Tracking Insulin- and Glucagon-Expressing Cells In Vitro and In Vivo Using a Double-Reporter Human Embryonic Stem Cell Line

Abstract

Human embryonic stem cell (hESC)-derived pancreatic α- and β-cells can be used to develop cell replacement therapies to treat diabetes. However, recent published differentiation protocols yield varying amounts of α- and β-cells amid heterogeneous cell populations. To visualize and isolate hESC-derived α- and β-cells, we generated a GLUCAGON-2A-mScarlet and INSULIN-2A-EGFP dual fluorescent reporter (INSEGFPGCGmScarlet) hESC line using CRISPR/Cas9. We established robust expression of EGFP and mScarlet fluorescent proteins in insulin- and glucagon-expressing cells, respectively, without compromising the differentiation or function of these cells. We also showed that the insulin- and glucagon-expressing bihormonal population at the maturing endocrine cell stage (stage 6) of our pancreatic islet differentiation lose insulin expression over time, while maintaining an α-like expression profile, suggesting these bihormonal cells are cell-autonomously fated to become α-like cells. We also demonstrated this cell line can be used to monitor hESC-derived insulin- and glucagon-expressing cells, and hESC-derived islet morphology in vivo, by transplanting them into the anterior chamber of the eye in mice. Together, the INSEGFPGCGmScarlet hESC line provides an efficient strategy for tracking populations of hESC-derived β- and α-like cells. Article Highlights Differentiation protocols used to generate stem cell–derived islet cells yield heterogenous cell populations. We generated a human embryonic stem cell line that reports insulin- and glucagon-expressing cells in vitro and in vivo without altering their differentiation or function. We showed some insulin- and glucagon-expressing bihormonal cells are cell-autonomously fated to become α-like cells. This reporter cell line can be used to further study and improve stem cell–derived islet differentiation and transplantation.