Understanding the variation of induced β-cell differentiation.

项目摘要 胰腺β细胞对于血糖的调节是必不可少的。糖尿病治疗的一个主要希望 是从患者来源的多能细胞产生大量功能性的可移植β细胞。 在过去的十年中，已经开发了一些体外方案来分化人多能干细胞， 细胞（hPSC）转化为功能性β样细胞，这也是研究人类 胰腺的发育，以揭示相关疾病的病因。然而，使用的主要限制 在β细胞分化系统的研究和治疗方面，目前的方案还不稳健。(i)的 分化产生异源细胞群体;（ii）分化效率在 不同的hPSC系，以及批次之间。(iii)由此产生的β样细胞仍然不完全等同于 在分子和生理水平上对来自人类胰岛的原代β细胞进行了研究。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一个可靠的地图， 在这个系统中的决定。重要的是，我们将首次绘制个体变异图， 在24个hPSC系的分化之间。为了确保可靠的比较，我们设计了一个汇集- 多路分离单细胞基因组方法，其允许同时定位多个hPSC系， 一个scRNA-seq或scATAC-seq实验。该策略最大限度地减少了批次差异， 降低了实验成本。在目标1中，我们将使用这种方法和scRNA-seq来绘制动态图， 以及在将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，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筛选和基因座特异性基因组编辑， 在基因和增强子水平验证关键分化调节因子。这个项目是建立在丰富的 一组已发表的和初步的数据，这已经导致了改进的分化方案和更好的 了解疾病遗传学。该项目的完成将提供全面的数据资源 转录组，表观基因组和三维基因组在β细胞分化，这将揭示 疾病的病因，并揭示新的治疗机会。

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.