Role of Neurogenin3 phosphorylation in the reprogramming of pancreatic organoids into endocrine cells

During pancreatic development, Neurogenin3 (Ngn3) promotes the differentiation of endocrine cells, including insulin-producing β-cells. Our previous work has shown that Ngn3 phosphorylation on multiple serine-proline (SP) sites increases protein stability and endocrine differentiation (Azzarelli et al., DevCell 2017). Here, we aim to exploit our recent data to investigate whether manipulation of Ngn3 phosphostatus might improve in vitro generation of β-cells for replacement therapies in diabetes. A potential source of β-cells comes from fate conversion of exocrine pancreatic cells into the endocrine lineage, by overexpression of 3 transcription factors, Ngn3, Pdx1 and MafA. Using this approach we show that pancreatic ductal cells grown in vitro as 3D organoids can be reprogrammed to express insulin. The efficiency can be strongly enhanced by substituting wild type Ngn3 with a the un(der)phosphorylated and more active Ngn3 form (6S-A Ngn3). Here we perform transcriptomic analysis of a low number of pancreatic organoid cells expressing wild type or phosphomutant Ngn3, alone or in various combination Pdx1 and/or MafA. The analysis revealed endocrine differentiation and in particular β-like cell gene expression in the conditions where Ngn3 was in combination with both Pdx1 and MafA. RNA-sequencing of pancreatic organoids infected with various combination of transcription factors: WTNgn3-Pdx1, WTNgn3-MafA, WTNgn3-Pdx1-MafA, 6SANgn3-Pdx1, 6SANgn3-MafA, 6SANgn3-Pdx1-MafA, Pdx1 only or MafA only expressing cells, in culture infected with either WTNgn3 or 6SANgn3. Samples are collected 8days after induction: 3 biological replicates and 1-2-3 technical replicates depending on the sample