New hPSC SOX9 and INS Reporter Cell Lines Facilitate the Observation and Optimization of Differentiation into Insulin-Producing Cells

Differentiation of human pluripotent stem cells into insulin-producing stem cell-derived beta cells harbors great potential for research and therapy of diabetes. SOX9 plays a crucial role during development of the pancreas and particularly in the development of insulin-producing cells as SOX9(+) cells form the source for NEUROG3(+) endocrine progenitor cells. For the purpose of easy monitoring of differentiation efficiencies into pancreatic progenitors and insulin-producing cells, we generated new reporter lines by knocking in a P2A-H-2K(k)-F2A-GFP2 reporter gene into the SOX9-locus and a P2A-mCherry reporter gene into the INS-locus mediated by CRISPR/CAS9-technology. The knock-ins enabled co-expression of the endogenous and reporter genes and report on the endogenous gene expression. Furthermore, FACS and MACS enabled the purification of pancreatic progenitors and insulin-producing cells. Using these cell lines, we established a new differentiation protocol geared towards SOX9(+) cells to efficiently drive human pluripotent stem cells into glucose-responsive beta cells. Our new protocol offers an alternative route towards stem cell-derived beta cells, pointing out the importance of Wnt/beta-catenin inhibition and the efficacy of EGF for the development of pancreatic progenitors, as well as the significance of 3D culture for the functionality of the generated beta cells. Graphic Abstract