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Transcription factor-based differentiation of pluripotent stem cells to investigate the role of glial cells in FUS-linked ALS

Boek - Dissertatie

ALS is a MN disease in which degeneration of both upper and lower MNs causes muscle weakness, paralysis and eventually death 2-3 years after symptom onset. Besides MNs, glial cells in the CNS are also involved as their supportive function towards neurons is affected in the disease. In 10% of the cases, ALS is familial and caused by - mostly autosomal dominant - genetic mutations of which C9ORF72, SOD1, TARDBP and FUS are the most commonly mutated genes. As no cure is available yet, deeper insights are needed of the pathophysiological mechanisms responsible for the MN death in ALS. Therefore, disease models are necessary which can be both in vivo animal models or in vitro cell models. In this thesis, human iPSCs were used allowing to model ALS in a human setting. Other studies already generated iPSC-derived MNs from sporadic or familial ALS patients recapitulating key ALS features like the presence of oxidative stress, protein aggregation and axonal transport deficits. To investigate the role of glial cells in ALS, iPSC-derived astrocytes and OPCs have been generated, but these studies did not include isogenic controls and lacked FUS mutant cells. Therefore, in this thesis FUS mutant iPSCs were used - with their respective isogenic controls - and subsequently differentiated into OPCs and astrocytes for disease modelling purposes.First, a new protocol was generated for fast and efficient differentiation of iPSCs towards astrocytes via the inducible overexpression of SOX9, the key TF during astrocyte development. Those iSOX9-astrocytes showed important functional characteristics such as glutamate uptake, cytokine/growth factor secretion and neuronal support. Next, FUS mutant iPSCs were differentiated to astrocytes via SOX9 overexpression and to OPCs via SOX10 overexpression as described previously by our lab. Before differentiating the cells, gene-editing approaches were applied on the iPSCs. The technique RMCE was used to insert the coding sequence of SOX9 or SOX10 under an inducible promotor in the safe harbour AAVS1 locus. In this way, the TF could be overexpressed in a clean and controlled way avoiding lentiviral transduction to which the FUS mutant cells were more vulnerable. Next to a patient-derived FUSR521H mutant iPSC line with its isogenic control made in a previous study, base editing was applied on healthy control iPSCs to insert a FUSP525L mutation generating another isogenic iPSC pair. Both FUS mutant iPSC lines and their isogenic controls could differentiate into OPCs and astrocytes. The FUSP525L mutant glial cells showed cytoplasmic mislocalisation of FUS, which is known to be present in FUS-linked ALS patients. Based on transcriptome analysis of FUSR521H mutant cells, ER stress and an effect on lipid metabolism was suggested due to the FUS mutation. However, upon validation, ER stress did not seem to be evidently present in FUS mutant glial cells compared to its isogenic control. After lipidomic analysis, FUSR521H mutant OPCs, but not astrocytes showed a decreased amount of membrane-associated lipids which can possibly contribute to affected myelin production.In conclusion, we set up TF-based differentiation of iPSCs as a tool to model the role of oligodendrocytes and astrocytes in FUS-linked ALS. Preliminary data suggested an effect on lipid metabolism in FUS mutant oligodendrocytes that could possibly contribute to MN degeneration in ALS.
Jaar van publicatie:2021
Toegankelijkheid:Embargoed