Induced Pluripotent Stem Cell-Derived Human Glutamatergic Neurons as a Platform for Mechanistic Assessment of Inducible Excitotoxicity in Drug Discovery

Abstract

Since the guiding principles of Replace, Reduce, and Refine were published, wider context-of-use for alternatives to animal testing have emerged. Induced pluripotent stem cell-derived human glutamatergic-enriched cortical neurons can be leveraged as 2-and 3-dimensional platforms to enable candidate drug screening. Uniquely so, 2-dimensional models are useful considering that they exhibit spontaneous firing, while, 3-dimensional models show spontaneous synchronized calcium transient oscillations. Here, the limitations of selected induced acute seizure models as well as the early utilization of fully differentiated glutamatergic neuron models for interrogation of inducible excitotoxicity following exposure to neuromodulators will be described. The context of use for candidate biomarkers of inducible seizure is also discussed.

Vesicular Glutamate Release from Feeder-FreehiPSC-Derived Neurons

“…On the other hand, dysregulated glutamate transmission is crucially involved in neuron damage in acute pathological conditions such as stroke or ischemia, as well as in chronic neuron damage in neurodegenerative diseases [81]; indeed, endpoints related to glutamatergic transmission are of primary relevance in the developmental neurotoxicity adverse outcome pathways [82]. Accordingly, a great deal of interest is focused on human-induced pluripotent stem cells towards glutamatergic neurons as a platform for mechanistic assessment of excitotoxicity or neurotoxicity, as seizure models, or in drug discovery [83][84][85]. It must be remembered that modes for neuronal communication through glutamatergic signaling are various, including exocytotic Ca 2+ -dependent vesicular glutamate release and Ca 2+ -independent modes, such as the function of transporters including the excitatory amino acid transporters [86,87] or the cystine-glutamate exchange transporter [88] and the efflux through receptor channels or accessory proteins [89,90].…”

“…Notably, the expression of V-Glut3 (present also in non-glutamatergic neurons; [96][97][98][99]) may indicate the ability of this protocol to differentiate hiPSCs into neurons co-releasing glutamate and other neurotransmitters such as acetylcholine, GABA, or serotonin; this may be subject to future research. The new methodological approach to achieve feeder-free neuronal differentiation from hiPSCs, avoiding inter-species contamination, may therefore represent a step-ahead not only in experimental neuroscience but also in neurotoxicology and neurodevelopmental toxicology as a platform for mechanistic assessment of excitotoxicity/neurotoxicity or for drug discovery [82][83][84][85]. In fact, our study is an attempt to develop a protocol allowing us to obtain a human neuron population suitable for in vitro modeling of brain disorders and new therapeutic approaches against the disorders.…”

Various facets of excitotoxicity

“…Primary cultures are limited in their lifespan, as they become senescent, and are subject to cell heterogeneity and animal background interference [67,69], which may be the cause of the diverse experimental conditions obtained in the field (Figure 2A and B). In addition, the study of alternative methods to primary cultures has been implemented in order to make research more appropriate to the animal user's guiding principles regarding the 3 R's-Replace, Reduce, and Refine [70].…”