P.165: Engineering Functionally Mature Human Pluripotent Stem Cell-derived Beta-Cells by Modifying the Beta-cell Niche

Abstract

Aim: Aim of the study is to engineer human pluripotent stem cell-derived beta-cells with physiological maturity by recapitulating endogenous intricate 3-D microenvironment niche. Methods: We use a 36-days long directed differentiation protocol to manoeuvre cells through different stages of pancreatic endocrine fate leading to insulin-producing beta-cells. We use a cocktail of growth factors, morphogens and small molecules to activate and repress different signaling pathways at each stage of differentiation. Differentiated cells are characterised using immunocytochemistry, Fluorescent Activated Cell Sorting, functional assays, state-of-the-art confocal imaging and in vivo implantation in animal models. Results: We have generated functionally active insulin-secreting human pluripotent stem cell-derived beta-cells in our lab. We further investigate the beta-cell niche in the differentiated spheroids and demonstrate the ameliorating effect of extracellular matrix proteins on structural polarity and glucose-stimulated insulin secretion index. Our study shows a tightly controlled basal insulin secretion under modified condition, suggesting enhanced beta-cell maturity, which needs further investigation. Conclusion: Human pluripotent stem cell-derived beta-cells provide a tangible alternative to conventional diabetes treatments. However, stem cell-derived beta-cells do not recapitulate the physiological aspects of human islets. In native islet beta-cell exist in an intricate 3-D structure. Work for the past 38-years suggests that inter-and intra-cellular interactions and the interaction of beta cells with basement membrane protein may play a vital role in beta-cell differentiation, proliferation, insulin gene synthesis and secretion. In the stem cell-derived spheroids beta-cell niche has neither been investigated nor deliberately controlled until very recently. Our study suggests that recapitulating endogenous interactions in differentiated spheroids may activate downstream signaling cascades required for glucose sensing and stimulated secretion. We are further analysing the effect of beta-cell niche on global gene expression pattern and thereby dissecting the underlying mechanism of favourably altered insulin secretion. We have incorporated Crispr-Cas9 genome editing tools to design fluorescently labelled human pluripotent stem cell lines for deep tissue live-cell imaging of implanted cells. This study will contribute significantly towards refining beta-cell differentiation methods, disease modeling, novel drug discovery, drug screening and developing cell replacement therapy for people living with diabetes. John and Ann Chong Fellowship. Diabetes Australia Grant. National Health and Medical Research Council Grant.