Gut cell plasticity during diabetes treatment

Project Summary There is irreversible destruction of insulin-secreting pancreatic ?-cells during type 1 diabetes. Consequently, individuals with this disease are dependent on continual insulin injections to manage their diabetes which is burdensome to patients. However, if one could trigger a patient's own cells to generate endogenous insulin it would essential cure these individuals of their diabetes. Elimination of the transcription factor Foxo1 in a population of intestinal endocrine progenitor (Neurogenin3+) cells prompts them to secrete functional insulin and reverse diabetes in mice. Interestingly, there are also more Ngn3+ cells in the intestines of these mice. Using a reporter mouse, the candidate has found that Foxo1 is expressed in Ngn3+ and some acid-secreting parietal cells in the stomach epithelium. While ablation of Foxo1 in both of these cell populations triggers the appearance of insulin immune-reactive cells, it also increases Ngn3 expression and parietal cell numbers. The candidate hypothesizes that elimination of Foxo1 in the stomach changes the cell plasticity of this organ. To further understand this process, using three specific aims, this research proposal will 1) study specific changes to gastric enteroendocrine and parietal cells when Foxo1 is deleted in mice and in human cells and 2) define molecular pathways important to insulin-cell conversion. To accomplish this, Foxo1 will be abolished, using cre recombinase technology, in primary stomach cultures (Aim1.1), in two separate populations of cells in mice (Aim1.2), and in human induced pluripotent cells (iPSC) (Aim2). Changes to cell type, distribution, proliferation, and death will be measured using immunohistochemistry and qPCR. Important to career development, new research skills (iPSC culture and genome modification by CRISPR) will be learned in Aim2 of this proposal. Using FACs to isolate cells, Aim3 will analyze Foxo1-deleted primary stomach cells and stomach insulin+ cells using RNA-seq. Unique multi-color fluorescent reporters will be used in both Aim2 and Aim3. Taken together, the proposed experiments will likely reveal new molecular targets/pathways important in the conversion of stomach cells to those that produce insulin, ultimately to discover a new avenue of treatment for type 1 diabetes. These experiments are complimented by a career development plan in which the candidate will learn new research techniques, broaden her scientific network through attending conferences, and develop her writing skills (through coursework, submitting manuscripts/R01 application) so that she is poised to become an independent investigator. Both the career development plan and research proposal will be completed at Columbia University, a prestigious institution.