Reprogramming of various cell types to a beta-like state by Pdx1, Ngn3 and MafA

Abstract

The three transcription factors, PDX1, NGN3 and MAFA, are very important in pancreatic development. Overexpression of these three factors can reprogram both pancreatic exocrine cells and SOX9-positive cells of the liver into cells resembling pancreatic beta cells. In this study we investigate whether other cell types can be reprogrammed. Eight cell types are compared and the results are consistent with the idea that reprogramming occurs to a greater degree for developmentally related cells (pancreas, liver) than for other types, such as fibroblasts. Using a line of mouse hepatocyte-derived cells we screened 13 compounds for the ability to increase the yield of reprogrammed cells. Three are active and when used in combination they can increase the yield of insulin-immunopositive cells by a factor of six. These results should contribute to the eventual ability to develop a new cure for diabetes based on the ability to reprogram other cells in the body to a beta cell phenotype.

Figure 1. Expression of various beta cell genes provoked by transduction with Ad-PNM.

A-H. Qualitative RT-PCRs showing the effect of Ad-PNM on the expression of a panel of beta cell genes. 30 cycles were used unless otherwise shown. The misaligned bands in the first two lanes of mouse fibroblast Mnx1 are primer dimers, not RNA.

Figure 2. Insulin-positive cells generated from different cell types.

A,B,C Expression of PDX1, NGN3, MAFA in ASH cells. The proteins are shown in green, DAPI in blue. D. Histogram of the percentage PDX1-positive cells which are insulin-positive in different cell types. E,E’ Rat hepatocytes, some PDX1-positive cells are insulin-positive. F,F’ Rat hepatocytes, insulin-positive cells are also C-peptide-positive. G Mouse ASH cells, some PDX1-positive cells are insulin-positive. H. Mouse ASH cells, showing cytoplasmic location of insulin. Inset, high magnification showing insulin-containing granules. I,I’ Mouse ASH cells showing co-expression of C-peptide with insulin. J Rat AR42j-B13 cells showing a high proportion of insulin-positive cells. K,K’ Rat AR42j-B13 cells showing co-expression of C-peptide with insulin. L. Rat AR42j-B13 showing insulin-containing granules. Scale bars 100µm, except H inset and L which are 25 µm. Blue color is DAPI throughout.

Figure 3. EdU labeling index in five cell types.

Those which generated insulin-immunopositive cells showed a reduction in EdU label. Errors are standard errors, n=3.

Figure 4. Effects of the small molecule combination.

A. ASH cells transduced with Ad-PNM, without and with the DNB combination. PDX1 is immunostained green and insulin red. Scale bar 100µm. B. Percentage of PDX1-positive cells that are also insulin-positive. Comparisons are by one way ANOVA with Games-Howell post hoc test, * indicates significant enhancement. Errors are standard errors, n=6.

Figure 5. Gene expression in ASH cells provoked by the small molecule combination, with and without Ad-PNM.

A. qRT-PCR for a panel of beta cell genes. Errors are standard errors, n=3. B. Insulin secretion in low and high glucose. Comparisons are by 1 way ANOVA with Tukey posthoc test and additional t tests.