UBC Theses and Dissertations
The cell cycle regulates mouse and human pancreas development Krentz, Nicole Angela Jane
Diabetes is caused by a loss or dysfunction of insulin-producing pancreatic beta-cells. A potential treatment for diabetes is to replace these cells through transplantation. As there is a shortage of donor tissue, efforts to generate an unlimited source of functional insulin-producing beta-cells from human embryonic stem cells (hESCs) are ongoing. During pancreas development, proliferating pancreatic progenitors activate Neurog3, exit the cell cycle, and differentiate. The overarching goal of this thesis was to understand the role of the cell cycle in regulating Neurog3 expression and endocrine cell fate. First, the length of each cell cycle phase of pancreatic progenitors was measured using cumulative EdU labelling, determining an increase in G1 length in Pdx1+ progenitors from 4.5±0.4 to 7.2±0.8 hours between embryonic day (E)11.5 and E13.5. Next, two mouse models were used to show that cell cycle lengthening within pancreatic progenitors stimulates endocrine differentiation. Kras heterozygous loss-of-function mice have increased endocrine cell genesis that was correlated with an increase in progenitor cell cycle length. Ectopic expression of the cyclin-dependent kinase inhibitor Cdkn1b in Sox9+ progenitor cells resulted in a 2.7-fold increase in the number of Neurog3+ cells. As Cdkn1b is an inhibitor of G1-S cyclin-dependent kinases (Cdks), the effect of directly inhibiting Cdk2, Cdk4 and Cdk6 on endocrine differentiation was investigated. Treating embryonic pancreata, ex vivo, for 24 hours with Cdk inhibitors resulted in a 3-fold increase in the number of Neurog3+ cells. To investigate the consequences of CDK inhibition on human endocrine differentiation, a NEUROG3-2A-eGFP (N5-5) knock-in reporter CyT49 hESC line was generated using CRISPR-Cas9. CDK inhibition increased the number of GFP+ endocrine progenitor cells 1.7-fold. These findings suggest that G1 lengthening is required for normal mouse and human organogenesis and that cyclin-dependent kinases act directly to reduce Neurog3 protein. In the final chapter, single-cell transcriptomics was used to profile the gene expression and cell populations present during mouse and human endocrine development. In conclusion, these studies show that progenitor cell-cycle G1 lengthening, through its actions on stabilization of Neurog3, is an essential determinant of normal endocrine cell genesis.
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