Efficient differentiation of AR42J cells towards insulin-producing cells using pancreatic transcription factors in combination with growth factors

Abstract

The AR42J-B13 rat pancreatic acinar cell line was used to identify pancreatic transcription factors and exogenous growth factors (GFs) that might facilitate the reprogramming of exocrine cells into islets. Adenoviruses were used to induce exogenous expression of the pancreatic transcription factors (TFs) Pdx1, MafA, Ngn3 and Pax4. Individually Pdx1, MafA and Pax4 had no effect on the expression of endocrine markers, whilst adeno-Ngn3 on its own increased the expression of Pax4, Ngn3 and NeuroD. In combination the four TFs had a significant effect on the expression of insulin 1 and 2 that was associated with a change in cell morphology from a rounded to a spindle-like shape. Amongst a range of growth factors, Betacellulin and Nicotinamide were shown to enhance the effects of the four TFs. The presence of adeno-Pax4 in the differentiation cocktail was important in limiting the expression of glucagon and in generating glucose sensitive insulin secretion. Further experiments asked whether the adenoviral TFs could be replaced by protein transduction domain (PTD)-containing TFs. The results showed that the PTD-TFs could mimic in part the effects of the adeno-TFs, but the resultant cells did not undergo the important morphological change associated with differentiation to endocrine lineages and levels of endogenous markers were very much lower. In summary, the results describe a cocktail of four TFs and two GFs that can be used to induce formation of glucose sensitive insulin secreting cells from ARJ42 cells, and demonstrate that it would be difficult to replace adenoviral transduction with PTD-TFS.

Introduction

The major therapeutic challenges in type 1 diabetes are, in the short term avoiding unexpected episodes of hypoglycaemia and in the long term avoiding the late complications of the disease that affect eye, kidney, and peripheral nerve function. Recent successes in islet transplantation (Shapiro et al., 2000), albeit limited (Ryan et al., 2005), have raised hope these challenges might be met. However, the dependence on the rare availability of cadaveric tissue has stimulated a drive towards generating an alternative source of islets. An understanding of the transcription factors that control the developing pancreas (Gittes, 2009) has provided protocols that reproducibly generate pancreatic progenitors from embryonic stem cells which can develop into functional β-cells when engrafted in mice (Kroon et al., 2008). At the same time the discovery that cells of one tissue type could be reprogrammed into those of other tissues (Takahashi and Yamanaka, 2006) has led to new therapeutic options. Thus acinar (Zhou et al., 2008) and liver (Yechoor et al., 2009, Yatoh et al., 2007) cells, which are developmentally closely related to pancreatic endocrine cells, have been reprogrammed to β-cells through a specific combination of pancreatic transcription factors that include Pdx1, Ngn3, NeuroD and MafA. Pdx1 is present in early progenitors that give rise to all cells of the pancreas, Ngn3 directs cells towards an endocrine fate, while later expression of NeuroD, MafA, and Pdx1 in a second wave of expression, act as β-cell differentiation factors (Bernardo et al., 2008). Because of the risk of inflammation and resultant pancreatitis in vivo reprogramming of acinar tissue using viral vectors is unrealistic for treatment of type 1 diabetes, however, the in vitro reprogramming of exocrine tissue may be of therapeutic value.

AR42J is a rat cell line that was originally derived from a chemically induced pancreatic tumour (Longnecker et al., 1979). AR42J cells can be induced to differentiate towards hepatocyte (Wallace et al., 2010) and endocrine pancreatic lineages (Mashima et al., 1996a). Here we used these cells in an attempt to optimise conditions for the efficient reprogramming of pancreatic exocrine cells towards functional pancreatic endocrine cells. Our strategy was to infect the cells with adenoviruses containing pancreatic transcription factors (TFs) and to measure effects on phenotypic markers that are characteristic of pancreatic endocrine lineages. An additional aim was to determine whether protein transduction domain (PTD) mediated uptake of TFs might provide a better alternative to the use of adenoviruses. The most commonly used PTD is the 11 amino acid peptide derived from the TAT protein of the TAT/HIV-1 transactivator protein (Gump and Dowdy, 2007). Recombinant proteins that have been engineered to contain the TAT sequence are able to directly cross the cell membrane, and enter the nucleus of a variety of cell types (Manceur et al., 2007, Krosl et al., 2005).