Transdiferencijacija alfa ćelija pankreasa miša u ćelije koje proizvode insulin ciljanom metilacijom DNK primenom Epi-CRISPR sistema

Abstract

Ekspresija gena za Arx (engl. Aristaless related homeobox) koji ima glavnu ulogu u održavanju identiteta alfa ćelija endokrinog pankreasa regulisana je metilacijom i predstavlja glavni target za ćelijsko reprogramiranje kao jedna od strategija u terapiji dijabetesa koji u osnovi ima poremećen izvor insulina usled propadanja beta ćelija. Cilj ove doktorske disertacije je podrazumevao ispitivanje sposobnosti transdiferencijacije alfa ćelija pankreasa miša u ćelije koje proizvode insulin nakon uvođenja metilacije u promotoru Arx-a. Nakon tranzijentne transfekcije αTC1-6 ćelijske linije pomoću epigenetičkog alata za ciljanu gensku represiju ispitivani su efekti uvedene metilacije na ekspresiju gena specifičnih za beta ćelije. Optimizacijom nukleofekcije αTC1-6 ćelija uspostavljeni su uslovi pomoću kojih je dostignuta efikasnost od 71,1% pri vijabilnosti ćelija od 80%. Visoka efikasnost uvođenja metilacije dCas9-Dnmt3a3L-KRAB fuzionim (EpiCRISPR) konstruktom je pokazana targetovanim bisulfitnim sekvenciranjem. Utišavanje Arx-a praćeno pokretanjem ekspresije Ins2 na 5. i 7. danu nakon transfekcije detektovano je metodom RT-qPCR-a i analizom transkriptoma. Proteinski nivo insulina detektovan je imunocitohemijskom metodom do 12. dana, a oslobađanje iz ćelija enzimskim imunoesejem na 7. danu nakon transfekcije. Pokretanje procesa transdiferencijacije αTC1-6 ćelija ispitivano je analizom prisustva markera beta ćelija. Rezultati su pokazali da jedna tranzijentna transfekcija može da inicira transdiferencijaciju ~1% alfa ćelija pankreasa u ćelije koje proizvode 35% više insulina u odnosu na lažno transfekovane (Mock) alfa ćelije. Delujući na plastičnu prirodu epigenoma, uspešno je iniciran proces direktnog reprogramiranja alfa ćelija pankreasa u ćelije koje proizvode insulin.

Aristaless-related homeobox (Arx) gene expression level is regulated by DNA methylation, plaing an important role in the maintenance of pancreatic alpha cell identity. Diabetes is characterized by a disturbed source of insulin, representing a good candidate for cell reprogramming strategy in diabetes therapy by Arx targeting. The aim of this doctoral dissertation was to examine the transdifferentiation ability of murine pancreatic alpha cells into insulin-producing cells induced by the targeted DNA methylation in the Arx promoter. The expression of beta specific marker was analiysed in transiently transfected αTC1-6 cells with a synthetic epigenetic tool for gene repression. The optimization of αTC1-6 cells nucleofection was established conditions by which was achieved an efficiency of 71.1% with an 80% of cell viability. The high efficiency of methylation induction by the dCas9-Dnmt3a3L-KRAB fusion (EpiCRISPR) construct was confirmed by targeted bisulfite sequencing. The Arx silencing followed by induction of Ins2 expression on 5 and 7 days after transfection was detected by RT-qPCR and transcriptome analysis. The insulin protein level was detected immunocytochemicaly until the 12th post-transfection day, and released insulin was detected by the enzyme immunoassay on the 7th post-transfection day. The initiation of the transdifferentiation process of αTC1-6 cells was examined by analyzing the presence of beta cell specific markers. The results showed that a single transient transfection initiate the transdifferentiation of ~1% of alpha cells into cells that produce 35% more insulin compared to mock-transfected cells. Acting on the epigenome plastic nature, the direct reprogramming of pancreatic alpha cells into insulin-producing cells was successfully initiated.