Development of human induced pluripotent stem cell-derived neural cultures for seizure-liability testing

Abstract

Drug-induced seizure is a major reason for compound attrition during drug development, hence testing the potential of novel agents to induce such neurotoxic events is a vital process. Currently, in vivo and in vitro animal assays are used for seizure-liability studies; yet controversy over the relevance, efficacy and cost of these methodologies has led to interest in the development of human based models, for increased translatability and data extrapolation. Human induced pluripotent stem cells (iPSCs) are a revolutionary platform for neurotoxicity testing. However, considerable variation in culturing protocols, growth media and analytical techniques exists, with no validated standard for drug-induced seizure-liability testing. In this thesis, this cutting-edge iPSC technology, in combination with concurrent morphological and functional analysis has considered several methods for generation of a robust, reproducible human seizure-liability model, capable of responding to ionic and pharmacological stimuli. Spontaneously differentiated neural cultures display electrical activity, but sporadic epileptiform activity, as observed with fluorescent calcium imaging. Moreover, weak functional activity and longevity and the absence of characteristic seizure-like activity was observed in isolated monocultures of neurons and astrocytes. Various co-culture protocols were developed and tested, displaying greater baseline activity, network interconnectivity and responses to pro-convulsant conditions than spontaneously differentiated cultures; highlighting the absolute requirement for both cell types to be present in culture. Final experiments introduced interneuronal populations to the established co-culture protocol; with preliminary results highly suggestive of providing a robust system which can be used for widespread seizure-liability assessment. This thesis provides the first comparison of iPSC-derived culture methods for seizure-liability testing, whilst factoring in several variables which currently exist in the literature; including growth medium, duration of differentiation and methods to control cell proliferation. In addition, a proposal for a validatory panel of pro-convulsant conditions for the inclusion of human iPSC-derived platforms in safety pharmacology studies is presented.