A Recombinase-Mediated Cassette Exchange Platform for a Triple Independent Inducible Expression System for Human...

Human pluripotent stem cells (hPSCs) and their differentiated derivatives represent valuable tools for studying development, modeling diseases, and advancing cell therapy. Recent improvements in genome engineering allow for precise modifications of hPSCs, further enhancing their utility in basic and translational research. Here we describe a Recombinase-Mediated Cassette Exchange (RMCE) platform in hPSCs that allows for the highly efficient, rapid, and specific integration of transgenes. The RCME-mediated DNA integration process is nearly 100% efficient, without negatively affecting the pluripotency or karyotypic stability of hPSCs. Taking advantage of this convenient system, we first established a dual inducible expression system based on the Tet-On and Cumate-On systems, allowing for the inducible expression of two transgenes independently. Secondly, we incorporated a Tet-on inducible system, driving the expression of three genes simultaneously. However, two genes also contain independent degron sequences, allowing for precise control over the expression of each gene individually. We demonstrated the utility of these systems in hPSCs, as well as their functionality after differentiation into cells that were representative of the three germ layers. Lastly, we used the triple inducible system to investigate the lineage commitment induced by the pancreatic transcription factors NKX6.1 and PDX1. We found that controlled dual expression, but not individual expression, biases hPSC embryoid body differentiation towards the pancreatic lineage by inducing the expression of the NeuroD program. In sum, we describe a novel genetic engineering platform that allows for the efficient and fast integration of any desired transgene(s) in hPSCs using RMCE. We anticipate that the ability to modulate the expression of three transgenes simultaneously will further accelerate discoveries using stem cell technology.