Endocrine Abstracts

Heterozygous mutations in the transcription factor, hepatocyte nuclear factor 1b (HNF1B), result in multisystem disease including diabetes due to beta-cell dysfunction and pancreatic hypoplasia. However, the mechanisms that underlie development of diabetes in HNF1B mutation carriers are still not fully understood due to lack of an appropriate animal model system. Human pluripotent stem cells (PSCs), which are capable of self-renewal and can differentiate into any cell type, are ideally suited to model human developmental diseases. The aim of this project was to develop a human PSC based model system to determine the molecular mechanisms by which HNF1B mutations cause pancreatic hypoplasia and diabetes.

HNF1B mutant PSC lines were produced using CRISPR-Cas9 genome editing. Isogenic HNF1B wild-type and homozygous and heterozygous mutant cell lines were then directed to differentiate along the pancreatic lineage. Cells were phenotyped at each stage to check for expression of appropriate markers using immunohistochemistry, flow cytometry and qPCR. The normal expression pattern of HNF1B in human pancreas development was analysed and showed upregulation of HNF1B at the foregut stage, and during pancreas specification. Homozygous knockout of HNF1B resulted in failure of foregut and pancreatic progenitor development, while heterozygous knockout of HNF1B resulted in impairment of pancreatic progenitor and endocrine cell differentiation as well as impaired insulin secretion upon glucose stimulation. RNA-sequencing analysis identified that the majority of top downregulated transcription factors and pathways in mutant compared with wild-type cells at the pancreatic progenitor stage, were those associated with pancreas development. Cell proliferation assays showed a significant decrease in the proliferation rate in HNF1B heterozygous and homozygous mutant cells compared with wild-type cells at the foregut stage, however, there was no change in the apoptosis rate.

HNF1B haploinsufficiency may therefore impair the expansion and maintenance of pancreatic progenitor cells in vivo during human pancreas development, resulting in reduced beta cell numbers at birth and diabetes later in life. This in vitro model provides further insights into the molecular mechanisms by which HNF1B regulates human pancreas development and function, as well as potentially identifying new genes and pathways that contribute to diabetes pathogenesis and providing novel therapeutic targets.