No abstract

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.

The emergence of the anterior-posterior body axis during early gastrulation constitutes a symmetry-breaking event, which is key to the development of bilateral organisms, and its mechanism remains poorly understood. Two-dimensional gastruloids constitute a simple and robust framework to study early developmental events in vitro. Although spontaneous symmetry breaking has been observed in three dimensional (3D) gastruloids, the mechanisms behind this phenomenon are poorly understood. We thus set out to explore whether a controllable 2D system could be used to reveal the mechanisms behind the emergence of asymmetry in patterned cellular structures. We first computationally simulated the emergence of organization in micro-patterned mouse pluripotent stem cell (mPSC) colonies using a Turing-like activator-repressor model with activator-concentration-dependent flux boundary condition at the colony edge. This approach allows the self-organization of the boundary conditions, which results in a larger variety of patterns than previously observed. We found that this model recapitulated previous results of centrosymmetric patterns in large colonies, and also that in simulated small colony sizes, patterns with spontaneous asymmetries emerged. Model analysis revealed reciprocal effects between diffusion and size of the colony, with model-predicted asymmetries in small pattern sizes being dominated by diffusion, and centrosymmetric patterns being size-dominated. To test these predictions, we performed experiments on micro-patterned mPSC colonies of different sizes stimulated with Bone Morphogenetic Protein 4 (BMP4), and used Brachyury (BRA)-GFP expressing cells as pattern readout. We found that while large colonies showed centrosymmetric BRA patterns, the probability of colony polarization increased with decreasing sizes, with a maximum polarization frequency of 35% at ~200μm. These results indicate that a simple molecular activator-repressor system can provide cells with collective features capable of initiating a body-axes plan, and constitute a theoretical foundation for the engineering of asymmetry in developmental systems.

During development, state transitions are coordinated through changes in the identity of molecular regulators in a cell state- and dose specific manner. The ability to rationally engineer such functions 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 discrete cell state, and upon state detection, produce fine-tuned effector proteins in a programmable manner. Effectively, these gene circuits convert a discrete (digital-like) cell state into an analog signal by merging AND-like logic integration of endogenous miRNAs (classifiers) with a miRNA-mediated output fine-tuning technology (miSFITs). Using an automated miRNA identification and model-guided circuit optimization approach, we were able to produce robust cell state specific and graded output production in undifferentiated hPSC. We further finely controlled the levels of endogenous BMP4 secretion, which allowed us to document the effect of endogenous factor secretion in comparison to exogenous factor addition on early tissue development using the hPSC-derived gastruloid system. Our work provides the first demonstration of a discrete-to-analog signal conversion circuit operating in living hPSC, and a platform for customized cell state-specific control of desired physiological factors, laying the foundation for programming cell compositions in hPSC-derived tissues and beyond.Graphical Abstract