Generation of fluorescent and bioluminescent induced pluripotent stem cells with their application in tracking organoid development

Islet organoids hold significant promise as a renewable source of insulin-producing cells for diabetes therapy; however, an efficient system for real-time tracking and dynamic capture of the developmental processes of islet organoids remains underdeveloped. Here, we report the generation of induced pluripotent stem cells (iPSCs) stably expressing enhanced green fluorescent protein (EGFP) and luciferase (Luc) via rational plasmid construction and lentiviral transduction. Using fluorescence and bioluminescence imaging, we systematically monitored the differentiation of these EGFP/Luc-iPSCs into islet organoids, demonstrating that the reporter iPSCs maintained pluripotency, stable fluorescent/bioluminescent signals, and uncompromised differentiation potential across multiple passages. The formed islet organoids consistently exhibited robust imaging signals, enabling noninvasive visualization of their spatiotemporal developmental dynamics. Our study established an innovative imaging platform that facilitates real-time, noninvasive monitoring of islet organoid morphogenesis, provides mechanistic insights into organoid differentiation pathways, and paves the way for advancing cell-based therapeutic strategies for diabetes.