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

干细胞生物学的重大进展为有机工程的创新铺平了道路。有机类化合物 3D组织来自人类诱导多能干细胞(HiPSCs)，是通过对患者进行重新编程而产生的- 特异性fic成体细胞，如fi成纤维细胞。虽然有机化合物作为研究开发的试验台显示出巨大的前景- 光学生物学，目前生产有机化合物的方法受到对外部输入的依赖，例如 作为生长因子和小分子，它们对细胞产生不精确的影响，并产生不成熟的有机物质，从而 没有如实概括体内的生理和功能。得到的有机化合物是大小受限的，Lim- 仅限于一小部分细胞类型，通常不会发育出成熟的组织，表现出完全的 发达的器官。虽然我们之前已经证明了基因程序使有机化合物能够产生 肝脏中所有必需的细胞类型，细胞比率的可变性仍然是实现可重复性、高 优质的有机化合物。此外，由于不能可靠地指导多血统物种fi阳离子，这一缺乏阻碍了进展 多步差分的精确计时，以及无法做出确定 结果单元格类型的比率。 为了克服这些障碍，我们将结合合成生物学、发育生物学和控制理论来 设计新颖的开环和闭环遗传控制器，分别从每个细胞内部引导分化 形成独特的新3D组织：内分泌和外分泌具有DefiNed比率的带血管的胰腺器官 细胞。我们将演示这些新的有机化合物如何作为更复杂和更全面的模型 研究发育生物学原理。这项工作将引领有机化合物合成的变革。 通过将fiELD从人工添加感应化学信号转变为细胞类型的有条件的、自我定时的异位 诱导分化的转录因子的表达。基于这样的前提：1)基因传感器可以 检测fic到分化阶段的细胞类型，以及2)至少在某些重要情况下，调节表达 谱系特定的转录因子可以引导分化进入下一阶段，我们的主要假设是 细胞谱系分叉决策的反馈调节可以导致更健壮和可重复性更强的亚群 与开环方法相比，有机化合物中的比例。我们的有机化合物将含有合成显影剂 自我计时和全球协调的程序，细胞协同工作以产生所需的比率。 我们将为可用于其他差异化步骤的程序化分支决策创建一个平台 在胰腺，以及更广泛地说，到其他器官和组织类型。我们将利用这个平台来表演 新的发育研究系统地改变内分泌和外分泌细胞的比例并测量 对外/内分泌细胞及其分化和功能的影响。能够精确地改变 在研究fi-LES的基因表达前，有机类分泌体及其对靶细胞的影响将提供 fit在胰腺发育和功能障碍研究中的无价优势。

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.