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1.
PLoS Genet ; 16(10): e1009069, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33057429

RESUMO

The genetic mechanisms that determine the size of the adult pancreas are poorly understood. Imprinted genes, which are expressed in a parent-of-origin-specific manner, are known to have important roles in development, growth and metabolism. However, our knowledge regarding their roles in the control of pancreatic growth and function remains limited. Here we show that many imprinted genes are highly expressed in pancreatic mesenchyme-derived cells and explore the role of the paternally-expressed insulin-like growth factor 2 (Igf2) gene in mesenchymal and epithelial pancreatic lineages using a newly developed conditional Igf2 mouse model. Mesenchyme-specific Igf2 deletion results in acinar and beta-cell hypoplasia, postnatal whole-body growth restriction and maternal glucose intolerance during pregnancy, suggesting that the mesenchyme is a developmental reservoir of IGF2 used for paracrine signalling. The unique actions of mesenchymal IGF2 are demonstrated by the absence of any discernible growth or functional phenotypes upon Igf2 deletion in the developing pancreatic epithelium. Additionally, increased IGF2 levels specifically in the mesenchyme, through conditional Igf2 loss-of-imprinting or Igf2r deletion, leads to pancreatic acinar overgrowth. Furthermore, ex-vivo exposure of primary acinar cells to exogenous IGF2 activates AKT, a key signalling node, and increases their number and amylase production. Based on these findings, we propose that mesenchymal Igf2, and perhaps other imprinted genes, are key developmental regulators of adult pancreas size and function.


Assuntos
Fator de Crescimento Insulin-Like II/genética , Mesoderma/crescimento & desenvolvimento , Pâncreas/crescimento & desenvolvimento , Comunicação Parácrina/genética , Células Acinares/metabolismo , Células Acinares/patologia , Aminoácidos/genética , Animais , Linhagem da Célula/genética , Cromo , Metilação de DNA/genética , Feminino , Citometria de Fluxo , Regulação da Expressão Gênica no Desenvolvimento/genética , Impressão Genômica/genética , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Camundongos , Ácidos Nicotínicos/genética , Pâncreas/citologia , Pâncreas/metabolismo , Gravidez , RNA Longo não Codificante/genética
2.
J Vis Exp ; (163)2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32955501

RESUMO

Islet transplantation (ITx) has the potential to become the standard of care in beta cell replacement medicine but its results remain inferior to those obtained with whole pancreas transplantation. The protocols currently used for human islet isolation are under scrutiny because they are based on the enzymatic digestion of the organ, whereby the pancreas is demolished, its connections to the body are lost and islets are irreversibly damaged. Islet damage is characterized by critical factors such as the destruction of the extracellular matrix (ECM), which represents the 3D framework of the islet niche and whose loss is incompatible with islet euphysiology. Researchers are proposing the use of ECM-based scaffolds derived from the mammalian pancreas to address this problem and ultimately improve islet viability, function, and lifespan. Currently available methods to obtain such scaffolds are harsh because they are largely detergent based. Thus, we propose a new, detergent-free method that creates less ECM damage and can preserve critical components of pancreatic ECM. The results show that the newly developed decellularization protocol allowed the achievement of complete DNA clearance while the ECM components were retained. The ECM obtained was tested for cytotoxicity and encapsulated with human pancreatic islets which showed a positive cellular behavior with insulin secretion when stimulated with glucose challenge. Collectively, we propose a new method for the decellularization of the human pancreas without the use of conventional ionic and non-ionic chemical detergents. This protocol and the ECM obtained with it could be of use for both in vitro and in vivo applications.


Assuntos
Matriz Extracelular/química , Pâncreas/ultraestrutura , Engenharia Tecidual/métodos , Tecidos Suporte/química , Humanos , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Pâncreas/citologia , Pâncreas/metabolismo , Solubilidade
3.
Nat Commun ; 11(1): 4516, 2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32908137

RESUMO

Acinar metaplasia is an initial step in a series of events that can lead to pancreatic cancer. Here we perform single-cell RNA-sequencing of mouse pancreas during the progression from preinvasive stages to tumor formation. Using a reporter gene, we identify metaplastic cells that originated from acinar cells and express two transcription factors, Onecut2 and Foxq1. Further analyses of metaplastic acinar cell heterogeneity define six acinar metaplastic cell types and states, including stomach-specific cell types. Localization of metaplastic cell types and mixture of different metaplastic cell types in the same pre-malignant lesion is shown. Finally, single-cell transcriptome analyses of tumor-associated stromal, immune, endothelial and fibroblast cells identify signals that may support tumor development, as well as the recruitment and education of immune cells. Our findings are consistent with the early, premalignant formation of an immunosuppressive environment mediated by interactions between acinar metaplastic cells and other cells in the microenvironment.


Assuntos
Células Acinares/patologia , Carcinoma Ductal Pancreático/genética , Regulação Neoplásica da Expressão Gênica , Pâncreas/patologia , Neoplasias Pancreáticas/genética , Lesões Pré-Cancerosas/genética , Animais , Animais Geneticamente Modificados , Biópsia , Carcinoma Ductal Pancreático/mortalidade , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/cirurgia , Diferenciação Celular , Modelos Animais de Doenças , Feminino , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Heterogeneidade Genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Estimativa de Kaplan-Meier , Masculino , Metaplasia/genética , Camundongos , Mutação , Pâncreas/citologia , Pâncreas/cirurgia , Pancreatectomia , Neoplasias Pancreáticas/mortalidade , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/cirurgia , Lesões Pré-Cancerosas/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , RNA-Seq , Análise de Célula Única , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Microambiente Tumoral/genética
4.
Proc Natl Acad Sci U S A ; 117(38): 23663-23673, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32900967

RESUMO

Cell stress and DNA damage activate the tumor suppressor p53, triggering transcriptional activation of a myriad of target genes. The molecular, morphological, and physiological consequences of this activation remain poorly understood in vivo. We activated a p53 transcriptional program in mice by deletion of Mdm2, a gene that encodes the major p53 inhibitor. By overlaying tissue-specific RNA-sequencing data from pancreas, small intestine, ovary, kidney, and heart with existing p53 chromatin immunoprecipitation (ChIP) sequencing, we identified a large repertoire of tissue-specific p53 genes and a common p53 transcriptional signature of seven genes, which included Mdm2 but not p21 Global p53 activation caused a metaplastic phenotype in the pancreas that was missing in mice with acinar-specific p53 activation, suggesting non-cell-autonomous effects. The p53 cellular response at single-cell resolution in the intestine altered transcriptional cell state, leading to a proximal enterocyte population enriched for genes within oxidative phosphorylation pathways. In addition, a population of active CD8+ T cells was recruited. Combined, this study provides a comprehensive profile of the p53 transcriptional response in vivo, revealing both tissue-specific transcriptomes and a unique signature, which were integrated to induce both cell-autonomous and non-cell-autonomous responses and transcriptional plasticity.


Assuntos
Especificidade de Órgãos/genética , Análise de Célula Única , Transcriptoma/genética , Proteína Supressora de Tumor p53 , Animais , Imunoprecipitação da Cromatina , Feminino , Intestino Delgado/citologia , Intestino Delgado/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Pâncreas/citologia , Pâncreas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteína Supressora de Tumor p53/análise , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
5.
Biochem Pharmacol ; 180: 114174, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32717227

RESUMO

Primary toxicity targets of alcohol and its metabolites in the pancreas are cellular energetics and endoplasmic reticulum (ER). Therefore, the role of AMP-Activated Protein Kinase (AMPKα) in amelioration of ethanol (EtOH)-induced pancreatic acinar cell injury including ER/oxidative stress, inflammatory responses, the formation of fatty acid ethyl esters (FAEEs) and mitochondrial bioenergetics were determined in human pancreatic acinar cells (hPACs) and AR42J cells incubated with/without AMPKα activator [5-aminoimidazole-4-carboxamide ribonucleotide (AICAR)]. EtOH treated hPACs showed concentration and time-dependent increases for FAEEs and inactivation of AMPKα, along with the upregulation of ACC1 and FAS (key lipogenic proteins) and downregulation of CPT1A (involved ß-oxidation of fatty acids). These cells also showed significant ER stress as evidenced by the increased expression for GRP78, IRE1α, and PERK/CHOP arm of unfolded protein response promoting apoptosis and activating p-JNK1/2 and p-ERK1/2 with increased secretion of cytokines. AR42J cells treated with EtOH showed increased oxidative stress, impaired mitochondrial biogenesis, and decreased ATP production rate. However, AMPKα activation by AICAR attenuated EtOH-induced ER/oxidative stress, lipogenesis, and inflammatory responses as well as the formation of FAEEs and restored mitochondrial function in hPACs as well as AR42J cells. Therefore, it is likely that EtOH-induced inactivation of AMPKα plays a crucial role in acinar cell injury leading to pancreatitis. Findings from this study also suggest that EtOH-induced inactivation of AMPKα is closely related to ER/oxidative stress and synthesis of FAEEs, as activation of AMPKα by AICAR attenuates formation of FAEEs, ER/oxidative stress and lipogenesis, and improves inflammatory responses and mitochondrial bioenergetics.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Células Acinares/enzimologia , Retículo Endoplasmático/enzimologia , Etanol/farmacologia , Estresse Oxidativo/fisiologia , Pâncreas/enzimologia , Células Acinares/efeitos dos fármacos , Aciltransferases/metabolismo , Adulto , Células Cultivadas , Relação Dose-Resposta a Droga , Retículo Endoplasmático/efeitos dos fármacos , Ácidos Graxos não Esterificados/metabolismo , Feminino , Humanos , Lipídeos , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo/efeitos dos fármacos , Pâncreas/citologia , Pâncreas/efeitos dos fármacos , Fenótipo
6.
Nat Commun ; 11(1): 3265, 2020 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-32601271

RESUMO

The culture of live pancreatic tissue slices is a powerful tool for the interrogation of physiology and pathology in an in vitro setting that retains near-intact cytoarchitecture. However, current culture conditions for human pancreatic slices (HPSs) have only been tested for short-term applications, which are not permissive for the long-term, longitudinal study of pancreatic endocrine regeneration. Using a culture system designed to mimic the physiological oxygenation of the pancreas, we demonstrate high viability and preserved endocrine and exocrine function in HPS for at least 10 days after sectioning. This extended lifespan allowed us to dynamically lineage trace and quantify the formation of insulin-producing cells in HPS from both non-diabetic and type 2 diabetic donors. This technology is expected to be of great impact for the conduct of real-time regeneration/developmental studies in the human pancreas.


Assuntos
Ilhotas Pancreáticas/citologia , Pâncreas/citologia , Técnicas de Cultura de Tecidos/métodos , Animais , Humanos , Estudos Longitudinais , Camundongos , Modelos Biológicos , Regeneração , Células-Tronco/citologia
7.
Anat Sci Int ; 95(4): 523-539, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32476103

RESUMO

Type 1 diabetes mellitus (T1DM) is a chronic metabolic disease caused by the destruction of pancreatic ß-cells. Human dental pulp stem cells represent a promising source for cell-based therapies, owing to their easy, minimally invasive surgical access, and high proliferative capacity. It was reported that human dental pulp stem cells can differentiate into a pancreatic cell lineage in vitro; however, few studies have investigated their effects on diabetes. Our study aimed to investigate the therapeutic potential of intravenous and intrapancreatic transplantation of human dental pulp stem cells in a rat model of streptozotocin-induced type 1 diabetes. Forty Sprague Dawley male rats were randomly categorized into four groups: control, diabetic (STZ), intravenous treatment group (IV), and intrapancreatic treatment group (IP). Human dental pulp stem cells (1 × 106 cells) or vehicle were injected into the pancreas or tail vein 7 days after streptozotocin injection. Fasting blood glucose levels were monitored weekly. Glucose tolerance test, rat and human serum insulin and C-peptide, pancreas histology, and caspase-3, vascular endothelial growth factor, and Ki67 expression in pancreatic tissues were assessed 28 days post-transplantation. We found that both IV and IP transplantation of human dental pulp stem cells reduced blood glucose and increased levels of rat and human serum insulin and C-peptide. The cells engrafted and survived in the streptozotocin-injured pancreas. Islet-like clusters and scattered human dental pulp stem cells expressing insulin were observed in the pancreas of diabetic rats with some difference in the distribution pattern between the two injection routes. RT-PCR analyses revealed the expression of the human-specific pancreatic ß-cell genes neurogenin 3 (NGN3), paired box 4 (PAX4), glucose transporter 2 (GLUT2), and insulin in the pancreatic tissues of both the IP and IV groups. In addition, the transplanted cells downregulated the expression of caspase-3 and upregulated the expression of vascular endothelial growth factor and Ki67, suggesting that the injected cells exerted pro-angiogenetic and antiapoptotic effects, and promoted endogenous ß-cell replication. Our study is the first to show that human dental pulp stem cells can migrate and survive within streptozotocin-injured pancreas, and induce antidiabetic effects through the differentiation and replacement of lost ß-cells and paracrine-mediated pancreatic regeneration. Thus, human dental pulp stem cells may have therapeutic potential to treat patients with long term T1DM.


Assuntos
Polpa Dentária/citologia , Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Tipo 1/terapia , Pâncreas/fisiologia , Transplante de Células-Tronco , Células-Tronco/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Caspase 3/metabolismo , Diferenciação Celular , Movimento Celular , Sobrevivência Celular , Modelos Animais de Doenças , Transportador de Glucose Tipo 2/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Antígeno Ki-67/metabolismo , Masculino , Proteínas do Tecido Nervoso/metabolismo , Fatores de Transcrição Box Pareados/metabolismo , Pâncreas/citologia , Pâncreas/metabolismo , Ratos , Ratos Sprague-Dawley , Regeneração , Estreptozocina
8.
Nat Commun ; 11(1): 2241, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32382023

RESUMO

The generation of pancreatic cell types from renewable cell sources holds promise for cell replacement therapies for diabetes. Although most effort has focused on generating pancreatic beta cells, considerable evidence indicates that glucagon secreting alpha cells are critically involved in disease progression and proper glucose control. Here we report on the generation of stem cell-derived human pancreatic alpha (SC-alpha) cells from pluripotent stem cells via a transient pre-alpha cell intermediate. These pre-alpha cells exhibit a transcriptional profile similar to mature alpha cells and although they produce proinsulin protein, they do not secrete significant amounts of processed insulin. Compound screening identified a protein kinase c activator that promotes maturation of pre-alpha cells into SC-alpha cells. The resulting SC-alpha cells do not express insulin, share an ultrastructure similar to cadaveric alpha cells, express and secrete glucagon in response to glucose and some glucagon secretagogues, and elevate blood glucose upon transplantation in mice.


Assuntos
Técnicas de Cultura de Células/métodos , Células Secretoras de Glucagon/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Células-Tronco Pluripotentes/citologia , Western Blotting , Diferenciação Celular/fisiologia , Linhagem Celular , Eletrofisiologia , Imunofluorescência , Humanos , Pâncreas/citologia
9.
Proc Natl Acad Sci U S A ; 117(20): 10876-10887, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32354994

RESUMO

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.


Assuntos
Pâncreas/citologia , Ductos Pancreáticos/citologia , Análise de Célula Única/métodos , Células-Tronco/citologia , Ativinas/metabolismo , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Diferenciação Celular , Diabetes Mellitus Tipo 1 , Diabetes Mellitus Tipo 2 , Feminino , Humanos , Células Secretoras de Insulina , Transplante das Ilhotas Pancreáticas , Masculino , Camundongos , Modelos Animais , Receptores Purinérgicos P2Y1/metabolismo , Transcriptoma
10.
Development ; 147(7)2020 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-32280064

RESUMO

Understanding the mechanisms that underlie the generation and regeneration of ß cells is crucial for developing treatments for diabetes. However, traditional research methods, which are based on populations of cells, have limitations for defining the precise processes of ß-cell differentiation and trans-differentiation, and the associated regulatory mechanisms. The recent development of single-cell technologies has enabled re-examination of these processes at a single-cell resolution to uncover intermediate cell states, cellular heterogeneity and molecular trajectories of cell fate specification. Here, we review recent advances in understanding ß-cell generation and regeneration, in vivo and in vitro, from single-cell technologies, which could provide insights for optimization of diabetes therapy strategies.


Assuntos
Diferenciação Celular , Linhagem da Célula , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/fisiologia , Regeneração/fisiologia , Análise de Célula Única/métodos , Animais , Rastreamento de Células/métodos , Rastreamento de Células/tendências , Humanos , Pâncreas/citologia , Pâncreas/fisiologia , Análise de Célula Única/tendências
11.
Adv Exp Med Biol ; 1236: 65-85, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32304069

RESUMO

The pancreas is a glandular organ responsible for diverse homeostatic functions, including hormone production from the endocrine islet cells to regulate blood sugar levels and enzyme secretion from the exocrine acinar cells to facilitate food digestion. These pancreatic functions are essential for life; therefore, preserving pancreatic function is of utmost importance. Pancreas dysfunction can arise either from developmental disorders or adult onset disease, both of which are caused by defects in shared molecular pathways. In this chapter, we discuss what is known about the molecular mechanisms controlling pancreas development, how disruption of these mechanisms can lead to developmental defects and disease, and how essential pancreas functions can be modeled using human pluripotent stem cells. At the core of understanding of these molecular processes are animal model studies that continue to be essential for elucidating the mechanisms underlying human pancreatic functions and diseases.


Assuntos
Modelos Animais , Organogênese , Pâncreas/embriologia , Pâncreas/patologia , Células Acinares/metabolismo , Células Acinares/patologia , Animais , Humanos , Pâncreas/citologia , Pâncreas Exócrino/citologia , Pâncreas Exócrino/embriologia , Pâncreas Exócrino/patologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/patologia
12.
Am J Physiol Gastrointest Liver Physiol ; 318(6): G1000-G1012, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32308041

RESUMO

Trypsinogen activation is the hallmark of acute pancreatitis (AP) independent of intra-acinar NF-κB activation and inflammation. We previously found that dopamine (DA) receptor 2 (DRD2) activation controls inflammation during AP via PP2A-dependent NF-κB activation. In this study, we sought to examine whether DRD2 signaling mediates trypsinogen activation and the underlying mechanisms. Pancreatic acinar cells were stimulated with cholecystokinin-8 in vitro. AP was induced by intraperitoneal injections of caerulein and LPS or l-arginine. Pancreatitis severity was assessed biochemically and histologically. We found that activation of DRD2 by quinpirole, a potent DRD2 agonist, resulted in the reduction of trypsinogen activation and the upregulation of HSP70 in vitro and in vivo. Mechanistically, we found that quinpirole induced dephosphorylation of heat shock factor 1 (HSF1), a master transcription factor of HSP70, leading to increased nuclear translocation of HSF1 in a PP2A-dependent pathway. Furthermore, DRD2 activation restored lysosomal pH and, therefore, maintained lysosomal cathepsin B activity in a HSP70-dependent manner. VER155008, a potent HSP70 antagonist, abolished the protective effects observed with DRD2 activation in vitro and in two experimental models of AP. Our data showed that besides controlling NF-κB activation, DRD2 activation prevented trypsinogen activation during acute pancreatitis via PP2A-dependent upregulation of HSP70 and further support that DRD2 agonist could be a promising therapeutic strategy for treating AP.NEW & NOTEWORTHY The current study demonstrated that activation of DRD2 by quinpirole protects against trypsinogen activation in the in vitro and in vivo setting of acute pancreatitis by upregulating HSP70 and restoring lysosomal degradation via a PP2A-dependent manner, therefore leading to reduced pancreatic injury. These findings provide a new mechanistic insight on the protective effect of DRD2 activation in acute pancreatitis.


Assuntos
Proteínas de Choque Térmico HSP72/metabolismo , Pancreatite/tratamento farmacológico , Quimpirol/farmacologia , Receptores de Dopamina D2/agonistas , Tripsinogênio/metabolismo , Animais , Ceruletídeo/farmacologia , Agonistas de Dopamina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico HSP72/genética , Fatores de Transcrição de Choque Térmico/genética , Fatores de Transcrição de Choque Térmico/metabolismo , Lisossomos , Camundongos , Camundongos Endogâmicos C57BL , Pâncreas/citologia , Pancreatite/metabolismo , Fosforilação , Proteína Fosfatase 2/metabolismo , Regulação para Cima
13.
Nat Biotechnol ; 38(9): 1061-1072, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32341565

RESUMO

Methods for differentiating human pluripotent stem cells to pancreatic and liver lineages in vitro have been limited by the inability to identify and isolate distinct endodermal subpopulations specific to these two organs. Here we report that pancreatic and hepatic progenitors can be isolated using the surface markers CD177/NB1 glycoprotein and inducible T-cell costimulatory ligand CD275/ICOSL, respectively, from seemingly homogeneous definitive endoderm derived from human pluripotent stem cells. Anterior definitive endoderm (ADE) subpopulations identified by CD177 and CD275 show inverse activation of canonical and noncanonical WNT signaling. CD177+ ADE expresses and synthesizes the secreted WNT, NODAL and BMP antagonist CERBERUS1 and is specified toward the pancreatic fate. CD275+ ADE receives canonical Wnt signaling and is specified toward the liver fate. Isolated CD177+ ADE differentiates more homogeneously into pancreatic progenitors and into more functionally mature and glucose-responsive ß-like cells in vitro compared with cells from unsorted differentiation cultures.


Assuntos
Endoderma/citologia , Endoderma/metabolismo , Células Secretoras de Insulina/citologia , Isoantígenos/metabolismo , Receptores de Superfície Celular/metabolismo , Adolescente , Adulto , Biomarcadores/metabolismo , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Citocinas/metabolismo , Feminino , Proteínas Ligadas por GPI/metabolismo , Humanos , Ligante Coestimulador de Linfócitos T Induzíveis/metabolismo , Células Secretoras de Insulina/metabolismo , Fígado/citologia , Fígado/metabolismo , Masculino , Pessoa de Meia-Idade , Pâncreas/citologia , Pâncreas/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Receptores CXCR4/metabolismo , Via de Sinalização Wnt/fisiologia , Adulto Jovem
14.
Biochem Biophys Res Commun ; 526(3): 592-598, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32247607

RESUMO

Extracellular acidification, playing a promoting role in the process of acute pancreatitis, has been reported to activate Cl- channels in several types of cells. However, whether extracellular acidification aggravates acute pancreatitis via activating Cl- channels remains unclear. Here, we investigated the effects of extracellular acidification on Cl- channels in rat pancreatic acinar AR42J cells using whole-cell patch-clamp recordings. We found that extracellular acidification induced a moderately outward-rectified Cl- current, with a selectivity sequence of I- > Br- ≥ Cl- > gluconate-, while intracellular acidification failed to induce the currents. The acid-sensitive currents were inhibited by Cl- channel blockers, 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt hydrate and 5-Nitro-2-(3-phenylpropylamino) benzoic acid. After ClC-3 was silenced by ClC-3 shRNA, the acid-sensitive Cl- currents were attenuated significantly, indicating that ClC-3 plays a vital role in the induction of acid-sensitive Cl- currents. Extracellular acid elevated the intracellular level of reactive oxygen species (ROS) significantly, prior to inducing Cl- currents. When ROS production was scavenged, the acid-sensitive Cl- currents were abolished. Whereas, the level of acid-induced ROS was unaffected with silence of ClC-3. Our findings above demonstrate that extracellular acidification induces a Cl- current in pancreatic acinar cells via promoting ROS generation, implying an underlying mechanism that extracellular acidification might aggravate acute pancreatitis through Cl- channels.


Assuntos
Células Acinares/metabolismo , Canais de Cloreto/metabolismo , Pâncreas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Células Acinares/citologia , Animais , Linhagem Celular , Cloretos/metabolismo , Espaço Extracelular/metabolismo , Concentração de Íons de Hidrogênio , Pâncreas/citologia , Técnicas de Patch-Clamp , Ratos
15.
Cell ; 180(6): 1198-1211.e19, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-32200801

RESUMO

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 ∼1% 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.


Assuntos
Técnicas de Cultura de Células/métodos , Receptor de Proteína C Endotelial/metabolismo , Ilhotas Pancreáticas/citologia , Animais , Diferenciação Celular/fisiologia , Linhagem Celular , Células Cultivadas , Diabetes Mellitus Experimental/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Feminino , Glucose/metabolismo , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/citologia , Ilhotas Pancreáticas/crescimento & desenvolvimento , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Nus , Organoides/crescimento & desenvolvimento , Organoides/metabolismo , Pâncreas/citologia , Pâncreas/metabolismo , Proteína C/metabolismo , Células-Tronco/citologia
16.
Int J Mol Sci ; 21(6)2020 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-32192184

RESUMO

The existence of telocytes (TCs) has not yet been established in the pancreases of aquatic reptiles. Here, we report TCs in the exocrine pancreas of Pelodiscus sinensis using transmission electron microscope (TEM), immunohistochemistry (IHC), and immunofluorescence (IF) techniques. TCs surrounded the acini and ducts of the connective tissue of the exocrine pancreas and between lobules and gland cells. The cells were located preferably close to the blood vessels, interlobular ducts, and nerve fibers. Ultrastructurally, TCs exhibited small and large bodies with thick and thin portions, podoms, and podomers, and prolongations that form dichotomous branching with hetero-cellular and homo-cellular junctions. The podom (thick) portions showed caveolae, mitochondria, rough endoplasmic reticulum, and vesicles. The nucleus carries heterochromatin and is irregular in shape. The shape of TCs depends on the number of telopodes (Tps) bearing long, short, spindle, triangular, and "beads on a string" shapes with twisted, tortuous prolongations and ramifications. Shed extracellular vesicles and exosomes were found frequently released from projections and Tps within connective tissue in the vicinity of the acini and collagen fibers. IHC and IF results showed CD34+, α-SMA+, and vimentin+, long and triangle-shaped TCs, consistent with the TEM findings. The presence of shaded vesicles from TCs might implicate their possible role in immune surveillance, tissue regeneration as well as regulatory functions in the reptilian pancreas.


Assuntos
Comunicação Celular , Pâncreas/citologia , Pâncreas/ultraestrutura , Telócitos/fisiologia , Telócitos/ultraestrutura , Tartarugas , Animais , Biomarcadores , Exossomos/metabolismo , Imunofluorescência , Imuno-Histoquímica , Pâncreas/fisiologia
17.
Metabolism ; 105: 154175, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32045582

RESUMO

PURPOSE: Abnormal glucagon concentrations are a feature of prediabetes but it is uncertain if α-cell dysfunction contributes to a longitudinal decline in ß-cell function. We therefore sought to determine if a decline in ß-cell function is associated with a higher nadir glucagon in the postprandial period or with higher fasting glucagon. METHODS: This was a longitudinal study in which 73 non-diabetic subjects were studied on 2 occasions 6.6 ±â€¯0.3 years apart using a 2-hour, 7-sample oral glucose tolerance test. Disposition Index (DI) was calculated using the oral minimal model applied to the measurements of glucose, insulin, C-peptide concentrations during the studies. We subsequently examined the relationship of glucagon concentrations at baseline with change in DI (used as a measure of ß-cell function) after adjusting for changes in weight and the baseline value of DI. RESULTS: After adjusting for covariates, nadir postprandial glucagon concentrations were not associated with changes in ß-cell function as quantified by DI. On the other hand, fasting glucagon concentrations during the baseline study were inversely correlated with longitudinal changes in DI. CONCLUSIONS: Defects in α-cell function, manifest as elevated fasting glucagon, are associated with a subsequent decline in ß-cell function. It remains to be ascertained if abnormal α-cell function contributes directly to loss of ß-cell secretory capacity in the pathogenesis of type 2 diabetes.


Assuntos
Glucagon/sangue , Células Secretoras de Insulina/fisiologia , Adulto , Glicemia/análise , Peptídeo C/sangue , Jejum , Feminino , Teste de Tolerância a Glucose , Humanos , Insulina/sangue , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Pâncreas/citologia , Pâncreas/crescimento & desenvolvimento , Período Pós-Prandial , Estado Pré-Diabético/metabolismo
18.
Cancer Immunol Immunother ; 69(5): 789-797, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32055919

RESUMO

CD160 is an Ig-like glycoprotein expressed by the majority of circulating natural killer cells and γδ T cells. Whether CD160 could regulate CD8+ T-cell functions remains unknown. In this study, we investigated the effects of CD160 on CD8+ T cells in pancreatic cancer. First, we found that the frequency of PD-1+ cells was comparable between CD160+ and CD160-CD8+ T cells, with the former presenting significantly higher PD-1 expression level. In contrast, the frequency of TIM-3+ cells was higher among CD160+ cells but the expression level was comparable between CD160+ and CD160-CD8+ T cells. The IFN-γ and IL-2-expressing CD8+ T cells, directly ex vivo, were highly enriched in the CD160+ subset. However, when CD160+ and CD160-CD8+ T cells were stimulated, the proliferation levels of CD160+ and CD160- cells were initially comparable, but were significantly lower in CD160+CD8+ T cells than in CD160-CD8+ T cells later on. The IFN-γ and IL-2 transcription levels were initially higher in CD160+CD8+ T cells, but eventually reduced in CD160+CD8+ T cells compared to CD160-CD8+ T cells. Also, CD160+CD8+ T cells presented lower cytotoxic capacity than CD160-CD8+ T cells. Interestingly, we observed that tumor-infiltrating CD8+ T cells were significantly enriched with the CD160+ subset in pancreatic cancer patients. In addition, patients with higher frequencies of tumor CD160+CD8+ T cells presented lower survival. Overall, these data demonstrated that tumor-infiltrating CD8+ T cells were enriched with the CD160+ subset in pancreatic cancer, with active effector responses directly ex vivo but limited potential for further activation.


Assuntos
Antígenos CD/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos do Interstício Tumoral/imunologia , Neoplasias Pancreáticas/imunologia , Receptores Imunológicos/metabolismo , Linfócitos T Citotóxicos/imunologia , Adulto , Idoso , Antígenos CD/imunologia , Biópsia , Linfócitos T CD8-Positivos/metabolismo , Feminino , Proteínas Ligadas por GPI/imunologia , Proteínas Ligadas por GPI/metabolismo , Humanos , Ativação Linfocitária , Linfócitos do Interstício Tumoral/metabolismo , Masculino , Pessoa de Meia-Idade , Pâncreas/citologia , Pâncreas/imunologia , Pâncreas/patologia , Pâncreas/cirurgia , Pancreatectomia , Neoplasias Pancreáticas/sangue , Neoplasias Pancreáticas/mortalidade , Neoplasias Pancreáticas/cirurgia , Receptores Imunológicos/imunologia , Análise de Sobrevida , Linfócitos T Citotóxicos/metabolismo , Fatores de Tempo
19.
Dev Cell ; 52(6): 731-747.e8, 2020 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-32059775

RESUMO

Notch signaling controls proliferation of multipotent pancreatic progenitor cells (MPCs) and their segregation into bipotent progenitors (BPs) and unipotent pro-acinar cells (PACs). Here, we showed that fast ultradian oscillations of the ligand Dll1 and the transcriptional effector Hes1 were crucial for MPC expansion, and changes in Hes1 oscillation parameters were associated with selective adoption of BP or PAC fate. Conversely, Jag1, a uniformly expressed ligand, restrained MPC growth. However, when its expression later segregated to PACs, Jag1 became critical for the specification of all but the most proximal BPs, and BPs were entirely lost in Jag1; Dll1 double mutants. Anatomically, ductal morphogenesis and organ architecture are minimally perturbed in Jag1 mutants until later stages, when ductal remodeling fails, and signs of acinar-to-ductal metaplasia appear. Our study thus uncovers that oscillating Notch activity in the developing pancreas, modulated by Jag1, is required to coordinate MPC growth and fate.


Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/metabolismo , Proteína Jagged-1/metabolismo , Pâncreas/citologia , Transdução de Sinais , Animais , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem da Célula , Células-Tronco Embrionárias/citologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteína Jagged-1/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Pâncreas/embriologia , Pâncreas/metabolismo , Periodicidade , Receptores Notch/genética , Receptores Notch/metabolismo , Fatores de Transcrição HES-1/genética , Fatores de Transcrição HES-1/metabolismo
20.
Nat Mater ; 19(7): 797-806, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32066931

RESUMO

Defining the interplay between the genetic events and microenvironmental contexts necessary to initiate tumorigenesis in normal cells is a central endeavour in cancer biology. We found that receptor tyrosine kinase (RTK)-Ras oncogenes reprogram normal, freshly explanted primary mouse and human cells into tumour precursors, in a process requiring increased force transmission between oncogene-expressing cells and their surrounding extracellular matrix. Microenvironments approximating the normal softness of healthy tissues, or blunting cellular mechanotransduction, prevent oncogene-mediated cell reprogramming and tumour emergence. However, RTK-Ras oncogenes empower a disproportional cellular response to the mechanical properties of the cell's environment, such that when cells experience even subtle supra-physiological extracellular-matrix rigidity they are converted into tumour-initiating cells. These regulations rely on YAP/TAZ mechanotransduction, and YAP/TAZ target genes account for a large fraction of the transcriptional responses downstream of oncogenic signalling. This work lays the groundwork for exploiting oncogenic mechanosignalling as a vulnerability at the onset of tumorigenesis, including tumour prevention strategies.


Assuntos
Reprogramação Celular/fisiologia , Matriz Extracelular/fisiologia , Oncogenes/fisiologia , Animais , Fenômenos Biomecânicos , Linhagem Celular Tumoral , Feminino , Regulação da Expressão Gênica , Humanos , Glândulas Mamárias Humanas/citologia , Glândulas Mamárias Humanas/metabolismo , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Microscopia/métodos , Oncogenes/genética , Pâncreas/citologia , Análise de Sequência de RNA
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