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

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.