Abstracts

Rationale: Despite a boom in approved anti-seizure medications (ASM) over the last 25 years the proportion of patients with drug-resistant epilepsy has not improved. In the current discovery paradigm, candidate ASMs are tested for efficacy against selected seizure types in acute-seizure animal models. However, drugs shown to be effective in animal models often fail to translate to efficacy in humans with drug resistant epilepsy. This discrepancy underscores the importance of early human-based assays in pre-clinical drug discovery. Drug screening using patient derived pluripotent stem cell technologies offers the possibility of personalized medicine for tailored selection of ASMs for individual patients. Here we present a human induced pluripotent stem cell (iPSC)-derived cortical neuron screening platform to test compounds with known anti-seizure properties.

Methods: We generated cortical neurons from iPSCs following activation of the Neurogenin 2 (NGN2) gene, and electrical activity was assessed by multi-electrode array (MEA) in multiwell format. The neurons were co-cultured with primary human astrocytes for synchronised network activity, and 4-aminopyridine (4-AP) was used to initiate epileptiform neural activity. ASMs were then assessed for their effects on both baseline and epileptiform neural activity. We assessed changes in MEA parameters including individual action potential (spike) data and burst properties in terms of duration, frequency and amplitude. We tested the effects of approved ASMs at range of concentrations (1 µM to 200 µM) with known mechanisms of action including Phenytoin, Carbamazepine, Tiagabine, Vigabatrin, Lamotrigine and Perampanel.

Results: NGN2 activation promoted the rapid differentiation of iPSCs into neurons as monitored by loss of pluripotent markers, transient PAX6 gene (neural progenitor) expression, and expression of pan-neuronal markers such as TUJ1, MAP2, ANK-G as assessed by IF and RNA-seq. Cultures also show increased burst activity following application of the GABA A receptor antagonist bicuculline, demonstrating presence of an inhibitory neural population. In the presence of astrocytes, synchronised network activity (burst across multiple electrodes) was observed after 4 weeks of in vitro culture (Figure 1). 4-AP produced reproducible epileptiform activity as demonstrated by increased burst rate and gain of overall neural excitability compared to baseline activity. All ASMs tested were able to mitigate 4-AP induced epileptiform activity in a concentration-dependent manner. A reduction in burst frequency was the principal component of this control (Figure 1).

Conclusions: We have developed a rapid human neuronal screening platform for ASM compounds. We have demonstrated that  approved ASMs are able to mitigate induced-epileptiform activity. This system can be used to screen pre-clinical compounds for the identification of new ASMs. As our platform is based on differentiation from human iPSCs, it can potentially be applied to drug-resistant patient derived iPSCs to allow personalized drug screening for individual patients.

Funding: Please list any funding that was received in support of this abstract.: Medical Research Future Fund (MRFF) Stem Cell Therapies Mission.