Synthetic gene circuits for cell state detection and protein tuning in human pluripotent stem cells

During development, cell state transitions are coordinated through changes in the identity of molecular regulators in a cell type‐ and dose‐specific manner. The ability to rationally engineer such transitions in human pluripotent stem cells (hPSC) will enable numerous applications in regenerative medicine. Herein, we report the generation of synthetic gene circuits that can detect a desired cell state using AND‐like logic integration of endogenous miRNAs (classifiers) and, upon detection, produce fine‐tuned levels of output proteins using an miRNA‐mediated output fine‐tuning technology (miSFITs). Specifically, we created an “hPSC ON” circuit using a model‐guided miRNA selection and circuit optimization approach. The circuit demonstrates robust PSC‐specific detection and graded output protein production. Next, we used an empirical approach to create an “hPSC‐Off” circuit. This circuit was applied to regulate the secretion of endogenous BMP4 in a state‐specific and fine‐tuned manner to control the composition of differentiating hPSCs. Our work provides a platform for customized cell state‐specific control of desired physiological factors in hPSC, laying the foundation for programming cell compositions in hPSC‐derived tissues and beyond. Synopsis Synthetic gene circuits with sense‐and‐respond capabilities were designed to detect cell states and perform cell‐state‐specific control of desired protein expression in human pluripotent stem cells (hPSCs). Circuits that detect the hPSC state (hPSC‐On) were created using automated design and a model‐guided circuit optimization process. The optimized circuits were merged with miSFITs fine‐tuning technology and showed hPSC‐specific actuation with precise control of the protein expression levels in hPSC. An empirical approach was used to create circuits that are repressed in hPSC (hPSC‐Off) and applied as proof of concept for cell composition control of the three germ layers. Graphical Abstract Synthetic gene circuits with sense‐and‐respond capabilities were designed to detect cell states and perform cell state‐specific control of desired protein expression in human pluripotent stem cells (hPSCs).