Identifying autophagy regulators in human cellular platforms using human pluripotent stem cell models

背景：自噬是一种细胞内降解途径，对细胞生存和机体健康至关重要。自噬损伤降低细胞活力，导致多种疾病的病理，如癌症和神经变性。相反，刺激自噬在各种转基因疾病模型中是有益的，特别是在神经退行性疾病的背景下。自噬受mTOR（雷帕霉素的机制靶点）和mTOR独立通路的调节，这些通路可受化学扰动的影响。尽管越来越需要调节这一过程的治疗效益，其在人体系统中的精确调节还没有很好地阐明。包括我们之前工作的新数据表明，自噬调节存在细胞类型特异性。为了开展与人类相关的生物学研究，我们正在利用人类胚胎干细胞（hESCs）分化为等基因人类细胞类型的能力，并利用基因组编辑技术建立自噬的遗传模型。本项目将利用这些人类细胞平台，在生理水平上通过化学遗传学研究人体系统中自噬的景观。问题：这个项目解决了自噬领域一个重要的新兴问题：在生理相关的等基因人类细胞类型中，自噬的分子调节因子是什么？这可以通过我们的人类多能干细胞自噬模型来实现，该模型可以分化成具有相同遗传背景的其他相关人类细胞类型，以比较细胞特异性效应。目的：确定hesc和hesc衍生等基因细胞类型（如神经元和巨噬细胞）自噬的调控因子和途径。方法：最初的目标是通过化学遗传学方法确定hESCs中自噬的分子调节因子。将在自噬报告细胞hESCs中进行高含量的基于图像的化学筛选，使用化学基因组学集约225种具有已知细胞靶点的化合物。最重要的撞击将通过严格的自噬分析及其对mTOR活性的影响来表征。高可信度的命中将通过慢病毒shRNA敲除（或CRISPR/Cas9基因敲除，如果可行）进行遗传验证，并将进一步评估其在自噬缺陷细胞中的通路特异性作用。随后，我们将通过各种细胞生物学和生化技术研究hesc中自噬调节的新机制，并扩展这一实验范式，以阐明hesc来源的神经元和巨噬细胞的自噬调节途径。如果时间允许，该筛选将在其他hesc来源的等基因细胞类型中进行，如成纤维细胞和肝细胞，以产生人类细胞类型特异性自噬调节的资源数据集。所有用于生成上述细胞类型的分化方案均已在实验室中成功实现。总的来说，我们的目标是获得人类组织特异性自噬调节的基本相关见解，这将具有生物医学开发的潜力。结果：阐明人体系统中自噬的调节因子将为这一重要的生物学过程提供基本的见解，并将揭示改善年龄相关病变（如神经退行性疾病）中缺陷自噬通量的潜在药物靶点。因此，该项目的成果将具有基础和生物医学意义。此外，多个高影响力的研究出版物，以及可能的专利申请和合作途径都是该项目的可能成果。

Background: Autophagy is an intracellular degradation pathway essential for cell survival and organismal health. Impairment of autophagy reduces cell viability and contributes to the pathology of diverse diseases such as cancer and neurodegeneration. On the contrary, stimulating autophagy is beneficial in various transgenic disease models, particularly in the context of neurodegenerative disorders. Autophagy is regulated by mTOR (mechanistic target of rapamycin) and mTOR-independent pathways that are amenable to chemical perturbations. Despite the growing need of modulating this process for therapeutic benefits, its precise regulation in the human system is not well elucidated. Emerging data including those from our previous work indicate that there is cell-type specificity of autophagy regulation. In order to undertake human-relevant biology, we are harnessing the power of human embryonic stem cells (hESCs) for differentiating into isogenic human cell types, and have employed genome editing technologies to establish genetic models of autophagy. This project will utilize these human cellular platforms to study the landscape of autophagy via chemical genetics in the human system at a physiological level.Question: This project addresses an important emerging issue in the field of autophagy: What are the molecular regulators of autophagy in physiologically-relevant isogenic human cell-types? This can be achieved via our human pluripotent stem cell models of autophagy that can be differentiated into other relevant human cell-types having the same genetic background to compare cell-specific effects.Aims: Identify the regulators and pathways governing autophagy in hESCs and hESC-derived isogenic cell-types such as neurons and macrophages.Methodology: The initial goal is to identify the molecular regulators of autophagy in hESCs via a chemical genetics approach. A high-content image-based chemical screen using a chemogenomics set of ~225 compounds with known cellular targets will be undertaken in autophagy reporter hESCs. The top significant hits will be characterized by rigorous autophagy assays and their effects on mTOR activity. The high-confidence hits will then be genetically validated by lentiviral shRNA knockdown (or gene knockout by CRISPR/Cas9 if feasible), and will be further assessed for their pathway-specific effects in autophagy-deficient cells. Subsequently, we will investigate novel mechanisms of autophagy regulation in hESCs by various cell biology and biochemical techniques, and extend this experimental paradigm in elucidating the autophagy-regulating pathways in hESC-derived neurons and macrophages. If time permits, this screen will be undertaken in additional hESC-derived isogenic cell-types, such as fibroblasts and hepatocytes, to generate a resource dataset of autophagy regulation in human cell-type specific manner. All the differentiation protocols for generating the cell-types mentioned above have been successfully implemented in the lab. Overall, we aim to gain fundamentally-relevant insights for human tissue-specific regulation of autophagy that will have the potential for biomedical exploitation.Outcome: Elucidating the regulators of autophagy in the human system will provide fundamental insights into this essential biological process, and will also reveal potential drug targets for improving defective autophagic flux in age-related pathologies like neurodegeneration. The outcome of this project will thus be of basic and biomedical relevance. In addition, multiple high-impact research publications, and possible patent applications and collaboration avenues are likely outcomes of this project.