Endocrine enriched genes in adult islet beta cells were identified and compared with that of induced beta cells (with M3 transcription factors) in adult. The control sample is non-beta pancreatic cells. Gene expression profile comparison of 3 samples, 3 independent repeats for each sample indicate a high degree of similarity between endogenous and induced beta cells in adult mouse.

Turn pancreatic exocrine cells into beta cells through adult cell reprogramming.

A strategy of re-expressing key developmental regulators in vivo was applied. Nine transcription factors were identified with beta-cell developmental phenotypes when mutated. These were delivered into the mouse pancreas exocrine cells of 2-month old adult mice using adenoviral vectors as mixtures. After one month, insulin positive cells were scored; GFP was coexpressed by the adenovirus and used to identify viral infected cells. Systematic removal of factors from viral pools was used to identify the optimal combination. Insulin positive cells were demonstrated to come from exocrine cells by lineage analysis and characterized by morphology and immunohistochemistry. Expression profiles of GFP+ cells were isolated by FACS (~70% GFP+ and ~22% insulin +) and compared to islets or non-GFP+ pancreatic cells by microarray analysis on Illumina Sentrix BeadChip Array MouseRef-8 v1.1.

The virus pool of nine transcription factors (Nkx2.2, Nk6.1, Pax4, NeuroD, Pax6, Isl1, Ngn3, Pdx1, MafA) led to a modest increase in insulin positive cells among viral infected cells. Removal of Nkx2.2, Nk6.1, or Pax4 did not prevent the generation of insulin producing cells. Reducing the pool to 6 factors identified Ngn3, Pdx1, and MafA as required; the combination of these 3 factors (M3) led to over 20% of infected cells being insulin positive. These cells had small dense granules (characteristic of insulin granules). Lineage tracing established these came from exocirne cells. Expression profile analysis indicates a strong overlap between the GFP+/insulin+ cells and islet cells. Immunohistochemistry demonstrated that these cell were predominately positive for beta cell markers (Glut2, GCK, Pcsk1, NeuroD, Nkx2.2, Nkx6.1) and negative for exocrine genes (amylase, Ptf1a, Krt19), mesenchymal genes (nestin, vimentin), neuronal Tubb3, or other hormones (glucagon, somatostatin, pancreatic peptide). Streptozotocin-induced diabetic mice infected with the M3 pool had improved glucose levels over controls.

Reprogramming of exocrine cell to beta cells can occur with first insulin+ cells at day 3 and up to 20% efficiency with an adenoviral pool of Ngn3, Pdx1, and MafA. Transient expression of the inducing factors is sufficient. Reprogramming does not appear to require extensive replication or reversion to a dedifferentiated state for an appreciable time.

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