Tracking insulin- and glucagon-expressing cells in vitro and in vivo using a double reporter human embryonic stem cell lineUntitled Item

Human embryonic stem cell (hESC)-derived pancreatic alpha and beta cells can be used to develop cell replacement therapies to treat diabetes. However, recent published differentiation protocols yield varying amounts of alpha and beta cells amidst heterogeneous cell populations. To visualize and isolate hESC-derived alpha and beta 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 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 alpha-like expression profile, suggesting these bihormonal cells are cell autonomously fated to become alpha-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 beta- and alpha-like cells.