Endocrine Abstracts

The shortage of donor material has driven research towards finding a replenishable supply of islets for transplantation. We have previously shown that the exocrine material that results as a by-product of the islet isolation procedure can be transdifferentiated towards functional islet-like structures by overexpression of the pancreatic transcription factors (TFs) Pdx1, MafA, Ngn3 and Pax4 followed by culture in the presence of betacellulin, nicotinamide and exendin-4. These cells secreted insulin in a glucose responsive manner and rescued diabetes in a streptozotocin-induced diabetic mouse model, but expressed 1% of the insulin levels of mature islets. Modification of the culture conditions to include laminin and a low glucose concentration has further enhanced transdifferentiation towards endocrine lineages.

A major increase in insulin production was observed upon siRNA knockdown of the α-cell TF ARX at the later stages of the protocol. Specific ELISAs for human proinsulin, insulin and C-peptide and ultrastructural analysis demonstrated that the newly transdifferentiated cells were able to efficiently store (33.5±7.3 pg/μg protein) and process insulin, secreting C-peptide in a regulated glucose-responsive manner, with insulin levels rising to 15% of those found in human islets. ARX knockdown further decreased glucagon expression. Removal of Pax4 abolished regulated glucose-response, supporting its critical role for functionality and maturation of the transdifferentiated cells. This population was monohormonal, comprising 40% C-peptide⁺, 4% glucagon⁺, and <2% somatostatin⁺ cells. Grafted in diabetic Scid/Bg mice, these cells had an immediate and prolonged (100 days) effect in normalising blood glucose levels and body weights.

We have shown for the first time that late inhibition of ARX, along with Pax4 overexpression, is crucial for the transdifferentiation of human exocrine cells towards mature beta-like cells. We estimate that ~3 billion of these cells would have an immediate and therapeutic effect following transplantation in people with type 1 diabetes.