Using induced pluripotent stem cells for modeling POLG mitochondrial disease : Stamceller som modellsystem for å behandle mitokondriesykdom

Background: More than 90% of the energy required to sustain life is provided by mitochondria through the process of oxidative phosphorylation (OXPHOS). The role of mitochondria, however, is not restricted to supplying cellular energy; they are also involved in many other cellular processes including differentiation and cell death. Mitochondrial functional impairment is also associated with a wide spectrum of devastating diseases known as mitochondrial diseases. Among the most common mitochondrial disorders are POLG-related diseases. These comprise a large number of different phenotypes with age of onset ranging from infancy to adulthood. These complicated and incurable disorders are caused by mutations in the POLG gene which encodes the catalytic subunit of DNA polymerase gamma (POLG). The enzyme POLG is involved in mitochondrial DNA (mtDNA) replication, and despite being present in all cells, the main disease manifestations show tissue and cell type-specificity. The patho-mechanisms underlying POLG-disease remain poorly documented mostly due to the lack of reliable animal models and limited access to affected tissues. Human induced pluripotent stem cells (iPSC) with the capacity of self-renewal and differentiation into all relevant tissues provide a promising tool for modeling POLG-related diseases and investigating possible treatments. Primary clinical experiments have shown that the high energy demanding tissues such as brain, liver and skeletal muscle are severely affected, however, cardiac tissue appear clinically unaffected. Understanding this paradox is important as it can increase our understanding of the tissue specific nature of these diseases. Aim: The main aim of this project was to model POLG-related disease using iPSCs derived from patient fibroblasts and differentiated into different cell types. We planned to differentiate them to cardiac and neuronal cells to investigate the impact of POLG mutations on mitochondrial function. Methods: In paper I, we modified neuronal differentiation protocols to generate neural stem cells (NSC), and investigate the impact of POLG mutation on mitochondrial function by comparing different mitochondrial parameters in control and mutant NSCs. We employed different methods including flow cytometry, PCR, western blotting and Liquid Chromatography/Mass Spectrophotometry (LC/MS) to investigate the mitochondrial content, mtDNA level, respiratory chain complexes and NAD+ metabolism, ROS generation and activation of mitophagy. In the second project, we established a high throughput method for differentiating cardiomyocytes in 96well plate format in order to monitor mitochondrial changes during early stages of cardiac differentiation (paper 2). In the third paper, we differentiated human pluripotent stem cells (PSC) towards mesoderm, cardiac progenitors and later to cardiomyocytes using the protocol established in paper II. We used this to investigate changes of mitochondrial content and mtDNA copy number during early stages of mesoderm differentiation using different methods including flowcytometry and qPCR. We also studied mitochondrial function and metabolic remodeling by seahorse analysis and flow cytometry. Results: The comparison of different mutant and control cell types including fibroblasts, iPSCs and NSCs in paper 1 showed that only NSCs manifested all the features that were seen in patient post-mortem tissues including mtDNA depletion and complex I deficiency. Using our iPSC-derived NSC model, we also showed the impact of POLG mutation on the overproduction of ROS and impairment of NAD+ metabolism, and how this led to increased cellular senescence. In the second part of the study, we established a high-throughput cardiomyocyte differentiation protocol with low variation of differentiation efficiency between wells and between runs of differentiation. We also showed that the differentiated cardiomyocytes generated by our micro plate format were fully functional and expressed the correct cardiomyocyte markers. We used this protocol in the third project to study the mitochondrial changes during early mesoderm differentiation towards cardiac lineage. We confirmed the previous reported metabolic remodeling during mesoderm differentiation, however, in contrast to previous studies, and our expectations, we showed that mitochondrial content and mtDNA copy number decreased during the early stages of cardiomyocyte differentiation. Conclusion: Our NSC model of POLG disease is the first that faithfully replicates the findings observed in patient post mortem tissues. Using this model, we showed that NSC developed mtDNA depletion and complex 1 deficiency and we confirmed the metabolic impact of this by demonstrating the changes in the NAD+/NADH ratio. We could also examine the downstream consequences of POLG mutation on aspects of mitochondrial function such as ROS production and show that the combined effects led to increased cellular senescence. Considering the regenerative capacity of NSCs, our aim is to use this robust model system for drug screening and identification of possible treatment for POLG diseases. In our work with cardiomyocytes, we showed that our method using the 96 well microplate format was robust and efficient with low inter well variation. This means that it too can be used for high throughput experiments such as studying the early stages of cardiac development and drug screening. In contrast to earlier reports, we detected a significant reduction in mitochondrial mass and mtDNA level during mesoderm differentiation towards cardiac lineage. Despite the remarkable mitochondrial reduction, we showed that differentiated cells nevertheless had a higher capacity to generate energy through OXPHOS. Overall our results suggested a unique mitochondrial remodeling process in which the metabolic switch from glycolysis to OXPHOS occurs without an increase in mitochondrial mass and mtDNA level. In the other words, metabolic remodeling is associated with mitochondrial maturation and increased mitochondrial activity rather than elevated mitochondrial mass and mtDNA level