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

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 (INS^EGFPGCG^mScarlet) 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 INS^EGFPGCG^mScarlet 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. [ABSTRACT FROM AUTHOR]