A triple independent inducible expression system for human pluripotent stem cells

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