Bryan James Black, The University of Texas at Dallas
Conference: Society for Neuroscience 2018, San Diego, CA
Abstract Title: Human induced pluripotent stem cell (hiPSC) - derived motor neurons co-cultured with primary astrocytes exhibit functional network sensitivity to botulinum neurotoxin
Abstract: Botulinum neurotoxin (BoNT) is the most toxic protein known to man. BoNTs act presynaptically to prevent neurotransmitter release, causing sustained paralysis, and, at sufficiently high doses, death by asphyxiation. Currently, there are no proven clinically effective treatments for reversing intoxication. The gold standard method for BoNT quantification is based on the mouse lethality assay which low-content, and incurs high animal burdens. These disadvantages have driven the development of alternative molecular, cellular, and tissue-based approaches. Substrate-integrated microelectrode arrays (MEAs) enable long-term non-invasive interrogation of phenotypic network activity in vitro. Recent technological advances have enabled more rapid neurotoxicological assessments via electrophysiological measures, but this approach is still limited due to the availability of physiologically relevant cells. Here, we report exploratory studies regarding BoNT/A sensitivity using a commercially available hiPSC motor neuron (hiPSC MN) – astrocyte co-culture model on multi-well MEA plates (Axion Biosystems, USA). Methods: Astrocytes were isolated and sub-cultured from primary embryonic whole-brains at a density of 50,000 cells/cm2. hiPSC MNs were provided by BrainXell, Inc. and cultured as specified by the vendor. hiPSC MNs were seeded at 100,000 cells/cm2 on astrocyte feeder layers three days following astrocyte culture. Spontaneous network activity was monitored using the Axion Maestro multi-well MEA recording system. Extracellular action potentials were defined as band pass filtered (300 – 5000 Hz) continuous data crossings of a 5.5σ adaptive threshold. Network activity was quantified as a combination of network bursting rates and cross-channel synchrony. 100 ng/ml BoNT/A was added on DIV 16 and recordings were collected every 24 hours following BoNT/A addition. Results: ChAT staining of homogeneous hiPSC MN cultures confirmed a highly purified homogeneous motor neuron population. Dual GFAP and β-III tubulin staining of astrocyte cultures confirmed high purity of GFAP positive astrocytes (>95%). Network activity, consistent with formation of functional synapses, emerged following 7 DIV. No network activity was observed in homogeneous control cultures of hiPSC MNs. BoNT/A (100 ng/ml) induced significant reduction in network bursting rate and synchrony following 24 h incubation. This reduction in network activity persisted at least 7 days following BoNT/A washout. No modulation of intrinsic spontaneous activity was observed in homogeneous control cultures of hiPSC MNs.