Polygenic risk score reveals mitochondrial transporter as new target in schizophrenia astrocytes: Optimizing iPSC-research for genetically complex diseases

The current thesis began with determining the most ideal study population for SCZ. We decided to use polygenic risk scores (PRS) to select SCZ cases and controls. Specifically, we selected 10 patients with high PRS and 10 controls with low PRS. The use of PRS leads to genetically more extreme groups of cases and controls which potentially increases statistical power by increasing the phenotypic differences between the two groups without a priori knowledge of potential causal pathways. In addition, we also selected patients carrying specific, rare alleles of copy number variants (CNVs) that have been shown to have a relatively large effect on the risk for SCZ. This allows us to confirm any findings from the PRS cases/controls in a different genetic background, which would provide more widespread support for a causal role of our findings. The next step in the optimization of our model was finding an appropriate reprogramming method to generate iPSCs for the selected subjects. Based on Chapter 3, we have chosen to reprogram the case and control fibroblasts episomally, as this does not lead to integration in the genome, and as it is a reliable but cost-efficient technique. Episomal reprogramming is widely used, and plasmids are freely available at Addgene at low cost. Still, in Chapter 3 we show that there is a difference between the episomal and lentiviral reprogramming when generating iPSCs from female donors: lentiviral reprogramming is able to induce X-chromosomal reactivation, while episomal reprogramming was not able to reactivate the inactive X-chromosome. However, there was no difference in pluripotency between the two methods. Since the use of episomal reprogramming did not compromise pluripotency and since the episomal plasmids do not integrate and therefore cannot alter the genetic architecture of our iPSCs, this was in our case the favored method to derive iPSCs from study subjects. In Chapter 4 we analyzed the variability of our iPSC-derived astrocytes. More specifically, we investigated the variability throughout reprogramming and differentiation, giving insight in the origin of variability of our protocol. Three healthy donors were selected for reprogramming, where we included three iPSC-lines per donor. These lines were differentiated into astrocytes, one line per donor was differentiated three times in parallel. This set up allowed us to determine the source of the variability between our iPSC-derived astrocytes. We found that the magnitude of variability between clones remains stable throughout the culturing process. Moreover, we show that iPSCs and astrocytes derived from the same donor (clones) are more strongly correlated than iPSCs and astrocytes derived from different donors. This confirmed previous reports that genetic background is the main source of variability between samples. Moreover, there was no cell type specific protein set responsible for the variation between clones. Accumulation of mutations after reprogramming and long-term cultures did not explain the variability between clones in the current sample. Based on the correlations seen in this chapter, power simulations revealed that the use of multiple clones per donors would result in very limited gain in power, while the inclusion of more donors had much more impact on the statistical power to detect phenotypic differences between groups. Using the optimized experimental set up described above, we generated iPSC-derived astrocytes using an established protocol. When cultured in isolation, astrocytes did not reveal any differences between patients and controls for any of the tested phenotypes. Transcriptome analysis revealed 10 DEGs between patients and controls, with SLC25A18 as most statistically significantly down regulated in patients. In addition, SCZ astrocytes did induce a decrease in VGAT puncta in comparison to VGLUT2 or synaptophysin puncta. This suggests an impaired E/I balance as typically reported in SCZ.