ESTUDI DE LA REPROGRAMACIÓ DE CÈL·LULES ACINARS A CÈL·LULES BETA MITJANÇANT FACTORS DE TRANSCRIPCIÓ

Abstract

Type 1 diabetes (T1D) is an autoimmune disease caused by the destruction of β_x000D_ cells, the insulin producing cells of the pancreas. T1D becomes clinically detectable_x000D_ when most β cells have been destroyed. Restoring the β cell mass in order to maintain a_x000D_ proper glucose control is one of the major challenges in regenerative medicine. The_x000D_ generation of new β cells from other cell types, such as acinar cells, by cellular_x000D_ reprogramming is a new therapeutic strategy with great potential. Furthermore, the use_x000D_ of acinar cells has several advantages over other differentiated cell types: they share a_x000D_ common embryonic origin with β cells, they are abundant and they are located in the_x000D_ pancreas. Several studies have shown that the expression of Pdx1, Ngn3 and MafA_x000D_ (PNM), three β cell specific transcription factors, using first-generation adenoviral_x000D_ vectors (FG-Ad) in acinar cells is able to reprogram said cells into insulin-producing_x000D_ cells. However, these studies indicate that FG-Ad vector transduction is necessary for_x000D_ the reprogramming process, and the functionality of these reprogrammed cells is not_x000D_ exactly the same as mature β cells._x000D_ To further investigate the molecular mechanisms involved in the reprogramming of_x000D_ acinar cells into β cells, three acinar cell lines (266-6, AR42J and AR42J-B13) were_x000D_ transduced by a FG-Ad vector expressing PNM. We observed that each cell line_x000D_ responded differently to PNM overexpression, and the greater reprogramming_x000D_ efficiency was observed in the AR42J-B13 (B13) cell line, evidenced by higher_x000D_ activation of β cell markers._x000D_ In a detailed characterization of the phenotype, it was observed that reprogrammed_x000D_ B13 cells presented many of the characteristics of adult β cells, such as the expression_x000D_ of pro-insulin processing genes and glucose sensors, and the production of mature_x000D_ insulin. However, insulin was not secreted efficiently to the culture media and was not_x000D_ regulated by the glucose levels._x000D_ The role of adenoviral vectors in the reprogramming process was also studied in_x000D_ this work. We found that adenoviral transduction per se negatively regulated the_x000D_ expression of exocrine genes, thus suggesting that these vectors promote a_x000D_ dedifferentiation effect in the acinar phenotype of B13 cells._x000D_ To identify microRNAs involved in the reprogramming process, a microRNA_x000D_ screening under different experimental conditions was performed. AR42J and B13 cells_x000D_ presented completely different microRNA expression patterns, and 60 microRNA were_x000D_ identified as differentially expressed comparing both cell lines. B13 cells showed signs_x000D_ of being in a more dedifferentiated state than AR42J cells, such as the suppression of_x000D_ the expression of the miR-200 family, which could facilitate their reprogramming. In_x000D_ addition, 11 microRNAs involved in the adenoviral transduction and 8 microRNAs_x000D_ associated with PNM overexpression have been identified._x000D_ In summary, in this work we have studied the mechanisms involved in the_x000D_ reprogramming of acinar cells into β cells, and microRNAs involved in this process_x000D_ have been identified. Such miRNAs hold great promise for improving the efficacy of_x000D_ reprogramming acinar cells into β cells for the treatment of diabetes

Date of Award	10 Jul 2015
Original language	Catalan
Supervisor	Eduard Ayuso López (Director)