AbstractMaturity-onset diabetes of the young 3 (MODY3) is the most common subtype of monogenic diabetes. MODY3 is caused by heterozygous mutations in the transcription factor hepatocyte nuclear factor 1α (HNF1α). The most prevalent HNF1α mutation in MODY3 is the insertion of a cytosine (C) in the poly-cytidine tract of codon 291(p291fsinsC), a frameshift mutation that induces an early stop codon and can result in the expression of a truncated form of HNF1α protein. Current mouse and human models for MODY3 are very limited and do not fully recapitulate the disease phenotypes. Due to a lack of appropriate disease models, the molecular mechanisms underlying the pathobiology of MODY3 due to p291fsinsC mutation have remained elusive.
In this thesis, I differentiated wild-type, MODY3 patient and CRISPR/Cas9 edited (with the p291fsinsC mutation) human induced pluripotent stem cells (hiPSCs) towards pancreatic progenitors in-vitro to study the disease mechanism. I also established progenitor organoids from these lines to circumvent limitations with current β-cell differentiation protocols and to study the mutational mechanism in more detail. We recently employed single-cell RNA-sequencing to investigate the heterogeneity within wild-type organoids and found that they are composed of multiple progenitor subpopulations and supporting mesenchymal cells. Once we established and characterized the wild-type organoids, I established and characterized mutant progenitor organoids and investigated their differentiation capacity towards β-like cells. Patient and CRISPR/Cas9-derived hiPSCs showed a severe reduction in PDX1+ and NKX6.1+ pancreatic progenitor formation which paralleled a reduction in β-cell differentiation in the mature organoids compared to wild-type hiPSCs. Mechanistically, we found that the truncated form of HNF1α protein interacts with HNF1β protein, thus impairing HNF1β-dependent developmental gene expression and stalling proper endocrine development. By inhibiting this protein interaction, we rescued HNF1β-mediated gene transactivation. Overexpression of HNF1β in the MODY3 patient progenitor organoids resulted in a partial rescue of the phenotype by an increase in PDX1+ cells, indicating that the truncated version of HNF1α protein has a dominant negative effect on both wild-type HNF1α and HNF1β proteins.
This study uncovers a novel mechanism of the p291fsinsC mutation during pancreas development in a new human organoid model of MODY3. The p291fsinsC mutation results in a previously undescribed developmental defect that can explain pancreatic defects observed in some MODY3 patients. Our organoid system could be used for investigating developmental defects associated with other mutations recurrent in monogenic or polygenic diabetes.
|Date of Award
|1 Oct 2021
|Rocio Sancho (Supervisor) & Fiona Watt (Supervisor)