Human iPSC-derived neuronal models for in vitro neurotoxicity assessment

Abstract

Neurotoxicity testing still relies on ethically debated, expensive and time consuming in vivo experiments, which are unsuitable for high-throughput toxicity screening. There is thus a clear need for a rapid in vitro screening strategy that is preferably based on human-derived neurons to circumvent interspecies translation. Recent availability of commercially obtainable human induced pluripotent stem cell (hiPSC)-derived neurons and astrocytes holds great promise in assisting the transition from the current standard of rat primary cortical cultures to an animal-free alternative. We therefore composed several hiPSC-derived neuronal models with different ratios of excitatory and inhibitory neurons in the presence or absence of astrocytes. Using immunofluorescent stainings and multi-well micro-electrode array (mwMEA) recordings we demonstrate that these models form functional neuronal networks that become spontaneously active. The differences in development of spontaneous neuronal activity and bursting behavior as well as spiking patterns between our models confirm the importance of the presence of astrocytes. Preliminary neurotoxicity assessment demonstrates that these cultures can be modulated with known seizurogenic compounds, such as picrotoxin (PTX) and endosulfan, and the neurotoxicant methylmercury (MeHg). However, the chemical-induced effects on different parameters for neuronal activity, such as mean spike rate (MSR) and mean burst rate (MBR), may depend on the ratio of inhibitory and excitatory neurons. Our results thus indicate that hiPSC-derived neuronal models must be carefully designed and characterized prior to large-scale use in neurotoxicity screening.

Role of Human-Induced Pluripotent Stem Cell-Derived Spinal Cord Astrocytes in the Functional Maturation of Motor Neurons in a Multielectrode Array System

“…We observed that astrocytes influenced hiPSC‐MN electrophysiology over time with increases in spiking and bursting activity that far exceeded the activity of hiPSC‐MN cultured in the absence of astrocytes. These findings are consistent with previous observations on human iPSC‐derived cortical neurons . Moreover, the spike frequencies we recorded are comparable to those reported by MEA studies with cortical hiPSC‐N or with rodent primary MN .…”

“…Consistent with the identity of these cells as iPSC‐MN, in our coculture platform we observed a robust and consistent response to AMPA receptor agonists/antagonists (KA and CNQX, respectively). As shown by others for hiPSC‐cortical neurons , this effect was maturation‐dependent, as it appeared only after 3–4 weeks in vitro, whereas it was absent at earlier stages of maturation. The response to the GABA antagonist bicuculline was less robust, a finding consistent with a lower representation of GABA receptors.…”

“…Previous literature on MEA applied to hiPSC studies have relied exclusively upon hiPSC‐derived cortical neurons, for in vitro modeling of epilepsy and seizures . Although recent MEA studies have used neurons cocultured with human iPSC‐derived astrocytes , less attention has been given to how astrocytes may influence neuronal electrophysiological activity—particularly in the context of human biology.…”

“…Previous MEA studies have focused on hiPSC‐derived cortical neurons, either alone or in coculture with primary rodent cortical astrocytes and more recently with hiPSC‐derived astrocytes . Despite an established body of evidence suggesting that astrocytes contribute to neuronal electrophysiological maturation by promoting synaptogenesis , MEA‐ and hiPSC‐based paradigms, still influenced by a traditionally neuron‐centered perspective, have not yet been used to model this unique glial contribution.…”

Optimized culture of retinal ganglion cells and amacrine cells from adult mice

“…In vitro culture of enriched neuronal populations allows for enhanced study of direct/intrinsic factors under a variety of conditions. Such cultures are commonly derived from either embryonic/neonatal tissues [1][2][3][4][5] or induced pluripotent stem cells (iPSCs) [6][7][8]. Embryonic/neonatal tissues have weak neuronal connections which allow for their isolation and culture without severe injury [9] and are particularly useful for the study of neuronal development [10][11][12][13].…”