Transgene expression level and inherent differences in target gene activation determine the rate and fate of neurogenin3-mediated islet cell differentiation in vitro

Abstract

A significant challenge in many areas of tissue engineering is a readily available source of cells. One approach to address this challenge is to direct the differentiation of expandable stem or progenitor cells or the transdifferentiation of an already differentiated cell type to the desired cell type. A variety of methods have been explored for directing cell differentiation, including the ectopic expression of transcriptional factors that are known to influence cell differentiation during development. One such transcription factor, neurogenin3 (Ngn3), plays a critical role in islet cell development in vivo. Ectopic expression of Ngn3 in various cell types has previously been shown to promote differentiation toward islet cell phenotypes, but the overall efficiency of this differentiation and the specific islet cell type produced vary widely between reports. The present work evaluates the hypotheses that cellular response is determined by (1) differentiation status of the starting cell, (2) basal expression of other transcriptional factors, and (3) level of ectopic Ngn3 expression. Retroviral vectors were used to express Ngn3 in primary adult pancreatic ductal epithelial cells (PDEC), embryonic and adult stem cells (ESC and ASC), and transformed mouse pancreatic adenocarcinoma (mPAC) cells in vitro. Changes in phenotypes were assessed using quantitative reverse transcription polymerase chain reaction (qRT-PCR), gene arrays, and immunohistochemistry. When Ngn3 was ectopically expressed in mouse and rat PDEC, downstream transcription factors (e.g., NeuroD, Nkx2.2, Isl-1) and endocrine hormones (most notably, ghrelin and somatostatin) were highly upregulated in a dose-dependent manner. In comparison to mPAC and mouse embryonic stem cells (mESC), PDEC displayed higher expression of most islet markers after normalization to Ngn3 levels. Differences in the basal expression and activation of transcription factors (e.g., Pax4, Pax6, and Nkx6.1) were observed between cell types, suggesting a mechanism by which precursors might preferentially generate different islet cell types.