Beta-cell Transdifferentiation

A challenging approach using phenotypic cell-based assays is to identify small molecules that can alter the endocrine specification in cell culture. Type 1 diabetes is an autoimmune disease characterized by the loss of insulin-producing beta cells in pancreatic islets of Langerhans. Islet transplantation into the liver can effectively cure the disease but is not an ideal treatment due to limited donor material and immunological complications. An alternative approach, not yet feasible, is to create new beta cells from patient material. These approaches include stepwise differentiation of undifferentiated stem or stem-like cells to beta cells and transdifferentiation, the heritable change of cell identity to an insulin-producing (beta-like) cell. The latter approach could result in a replacement source for the deficient cell type directly from patient material (either in vivo or ex vivo). Increasing beta-cell mass by chemically induced transdifferentiation is a speculative but exciting approach to treating diabetes, one that is significantly different from currently available small-molecule drugs that increase insulin secretion in existing beta cells and therefore are ineffective in the later stages of type 1 diabetes, in which most beta-cell mass has been lost.

Beta-Cell Transdifferentiation from Alpha Cells. We are developing methods to identify small molecules that increase beta-cell mass by inducing transdifferentiation of alpha cells. Cell-type specification in the pancreas is regulated by a set of master regulatory transcription factors, which control the transition from one progenitor cell state to the next, ultimately yielding mature endocrine cell types in islets. Recently, it has been demonstrated that misexpression of these master regulatory transcription factors causes direct transdifferentiation between cell types. For example, ectopic overexpression of a single transcription factor (Arx) is sufficient to cause in vivo transdifferentiation of beta cells to alpha cells in the adult mouse pancreas. Similarly, viral delivery of three transcription factors (Pdx1, Ngn3, MafA) to an adult mouse pancreas causes the transdifferentiation of acinar cells to beta cells. Conversion of alpha cells to beta cells in vivo has recently been achieved in mature mouse alpha cells by ectopic overexpression of Pax4. Since a single gene is sufficient to induce transdifferentiation of alpha cells to beta cells, we are seeking to determine whether a small molecule could have the same effect. To that end, we have developed a high-content cell-based assay to detect insulin protein expression in the mouse alpha cell line αTC1. Normal mouse alpha cells are insulin-negative, but have been shown to adopt a beta-cell phenotype after extreme beta-cell loss. The alpha cell line we used expresses small but detectable levels of insulin, despite being a sub-clone selected for low insulin protein.
Beta-Cell Transdifferentiation from Exocrine Cells. The ability to induce cellular transdifferentiation, particularly to a beta cell-like phenotype, is both of great interest biologically and as a potential benefit to patients with type 1 diabetes. Other pancreatic cells, such as endocrine alpha cells, exocrine acinar cells and ductal cells have shown some extent of plasticity in their ability to adopt an insulin-producing phenotype. Recently, the expression of three transcription factors (Pdx1, Ngn3, and MafA) was shown to reprogram mouse acinar cells towards a beta-cell phenotype in vivo, enabling these cells to synthesize and secrete insulin in a glucose-regulated manner and restoring glucose homeostasis, even after chemical destruction of the original population of endogenous beta cells. Our aim has been to identify small-molecule inducers of gene expression of at least one of these transcription factors. Previously, we developed a method for high-throughput real-time PCR-based screening; because of our interest in using human biology as much as possible, we use the PANC-1 ductal adenocarcinoma cell line.