RESUMEN
IMPLICATIONS: The study identifies an opportunity to discover a PKA-independent pathway downstream of oncogene GNAS for managing IPMN lesions and their progression to PDAC.
Asunto(s)
Proliferación Celular , Cromograninas , Proteínas Quinasas Dependientes de AMP Cíclico , Subunidades alfa de la Proteína de Unión al GTP Gs , Organoides , Neoplasias Pancreáticas , Humanos , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Organoides/metabolismo , Organoides/patología , Cromograninas/genética , Cromograninas/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Carcinoma Ductal Pancreático/patología , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Conductos Pancreáticos/metabolismo , Conductos Pancreáticos/patología , Conductos Pancreáticos/citología , Células Acinares/metabolismo , Células Acinares/patologíaRESUMEN
BACKGROUND & AIMS: Acinar to ductal metaplasia (ADM) occurs in the pancreas in response to tissue injury and is a potential precursor for adenocarcinoma. The goal of these studies was to define the populations arising from ADM, the associated transcriptional changes, and markers of disease progression. METHODS: Acinar cells were lineage-traced with enhanced yellow fluorescent protein (EYFP) to follow their fate post-injury. Transcripts of more than 13,000 EYFP+ cells were determined using single-cell RNA sequencing (scRNA-seq). Developmental trajectories were generated. Data were compared with gastric metaplasia, KrasG12D-induced neoplasia, and human pancreatitis. Results were confirmed by immunostaining and electron microscopy. KrasG12D was expressed in injury-induced ADM using several inducible Cre drivers. Surgical specimens of chronic pancreatitis from 15 patients were evaluated by immunostaining. RESULTS: scRNA-seq of ADM revealed emergence of a mucin/ductal population resembling gastric pyloric metaplasia. Lineage trajectories suggest that some pyloric metaplasia cells can generate tuft and enteroendocrine cells (EECs). Comparison with KrasG12D-induced ADM identifies populations associated with disease progression. Activation of KrasG12D expression in HNF1B+ or POU2F3+ ADM populations leads to neoplastic transformation and formation of MUC5AC+ gastric-pit-like cells. Human pancreatitis samples also harbor pyloric metaplasia with a similar transcriptional phenotype. CONCLUSIONS: Under conditions of chronic injury, acinar cells undergo a pyloric-type metaplasia to mucinous progenitor-like populations, which seed disparate tuft cell and EEC lineages. ADM-derived EEC subtypes are diverse. KrasG12D expression is sufficient to drive neoplasia when targeted to injury-induced ADM populations and offers an alternative origin for tumorigenesis. This program is conserved in human pancreatitis, providing insight into early events in pancreas diseases.
Asunto(s)
Células Acinares/metabolismo , Carcinoma Ductal Pancreático/genética , Metaplasia/genética , Conductos Pancreáticos/metabolismo , Neoplasias Pancreáticas/genética , Células Acinares/citología , Plasticidad de la Célula/genética , Células Enteroendocrinas/citología , Células Enteroendocrinas/metabolismo , Perfilación de la Expresión Génica , Humanos , Metaplasia/metabolismo , Mucina 5AC/genética , Páncreas/citología , Páncreas/metabolismo , Conductos Pancreáticos/citología , Pancreatitis/genética , Pancreatitis/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Análisis de la Célula IndividualRESUMEN
The recapitulation of human developmental processes and pathological manifestations requires access to specific cell types and precursor stages during embryogenesis and disease. Here, we describe a scalable in vitro differentiation protocol to guide human pluripotent stem cells stepwise into pancreatic duct-like organoids. The protocol mimics pancreatic duct development and was successfully used to model the onset and progression of pancreatic ductal adenocarcinoma; the approach is suitable for multiple downstream applications. However, the protocol is cost- and time-intensive. For complete details on the use and execution of this protocol, please refer to Breunig et al. (2021).
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Técnicas de Cultivo de Célula/métodos , Conductos Pancreáticos/citología , Células Madre Pluripotentes/citología , Diferenciación Celular/fisiología , Línea Celular , Humanos , Organoides/citologíaRESUMEN
The production of pancreatic ß cells is the most challenging step for curing diabetes using next-generation treatments. Adult pancreatic endocrine cells are thought to be maintained by the self-duplication of differentiated cells, and pancreatic endocrine neogenesis can only be observed when the tissue is severely damaged. Experimentally, this can be performed using a method named partial duct ligation (PDL). As the success rate of PDL surgery is low because of difficulties in identifying the pancreatic duct, we previously proposed a method for fluorescently labeling the duct in live animals. Using this method, we performed PDL on neurogenin3 (Ngn3)-GFP transgenic mice to determine the origin of endocrine precursor cells and evaluate their potential to differentiate into multiple cell types. Ngn3-activated cells, which were marked with GFP, appeared after PDL operation. Because some GFP-positive cells were aligned proximally to the duct, we hypothesized that Ngn3-positive cells arise from the pancreatic duct. Therefore, we next developed an in vitro pancreatic duct culture system using Ngn3-GFP mice and examined whether Ngn3-positive cells emerge from this duct. We observed GFP expressions in ductal organoid cultures. GFP expressions were correlated with Ngn3 expressions and endocrine cell lineage markers. Interestingly, tuft cell markers were also correlated with GFP expressions. Our results demonstrate that in adult mice, Ngn3-positive endocrine precursor cells arise from the pancreatic ducts both in vivo and in vitro experiments indicating that the pancreatic duct could be a potential donor for therapeutic use.
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Antígenos de Diferenciación/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Células Secretoras de Insulina/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Conductos Pancreáticos/metabolismo , Células Madre/metabolismo , Animales , Antígenos de Diferenciación/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células Secretoras de Insulina/citología , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Organoides/citología , Organoides/metabolismo , Conductos Pancreáticos/citología , Células Madre/citologíaRESUMEN
Creating in vitro models of diseases of the pancreatic ductal compartment requires a comprehensive understanding of the developmental trajectories of pancreas-specific cell types. Here we report the single-cell characterization of the differentiation of pancreatic duct-like organoids (PDLOs) from human induced pluripotent stem cells (hiPSCs) on a microwell chip that facilitates the uniform aggregation and chemical induction of hiPSC-derived pancreatic progenitors. Using time-resolved single-cell transcriptional profiling and immunofluorescence imaging of the forming PDLOs, we identified differentiation routes from pancreatic progenitors through ductal intermediates to two types of mature duct-like cells and a few non-ductal cell types. PDLO subpopulations expressed either mucins or the cystic fibrosis transmembrane conductance regulator, and resembled human adult duct cells. We also used the chip to uncover ductal markers relevant to pancreatic carcinogenesis, and to establish PDLO co-cultures with stellate cells, which allowed for the study of epithelial-mesenchymal signalling. The PDLO microsystem could be used to establish patient-specific pancreatic duct models.
Asunto(s)
Diferenciación Celular , Dispositivos Laboratorio en un Chip , Organoides/citología , Conductos Pancreáticos/citología , Animales , Biomarcadores de Tumor/metabolismo , Reprogramación Celular , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Filaminas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/trasplante , Ratones , Ratones Endogámicos NOD , Ratones SCID , Mucinas/metabolismo , Organoides/metabolismo , Conductos Pancreáticos/metabolismo , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/mortalidad , Análisis de la Célula Individual , Tasa de SupervivenciaRESUMEN
AIMS/HYPOTHESIS: Islets are thought to be stably present in the adult human pancreas to maintain glucose homeostasis. However, identification of the pancreatic intraepithelial neoplasia (PanIN)-islet complex in mice and the presence of PanIN lesions in adult humans suggest that similar remodelling of islet structure and environment may occur in the human pancreas. To identify islet remodelling in a clinically related setting, we examine human donor pancreases with 3D histology to detect and characterise the human PanIN-islet complex. METHODS: Cadaveric donor pancreases (26-65 years old, n = 10) were fixed and sectioned (350 µm) for tissue labelling, clearing and microscopy to detect local islet remodelling for 3D analysis of the microenvironment. The remodelled microenvironment was subsequently examined via microtome-based histology for clinical assessment. RESULTS: In nine pancreases, we identified the unique peri-lobular islet aggregation associated with the PanIN lesion (16 lesion-islet complexes detected; size: 3.18 ± 1.34 mm). Important features of the lesion-islet microenvironment include: (1) formation of intra-islet ducts, (2) acinar atrophy, (3) adipocyte association, (4) inflammation (CD45+), (5) stromal accumulation (α-SMA+), (6) increase in Ki-67 proliferation index but absence of Ki-67+ alpha/beta cells and (7) in-depth and continuous duct-islet cell contacts, forming a cluster. The duct-islet cell cluster and intra-islet ducts suggest likely islet cell neogenesis but not replication. CONCLUSIONS/INTERPRETATION: We identify local islet remodelling associated with PanIN-islet complex in the adult human pancreas. The tissue remodelling and the evidence of inflammation and stromal accumulation suggest that the PanIN-islet complex is derived from tissue repair after a local injury.
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Islotes Pancreáticos/citología , Conductos Pancreáticos/citología , Actinas/metabolismo , Adipocitos/metabolismo , Adulto , Anciano , Proliferación Celular , Microambiente Celular , Femenino , Humanos , Imagenología Tridimensional , Islotes Pancreáticos/fisiología , Antígeno Ki-67/metabolismo , Antígenos Comunes de Leucocito/metabolismo , Masculino , Microscopía Confocal , Persona de Mediana Edad , Conductos Pancreáticos/fisiología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Donantes de TejidosRESUMEN
We describe our initial studies in the development of an orthotopic, genetically defined, large animal model of pancreatic cancer. Primary pancreatic epithelial cells were isolated from pancreatic duct of domestic pigs. A transformed cell line was generated from these primary cells with oncogenic KRAS and SV40T. The transformed cell lines outperformed the primary and SV40T immortalized cells in terms of proliferation, population doubling time, soft agar growth, transwell migration and invasion. The transformed cell line grew tumors when injected subcutaneously in nude mice, forming glandular structures and staining for epithelial markers. Future work will include implantation studies of these tumorigenic porcine pancreatic cell lines into the pancreas of allogeneic and autologous pigs. The resultant large animal model of pancreatic cancer could be utilized for preclinical research on diagnostic, interventional, and therapeutic technologies.
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Antígenos Transformadores de Poliomavirus/fisiología , Transformación Celular Neoplásica/genética , Células Epiteliales/patología , Genes ras , Conductos Pancreáticos/citología , Neoplasias Pancreáticas/patología , Animales , Antígenos Transformadores de Poliomavirus/genética , División Celular , Línea Celular Transformada , Células Epiteliales/trasplante , Xenoinjertos , Masculino , Ratones , Ratones Desnudos , Modelos Animales , Mutación Missense , Invasividad Neoplásica , Trasplante de Neoplasias , Neoplasias Pancreáticas/genética , Mutación Puntual , PorcinosRESUMEN
To study disease development, an inventory of an organ's cell types and understanding of physiologic function is paramount. Here, we performed single-cell RNA-sequencing to examine heterogeneity of murine pancreatic duct cells, pancreatobiliary cells, and intrapancreatic bile duct cells. We describe an epithelial-mesenchymal transitory axis in our three pancreatic duct subpopulations and identify osteopontin as a regulator of this fate decision as well as human duct cell dedifferentiation. Our results further identify functional heterogeneity within pancreatic duct subpopulations by elucidating a role for geminin in accumulation of DNA damage in the setting of chronic pancreatitis. Our findings implicate diverse functional roles for subpopulations of pancreatic duct cells in maintenance of duct cell identity and disease progression and establish a comprehensive road map of murine pancreatic duct cell, pancreatobiliary cell, and intrapancreatic bile duct cell homeostasis.
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Perfilación de la Expresión Génica , Heterogeneidad Genética , Conductos Pancreáticos/citología , Análisis de la Célula Individual , Transcriptoma , Animales , Línea Celular , Separación Celular , Daño del ADN , Bases de Datos Genéticas , Modelos Animales de Enfermedad , Transición Epitelial-Mesenquimal , Femenino , Geminina/genética , Geminina/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Ratones Endogámicos C57BL , Ratones Transgénicos , Morfogénesis , Osteopontina/genética , Osteopontina/metabolismo , Conductos Pancreáticos/metabolismo , Pancreatitis Crónica/genética , Pancreatitis Crónica/metabolismo , Pancreatitis Crónica/patología , Fenotipo , RNA-SeqRESUMEN
The adult pancreatic ductal system was suggested to harbor facultative beta-cell progenitors similar to the embryonic pancreas, and the appearance of insulin-positive duct cells has been used as evidence for natural duct-to-beta-cell reprogramming. Nevertheless, the phenotype and fate of these insulin-positive cells in ducts have not been determined. Here, we used a cell-tagging dye, CFDA-SE, to permanently label pancreatic duct cells through an intraductal infusion technique. Representing a time when significant increases in beta-cell mass occur, pregnancy was later induced in these CFDA-SE-treated mice to assess the phenotype and fate of the insulin-positive cells in ducts. We found that a small portion of CFDA-SE-labeled duct cells became insulin-positive, but they were not fully functional beta-cells based on the in vitro glucose response and the expression levels of key beta-cell genes. Moreover, these insulin-positive cells in ducts expressed significantly lower levels of genes associated with extracellular matrix degradation and cell migration, which may thus prevent their budding and migration into preexisting islets. A similar conclusion was reached through analysis of the Gene Expression Omnibus database for both mice and humans. Together, our data suggest that the contribution of duct cells to normal beta-cells in adult islets is minimal at best.
Asunto(s)
Movimiento Celular , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Conductos Pancreáticos/citología , Animales , Biomarcadores , Diferenciación Celular , Biología Computacional/métodos , Matriz Extracelular , Femenino , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Inmunofenotipificación , Ratones , Ratones Transgénicos , EmbarazoRESUMEN
AIMS: We investigated the in vitro differentiation of adult rat PDESCs into ß-like cells through supplementation of different combinations of GABA, BMP7, and Activin A in basic culture media. MATERIALS AND METHODS: The PDESCs were cultured using different inducement combinations for 28 days and microscopy, dithizone (DTZ) staining, immunohistochemical staining, real-time PCR, and glucose-stimulated insulin secretion (GSIS) assay were used to delineate the differentiation inducement potential of these combinations. KEY FINDINGS: The results show that after 28 days, the PDESCs were differentiated into ICCs containing insulin-secreting ß-like cells in different groups treated with A + B, A + G, B + G, and A + B + G but not in the control group. Upon DTZ staining the cells in ICCs were stained crimson red, demonstrating the presence of ß-like cells in ICCs and the immunohistochemistry showed the expression of Pdx1 and insulin in ICCs. Further, on 28 d the expression of Pdx1 and insulin mRNA was high in inducement groups as compared to the control group and ß-like cells in ICCs also secreted insulin and C-peptide upon glucose stimulation. Thus, the supplementation of GABA, BMP7, and Activin A in different combinations in basic culture media can induce the in vitro differentiation of PDESCs into ICCs containing ß-like cells. SIGNIFICANCE: The in vitro development of ß-like cells is a herald for cell therapy of diabetic patients and our results are a step closer towards finding the cure for diabetes.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Células Madre/metabolismo , Activinas/metabolismo , Activinas/farmacología , Animales , Proteína Morfogenética Ósea 7/metabolismo , Proteína Morfogenética Ósea 7/farmacología , Técnicas de Cultivo de Célula/métodos , Línea Celular , Células Cultivadas , Medios de Cultivo/química , Insulina/metabolismo , Secreción de Insulina , Conductos Pancreáticos/citología , Conductos Pancreáticos/metabolismo , Ratas , Ratas Sprague-Dawley , Células Madre/citología , Ácido gamma-Aminobutírico/metabolismo , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related death in the United States. Despite the high prevalence of Kras mutations in pancreatic cancer patients, murine models expressing the oncogenic mutant Kras (Krasmut) in mature pancreatic cells develop PDAC at a low frequency. Independent of cell of origin, a second genetic hit (loss of tumor suppressor TP53 or PTEN) is important for development of PDAC in mice. We hypothesized ectopic expression and elevated levels of oncogenic mutant Kras would promote PanIN arising in pancreatic ducts. To test our hypothesis, the significance of elevating levels of K-Ras and Ras activity has been explored by expression of a CAG driven LGSL-KrasG12V allele (cKras) in pancreatic ducts, which promotes ectopic Kras expression. We predicted expression of cKras in pancreatic ducts would generate neoplasia and PDAC. To test our hypothesis, we employed tamoxifen dependent CreERT2 mediated recombination. Hnf1b:CreERT2;KrasG12V (cKrasHnf1b/+) mice received 1 (Low), 5 (Mod) or 10 (High) mg per 20 g body weight to recombine cKras in low (cKrasLow), moderate (cKrasMod), and high (cKrasHigh) percentages of pancreatic ducts. Our histologic analysis revealed poorly differentiated aggressive tumors in cKrasHigh mice. cKrasMod mice had grades of Pancreatic Intraepithelial Neoplasia (PanIN), recapitulating early and advanced PanIN observed in human PDAC. Proteomics analysis revealed significant differences in PTEN/AKT and MAPK pathways between wild type, cKrasLow, cKrasMod, and cKrasHigh mice. In conclusion, in this study, we provide evidence that ectopic expression of oncogenic mutant K-Ras in pancreatic ducts generates early and late PanIN as well as PDAC. This Ras rheostat model provides evidence that AKT signaling is an important early driver of invasive ductal derived PDAC.
Asunto(s)
Carcinoma Ductal Pancreático , Tasa de Mutación , Neoplasias Pancreáticas , Proteínas Proto-Oncogénicas p21(ras) , Animales , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica , Ratones , Ratones Transgénicos , Conductos Pancreáticos/citología , Conductos Pancreáticos/metabolismo , Conductos Pancreáticos/patología , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Lesiones Precancerosas/genética , Lesiones Precancerosas/metabolismo , Lesiones Precancerosas/patología , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Recombinación GenéticaRESUMEN
Extracellular vesicles (EV) are considered as a promising diagnostic tool for pancreatic ductal adenocarcinoma (PDAC), a disease with a poor 5-year survival that has not improved in the past years. PDAC patient-derived 3D organoids maintain the intratumoral cellular heterogeneity, characteristic for the tumor in vivo.Thus, they represent an ideal in vitro model system to study human cancers. Here we show that the miRNA cargo of EVs from PDAC organoids largely differs among patients. However, we detected a common set of EV miRNAs that were present in matched organoids and blood plasma samples of individual patients. Importantly, the levels of EV miR-21 and miR-195 were higher in PDAC blood EV preparations than in healthy controls, albeit we found no difference compared to chronic pancreatitis (CP) samples. In addition, here we report that the accumulation of collagen I, a characteristic change in the extracellular matrix (ECM) in both CP and PDAC, largely increases EV release from pancreatic ductal organoids. This provides a possible explanation why both CP and PDAC patient-derived plasma samples have an elevated amount of CD63 + EVs. Collectively, we show that PDAC patient-derived organoids represent a highly relevant model to analyze the cargo of tumor cell-derived EVs. Furthermore, we provide evidence that not only driver mutations, but also changes in the ECM may critically modify EV release from pancreatic ductal cells.
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Carcinoma Ductal Pancreático/patología , Vesículas Extracelulares/genética , MicroARNs/metabolismo , Organoides/metabolismo , Neoplasias Pancreáticas/patología , Animales , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Estudios de Casos y Controles , Células Cultivadas , Colágeno Tipo I/metabolismo , Colágeno Tipo I/farmacología , Citocinas/farmacología , Matriz Extracelular/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , MicroARNs/sangre , Organoides/citología , Organoides/efectos de los fármacos , Conductos Pancreáticos/citología , Conductos Pancreáticos/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Pancreatitis/genética , Pancreatitis/metabolismo , Pancreatitis/patologíaRESUMEN
The goal of this study was to research long-term saturated fatty acid overexposure that can induce differentiation of pancreatic duct cells into adipocytes and also into ß-cells. The important findings can be summarized as follows: (i) adipogenesis and early stage ß-cell differentiation were stimulated in duct cells under lipotoxicity and glucolipotoxicity conditions, (ii) miR-375 expression was upregulated while its target Erk1 was downregulated and miR-375 inhibitor upregulated Erk1 while expression of adipogenesis markers was downregulated in duct cells under both conditions, (iii) apoptosis was induced in ß and duct cells under both conditions, (iv) lipotoxicity induced proliferation of co-cultured ß-cells. These findings suggest that long-term saturated fatty acid overexposure may cause intrapancreatic fat accumulation by inducing differentiation of duct cells into adipocytes and it may contributes to ß-cell compensation by stimulating the early stage of ß-cell differentiation in duct cells. In addition, miR-375 may have the potential to be a new target in the treatment of Type 2 diabetes, and NAFPD due to its role in the adipogenesis of duct cells.
Asunto(s)
Adipogénesis/genética , MicroARNs/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Ácido Palmítico/farmacología , Conductos Pancreáticos/citología , Adipogénesis/efectos de los fármacos , Animales , Muerte Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Glucosa/toxicidad , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Lípidos/toxicidad , Masculino , MicroARNs/genética , Modelos Biológicos , Necrosis , Ratas Wistar , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
To cure the epidemic of diabetes, in vitro produced ß-like cells are lauded for cell therapy of diabetic patients. In this regard, we investigated the effects of different concentrations of bone morphogenetic protein 7 (BMP7) on the differentiation of rat pancreatic ductal epithelial-like stem cells (PDESCs) into ß-like cells. For inducement of the differentiation, PDESCs were cultured in the basal media (H-DMEM + 10 % FBS + 1% penicillin-streptomycin) supplemented with 5 ng/mL, 10 ng/mL, 15 ng/mL, and 20 ng/mL of BMP7 for 28 days. To corroborate the identity of induced cells, they were examined through cell morphology, dithizone (DTZ) staining, immunofluorescence staining, real-time polymerase chain reaction (qPCR), and glucose-stimulated insulin secretion assay (GSIS). The enrichment of induced cells was high among 5 ng/mL, 10 ng/mL, 15 ng/mL, and 20 ng/mL of BMP7 supplemented groups as compared to the control group. Further, the induced cells were positive, while, the control group cells were negative to DTZ staining and the differentiated cells also have shown the upregulated expression of ß cell-specific marker genes (Ins1 and Pdx1). The GSIS assay of inducement groups for insulin and C-peptide secretion has shown significantly higher values as compared to the control group (P < 0.01). Hence, the addition of BMP7 to basal medium has effectually induced differentiation of adult rat PDESCs into islet like-cell clusters containing insulin-secreting ß-like cells.
Asunto(s)
Proteína Morfogenética Ósea 7/farmacología , Diferenciación Celular , Células Secretoras de Insulina/citología , Conductos Pancreáticos/citología , Células Madre/citología , Animales , Péptido C/metabolismo , Agregación Celular/efectos de los fármacos , Recuento de Células , Diferenciación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Ditizona , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Glucosa/farmacología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Insulina/genética , Insulina/metabolismo , Secreción de Insulina/efectos de los fármacos , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Ratas Sprague-Dawley , Células Madre/efectos de los fármacos , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
OBJECTIVES: Chronic pancreatitis is the end stage of a pathologic inflammatory syndrome with multiple etiological factors, including genetic. We hypothesized that some pancreatitis etiology originates in pancreatic acinar or duct cells and requires both injury and compensatory mechanism failure. METHODS: One hundred pancreatitis patients were assessed using a DNA sequencing panel for pancreatitis. Cooccurrence of variants within and between genes was measured. Gene coexpression was confirmed via published single-cell RNA sequencing. RESULTS: One hundred and twenty-one variants were identified in 2 or more patients, 15 of which were enriched compared with reference populations. Single cell RNA-sequencing data verified coexpression of GGT1, CFTR, and PRSS1 in duct cells, PRSS1, CPA1, CEL, CTRC, and SPINK1 in acinar cells, and UBR1 in both. Multiple-risk variants with injury/stress effects (CEL, CFTR, CPA1, PRSS1) and impaired cell protection (CTRC, GGT1, SPINK1, UBR1) cooccur within duct cells, acinar cells, or both. CONCLUSIONS: Pancreatitis is a complex disorder with genetic interactions across genes and cell types. These findings suggest a new, non-Mendelian genetic risk/etiology paradigm where a combination of nonpathogenic genetic risk variants in groups of susceptibility genes and injury/dysfunction response genes contribute to acquired pancreatic disease.
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Predisposición Genética a la Enfermedad/genética , Páncreas/metabolismo , Enfermedades Pancreáticas/genética , Pancreatitis Crónica/genética , Polimorfismo de Nucleótido Simple , Células Acinares/citología , Células Acinares/metabolismo , Estudios de Cohortes , Redes Reguladoras de Genes , Humanos , Desequilibrio de Ligamiento , Páncreas/patología , Enfermedades Pancreáticas/diagnóstico , Conductos Pancreáticos/citología , Conductos Pancreáticos/metabolismo , Pancreatitis Crónica/diagnóstico , Fenotipo , RNA-Seq/métodos , Análisis de la Célula Individual/métodosRESUMEN
We have described multipotent progenitor-like cells within the major pancreatic ducts (MPDs) of the human pancreas. They express PDX1, its surrogate surface marker P2RY1, and the bone morphogenetic protein (BMP) receptor 1A (BMPR1A)/activin-like kinase 3 (ALK3), but not carbonic anhydrase II (CAII). Here we report the single-cell RNA sequencing (scRNA-seq) of ALK3bright+-sorted ductal cells, a fraction that harbors BMP-responsive progenitor-like cells. Our analysis unveiled the existence of multiple subpopulations along two major axes, one that encompasses a gradient of ductal cell differentiation stages, and another featuring cells with transitional phenotypes toward acinar tissue. A third potential ducto-endocrine axis is revealed upon integration of the ALK3bright+ dataset with a single-cell whole-pancreas transcriptome. When transplanted into immunodeficient mice, P2RY1+/ALK3bright+ populations (enriched in PDX1+/ALK3+/CAII- cells) differentiate into all pancreatic lineages, including functional ß-cells. This process is accelerated when hosts are treated systemically with an ALK3 agonist. We found PDX1+/ALK3+/CAII- progenitor-like cells in the MPDs of types 1 and 2 diabetes donors, regardless of the duration of the disease. Our findings open the door to the pharmacological activation of progenitor cells in situ.
Asunto(s)
Páncreas/citología , Conductos Pancreáticos/citología , Análisis de la Célula Individual/métodos , Células Madre/citología , Activinas/metabolismo , Animales , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/metabolismo , Diferenciación Celular , Diabetes Mellitus Tipo 1 , Diabetes Mellitus Tipo 2 , Femenino , Humanos , Células Secretoras de Insulina , Trasplante de Islotes Pancreáticos , Masculino , Ratones , Modelos Animales , Receptores Purinérgicos P2Y1/metabolismo , TranscriptomaRESUMEN
OBJECTIVES: Transcription factor Forkhead box protein M1 (FOXM1) plays critical roles in the progression of cancer including epithelial-to-mesenchymal transition (EMT). The aim of this study is to characterize the regulatory mechanisms of FOXM1 in EMT via pancreatic cancer metabolism. METHODS: We investigated the regulation of EMT via mitochondrial respiration by FOXM1 using pancreatic cancer cell lines HPAC and PANC-1 and normal human pancreatic duct epithelial cells. RESULTS: Forkhead box protein M1 and Snail were strongly expressed in HPAC and PANC-1. Epithelial-to-mesenchymal transition-modulated claudin-1 level was lower in PANC-1 than in HPAC. In both cell lines in low-glucose medium, FOXM1 and Snail were decreased and claudin-1 was increased. Knockdown of FOXM1 increased claudin-1 and decreased Snail in both cell lines. Low-glucose medium and downregulation of FOXM1 inhibited the cell migration in both cell lines. In both cell lines, mitochondrial respiration was at higher levels in low-glucose medium than in high-glucose medium. Downregulation of FOXM1 induced mitochondrial respiration in high-glucose medium. In normal human pancreatic duct epithelial cells, FOXM1 and Snail were low and claudin-1 was highly expressed, whereas overexpression of FOXM1 decreased claudin-1. CONCLUSIONS: Glucose-dependent FOXM1 promoted EMT via Snail and pancreatic cancer metabolism.
Asunto(s)
Metabolismo Energético/genética , Transición Epitelial-Mesenquimal/genética , Proteína Forkhead Box M1/genética , Neoplasias Pancreáticas/genética , Factores de Transcripción de la Familia Snail/genética , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Células Cultivadas , Claudina-1/genética , Claudina-1/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Proteína Forkhead Box M1/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glucosa , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/genética , Conductos Pancreáticos/citología , Conductos Pancreáticos/efectos de los fármacos , Conductos Pancreáticos/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Factores de Transcripción de la Familia Snail/metabolismoRESUMEN
Pancreatic ductal adenocarcinoma (PDA) is an aggressive malignancy typified by a highly stromal and weakly immunogenic tumor microenvironment that promotes tumor evolution and contributes to therapeutic resistance. Here, we demonstrate that PDA tumor cell-derived proinflammatory cytokine IL1ß is essential for the establishment of the protumorigenic PDA microenvironment. Tumor cell-derived IL1ß promoted the activation and secretory phenotype of quiescent pancreatic stellate cells and established an immunosuppressive milieu mediated by M2 macrophages, myeloid-derived suppressor cells, CD1dhiCD5+ regulatory B cells, and Th17 cells. Loss of tumor cell-derived IL1 signaling in tumor stroma enabled intratumoral infiltration and activation of CD8+ cytotoxic T cells, attenuated growth of pancreatic neoplasia, and conferred survival advantage to PDA-bearing mice. Accordingly, antibody-mediated neutralization of IL1ß significantly enhanced the antitumor activity of α-PD-1 and was accompanied by increased tumor infiltration of CD8+ T cells. Tumor cell expression of IL1ß in vivo was driven by microbial-dependent activation of toll-like receptor 4 (TLR4) signaling and subsequent engagement of the NLRP3 inflammasome. Collectively, these findings identify a hitherto unappreciated role for tumor cell-derived IL1ß in orchestrating an immune-modulatory program that supports pancreatic tumorigenesis. SIGNIFICANCE: These findings identify a new modality for immune evasion in PDA that depends on IL1ß production by tumor cells through TLR4-NLRP3 inflammasome activation. Targeting this axis might provide an effective PDA therapeutic strategy.
Asunto(s)
Carcinogénesis/inmunología , Carcinoma Ductal Pancreático/inmunología , Interleucina-1beta/metabolismo , Neoplasias Pancreáticas/inmunología , Escape del Tumor/inmunología , Animales , Antineoplásicos Inmunológicos/farmacología , Antineoplásicos Inmunológicos/uso terapéutico , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral/trasplante , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Células Epiteliales , Femenino , Humanos , Inflamasomas/inmunología , Inflamasomas/metabolismo , Interleucina-1beta/antagonistas & inhibidores , Interleucina-1beta/inmunología , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/inmunología , Ratones , Ratones Transgénicos , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Conductos Pancreáticos/citología , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/patología , Cultivo Primario de Células , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/inmunología , Transducción de Señal/inmunología , Receptor Toll-Like 4/metabolismo , Escape del Tumor/efectos de los fármacos , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunologíaRESUMEN
Restoring ß-cell mass by the transplantation of pancreatic islets is an effective diabetes treatment, but it is limited by the shortage of donor organs. CD133-expressing pancreatic ductal epithelial cells (PDECs) have the ability to generate insulin-producing cells. The expansion of these cells is dependent on extrinsic niche factors, but few of those signals have been identified. In this study, CD133-expressing PDECs were purified by sorting from adult wild-type C57BL/6 mice and TGFßRIInull/null mice. Furthermore, using immunofluorescence and transplantation assays, we found that the inhibition of the transforming growth factor-ß (TGF-ß) pathway promoted the expansion of CD133-expressing PDECs for many generations and maintained the ability of CD133-expressing PDECs to generate insulin-producing cells. Moreover, western blot, qRT-PCR, and dual luciferase assays using TGF-ß inhibitors were performed to identify the mechanisms by which TGF-ß signaling regulates proliferation and differentiation. The results showed that the inhibition of TGF-ß signaling enhanced Id2 binding to the promoter region of the cell proliferation repressor p16 and promoted the expansion of CD133-expressing PDECs, and the increased Id2 binding to NeuroD1 decreased the transcription of Pax6 to maintain CD133-expressing PDECs in the Pdx1-expression stage. Taken together, the effect of TGF-ß antagonists on CD133-expressing PDECs reveals a novel paradigm of signaling that explains the balance between the expansion and differentiation of pancreatic duct epithelial progenitors.
Asunto(s)
Antígeno AC133/metabolismo , Células Epiteliales/metabolismo , Células Secretoras de Insulina/citología , Conductos Pancreáticos/citología , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Antígeno AC133/genética , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Transdiferenciación Celular , Células Cultivadas , Células Epiteliales/citología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Proteína 2 Inhibidora de la Diferenciación/metabolismo , Células Secretoras de Insulina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Factor de Transcripción PAX6/genética , Factor de Transcripción PAX6/metabolismo , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
Cystic fibrosis (CF) is a genetic disorder caused by defective CF Transmembrane Conductance Regulator (CFTR) function. Insulin producing pancreatic islets are located in close proximity to the pancreatic duct and there is a possibility of impaired cell-cell signaling between pancreatic ductal epithelial cells (PDECs) and islet cells as causative in CF. To study this possibility, we present an in vitro co-culturing system, pancreas-on-a-chip. Furthermore, we present an efficient method to micro dissect patient-derived human pancreatic ducts from pancreatic remnant cell pellets, followed by the isolation of PDECs. Here we show that defective CFTR function in PDECs directly reduced insulin secretion in islet cells significantly. This uniquely developed pancreatic function monitoring tool will help to study CF-related disorders in vitro, as a system to monitor cell-cell functional interaction of PDECs and pancreatic islets, characterize appropriate therapeutic measures and further our understanding of pancreatic function.