Enfermedades Metabólicas/prevención & control , Ratones , Modelos Animales , Páncreas/metabolismo , Sarcopenia/metabolismoRESUMEN
The endoderm germ layer contributes to the respiratory and gastrointestinal (GI) lineages during development, giving rise to an array of specialized epithelial cell types lining organs, including the thyroid, thymus, lungs, liver, biliary system, pancreas, and intestines. This SnapShot timelines and summarizes key stages following gastrulation, including endoderm patterning, organ specification, and organogenesis. A lineage tree of the developing endocrine pancreas is outlined to further illustrate this process.
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Tracto Gastrointestinal/embriología , Animales , Tracto Gastrointestinal/citología , Tracto Gastrointestinal/metabolismo , Humanos , Organogénesis , Páncreas/citología , Páncreas/embriología , Páncreas/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Pancreatic cancer is one of the most lethal malignancies due to its late diagnosis and limited response to treatment. Tractable methods to identify and interrogate pathways involved in pancreatic tumorigenesis are urgently needed. We established organoid models from normal and neoplastic murine and human pancreas tissues. Pancreatic organoids can be rapidly generated from resected tumors and biopsies, survive cryopreservation, and exhibit ductal- and disease-stage-specific characteristics. Orthotopically transplanted neoplastic organoids recapitulate the full spectrum of tumor development by forming early-grade neoplasms that progress to locally invasive and metastatic carcinomas. Due to their ability to be genetically manipulated, organoids are a platform to probe genetic cooperation. Comprehensive transcriptional and proteomic analyses of murine pancreatic organoids revealed genes and pathways altered during disease progression. The confirmation of many of these protein changes in human tissues demonstrates that organoids are a facile model system to discover characteristics of this deadly malignancy.
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Carcinoma Ductal Pancreático/patología , Modelos Biológicos , Técnicas de Cultivo de Órganos , Organoides/patología , Neoplasias Pancreáticas/patología , Animales , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Páncreas/metabolismo , Páncreas/patologíaRESUMEN
Pancreatic intraepithelial neoplasias (PanINs) are the most common precursors of pancreatic cancer, but their small size and inaccessibility in humans make them challenging to study1. Critically, the number, dimensions and connectivity of human PanINs remain largely unknown, precluding important insights into early cancer development. Here, we provide a microanatomical survey of human PanINs by analysing 46 large samples of grossly normal human pancreas with a machine-learning pipeline for quantitative 3D histological reconstruction at single-cell resolution. To elucidate genetic relationships between and within PanINs, we developed a workflow in which 3D modelling guides multi-region microdissection and targeted and whole-exome sequencing. From these samples, we calculated a mean burden of 13 PanINs per cm3 and extrapolated that the normal intact adult pancreas harbours hundreds of PanINs, almost all with oncogenic KRAS hotspot mutations. We found that most PanINs originate as independent clones with distinct somatic mutation profiles. Some spatially continuous PanINs were found to contain multiple KRAS mutations; computational and in situ analyses demonstrated that different KRAS mutations localize to distinct cell subpopulations within these neoplasms, indicating their polyclonal origins. The extensive multifocality and genetic heterogeneity of PanINs raises important questions about mechanisms that drive precancer initiation and confer differential progression risk in the human pancreas. This detailed 3D genomic mapping of molecular alterations in human PanINs provides an empirical foundation for early detection and rational interception of pancreatic cancer.
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Heterogeneidad Genética , Genómica , Imagenología Tridimensional , Neoplasias Pancreáticas , Lesiones Precancerosas , Análisis de la Célula Individual , Adulto , Femenino , Humanos , Masculino , Células Clonales/metabolismo , Células Clonales/patología , Secuenciación del Exoma , Aprendizaje Automático , Mutación , Páncreas/anatomía & histología , Páncreas/citología , Páncreas/metabolismo , Páncreas/patología , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Lesiones Precancerosas/genética , Lesiones Precancerosas/patología , Flujo de Trabajo , Progresión de la Enfermedad , Detección Precoz del Cáncer , Oncogenes/genéticaRESUMEN
Replenishing insulin-producing pancreatic ß cell mass will benefit both type I and type II diabetics. In adults, pancreatic ß cells are generated primarily by self-duplication. We report on a mouse model of insulin resistance that induces dramatic pancreatic ß cell proliferation and ß cell mass expansion. Using this model, we identify a hormone, betatrophin, that is primarily expressed in liver and fat. Expression of betatrophin correlates with ß cell proliferation in other mouse models of insulin resistance and during gestation. Transient expression of betatrophin in mouse liver significantly and specifically promotes pancreatic ß cell proliferation, expands ß cell mass, and improves glucose tolerance. Thus, betatrophin treatment could augment or replace insulin injections by increasing the number of endogenous insulin-producing cells in diabetics.
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Proliferación Celular , Células Secretoras de Insulina/metabolismo , Páncreas/citología , Hormonas Peptídicas/metabolismo , Tejido Adiposo Blanco/metabolismo , Secuencia de Aminoácidos , Proteína 8 Similar a la Angiopoyetina , Proteínas Similares a la Angiopoyetina , Animales , Femenino , Glucosa/metabolismo , Humanos , Resistencia a la Insulina , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Páncreas/metabolismo , Hormonas Peptídicas/química , Hormonas Peptídicas/genética , Péptidos/administración & dosificación , Receptor de Insulina/antagonistas & inhibidores , Alineación de SecuenciaRESUMEN
Protein secretion in eukaryotes and prokaryotes involves a universally conserved protein translocation channel formed by the Sec61 complex. Unrelated small-molecule natural products and synthetic compounds inhibit Sec61 with differential effects for different substrates or for Sec61 from different organisms, making this a promising target for therapeutic intervention. To understand the mode of inhibition and provide insight into the molecular mechanism of this dynamic translocon, we determined the structure of mammalian Sec61 inhibited by the Mycobacterium ulcerans exotoxin mycolactone via electron cryo-microscopy. Unexpectedly, the conformation of inhibited Sec61 is optimal for substrate engagement, with mycolactone wedging open the cytosolic side of the lateral gate. The inability of mycolactone-inhibited Sec61 to effectively transport substrate proteins implies that signal peptides and transmembrane domains pass through the site occupied by mycolactone. This provides a foundation for understanding the molecular mechanism of Sec61 inhibitors and reveals novel features of translocon function and dynamics.
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Macrólidos/farmacología , Microsomas/química , Ribosomas/química , Canales de Translocación SEC/química , Animales , Sitios de Unión , Sistema Libre de Células/metabolismo , Perros , Expresión Génica , Células HCT116 , Células HEK293 , Humanos , Macrólidos/química , Macrólidos/aislamiento & purificación , Microsomas/metabolismo , Simulación de Dinámica Molecular , Mutación , Mycobacterium ulcerans/química , Mycobacterium ulcerans/patogenicidad , Páncreas/química , Páncreas/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , Ribosomas/metabolismo , Canales de Translocación SEC/antagonistas & inhibidores , Canales de Translocación SEC/genética , Canales de Translocación SEC/metabolismo , Homología Estructural de Proteína , Especificidad por SustratoRESUMEN
Proper embryonic development depends on the timely progression of a genetic program. One of the key mechanisms for achieving precise control of developmental timing is to use gene expression oscillations. In this Review, we examine how gene expression oscillations encode temporal information during vertebrate embryonic development by discussing the gene expression oscillations occurring during somitogenesis, neurogenesis, myogenesis and pancreas development. These oscillations play important but varied physiological functions in different contexts. Oscillations control the period of somite formation during somitogenesis, whereas they regulate the proliferation-to-differentiation switch of stem cells and progenitor cells during neurogenesis, myogenesis and pancreas development. We describe the similarities and differences of the expression pattern in space (i.e. whether oscillations are synchronous or asynchronous across neighboring cells) and in time (i.e. different time scales) of mammalian Hes/zebrafish Her genes and their targets in different tissues. We further summarize experimental evidence for the functional role of their oscillations. Finally, we discuss the outstanding questions for future research.
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Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , Somitos , Animales , Desarrollo Embrionario/genética , Humanos , Somitos/metabolismo , Somitos/embriología , Desarrollo de Músculos/genética , Neurogénesis/genética , Neurogénesis/fisiología , Páncreas/embriología , Páncreas/metabolismo , Diferenciación Celular/genéticaRESUMEN
Studies based on single cells have revealed vast cellular heterogeneity in stem cell and progenitor compartments, suggesting continuous differentiation trajectories with intermixing of cells at various states of lineage commitment and notable degrees of plasticity during organogenesis1-5. The hepato-pancreato-biliary organ system relies on a small endoderm progenitor compartment that gives rise to a variety of different adult tissues, including the liver, pancreas, gall bladder and extra-hepatic bile ducts6,7. Experimental manipulation of various developmental signals in the mouse embryo has underscored important cellular plasticity in this embryonic territory6. This is reflected in the existence of human genetic syndromes as well as congenital malformations featuring multi-organ phenotypes in liver, pancreas and gall bladder6. Nevertheless, the precise lineage hierarchy and succession of events leading to the segregation of an endoderm progenitor compartment into hepatic, biliary and pancreatic structures have not yet been established. Here we combine computational modelling approaches with genetic lineage tracing to accurately reconstruct the hepato-pancreato-biliary lineage tree. We show that a multipotent progenitor subpopulation persists in the pancreato-biliary organ rudiment, contributing cells not only to the pancreas and gall bladder but also to the liver. Moreover, using single-cell RNA sequencing and functional experiments we define a specialized niche that supports this subpopulation in a multipotent state for an extended time during development. Together these findings indicate sustained plasticity underlying hepato-pancreato-biliary development that might also explain the rapid expansion of the liver while attenuating pancreato-biliary growth.
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Sistema Biliar/citología , Linaje de la Célula , Hígado/citología , Páncreas/citología , Nicho de Células Madre , Animales , Sistema Biliar/embriología , Sistema Biliar/metabolismo , Linaje de la Célula/genética , Rastreo Celular , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Femenino , Hígado/embriología , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Páncreas/embriología , Páncreas/metabolismo , RNA-Seq , Transducción de Señal , Análisis de la Célula Individual , Nicho de Células Madre/genéticaRESUMEN
Tissue damage increases the risk of cancer through poorly understood mechanisms1. In mouse models of pancreatic cancer, pancreatitis associated with tissue injury collaborates with activating mutations in the Kras oncogene to markedly accelerate the formation of early neoplastic lesions and, ultimately, adenocarcinoma2,3. Here, by integrating genomics, single-cell chromatin assays and spatiotemporally controlled functional perturbations in autochthonous mouse models, we show that the combination of Kras mutation and tissue damage promotes a unique chromatin state in the pancreatic epithelium that distinguishes neoplastic transformation from normal regeneration and is selected for throughout malignant evolution. This cancer-associated epigenetic state emerges within 48 hours of pancreatic injury, and involves an 'acinar-to-neoplasia' chromatin switch that contributes to the early dysregulation of genes that define human pancreatic cancer. Among the factors that are most rapidly activated after tissue damage in the pre-malignant pancreatic epithelium is the alarmin cytokine interleukin 33, which recapitulates the effects of injury in cooperating with mutant Kras to unleash the epigenetic remodelling program of early neoplasia and neoplastic transformation. Collectively, our study demonstrates how gene-environment interactions can rapidly produce gene-regulatory programs that dictate early neoplastic commitment, and provides a molecular framework for understanding the interplay between genetic and environmental cues in the initiation of cancer.
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Transformación Celular Neoplásica/genética , Epigénesis Genética , Interacción Gen-Ambiente , Páncreas/metabolismo , Páncreas/patología , Adenocarcinoma/genética , Adenocarcinoma/patología , Animales , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Transformación Celular Neoplásica/patología , Cromatina/genética , Cromatina/metabolismo , Cromatina/patología , Modelos Animales de Enfermedad , Femenino , Genómica , Humanos , Interleucina-33/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The pancreas is an essential organ for proper nutrient metabolism and has both endocrine and exocrine function. In the past two decades, knowledge of how the pancreas develops during embryogenesis has significantly increased, largely from developmental studies in model organisms. Specifically, the molecular basis of pancreatic lineage decisions and cell differentiation is well studied. Still not well understood are the mechanisms governing three-dimensional morphogenesis of the organ. Strategies to derive transplantable ß-cells in vitro for diabetes treatment have benefited from the accumulated knowledge of pancreas development. In this review, we provide an overview of the current understanding of pancreatic lineage determination and organogenesis, and we examine future implications of these findings for treatment of diabetes mellitus through cell replacement.
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Organogénesis , Páncreas/citología , Páncreas/embriología , Animales , Diferenciación Celular , Humanos , Páncreas/metabolismo , Transducción de SeñalRESUMEN
Pancreatic ductal adenocarcinoma (PDA) tumor cells are deprived of oxygen and nutrients and therefore must adapt their metabolism to ensure proliferation. In some physiological states, cells rely on ketone bodies to satisfy their metabolic needs, especially during nutrient stress. Here, we show that PDA cells can activate ketone body metabolism and that ß-hydroxybutyrate (ßOHB) is an alternative cell-intrinsic or systemic fuel that can promote PDA growth and progression. PDA cells activate enzymes required for ketogenesis, utilizing various nutrients as carbon sources for ketone body formation. By assessing metabolic gene expression from spontaneously arising PDA tumors in mice, we find HMG-CoA lyase (HMGCL), involved in ketogenesis, to be among the most deregulated metabolic enzymes in PDA compared to normal pancreas. In vitro depletion of HMGCL impedes migration, tumor cell invasiveness, and anchorage-independent tumor sphere compaction. Moreover, disrupting HMGCL drastically decreases PDA tumor growth in vivo, while ßOHB stimulates metastatic dissemination to the liver. These findings suggest that ßOHB increases PDA aggressiveness and identify HMGCL and ketogenesis as metabolic targets for limiting PDA progression.
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Cuerpos Cetónicos , Neoplasias Pancreáticas , Ácido 3-Hidroxibutírico/metabolismo , Animales , Cuerpos Cetónicos/metabolismo , Ratones , Oxo-Ácido-Liasas , Páncreas/metabolismoRESUMEN
Hormone secretion from pancreatic islets is essential for glucose homeostasis, and loss or dysfunction of islet cells is a hallmark of type 2 diabetes. Maf transcription factors are crucial for establishing and maintaining adult endocrine cell function. However, during pancreas development, MafB is not only expressed in insulin- and glucagon-producing cells, but also in Neurog3+ endocrine progenitor cells, suggesting additional functions in cell differentiation and islet formation. Here, we report that MafB deficiency impairs ß cell clustering and islet formation, but also coincides with loss of neurotransmitter and axon guidance receptor gene expression. Moreover, the observed loss of nicotinic receptor gene expression in human and mouse ß cells implied that signaling through these receptors contributes to islet cell migration/formation. Inhibition of nicotinic receptor activity resulted in reduced ß cell migration towards autonomic nerves and impaired ß cell clustering. These findings highlight a novel function of MafB in controlling neuronal-directed signaling events required for islet formation.
Asunto(s)
Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Islotes Pancreáticos , Ratones , Adulto , Animales , Humanos , Glucagón/genética , Glucagón/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Islotes Pancreáticos/metabolismo , Insulina/metabolismo , Páncreas/metabolismo , Factor de Transcripción MafB/genética , Factor de Transcripción MafB/metabolismoRESUMEN
Type 1 diabetes (T1D) onset is characterized by an autoimmune attack on ß islet cells within the pancreas, preventing the insulin secretion required to maintain glucose homeostasis. Targeted modulation of key immunoregulatory cell populations is a promising strategy to restore tolerance to ß cells. This strategy can be used to prevent T1D onset or reverse T1D with transplanted islets. To this end, drug delivery systems can be employed to transport immunomodulatory cargo to specific cell populations that inhibit autoreactive T cell-mediated destruction of the ß cell mass. The rational engineering of biomaterials into nanoscale and microscale drug carriers can facilitate targeted interactions with immune cells. The physicochemical properties of the biomaterial, the delivered immunomodulatory agent, and the target cell populations are critical variables in the design of these delivery systems. In this review, we discuss recent biomaterials-based drug delivery approaches to induce islet tolerance and the need to consider both immune and metabolic markers of disease progression.
Asunto(s)
Diabetes Mellitus Tipo 1 , Células Secretoras de Insulina , Islotes Pancreáticos , Humanos , Materiales Biocompatibles/metabolismo , Islotes Pancreáticos/metabolismo , Páncreas/metabolismo , Tolerancia InmunológicaRESUMEN
The invasion of nerves by cancer cells, or perineural invasion (PNI), is potentiated by the nerve microenvironment and is associated with adverse clinical outcomes. However, the cancer cell characteristics that enable PNI are poorly defined. Here, we generated cell lines enriched for a rapid neuroinvasive phenotype by serially passaging pancreatic cancer cells in a murine sciatic nerve model of PNI. Cancer cells isolated from the leading edge of nerve invasion showed a progressively increasing nerve invasion velocity with higher passage number. Transcriptome analysis revealed an upregulation of proteins involving the plasma membrane, cell leading edge, and cell movement in the leading neuroinvasive cells. Leading cells progressively became round and blebbed, lost focal adhesions and filipodia, and transitioned from a mesenchymal to amoeboid phenotype. Leading cells acquired an increased ability to migrate through microchannel constrictions and associated more with dorsal root ganglia than nonleading cells. ROCK inhibition reverted leading cells from an amoeboid to mesenchymal phenotype, reduced migration through microchannel constrictions, reduced neurite association, and reduced PNI in a murine sciatic nerve model. Cancer cells with rapid PNI exhibit an amoeboid phenotype, highlighting the plasticity of cancer migration mode in enabling rapid nerve invasion.
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Amoeba , Tejido Nervioso , Neoplasias Pancreáticas , Ratones , Animales , Neoplasias Pancreáticas/genética , Nervio Ciático/metabolismo , Páncreas/metabolismo , Tejido Nervioso/metabolismo , Movimiento Celular/genética , Invasividad Neoplásica , Microambiente TumoralRESUMEN
BACKGROUND & AIMS: Acinar cells produce digestive enzymes that impede transcriptomic characterization of the exocrine pancreas. Thus, single-cell RNA-sequencing studies of the pancreas underrepresent acinar cells relative to histological expectations, and a robust approach to capture pancreatic cell responses in disease states is needed. We sought to innovate a method that overcomes these challenges to accelerate study of the pancreas in health and disease. METHODS: We leverage FixNCut, a single-cell RNA-sequencing approach in which tissue is reversibly fixed with dithiobis(succinimidyl propionate) before dissociation and single-cell preparation. We apply FixNCut to an established mouse model of acute pancreatitis, validate findings using GeoMx whole transcriptome atlas profiling, and integrate our data with prior studies to compare our method in both mouse and human pancreas datasets. RESULTS: FixNCut achieves unprecedented definition of challenging pancreatic cells, including acinar and immune populations in homeostasis and acute pancreatitis, and identifies changes in all major cell types during injury and recovery. We define the acinar transcriptome during homeostasis and acinar-to-ductal metaplasia and establish a unique gene set to measure deviation from normal acinar identity. We characterize pancreatic immune cells, and analysis of T-cell subsets reveals a polarization of the homeostatic pancreas toward type-2 immunity. We report immune responses during acute pancreatitis and recovery, including early neutrophil infiltration, expansion of dendritic cell subsets, and a substantial shift in the transcriptome of macrophages due to both resident macrophage activation and monocyte infiltration. CONCLUSIONS: FixNCut preserves pancreatic transcriptomes to uncover novel cell states during homeostasis and following pancreatitis, establishing a broadly applicable approach and reference atlas for study of pancreas biology and disease.
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Células Acinares , Modelos Animales de Enfermedad , Homeostasis , Pancreatitis , Análisis de la Célula Individual , Transcriptoma , Animales , Pancreatitis/genética , Pancreatitis/inducido químicamente , Pancreatitis/patología , Pancreatitis/metabolismo , Humanos , Células Acinares/metabolismo , Células Acinares/patología , Ratones , Páncreas/patología , Páncreas/metabolismo , Perfilación de la Expresión Génica/métodos , RNA-Seq , Enfermedad Aguda , Páncreas Exocrino/metabolismo , Páncreas Exocrino/patología , Macrófagos/metabolismo , Metaplasia/genética , Metaplasia/patología , Ratones Endogámicos C57BLRESUMEN
Hypoxia can occur in pancreatic ß-cells in type 2 diabetes. Although hypoxia exerts deleterious effects on ß-cell function, the associated mechanisms are largely unknown. Here, we show that the transcriptional repressor basic helix-loop-helix family member e40 (BHLHE40) is highly induced in hypoxic mouse and human ß-cells and suppresses insulin secretion. Conversely, BHLHE40 deficiency in hypoxic MIN6 cells or ß-cells of ob/ob mice reverses defects in insulin secretion. Mechanistically, BHLHE40 represses the expression of Mafa, encoding the transcription factor musculoaponeurotic fibrosarcoma oncogene family A (MAFA), by attenuating the binding of pancreas/duodenum homeobox protein 1 (PDX1) to its enhancer region. Impaired insulin secretion in hypoxic ß-cells was recovered by MAFA re-expression. Collectively, our work identifies BHLHE40 as a key hypoxia-induced transcriptional repressor in ß-cells that inhibit insulin secretion by suppressing MAFA expression.