4 research outputs found
Late-stage differentiation of embryonic pancreatic 尾-cells requires Jarid2.
Jarid2 is a component of the Polycomb Repressor complex 2 (PRC2), which is responsible for genome-wide H3K27me3 deposition, in embryonic stem cells. However, Jarid2 has also been shown to exert pleiotropic PRC2-independent actions during embryogenesis. Here, we have investigated the role of Jarid2 during pancreas development. Conditional ablation of Jarid2 in pancreatic progenitors results in reduced endocrine cell area at birth due to impaired endocrine cell differentiation and reduced prenatal proliferation. Inactivation of Jarid2 in endocrine progenitors demonstrates that Jarid2 functions after endocrine specification. Furthermore, genome-wide expression analysis reveals that Jarid2 is required for the complete activation of the insulin-producing 尾-cell differentiation program. Jarid2-deficient pancreases exhibit impaired deposition of RNAPII-Ser5P, the initiating form of RNAPII, but no changes in H3K27me3, at the promoters of affected endocrine genes. Thus, our study identifies Jarid2 as a fine-tuner of gene expression during late stages of pancreatic endocrine cell development. These findings are relevant for generation of transplantable stem cell-derived 尾-cells
Desenvolupament de noves estrat猫gies per generar c猫l路lules productores d'insulina
[cat] La diabetis tipus I 茅s una malaltia autoimmune que resulta de la destrucci贸 de les c猫l路lules 尾 pancre脿tiques i es caracteritza per alts nivells de glucosa en sang. El control de la gluc猫mia gr脿cies a l'administraci贸 d'insulina ex貌gena no 茅s sempre prec铆s i no evita les complicacions secund脿ries que ocasiona la malaltia. En la recerca en diabetis, s'est脿 apostant fort per la reprogramaci贸 cel路lular amb l鈥檕bjectiu de generar noves c猫l路lules productores d鈥檌nsulina a partir d'altres tipus cel路lulars. En els darrers anys, s鈥檋a estat treballant en la generaci贸 de c猫l路lules 尾 substitutes a partir de c猫l路lules mare o pluripotents indu茂des iPSC (protocols de diferenciaci贸 dirigida) i en la reprogramaci贸 directa de c猫l路lules som脿tiques d鈥檃ltres llinatges cap a c猫l路lula productora d鈥檌nsulina (protocols de transdiferenciaci贸). En aquesta tesi, hem volgut desenvolupar noves estrat猫gies per generar c猫l路lules productores d鈥檌nsulina mitjan莽ant aquestes dues aproximacions.
L鈥檈structura de la cromatina pot esdevenir un escull important per aconseguir la diferenciaci贸 terminal (i maduresa funcional) de les c猫l路lules productores d鈥檌nsulina generades tant en protocols de diferenciaci贸 dirigida com en protocols de reprogramaci贸 directa. La metilaci贸 de les histones 茅s una de les modificacions posttraduccionals de les histones m茅s estudiada. Concretament, la trimetilaci贸 de la lisina 27 de la histona 3 (H3K27me3) 茅s una marca repressora molt important durant el desenvolupament. Alteracions d鈥檃questa marca en gens de diferenciaci贸 claus podria estar relacionat amb la immaduresa de les c猫l路lules 尾 obtingudes en protocols. D鈥檃ltra banda, resultats previs del grup havien demostrat que l鈥檃ctivitat transdiferenciadora de Neurogenina3 (Ngn3), factors de transcripci贸 (FT) clau pel desenvolupament de les c猫l路lules endocrines durant la embriog猫nesis, s鈥檃ssociava a la p猫rdua de H3K27me3 en zones promotores de gens diana d鈥檃quest factor. Totes aquestes evid猫ncies ens van portar a postular, en la primera part de la tesi, que la modulaci贸 de l鈥檃ctivitat de les prote茂nes modificadores de la cromatina reguladores de la marca H3K27me3 podria servir per a millorar l鈥檃ctivaci贸 del programa endocrino-pancre脿tic en un protocol de diferenciaci贸 dirigida descrit.
Per altra banda, la transdiferenciaci贸 implica la conversi贸 directa d'un tipus cel路lular en un altre sense passar per un estadi pluripotent, reduint el perill de tumorog猫nesis que t茅 associat l鈥櫭簊 de c猫l路lules pluripotents. Durant aquest proc茅s, el programa de desenvolupament natural s鈥檃ctiva mitjan莽ant FTs espec铆fics de la c猫l路lula dest铆. Aquests FT necessiten establir una xarxa transcripcional nova similar al de la c猫l路lula que es vol obtenir. S'ha vist que la activaci贸 de la xarxa transcripcional endocrina regulada per la pres猫ncia de FT claus de llinatge endocr铆 pot promoure la conversi贸 de c猫l路lules som脿tiques a c猫l路lules 尾 pancre脿tiques. El Dr. Melton va ser el primer en veure com la introducci贸 de 3 FT claus en el desenvolupament de la c猫l路lula 尾 Pdx1, Ngn3 i MafA (PNM) en les c猫l路lules exocrines d贸na lloc a c猫l路lules productores d鈥檌nsulina. Posteriorment, altres investigadors han aconseguit obtenir c猫l路lules productores d鈥檌nsulina a partir d鈥檃ltres tipus cel路lulars, com c猫l路lules ductals, hep脿tiques, intestinals o estomacals mitjan莽ant la mateixa combinaci贸 de FTs. Totes aquestes c猫l路lules comparteixen un origen embrionari proper amb les c猫l路lules 尾 pancre脿tiques permetent que mantinguin una certa relaci贸 en el perfil d鈥檃ctivitat g猫nica. Per貌 la dificultat per l鈥檕btenci贸 d鈥檃questes c猫l路lules fa que sigui una aproximaci贸 inviable per 煤s terap猫utic. Fet que fa plantejar-nos en la necessitar de buscar una font cel路lular alternativa, de f脿cil obtenci贸 i capa莽 de generar noves c猫l路lules 尾 com el fibroblast. En la segona part de la tesi, hem investigat estrat猫gies de reprogramaci贸 directa basades en la introducci贸 d鈥檜n grup redu茂t de FT per convertir fibroblasts humans cap a c猫l路lules productores d鈥檌nsulina.[eng] Loss of insulin-producing 尾-cells causes diabetes. Cell replacement therapies have emerged as promising alternatives to daily insulin injections for treatment of diabetic patients. At the moment, Scientifics has been working on the generation of 尾-substituted cells from stem cells or pluripotent induced iPSC (differentiation protocols) or from adult specific cells (transdifferentiation protocols). In this thesis, I developed new strategies for generating insulin-producing cells through these two approaches.
The structure of chromatin can become an important point to achieve terminal differentiation (and functional maturity) of insulin-producing cells generated both in targeted differentiation protocols and in transdifferentiation protocols. Specifically, trimethylation of lysine 27 of histone 3 (H3K27me3) is a very important repressive mark during development. Alterations of this mark in key differentiation genes could be related to the immaturity of 尾 cells obtained in protocols. In the first part of this thesis, I described that the modulation of the activity of the regulating chromatin-modifying proteins of the H3K27me3 mark could serve to improve the activation of the endocrine-pancreatic program in a described differentiation protocols.
Direct reprogramming of somatic cells bypassing a pluripotent state has emerged as an alternative pathway to stem cells to generate clinically relevant cell types for cell replacement therapies. From a therapeutic standpoint, the choice of cell source for generation of replacement cells is pivotal. Skin fibroblasts are a good option as they are easily accessible and further permit autologous transplantation. To date, the few reported attempts to generate 尾-like cells from skin cells have been unsatisfactory. In the second part of this thesis I designed a reprogramming protocol based on the introduction of 5 transcription factors specific to the beta cell (尾-TFs) into skin fibroblasts that is able to activate key 尾 cell genes and down-regulate the fibroblast transcriptional program in human fibroblasts. Furthermore, I showed that the converted fibroblasts exhibit functional characteristics similar to 尾 cells both in vitro and in vivo. These findings provide a proof-of-concept and the molecular basis for a new path to generate 尾-like cells for cell replacement strategies to treat insulin-dependent diabetes
Late-stage differentiation of embryonic pancreatic 尾-cells requires Jarid2.
Jarid2 is a component of the Polycomb Repressor complex 2 (PRC2), which is responsible for genome-wide H3K27me3 deposition, in embryonic stem cells. However, Jarid2 has also been shown to exert pleiotropic PRC2-independent actions during embryogenesis. Here, we have investigated the role of Jarid2 during pancreas development. Conditional ablation of Jarid2 in pancreatic progenitors results in reduced endocrine cell area at birth due to impaired endocrine cell differentiation and reduced prenatal proliferation. Inactivation of Jarid2 in endocrine progenitors demonstrates that Jarid2 functions after endocrine specification. Furthermore, genome-wide expression analysis reveals that Jarid2 is required for the complete activation of the insulin-producing 尾-cell differentiation program. Jarid2-deficient pancreases exhibit impaired deposition of RNAPII-Ser5P, the initiating form of RNAPII, but no changes in H3K27me3, at the promoters of affected endocrine genes. Thus, our study identifies Jarid2 as a fine-tuner of gene expression during late stages of pancreatic endocrine cell development. These findings are relevant for generation of transplantable stem cell-derived 尾-cells