<p>  </p> <p>Traditionally, the exocrine and endocrine cellular compartments of the pancreas have been considered distinct functional systems. However, recent studies suggest a more intricate relationship between the exocrine and endocrine, which may impact pancreatic growth and health. Additionally, translational control mechanisms have been linked to organ development. Our lab has shown that the mRNA translation factor eukaryotic initiation factor 5A (eIF5A), when in its post-translationally modified “hypusinated” form, plays a role in pancreas development. The hypusination of eIF5A requires the rate-limiting enzyme deoxyhypusine synthase (<em>Dhps</em>) to post-translationally modify a critical lysine residue which in turn produces the active form of eIF5A that functions in mRNA translation. When we generated animals with a deletion of <em>Dhps</em> in the pancreatic progenitor cells, there was no alteration in islet mass but significant exocrine insufficiency at embryonic (E) day 18.5 concomitant with downregulation of proteins required for exocrine pancreas development and function. Resultantly these animals died by 6 weeks-of-age. These observations prompted the question, is the phenotype caused by the absence of hypusinated eIF5A or the increase of unhypusinated eIF5A? To address this, we generated a mouse model wherein <em>Eif5a</em> is deleted in the pancreas (eIF5A∆PANC) and these mutant animals also display exocrine insufficiency. Interestingly, beta cell mass is increased at E18.5, and the mutant animals maintain euglycemia and survive up to 2 years. Ongoing analyses are interrogating the differences between these animal models with the goal to determine if mRNA translation facilitates cellular communication between the exocrine and endocrine pancreas.</p>

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Pancreatic acinar cells display plasticity to acquire distinct differentiation programs, being involved in diseases as chronic pancreatitis and pancreatic ductal adenocarcinoma. This work shows that acinar cells cultured in suspension undergo dedifferentiation, acquiring a pancreatic embryonic progenitor phenotype. Dedifferentiated cells turn on a senescent program, associated with activation of p53 and Ras pathways. A similar progenitor‐like phenotype with activation of senescence is present in experimental chronic pancreatitis. Acinar cultures lacking p53 overcome growth arrest and lose the pancreatic phenotype, undergoing an epithelial‐mesenchymal transition, while maintaining the expression of pre‐pancreatic endoderm and stem cell markers. In experimental acute pancreatitis, absence of p53 results in increased acinar cell proliferation and delayed regeneration. Our findings support a role for acinar cell dedifferentiation in the initiation of pancreatic diseases. A p53‐ dependent control of cell growth and epithelial differentiation constitutes a tumor suppressive mechanism that may limit PDAC development. / Las células pancreáticas acinares poseen plasticidad que les permite adquirir distintos programas de diferenciación, estando implicadas en enfermedades como la pancreatitis crónica y el adenocarcinoma ductal pancreático. En este trabajo hemos demostrado que las células acinares cultivadas en suspensión se desdiferencian, adquiriendo un fenotipo de progenitores pancreáticos embrionarios. En estas células se induce un programa de senescencia asociado con la activación de las vías de p53 y Ras. Un fenotipo similar se evidencia en modelos de pancreatitis crónica experimental. Cultivos acinares en los que se ha inactivado p53 sobrepasan el bloqueo de crecimiento y pierden el fenotipo pancreático, presentando una transición epitelio‐mesenquimal y manteniendo la expresión de marcadores de endodermo pre‐pancreático y de células madre. Durante la inducción de una pancreatitis aguda experimental, la ausencia de p53 resulta en un incremento de la proliferación acinar y en un retraso en la regeneración. Nuestros resultados demuestran que la desdiferenciación de las células acinares participa en el desarrollo de enfermedades pancreáticas. El control del crecimiento celular y de la diferenciación pancreática epitelial dependiente de p53 constituye un mecanismo de supresión tumoral que puede limitar el desarrollo del PDAC.

A pesar de la abundancia de estudios realizados sobre el papel de las células acinares en las patologías exocrinas del páncreas (i.e. pancreatitis y cáncer), el estudio de las modificaciones producidas durante la diferenciación acinar en dichas patologías, se ha visto limitado por la escasez de modelos celulares no tumorales. Resultados previos de nuestro laboratorio, muestran que las células mES (células madre embrionarias de ratón )- pluripotentes y con la capacidad para generar tipos celulares especializados- pueden desarrollar un fenotipo acinar in vitro. Los objetivos de esta tesis han sido aumentar el contenido de enzimas digestivos así como las propiedades funcionales de las células generadas. Para ello se sobreexpresaron de forma estable p48, RBPJL y Mist1en células madre por transducción lentiviral de estos genes. Obtuvimos, gracias a una estrategia de infección en múltiples etapas, líneas celulares transgénicas mES que expresaban de forma constitutiva RBPJL y/o Mist1. La superimposición de la expresión de p48 por infección lentiviral en células en proceso de diferenciación dio lugar a una fuerte expresión de enzimas digestivos, con un patrón de regulación similar al que acontece in vivo durante el desarrollo pancreático. En esta inducción, tanto p48 como RPBJL son indispensables. Por otro lado, hemos mostrado un aumento elevado en la producción de varios componentes de la maquinaria secretota dependiente de Mist1. Además, hay que hacer notar ,que las células p48/RBPJL/Mist1 exhiben una regulada-secreción en respuesta a los secretagogos acinares y una mejor actividad de que la línea celular acinar 266-6. La expresión combinada de genes clave implicados en el desarrollo pancreático en células ES es un prometedor abordaje que nos llevará a una comprensión de los sutiles procesos del desarrollo exocrino pancreático. / Despite known involvement of acinar cells in pancreatic exocrine pathologies (i.e pancreatitis and pancreatic cancer), the lack of normal cell-based models has limited the study of the alterations that occur in the acinar differentiation program. Our previous data showed that mES (murine embryonic stem) cells, which are pluripotent and have the ability to generate specialized cell types, can acquire an acinar phenotype in vitro. The aim of this work was to increase the digestive enzyme content of the generated cells as well as their functional properties based on stable overexpression of p48, RBPJL and Mist1 by lentiviral gene transduction. Thus, we engineered transgenic mES cell lines constitutively expressing RBPJL and/or Mist1 using a multi-step infection strategy. The superimposition of p48 expression by lentiviral infection of differentiating cells resulted in a strong expression of digestive enzymes, with a pattern of regulation similar to what occurs in vivo during pancreatic development. In this induction, both p48 and RPBJL are indispensable. On the other hand, we showed a high increase in the production of several components of the secretory machinery which was dependent of Mist1. Importantly, p48/RBPJL/Mist1 cells exhibited a regulated-secretory in response to acinar secretagogues and a better secretion activity than the 266-6 acinar cell line. Combined expression of key genes involved in pancreatic development in ES cells may be a promising approach to better understand subtle steps of pancreatic exocrine development.

Tot i que es coneix l’involucrament de les cèl·lules pancreàtiques acinars en patologies exocrines (pancreatitis i càncer de pàncrees), la manca de models normals basats en cèl·lules ha limitat l’estudi de les alteracions que succeeixen en el programa de diferenciació pancreàtica. Hem demostrat prèviament que les cèl·lules mare embrionàries murines, que són pluripotents, poden adquirir un fenotip acinar in vitro. Això es va aconseguir, en part, amb una combinació de senyals que provenien del cultiu de pàncrees fetals que no era, però, específic del llinatge pancreàtic. L’objectiu d’aquest treball ha estat el de desenvolupar un protocol selectiu pel llinatge acinar basat en l’activació seqüencial de vies de senyalització que recapitulin el desenvolupament pancreàtic in vivo, a través de la formació definitiva de l’endoderm, l’especificació pancreàtica i acinar i l’expansió/diferenciació de progenitors acinars. El tractament de cossos embrionaris amb Activina A va promoure l’expressió de gens d’endoderm com està prèviament descrit. El tractament subsegüent amb àcid Retinoic, FGF10 i Ciclopamina, un inhibidor de la via de Hedgehog, va resutar en la inducció dels marcadors de progenitors pancreàtics Pdx1, Ptf1a i Cpa1 però també d’aquells expressats en el llinatge pancreàtic, que van ser reduïts amb la inhibició de BMPs. Les cèl·lules van ser a continuació cultivades en Matrigel utilitzant un sistema de cultiu en 3D en presència de fol·listatina, dexametasona i KGF comportant una inducció significativa dels nivells de mRNA i proteïna de marcadors acinars i una disminució de l’expressió dels de marcadors acinars. A més, es va veure que Amyl es secretava en el medi. Aquestes dades indiquen que l’activació selectiva del programa de diferenciació acinar en cèl·lules mare embrionàries es pot dur a terme mitjançant una inducció esgraonada de vies de senyalització involucrades en el desenvolupament pancreàtic exocrí proporcionant una eina potencial per estudiar la diferenciació pancreàtica i malalties relacionades amb el pàncrees. / Despite known involvement of pancreatic acinar cells in exocrine pathologies (pancreatitis and pancreatic cancer), the lack of normal cell-based models has limited the study of the alterations that occur in the acinar differentiation program. We have previously shown that mESC (murine embryonic stem cells), which are pluripotent, can acquire an acinar phenotype in vitro. This was achieved, in part, by a combination of signals provided by the culture of foetal pancreases which was, however, no specific for the acinar lineage. The aim of this work was to develop a protocol selective for the acinar lineage based on the sequential activation of signaling pathways that recapitulate pancreatic development in vivo, through the definitive endoderm formation, the pancreatic and acinar specification and the expansion/differentiation of acinar progenitors. Treatment of embryoid bodies with Activin A enhanced the expression of endodermal genes as previously described. Subsequent treatment with Retinoic acid, FGF10 and Cyclopamine, an inhibitor of the Hedgehog pathway, resulted in the enhancement of pancreatic progenitor markers Pdx1, Ptf1a and Cpa1 but also of those expressed in the hepatic lineage, which were reduced by BMPs inhibition. Cells were further cultured in Matrigel using a 3D culture system in the presence of follistatin, dexamethasone, and KGF leading to a significant enhancement of the mRNA and protein levels of acinar markers while decreasing the expression of endocrine ones. Moreover, active Amyl was released into the medium. These data indicate that the selective activation of the acinar differentiation program in ES cells can be achieved by stepwise induction of signaling pathways involved in pancreatic exocrine development providing a potential tool for studying pancreatic differentiation and pancreas-related diseases.

Background: Replacement of dysfunctional β-cells in the islets of Langerhans by transdifferentiation of pancreatic acinar cells has been proposed as a regenerative therapy for diabetes. Adult acinar cells spontaneously revert to a multipotent state upon tissue dissociation in vitro and can be stimulated to redifferentiate into β-cells. Despite accumulating evidence that contact-mediated signals are involved, the mechanisms regulating acinar-to-islet cell transdifferentiation remain poorly understood. Results: In this study, we propose that the crosstalk between two contact-mediated signaling mechanisms, lateral inhibition and lateral stabilization, controls cell fate stability and transdifferentiation of pancreatic cells. Analysis of a mathematical model combining gene regulation with contact-mediated signaling reveals the multistability of acinar and islet cell fates. Inhibition of one or both modes of signaling results in transdifferentiation from the acinar to the islet cell fate, either by dedifferentiation to a multipotent state or by direct lineage switching. Conclusions: This study provides a theoretical framework to understand the role of contact-mediated signaling in pancreatic cell fate control that may help to improve acinar-to-islet cell transdifferentiation strategies for β-cell neogenesis.

Background: Replacement of dysfunctional β-cells in the islets of Langerhans by transdifferentiation of pancreatic acinar cells has been proposed as a regenerative therapy for diabetes. Adult acinar cells spontaneously revert to a multipotent state upon tissue dissociation in vitro and can be stimulated to redifferentiate into β-cells. Despite accumulating evidence that contact-mediated signals are involved, the mechanisms regulating acinar-to-islet cell transdifferentiation remain poorly understood. Results: In this study, we propose that the crosstalk between two contact-mediated signaling mechanisms, lateral inhibition and lateral stabilization, controls cell fate stability and transdifferentiation of pancreatic cells. Analysis of a mathematical model combining gene regulation with contact-mediated signaling reveals the multistability of acinar and islet cell fates. Inhibition of one or both modes of signaling results in transdifferentiation from the acinar to the islet cell fate, either by dedifferentiation to a multipotent state or by direct lineage switching. Conclusions: This study provides a theoretical framework to understand the role of contact-mediated signaling in pancreatic cell fate control that may help to improve acinar-to-islet cell transdifferentiation strategies for β-cell neogenesis.