Understanding the variation of induced β-cell differentiation.

了解诱导β细胞分化的变化。

• 批准号: 10646289
• 负责人: Fulai Jin
• 金额: $ 59.38万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10646289
• 关键词: 3-Dimensional; Address; Adult; Affect; Beta Cell; Biological Assay; CRISPR screen; Cell Differentiation process; Cell Line; Cell Lineage; Cells; Chromatin; Data; Development; Diabetes Mellitus; Disease; Enhancers; Ensure; Epigenetic Process; Ethics; Etiology; Fetus; Genes; Genetic Diseases; Genetic Variation; Genome; Genomic approach; Genomics; Goals; Hi-C; Human; In Vitro; Individual; Intuition; Lead; Legal; Maps; Metabolic; Metabolism; Methods; Mitochondrial DNA; Modeling; Molecular; Mus; Names; Nature; Obesity; Pancreas; Patients; Physiological; Population; Protocols documentation; Publishing; Reporting; Research; Resolution; SOX17 gene; Series; Sorting; Structure of beta Cell of islet; System; TCF7L2 gene; Technology; Therapeutic; Time; Tissues; Transplantation; Untranslated RNA; Variant; Visualization software; Work; blood glucose regulation; cost; data resource; data sharing; data visualization; diabetes mellitus therapy; differentiation protocol; endodermal progenitor; epigenetic variation; epigenome; experimental study; gain of function; genome editing; genome wide association study; human embryonic stem cell; human pluripotent stem cell; improved; in vivo; individual variation; induced pluripotent stem cell; innovation; insight; islet; loss of function; novel therapeutics; pancreas development; prevent; promoter; risk variant; single-cell RNA sequencing; stem cells; tool; trait; transcriptome; web site

Project Abstract Pancreatic β-cells is essential for the regulation of blood glucose. One major hope for diabetes therapy is to generate a large number of functional, transplantable beta-cells from patient-derived pluripotent cells. In the past decade, a few in vitro protocols have been developed to differentiate human pluripotent stem cells (hPSCs) into functional β-like cells, which also serve as fantastic tools for the study of human pancreatic development to reveal the etiology of relevant diseases. However, the major limitations to use β-cell differentiation system in research and therapeutics is that the protocol is still not robust. (i) The differentiation generates heterogenous cell populations; (ii) Differentiation efficiency is variable between different hPSC lines, and also between batches. (iii) The resulting β-like cells are still not quite equivalent to primary β-cells from human islets at molecular and physiological levels. To address this problem, we propose to use the latest single cell and low-input genomic technology to generate a reliable map of lineage determination in this system. Importantly, we will for the first time map the individual variation between the differentiation of 24 hPSC lines. To ensure robust comparison, we have devised a pooling- demultiplexing single cell genomic approach that allows simultaneous mapping of many hPSC lines in one scRNA-seq or scATAC-seq experiment. This strategy minimizes the batch variation and significantly reduces the experimental cost. In Aim 1, we will use this approach and scRNA-seq to map the dynamics and variation of single cell transcriptome while differentiating 24 hPSC lines towards pancreatic β-cells. In Aim 2, we will map the dynamics and variation of open chromatin using scATAC-seq, and we will also use a low-input Hi-C technology to reveal the dynamic 3D genome during β-cell differentiation. In aim 3, we will perform high-throughput CRISPR screen and locus-specific genome editing to discover and validate key differentiation regulators at both gene and enhancer levels. This project is built upon a rich set of published and preliminary data, which already led to improved differentiation protocol and better understanding of disease genetics. Completion of this project will deliver a comprehensive data resource of transcriptome, epigenome, and 3D genome during the β-cell differentiation, which will shed light on the disease etiology, and reveal novel therapeutic opportunities.

项目摘要 胰腺β细胞对于血糖的调节至关重要。糖尿病治疗的一大希望 目的是从患者来源的多能细胞中产生大量功能性的、可移植的β细胞。 在过去的十年中，已经开发了一些体外方案来区分人类多能干细胞 细胞（hPSC）转化为功能性 β 样细胞，这也是研究人类的绝佳工具 胰腺发育揭示相关疾病的病因。然而，使用的主要限制 β细胞分化系统在研究和治疗方面的问题在于该方案仍然不健全。 （一） 分化产生异质细胞群； (ii) 分化效率在 不同的 hPSC 系，以及批次之间。 (iii) 所得的 β 样细胞仍然不太等效 在分子和生理水平上对来自人类胰岛的初级β细胞进行研究。为了解决这个问题，我们 建议使用最新的单细胞和低输入基因组技术来生成可靠的图谱 该系统中的谱系确定。重要的是，我们将首次绘制个体差异图 24 个 hPSC 系之间的分化。为了确保稳健的比较，我们设计了一个池化- 解复用单细胞基因组方法允许同时对许多 hPSC 系进行定位 一项 scRNA-seq 或 scATAC-seq 实验。该策略最大限度地减少了批次差异并显着 降低实验成本。在目标 1 中，我们将使用这种方法和 scRNA-seq 来绘制动态图 以及将 24 个 hPSC 系分化为胰腺 β 细胞时单细胞转录组的变化。 在目标 2 中，我们将使用 scATAC-seq 绘制开放染色质的动态和变化图，并且我们还将 使用低输入 Hi-C 技术揭示 β 细胞分化过程中的动态 3D 基因组。在目标 3 中， 我们将进行高通量 CRISPR 筛选和位点特异性基因组编辑来发现和 在基因和增强子水平上验证关键的分化调节因子。该项目建立在丰富的 一组已发布的初步数据，这些数据已经导致了分化方案的改进和更好的 了解疾病遗传学。该项目的完成将提供全面的数据资源 β细胞分化过程中转录组、表观基因组和 3D 基因组的研究，这将有助于揭示 疾病的病因学，并揭示新的治疗机会。