NeuroD and Reaggregation Induce Beta-cell Specific Gene Expression in Cultured Hepatocytes

Background: Our goal was to convert adult mouse hepatocytes to pancreatic beta-cells.

Methods And Results: To facilitate conversion, cultured primary hepatocytes were dedifferentiated by the removal of dexamethasone (Dex) from the culture media. Removal of Dex caused detachment of hepatocytes from the culture dish, but the addition of betacellulin prevented this from happening. With the combination of lack of Dex and addition of betacellulin, albumin mRNA levels decreased. Cultured hepatocytes had a faint expression of insulin 2 mRNA, Nkx 6.1 and Pax 6 mRNA. Dedifferentiated hepatocytes were transduced with adenoviruses expressing NeuroD1, Ngn 3, or Pax 4. NeuroD1 transduction increased the insulin 2 mRNA but caused detachment of cells. However, when hepatocytes were allowed to reaggregate for 4 and 6 days in hydrophobic plates after transduction with NeuroD1, further increases of insulin 2 mRNA were found along with induction of PDX-1, IAPP, NeuroD1, Ngn3, Pax 4, Isl-1, PC1, PC2 and islet glucokinase. Additionally, glucagon, pancreatic polypeptide and somatostatin expression were induced, but neither elastase 1 nor insulin 1 mRNA could be detected. Ngn 3 and Pax 4 had effects similar to NeuroD1, but did not increase insulin 2 mRNA as much as NeuroD1.

Conclusion: We conclude that the combination of NeuroD1 and reaggregation promotes cultured dedifferentiated hepatocytes to differentiate towards a pancreatic beta-cell phenotype.