Exendin-4 Enhances GSIS By Upregulating Genes Related to Maturation, Glucose-Sensing Apparatus and Mitochondrial Oxidative Phosphorylation Machinery in hPSC-Derived Islets Without Increasing β-Cell Number

Emerging β-cell replacement with human pluripotent stem cell (hPSC)–derived β-cells could provide remedial cell therapy for diabetes. Most in vitro differentiation protocols generated hPSC?derived β-cells with immature phenotypes such as impaired or weakened glucose?stimulated insulin secretion (GSIS) relative to primary β-cells. Evidence has shown the effectiveness of exendin?4 in increasing β-cell mass and function in vivo. This study investigates the effect of exendin?4 on maturation and functionality of hPSC?derived β?cells. Differentiation of two hPSC cell lines (HUES8 and iPSC824) into islets was carried out using a 3D differentiation protocol. 50 nM Exendin?4 was added to the suspension culture during the last three days of differentiation. The gene expression patterns in both cell lines showed that expression of the pluripotent marker OCT4 was lost upon initiation of differentiation. The definitive endoderm marker SOX17 was transiently and significantly increased at Stage 1. PDX1, a marker of the pancreatic progenitor 1 started being significantly upregulated at Stage 3; while the β-cell specific markers including insulin, NKX6-1 were strongly induced at stage 5, and 6. In HUES8 there was no difference in c?peptide secretion between low (2.8mM) and high (20 mM) glucose; suggesting a lack of functional β-cells. While flow cytometry data showed no significant difference between control and exendin-4 treated groups in NKX6.1+/insulin+ (β-cell markers), the addition of exendin?4 significantly enhanced GSIS in both cell lines. This was associated with increased expression of maturation (NeuroD1, Six2, MAFA) glucose?sensing (Glut2, GCK) and mitochondrial oxidative phosphorylation machinery (NDUSF1, NDUSF2) genes. In summary, we demonstrated for the first time in 3D differentiation of hPSC-derived β-cells that addition of exendin-4 enhances GSIS without increasing β-cell number.