HNF1A-MODY is the most prevalent form of monogenic diabetes in humans and results from a haploinsufficiency of the transcription factor hepatocyte nuclear factor 1 alpha (HNF1A). The molecular mechanisms resulting in a diabetic phenotype remain unclear as haploinsufficient mouse models of HNF1A- MODY have failed to produce any phenotypes. The recent advances in the human induced pluripotent stem cell (iPSC) field and human hepatic and pancreatic endocrine differentiation fields provide a powerful set of tools for assessing diseases, such as HNF1A-MODY, at the molecular and cellular level. HNF1A is particularly known for its expression in the hepatocyte and the pancreatic islet. We hypothesize that the pathophysiology of HNF1A-MODY results from gene expression changes in the hepatocyte and in the pancreatic cell. Two iPSC lines have been generated from HNF1A-MODY patients with a R272H mutation and a T564I mutation. These clones express pluripotency factors and are capable of directed differentiation to the hepatic lineage. Lacking direct sibling or parental controls, cells generated from these mutant iPSCs will be compared to two control iPSC lines lacking mutations in HNF1A.
Aim 1 will first utilize microarrays to identify changes in gene transcription in the mutant HNF1A-MODY hepatocytes versus control hepatocytes. A targeted bioinformatics assessment will identify altered key targets relating to glucose homeostasis and insulin signaling in the hepatocyte. These targets will be confirmed by qRT-PCR and Western blots. Key targets will be validated for deficiencies at the functional level, such as assays for measuring glucose uptake or oxidative phosphorylation capacity.
Aim 2 will first generate functional pancreatic beta cells by a combined in vitro and in vivo differentiation protocol in mice. Engrafted HNF1A-MODY beta cells will be assayed compared to control beta cells for: fasting glucose levels, basal insulin secretion, and glucose-stimulated insulin secretion. Hyperglycemia, decreased basal insulin secretion, and diminished glucose-stimulated insulin secretion are the respective anticipated results based on the clinical profile of HNF1A-MODY. Grafts will be removed and beta cells will be purified for downstream analyses. First, functional pancreatic endocrine cells from HNF1A-MODY and control iPSCs will be processed via various assays to quantitate absolute insulin expression, insulin cleavage, insulin exocytosis, and calcium signaling. Second, mRNA isolated from mutant and control beta cells will be utilized for microarrays to identify changes in gene expression. A targeted bioinformatics approach will identify altered key targets relating to glucose homeostasis and to insulin processing. This proposal will establish which specific molecular mechanisms relating to glucose homeostasis and insulin signaling or production are altered in the HNF1A-haploinsufficient hepatocyte and pancreatic beta cell.
Increasing numbers of Americans are being diagnosed with diabetes: up to a third of the population is expected to have diabetes by 2050. The potential for damage to human tissues resulting in amputations, infections, kidney disease, and death remain much too high. In order to develop new strategies to prevent diabetes from becoming worse with time, the scientific community needs to understand the specific cellular mechanisms causing diabetes.
|Noto, Fallon K; Determan, Megan R; Cai, Jun et al. (2014) Aneuploidy is permissive for hepatocyte-like cell differentiation from human induced pluripotent stem cells. BMC Res Notes 7:437|