Establishing human pluripotent stem cell-based platforms with autophagy readouts to investigate the role of autophagy in cellular homeostasis and disease

Seranova, Elena ORCID: 0000-0002-5395-1737 (2021). Establishing human pluripotent stem cell-based platforms with autophagy readouts to investigate the role of autophagy in cellular homeostasis and disease. University of Birmingham. Ph.D.

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Abstract

Autophagy is a vital homeostatic pathway essential for cellular survival and human health. It primarily functions as an intracellular degradation process for the turnover of unwanted cytoplasmic materials such as aggregation-prone proteins, damaged organelles and invading pathogens. Defective autophagy contributes to the pathology of a myriad human diseases including neurodegeneration and infection, and thus therapeutic exploitation of autophagy presents an attractive opportunity. The work described in this thesis involves establishment of human embryonic stem cell (hESC) models of autophagy for studying its homeostatic role and developing screening platforms in physiologically-relevant human cell-types of interest. The autophagy-deficient (ATG5–/–) hESC-based system was used to address how loss of autophagy leads to cytotoxicity; an important question relevant to neurodegeneration. The ATG5–/– hESCs and hESC-derived neurons exhibited basal cytotoxicity accompanied by a specific metabolic defect. Depletion of NADH was found to be mediating cytotoxicity during autophagy deficiency via mitochondrial depolarisation, since boosting cellular NADH levels improved cell viability and mitochondrial membrane potential in ATG5–/– neurons. This intervention was also cytoprotective in a human induced pluripotent stem cell (hiPSC)-based disease model of a neurodegenerative lysosomal storage disorder associated with autophagy dysfunction, and thus could be relevant for related neurodegenerative conditions. The autophagy reporter (mCherry-EGFP-LC3) hESC-based system was developed for identifying potent autophagy inducers enhancing pathogen clearance in macrophages; relevant for combating infection. The hESC-derived macrophages were characterised to confirm their cellular identity. These isogenic platforms can be of basic and biomedical relevance for mechanistic studies of autophagy regulation including human cell-type specific effects, and drug discovery of autophagy modulators for therapeutic applications in human diseases.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):