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

干细胞生物学的重大进展为类器官工程的创新铺平了道路。类器官 是源自通过重编程患者产生的人诱导多能干细胞（hiPSC）的3D组织， 特定的成体细胞，如成纤维细胞。虽然类器官显示出作为研究发育的试验平台的巨大潜力， 在视觉生物学中，目前用于类器官产生的方法受限于它们对外部输入的依赖，例如 作为生长因子和小分子，它们不精确地影响细胞，并产生不成熟的类器官， 不能忠实地概括体内生理学和功能。由此产生的类器官是大小受限的，lim- 局限于一小部分细胞类型，并且通常不发育成熟的组织，其表现出完全的免疫功能。 发达的器官虽然我们以前已经证明了基因程序，使类器官产生 尽管在肝脏中存在所有必需的细胞类型，但细胞比率的可变性仍然是实现可重复的、高水平的 高质量的类器官此外，由于无法可靠地指导多谱系特异性，缺乏 多步微分的精确定时，以及无法做出确定 所产生的细胞类型的比率。 为了克服这些障碍，我们将结合联合收割机、发育生物学和控制理论， 设计新颖的开环和闭环遗传控制器，从每个细胞内单独指导分化 形成独特的新3D组织：血管化的胰腺类器官，内分泌和外分泌的比例确定 细胞我们将展示这些新的类器官如何作为更复杂和全面的模型 来研究发育生物学原理这项工作将引领类器官合成的转变 通过将领域从人工添加诱导化学信号转移到细胞类型条件性， 诱导分化的转录因子的表达。建立在1）基因传感器可以 检测特定于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 谱系特异性转录因子可以指导分化到下一阶段，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鲁棒和可再生的亚群 与开环方法相比，类器官中的比率。我们的类器官将包含合成发育 这些程序是自动定时和全局协调的，细胞一起工作以产生必要的比例。 我们将创建一个可用于其他差异化步骤的程序化分叉决策平台 在胰腺中，并且更广泛地应用于其他类器官和组织类型。我们将利用这个平台， 新的发展研究，系统地改变内分泌细胞与外分泌细胞的比例，并测量 对外分泌/内分泌细胞及其分化和功能的影响。能够精确地改变 在研究基因表达谱的同时，类器官分泌组及其对靶细胞的影响将提供 在胰腺发育和功能障碍的研究中有着不可估量的贝内。

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.