Tajdivand, Helen; (2020) Generation of Mature, Functional b cells from Human Induced Pluripotent Stem Cells for the Potential Treatment of Type 1 Diabetes Mellitus. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Diabetes Mellitus has reached pandemic levels affecting 424.9 million individuals aged 20-79. This represents an increasing burden on health care costs, and the quality and quantity of patients’ lives. Type 1 Diabetes Mellitus (T1DM) occurs due to Insulin deficiency following autoimmune destruction of Insulin producing β-cells in the pancreatic islets of Langerhans. Current therapies are insufficient: exogenous Insulin administration fails to accurately control glucose homeostasis; replacement of β-cells by transplantation is limited by donor shortage and the need for life-long immunosuppression. By generating autologous β-cells from patient-derived human induced pluripotent stem cells (hiPSCs), regenerative medicine could overcome these limitations. Unfortunately, thus far, current methods produce β-like cells that lack full maturity and functionality. The aims of this project were two-fold: (1), develop a human pancreasderived hydrogel scaffold (Pancrigel) to provide an in vivo-like niche that would (2), enhance differentiation of mature, functional b-cells from hiPSCs. HiPSCs were successfully reprogrammed from a volunteer’s peripheral blood using self-replicating RNA vectors. These hiPSCs were shown to be healthy, pluripotent, karyotypically normal and could differentiate into b-like-cells expressing mature markers (MAFA, NKX6.1, NEUROD1), and secreted Insulin in response to sequential glucose stimuli (2mM and 20mM). Successful decellularisation of human pancreas resulted in 95% DNA depletion, 66% and 63.5% preservation of collagen and sGAG respectively, with good laminin and fibronectin preservation, from which Pancrigel was produced. The Pancrigel supported attachment and proliferation of hiPSCs which were able to differentiate to b-like cells with some mature features and functions including being able to release Insulin at a similar level to freshly isolated human pancreatic islets (b-like cells: 0.0161; islets: 0.022 µU Insulin/1000 cells). Overall, this project provides a potential system for generation of autologous, functional b-like cells, using a bespoke Pancrigel niche, which could be modified to evade auto-immunity, and be employed for T1DM therapy.

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