PROJECT SUMMARY Biological stochasticity is intrinsic to the growth and developmental processes of all multi-cellular organisms. Nature utilizes regulatory networks including feedback loops to tightly regulate the ratio of stochastic versus deterministic gene expression in order to generate robust and reproducible functionality under dynamic conditions. Stochasticity can affect cellular fate decisions from bacterial persistence, HIV latency, cancer metastasis, and cellular reprograming, suggesting that the active modulation and engineering of stochastic control by changing levels of noise in gene expression can strongly influence multi-cellular patterning. We have previously identified a novel class of noise modulating compounds that exogenously control gene expression fluctuations and enhance control of single-cell fate-determination and decision-making in HIV viral circuitry (Dar et al., Science 2014). This research aims to establish the fundamentals of engineering noise in the expression of pluripotent transcription factors during stem cell aggregate differentiation and differentiation into cardiovascular organoids. In addition to exogenous treatments, synthetic gene circuits that endogenously tune both the mean abundance and noise levels of targeted pluripotent factors, such as Nanog, Oct4, and Sox2, will be implemented. A library of stem cell aggregates will be created with diverse stochastic backgrounds and differentiated into cardiovascular organoid for further characterization. This research will establish a foundation towards control of heterogeneous growth and development in other multi-cellular systems by providing fundamentals for the stochastic engineering and synthesis of complex tissue patterning. This highly interdisciplinary effort includes scientific areas relevant to the mission of the NIH such as biological, clinical, physical, chemical, computational, engineering, and mathematical sciences. The proposed areas of research combine tissue engineering, systems and synthetic biology, single-cell biophysics, and pharmaceutical sciences. The research will train and support two faculty members, a postdoc, and a graduate student for the three year term.