Abstract
Directed differentiation of human pluripotent stem cells (hPSCs) into functional insulin-producing cells (IPCs) holds great promise for cell therapy for diabetic patients. Despite recent advances in developing beta cell differentiation protocols, it is becoming clear that the hPSC-derived beta-like cells are functionally immature, and the efficiencies of differentiation can be variable depending on the hPSC lines used. Therefore, advanced methodologies are highly desirable for the development and refinement of beta cell differentiation protocols from hPSCs. In this report, we first derived and validated a Pdx1-mRFP/insulin-hrGFP dual-reporter cell line from MRC5-iPSCs. Then, using this dual-reporter cell line, we developed and optimized an in vitro beta cell differentiation protocol through real-time monitoring expression of Pdx1 and insulin. We demonstrated that DNA demethylation could increase the efficiency of beta cell differentiation. Furthermore, three-dimensional induction not only significantly increased the efficiency of pancreatic progenitor specification and the yield of IPCs, but also produced more mature IPCs. The current study indicates that this dual-reporter cell line is of great value for developing and optimizing the beta cell differentiation protocols. It will facilitate the development of novel protocols for generating IPCs from hPSCs and the investigation of beta cell differentiation mechanisms.