The use of human pluripotent stem cells to model HNF1B-associated diabetes

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 model system. Human induced pluripotent stem cells (hiPSCs), which are capable of self-renewal and can differentiate into any cell type, provide an advantageous alternative to model human developmental diseases. The aim of this project was to develop a hiPSC based model system to determine the molecular mechanisms by which HNF1B mutations cause pancreatic hypoplasia and diabetes. HNF1B mutant hiPSC lines were produced using CRISPR-Cas9 genome editing. Isogenic HNF1B wild-type, homozygous and heterozygous mutant hiPSC lines were directed to differentiate along the pancreatic lineage and cells were phenotyped at each stage of the differentiation process to check for appropriate expression of lineage markers. The normal expression pattern of HNF1B in human pancreas development was analysed and showed up-regulation 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. Cell proliferation analyses showed a significant decrease in the proliferation rate in HNF1B heterozygous and homozygous mutant cells compared with wild-type cells at the foregut stage while no change in the apoptosis rate could be detected. RNA-sequencing and ATAC-sequencing, were used to further define the molecular mechanisms controlled by HNF1B and the effect HNF1B on modulation of chromatin accessibility during pancreas development. These results provide further insights into the molecular mechanisms by which HNF1B regulates human pancreas development and function, revealing that HNF1B haploinsufficiency impairs the expansion and maintenance of pancreatic progenitor cells in vitro. In vivo, this would likely result in reduced beta cell numbers at birth and diabetes later in life in patients with HNF1B-associated disease. These mechanisms suggest that the capacity to produce pancreatic progenitor cells during embryonic life could determine individual susceptibility to diabetes.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2018

Author dc:creator

• Ranna El Khairi, Ranna

Advisor dc:contributor.advisor

• Vallier, Ludovic

• HNF1B
• monogenic diabetes
• stem cells
• pancreas development

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/0b616f4e-ae09-4d4d-8e6c-bee96ee8b568/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

en

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.32005

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/284631