Cell-State Specific Digital to Analog Signal Conversion in Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSC), due to their ability to self-replicate and differentiate in vitro, are an ideal cell source for generation of cell-based therapeutics and as a model system for drug screening and basic research. Controlling hPSC through synthetic gene circuits has the potential to tackle key challenges in the field and can increase our understanding of basic cellular and molecular mechanisms. To achieve advanced cell state control, regulatory- proteins or RNAs often need to be i) restricted to a desired cell state- and ii) produced at defined physiological amounts. While previous synthetic circuits have achieved either digital cell-state specific actuation or analogue output tuning, none have combined these functionalities in hPSC or other human cells. Here, we have created a first generation of digital to analog converter in hPSC that operate entirely on endogenous miRNAs. Our digital to analog converter is based on a bow-tie framework composed of a digital input module, that detects multiple endogenous miRNAs, integrates them in an AND-like logic function and, in turn, controls an analog output module controlled by a library of miRNA17 target site variants. To implement this circuit in hPSC, we used a previously established automated design toolbox to search for a set of miRNAs and a circuit topology that maximizes output expression in hPSC compared to other cell types in the testing space using published miRNA Seq data. We then optimize logic processing of the identified miRNAs using a model-guided approach and finally validate cell-state specific analog processing in hPSC. We then apply the circuit to fine tune BMP4, a morphogen that is required to induce peri-gastrulation like pattern formation in vitro and demonstrate BMP4-dose dependent pattern formation in the absence of BMP4 in the culture media.