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

Mol Cell Endocrinol. 2012 Jul 6;358(1):69-80. doi: 10.1016/j.mce.2012.02.024. Epub 2012 Mar 10.

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.

MeSH terms

  • Animals
  • Basic Helix-Loop-Helix Transcription Factors / biosynthesis
  • Basic Helix-Loop-Helix Transcription Factors / metabolism*
  • Betacellulin
  • Cell Differentiation
  • Cell Line
  • Glucagon / biosynthesis
  • Homeodomain Proteins / metabolism*
  • Insulin / biosynthesis
  • Insulin / metabolism
  • Insulin Secretion
  • Insulin-Secreting Cells / cytology*
  • Insulin-Secreting Cells / metabolism*
  • Intercellular Signaling Peptides and Proteins / metabolism
  • Lectins, C-Type / metabolism*
  • Membrane Glycoproteins / metabolism*
  • Nerve Tissue Proteins / biosynthesis
  • Nerve Tissue Proteins / metabolism*
  • Niacinamide / metabolism
  • Paired Box Transcription Factors / metabolism*
  • Pancreas / cytology
  • Pancreas / metabolism
  • Rats
  • Trans-Activators / metabolism*

Substances

  • Basic Helix-Loop-Helix Transcription Factors
  • Betacellulin
  • Btc protein, rat
  • Homeodomain Proteins
  • Insulin
  • Intercellular Signaling Peptides and Proteins
  • Klrg1 protein, rat
  • Lectins, C-Type
  • Membrane Glycoproteins
  • Nerve Tissue Proteins
  • Neurog3 protein, rat
  • Paired Box Transcription Factors
  • Pax4 protein, rat
  • Trans-Activators
  • pancreatic and duodenal homeobox 1 protein
  • NeuroD protein
  • Niacinamide
  • Glucagon