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

具有自适应多谱系群体控制的基因编程胰腺类器官

• 批准号: 10470862
• 负责人: RON WEISS
• 金额: $ 64.65万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10470862
• 关键词: 3-Dimensional; Address; Adult; Affect; Biological; Biology; Blood Vessels; Cell Lineage; Cells; Chemicals; Complex; DIF factor; Development; Developmental Biology; Ectopic Expression; Endocrine; Endoderm; Endoderm Cell; Endoribonucleases; Engineering; Exhibits; Feedback; Fibroblasts; Functional disorder; Gene Expression; Genes; Genetic; Genetic Engineering; Growth Factor; Hepatic; Human; In Vitro; Individual; Investigation; Lead; Liver; Manuals; Measures; Mesoderm; Methods; Modeling; Organ; Organogenesis; Organoids; Output; Pancreas; Patients; Physiology; Population; Population Control; Production; Protocols documentation; Regulation; Reproducibility; Research; Signal Transduction; Specific qualifier value; Structure; System; Techniques; Tissues; Work; base; cell type; control theory; design; developmental plasticity; in vivo; induced pluripotent stem cell; innovation; novel; pancreas development; progenitor; programs; ratiometric; recombinase; sensor; single-cell RNA sequencing; small molecule; stem cell biology; stem cell differentiation; synthetic biology; transcription factor

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.

干细胞生物学的主要进步为器官工程创新铺平了道路。器官 是由人类诱导的多能干细胞（HIPSC）得出的3D组织，该组织通过重编程患者而产生 虽然器官在研究开发的测试中表现出巨大的希望 - Opmental Biology，类器官生产的当前方法受到对外部投入的依赖的限制，例如 作为生长因子和小分子，影响细胞的暗示并引起未成熟的类器官 不忠实地概括体内生理和功能。由此产生的类器官是尺寸约束的， 到一系列的细胞类型中，通常不会形成成熟的组织，表现出完全 发达的器官。虽然我们以前展示了遗传程序，使类器官能够生成 肝脏中所有​​必要的细胞类型，细胞比率的变异性仍然是实现可再现的高度挑战 优质的类器官。此外，由于无法可靠的指南多部规范而阻碍了进度 多步分化的精确时机，以及无法做出强大的分叉决定来决定 所得细胞类型的比率。 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 设计新颖的开放和闭环遗传控制器，它们单独指导与每个单元格内的分化 形成独特的新3D组织：具有定义的内分泌比定义的血管化胰腺机体 细胞。我们将展示这些新的类型器可以用作更复杂和全面的模型 用于研究发育生物学原理。这项工作将带头构成器官合成的转换 通过将电场从手动添加归纳化学信号转移到有条件的，自切的ecopic 影响分化的转录因子的表达。以1）基因传感器可以的前提为基础 检测特定于分化阶段的细胞类型，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与下一阶段的区分，我们的主要假设是 细胞谱系分叉决策的反馈调节可能会导致更健壮和可重复的亚群 与开放环方法相比，类器官的比率。我们的类器官将包含合成的发育 经过自定位且全球策划的程序，细胞共同工作以生成必要的比率。 我们将创建一个用于编程分叉决策的平台，可用于其他差异步骤 在胰腺中，更广泛地针对其他器官和组织类型。我们将使用此平台执行 新的发育研究与系统地改变了内分泌与外分泌细胞的比率，并测量 外分/内分泌细胞及其分化和功能的后果。精确改变的能力 在研究基因表达方面的比率时，器官分泌组及其对靶细胞的影响将提供 胰腺发育和功能障碍的投资中有价值的好处。