ChemPerturb-Seq Screen to Identify Small Molecules Enhancing Human Beta Cell Survival After Subcutaneous Transplantation

Although high throughput/content chemical screens have been employed to characterize cellular response to small molecules treatment, traditional chemical screens have only focused on a single assay per screen, making them labor intensive and costly. Here, we combined a chemical screen with scRNA-seq to perform Chemical Perturb-Seq (ChemPerturb-Seq), enabling a systematic analysis of cellular responses and molecular changes of human beta cells upon individual hormone treatments. Furthermore, we developed an AI-powered website, ChemPerturbDB, which provides user friendly open-access analysis of this extensive dataset. Next, we performed an in vivo barcoded screen and developed a hormone cocktail, including Insulin growth factor-1, Lipotropin, and Prostaglandin E2. Pre-conditioning human beta cells and primary islets with this hormone cocktail significantly enhanced their function and survival when transplanted subcutaneously to female, but not to male mice. Combining scRNA-seq and ChemPerturb-seq, we identified two additional moleculeshormones, serotonin and histamine, that promote the function of human islets when transplanted subcutaneously to male mice. Together, we not only generated a comprehensive ChemPerturb-Seq dataset, which can be utilized to systematically investigate the effects of hormones on human beta cells, but also developed hormone cocktails that enhance the survival of human beta cells following subcutaneous transplantation. With further validation, this, which could be applied to improve the current FDA-approved islet transplantation procedure. Multiplexed scRNA-seq analysis of human EndoC-betaH1 cells treated with different small molecules from an in-house small molecule screening library

尽管高通量/高内涵化学筛选已被用于表征细胞对小分子处理的应答，但传统化学筛选仅针对单重检测，导致实验耗时耗力且成本高昂。本研究将化学筛选与单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）相结合，建立了化学扰动测序（Chemical Perturb-Seq, ChemPerturb-Seq）技术，从而能够系统分析人类β细胞在单个激素处理后的细胞应答与分子变化。此外，本研究还开发了一款人工智能赋能的网站ChemPerturbDB，可为该大规模数据集提供友好的开放获取分析功能。随后，我们开展了体内条形码筛选，并开发了一种激素鸡尾酒疗法，其组分包括胰岛素样生长因子-1（Insulin growth factor-1）、促脂素（Lipotropin）和前列腺素E2（Prostaglandin E2）。用该激素鸡尾酒预培养人类β细胞与原代胰岛，可在将其皮下移植至雌性小鼠时显著提升其功能与存活能力，但对雄性小鼠无此效果。结合单细胞RNA测序与ChemPerturb-seq技术，我们还鉴定出另外两种分子激素——5-羟色胺（serotonin）与组胺（histamine），它们可在将人类胰岛皮下移植至雄性小鼠时提升胰岛功能。综上，本研究不仅构建了可用于系统探究激素对人类β细胞影响的完整ChemPerturb-Seq数据集，还开发了可提升人类β细胞皮下移植后存活率的激素鸡尾酒疗法。经进一步验证后，该疗法可用于优化目前经美国食品药品监督管理局（Food and Drug Administration, FDA）批准的胰岛移植流程。本研究还针对用内部小分子筛选库中的不同小分子处理的人类EndoC-βH1细胞开展了多重单细胞RNA测序分析。