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Stem cells from patients with Congenital Hyperinsulinism

Kellaway, Sophie

[Thesis]. Manchester, UK: The University of Manchester; 2016.

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Abstract

Diabetes and congenital hyperinsulinism (CHI) are severe diseases affecting the pancreas. Current models for testing drugs to treat these diseases are in vivo in rodents or isolated rodent islets. Differences between the human and rodent pancreas, and ethical issues, mean that in vitro human models are needed. To develop a novel in vitro model for pancreatic diseases, mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs) were derived from the pancreas of patients with CHI. MSCs from three forms of CHI were phenotypically normal for MSCs, and maintained the CHI-causing mutation. When compared to MSCs from bone marrow, the CHI pancreatic MSCs expressed pancreas-specific gene ISL1 and showed promoter hypomethylation of other pancreatic genes, including PDX1. The CHI pMSCs could be differentiated to cells resembling immature beta-cells, with some beta-cell gene expression (INS, PDX1), but no glucose responsive insulin secretion. CHI associated hypersecretion of insulin was not seen as the ATP-sensitive potassium KATP channels were not being expressed. Addition of the Wnt inhibitor DKK1 markedly enhanced differentiation via induction of neuronal genes. Alongside high insulin secretion, CHI also features increased proliferation. CHI MSCs were also hyperproliferative, and showed alterations to the cell cycle. These changes were related to p27Kip1 localisation, a known affected protein in CHI tissue, and CDK1, a novel regulator for CHI. iPSCs were also derived from focal CHI MSCs and were also phenotypically normal, but did not maintain the pancreatic hypomethylation present in MSCs. The CHI iPSCs were efficiently differentiated to definitive endoderm and PDX1 positive cells. Terminally differentiated iPSCs were endocrine, but were not mature beta-cells. In conclusion, authentic MSCs and iPSCs were derived for the first time from patients with CHI. These stem cells could be differentiated towards beta-cells, but mature glucose responsive beta-cells were not produced. MSC derived beta-like cells secreted insulin but did not have KATP channels, whereas iPSC derived beta-like cells had KATP channel gene expression but not INS. With further optimisation to resolve these, CHI stem cell derived beta-cells may be used for in vitro modelling. Further, the undifferentiated MSCs only show hyperproliferation associated with p27Kip1 and CDK1 and so can be a useful resource for modelling hyperproliferation seen in CHI.