Engineering a multistable MINPA gene network to regulate cell fate determination

Engineering a multistable MINPA gene network to regulate cell fate determination Engineering of a multistable MINPA gene network in Escherichia coli Cardiac cells derived from induced pluripotent stem cells have the potential to be used to treat cardiovascular injuries. However, successful stem cell therapies require accurate and quantitative understanding of the cell differentiation process. Cell differentiation is a common biological manifestation of cellular state determination in a multistable system, a system with two or more mutually exclusive states. Nonlinear multistability has long been proposed as the underlying mechanism that cells use to maintain pluripotency and guide differentiation. So fundamental understanding of the state determination and transition dynamics in a multistable system can help reveal the principles of cell fate choice and identify optimal routes of cell reprogramming. Here, we propose to use synthetic biology approaches to engineer a multistable (at least three stable steady states) topology, a mutual inhibitory network with positive autoregulations (MINPA) in Escherichia coli. Our preliminary results indicated that the assembled MINPA could support four stable steady states with induction in living cells. Based on that, we plan to develop a more elaborate model to help quantitatively understand the three-dimensional landscape of MINPA system and guide experimental design. As a hallmark of multistability, hysteresis will be performed experimentally to calibrate model parameters and identify the multistable region. Next, mathematical analysis with experimental validation will be combined to realize manipulation of states transitions including single state transition from each other and achievement of multistability from different initial states. Finally, we will investigate the temporal dynamics using microfluidics platform to visualize and verify the role of mutual inhibition in MINPA in determining state transition routes at the single-cell level. Our well-developed experimental and computational tools with established microfluidics platforms can help realize our goal of understanding the fundamental mechanisms generating multistability and state transition dynamics. Results from the proposed forward engineering studies can help reveal the underlying principles of cell decision-making and cell fate choice in the high-dimensional, multistable cellular systems. This will, in turn, lead to potential biotechnology applications and new perspectives on stem cell differentiation related to heart development, cardiac tissue engineering, and therapeutic intervention.