Genetically Programmed Pancreatic Organoids with Self-Adaptive Multi-Lineage Population Control

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Major advancements in stem cell biology have paved the way for innovation in organoid engineering. Organoids are 3D tissues derived from human induced pluripotent stem cells (hiPSCs) generated by reprogramming patient- specific adult cells, such as fibroblasts. While organoids show great promise as testbeds for investigating devel- opmental biology, current methods for organoid production are limited by their reliance on external inputs, such as growth factors and small molecules, which affect cells imprecisely and give rise to immature organoids that do not faithfully recapitulate in vivo physiology and functionality. The resulting organoids are size-constrained, lim- ited to a small set of cell types, and do not generally develop mature tissue that exhibits the functionality of fully developed organs. While we have previously demonstrated genetic programs that enable organoids to generate all requisite cell types in liver, variability in cell ratios remains an open challenge for achieving reproducible, high quality organoids. Further, progress is blocked by the inability to reliably guide multi-lineage specification, the lack of precise timing of multistep differentiation, and the inability to make robust bifurcation decisions that determine the ratios of the resulting cell types. To overcome these obstacles, we will combine synthetic biology, developmental biology, and control theory to design novel open and closed loop genetic controllers that individually guide differentiation from within each cell to form unique new 3D tissue: vascularized pancreatic organoids with defined ratios of endocrine and exocrine cells. We will demonstrate how these new organoids can serve as more sophisticated and comprehensive models for investigating developmental biology principles. This work will spearhead a transformation in organoid synthesis by shifting the field from manual addition of inductive chemical signals to cell type conditional, self-timed ectopic expression of transcription factors that induce differentiation. Building upon the premise that 1) gene sensors can detect cell types specific to differentiation stages, and 2) at least in certain important cases, regulated expression of lineage-specifying transcription factors can guide differentiation to the next stage, our main hypothesis is that feedback regulation of cell lineage bifurcation decisions can lead to more robust and reproducible sub-population ratios in organoids in comparison to open loop approaches. Our organoids will contain synthetic developmental programs that are self-timed and globally-orchestrated, with cells working together to generate the requisite ratios. We will create a platform for programmed bifurcation decisions that can be used for other differentiation steps in the pancreas, and more broadly to other organoid and tissue types. We will use this platform to perform novel developmental studies to systematically vary the ratio of endocrine to exocrine cells and measure the consequences on exocrine/endocrine cells and their differentiation and function. The ability to precisely vary the ratio while studying gene expression profiles, the organoid secretome, and its affects on target cells will provide invaluable benefit in the investigation of pancreas development and dysfunction.