Reprogramming of different cell types into pancreatic beta cells by using transcription factor genes Pdx1, Ngn3 and MafA

Abstract

In this dissertation we studied the effect of the pancreatic transcription factor genes Pdx1, Ngn3 and MafA, all of which were cloned into single adenoviral construct. First, we investigated the reprogramming competency to Pdx1, Ngn3 and MafA of different cell types at different developmental stages from mouse and rat. Second, we looked at the effect of these three genes on a pancreatic rat exocrine cell line AR42J-B13 to see if this gene combination provides a true beta cell reprogramming event. Next, we investigated the effect of some small molecules to see if they increased the reprogramming efficiency of this gene combination. Finally we tried to sort the small fraction of insulin-positive cells formed after Pdx1, Ngn3, MafA transduction to enrich the insulin-positive cell population hence getting more accurate data from them.Transduction of different cell types with Ad-PNM showed that the Pdx1, Ngn3 and MafA gene combination is enough to stimulate the expression of Insulin genes with varying intensities depending on cell type. Rat cell lines responded to Ad-PNM in a better way than mouse cell lines by activating more beta cell genes. The number of responding cells to Ad-PNM was also higher for the rat cell lines than the mouse cell lines. Moreover progenitor-like cells as well as the cells developmentally related to beta cells are prone to be reprogrammed into beta-like state. Supporting this final statement, embryonic mouse heptoblasts from Pdx1-GFP transgenic mice gave the most promising result by activating the endogenous Pdx1 as well as many other important beta cell genes upon transduction with Ad-PNM.Further characterization of the effect of Ad-PNM on the most resposive cell type AR42J-B13 cells showed that after Ad-PNM the cells became post-mitotic, began to express Insulin genes together with many other beta cell genes, produced mature insulin hormone, changed the epigenetic state of their chromatin, and rescued diabetic mice if transplanted into the kidney capture. However, some important beta cell genes were not activated thereby making these cells unresponsive to glucose which is an indispensable beta cell quality. It is obvious that even though the changes are dramatic, the combination of Pdx1, Ngn3 and MafA did not provide a true beta cell reprogramming in vitro at least for this cell system. Among some of the small molecules which had been reported to favor beta cell formation, regeneration and/or survival, three of them including DAPT, BIX-01294 and NECA increased the effect of Ad-PNM on mouse hepatocyte-derived small cells. Moreover when all three were used together they showed a better efficiency. Even though there was a significant increase in the number of insulin positive cells, the fraction of the Ad-PNM-responding cells was still low after the small molecules. For that reason the effect of small molecules on reprogramming efficiency of Ad-PNM was compared by counting the fraction of insulin-positive cells out of Ad-PNM-bearing cells between control and experimental cell groups instead of performing qRT-PCR.Of the attempts that we performed to increase the low fraction of insulin-positive cells via different sorting techniques to get more accurate and reliable results from Ad-PNM responding cells, only fluorescence-activated cell sorting of insulin-immunostained cells worked. Even though these cells were enriched through the FACS, because the cells were nonviable not alive we could not perform any downstream application that necessitate living cells. This system allows us to do qRT-PCR only.