Pluripotent stem cell models of mitochondrial disease

Summary In this research project, the hESCs generated by CRISPR/Cas9 mediated gene disruption, or iPCs from mitochondrial disease patient fibroblasts, will be validated as mitochondrial disease models, followed by confirmation of the impact on the targeted gene and pathway. Selected cell lines will then be differentiated to cardiomyocyte and/or neural lineages to enable comparison (with control cells) of the impact of the gene knockout on various aspects of mitochondrial and cellular function.

Project Details

Mitochondria are our cellular power plants that burn sugars, fats and proteins to generate energy. Each week in Australia a child is born with a mitochondrial disorder. Many of these children die in the first years of life and most suffer from severe disease, particularly affecting their brain and/or heart. Access to these tissues from patients is limited, making it difficult to assess the impact on mitochondrial and other pathways contributing to disease pathology.   This project will involve the generation and characterization of human pluripotent stem cell models of mitochondrial energy generation disorders. These models include human Embryonic Stem Cells (hESCs) with knockout-type mutations generated by CRISPR/Cas9 gene editing in genes known to cause mitochondrial disease, and human Induced Pluripotent Stem Cells (iPSCs) generated from mitochondrial disease patient cell lines. These pluripotent cell lines can then be differentiated into cardiac and neural cell lineages relevant to mitochondrial disease, thus enabling the study of the phenotypic rescue of novel defects, disease pathogenicity and treatment approaches.    The project aims are:     1) Characterize pathogenic pathways in relevant cell lineages by assessing the impact of OXPHOS (energy generation) defects on the mitochondrial and cellular proteome of cardiomyocytes and neural cells generated from hESCs or iPSCs, as well as the impact on mitochondrial function and cellular physiology.     3) Define the impact of targeted therapeutic strategies in these models on the cellular proteome and other markers of cellular homeostasis.     In this research project, the hESCs generated by CRISPR/Cas9 mediated gene disruption, or iPCs from mitochondrial disease patient fibroblasts, will be validated as mitochondrial disease models, followed by confirmation of the impact on the targeted gene and pathway. Selected cell lines will then be differentiated to cardiomyocyte and/or neural lineages to enable comparison (with control cells) of the impact of the gene knockout on various aspects of mitochondrial and cellular function. These may include respiration, ATP synthesis, reactive oxygen species, mitochondrial membrane potential, redox balance, cellular stress response and quantitative proteomics.