Tracking insulin- and glucagon-expressing bihormonal cells during differentiation using anINSULINandGLUCAGONdouble reporter human embryonic stem cell line

Summary 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 EGFP GCG 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) lose insulin expression over time, while maintaining an alpha-like expression profile, suggesting these bihormonal cells are preferentially fated to become alpha-like cells in vitro . Together, the INS EGFP GCG mScarlet hESC line provides an efficient strategy for tracking populations of hESC-derived beta- and alpha-like cells.