Abstract
Valproate (VPA) is a widely prescribed drug, with primary indications of bipolar disorder, epilepsy, and migraines. VPA can induce teratogenicity and hepatotoxicity. Despite decades of clinical usage, the mechanisms of action for this neuropsychiatric drug are poorly understood. One established property of VPA is direct inhibition of histone deacetylases, altering transcription across the genome. This project developed an experimental workflow involving undifferentiated human induced pluripotent stem cells (iPSCs) as a novel cellular model for studying transcriptome-wide impacts of VPA exposure via nanopore RNA-sequencing. Undifferentiated iPSCs may model the teratogenicity of VPA and were selected for this project as a pilot study preceding possible future work in other cell types differentiated from iPSCs. For this pilot study, it was hypothesised that acute exposure of undifferentiated human iPSCs to VPA would result in gene expression changes that reflect therapeutic and adverse activities of VPA. A therapeutically relevant dose (0.5 mM) of VPA was administered for 4 h and 24 h durations to undifferentiated KOLF2.1J, a well-characterised human iPSC line. Undifferentiated KOLF2.1J were screened for VPA-induced effects on key pluripotency markers. This project optimised methods of culturing undifferentiated KOLF2.1J in the presence of VPA, and purifying RNA from adherent cells. High-quality RNA samples were collected from four independent VPA exposure experiment replicates. Issues during several attempts to reverse-transcribe these RNA samples led to a preponderance of short-length transcripts in the resulting nanopore-sequenced dataset. Differential expression analysis of this dataset through a bioinformatic pipeline was supplemented by sensitivity testing and further filtration. This analysis did not identify any genes with statistically significant VPA-induced differential expression, though the top candidate genes tending towards significance (p < 0.05 < p-adjusted) suggested may be interesting to test with greater sensitivity in future validation datasets. Some statistically significant (p < 0.05) differential exon usage on the transcript level suggested that both durations of VPA exposure may promote alternate mRNA isoforms for several transcripts. However, the statistical significance of these mRNA isoform switching effects did not persist when grouped by gene then subjected to post-analysis corrections (α=0.05). Validation is necessary, especially due to dataset limitations. Overall, this project demonstrated the potential of KOLF2.1J as a cellular model for exploring gene expression effects of VPA. Future research may adapt the methods optimised during this project to study VPA in KOLF2.1J cultures differentiated to specific cell types or three-dimensional organoids, for versatile modelling and enhanced exploration of pharmacogenomic and neuropsychiatric imperatives.