Analysis of Motor Neurons Differentiated from Human Induced Pluripotent Stem Cells for the Use in Cell-Based Botulinum Neurotoxin Activity Assays

Abstract

Botulinum neurotoxins (BoNTs) are potent neurotoxins produced by bacteria, which inhibit neurotransmitter release, specifically in their physiological target known as motor neurons (MNs). For the potency assessment of BoNTs produced for treatment in traditional and aesthetic medicine, the mouse lethality assay is still used by the majority of manufacturers, which is ethically questionable in terms of the 3Rs principle. In this study, MNs were differentiated from human induced pluripotent stem cells based on three published protocols. The resulting cell populations were analyzed for their MN yield and their suitability for the potency assessment of BoNTs. MNs produce specific gangliosides and synaptic proteins, which are bound by BoNTs in order to be taken up by receptor-mediated endocytosis, which is followed by cleavage of specific soluble N-ethylmaleimide-sensitive-factor attachment receptor (SNARE) proteins required for neurotransmitter release. The presence of receptors and substrates for all BoNT serotypes was demonstrated in MNs generated in vitro. In particular, the MN differentiation protocol based on Du et al. yielded high numbers of MNs in a short amount of time with high expression of BoNT receptors and targets. The resulting cells are more sensitive to BoNT/A1 than the commonly used neuroblastoma cell line SiMa. MNs are, therefore, an ideal tool for being combined with already established detection methods.

hiPSC-Derived Neurons Provide a Robust and Physiologically Relevant In Vitro Platform to Test Botulinum Neurotoxins

“…We have recently highlighted the potential of three additional hiPSC-derived neuronal models, including a Motor Neuron model, to provide a sensitive platform for studying BoNT intoxication mechanisms and for BoNT potency determination ( Nicoleau et al, 2018a ; Nicoleau et al, 2018b ). The potential of hiPSC-derived Motor Neurons for BoNT testing has been confirmed more recently by two studies ( Pellett et al, 2019 ; Schenke et al, 2020 ). However, most assays using hiPSC-derived neurons for BoNT study and testing used so far have been biochemical assays focusing on the quantification of one specific BoNT target which does not allow the comparison of BoNTs cleaving different SNARE.…”

“…Another study, published more recently, has also reported results of BoNT/A testing on hiPSC- derived Motor Neurons ( Schenke et al, 2020 ). In that study, EC 50 for BoNT/A was estimated at 0.046 pM while in our study, EC 50 was estimated at 0.39 pM.…”

“…So far, two studies have reported BoNT testing on monocultures of human iPSC-derived Motor Neurons ( Pellett et al, 2019 ; Schenke et al, 2020 ). In both studies, the sensitivity of hiPSC-derived Motor neurons to BoNT/A or BoNT/E was high which is aligned with our findings.…”

Connexin43 in Germ Cells Seems to Be Dispensable for Murine Spermatogenesis

“…RNA extraction, reverse transcription and quantification gene of interest expression were performed as described in Schenke et al [ 71 ] from testis samples of four mice per genotype with three technical raplicates each. Relative gene expression levels were calculated according to the 2 ∆∆CT method [ 72 ].…”

Emerging Opportunities in Human Pluripotent Stem-Cells Based Assays to Explore the Diversity of Botulinum Neurotoxins as Future Therapeutics

“…Many studies confirmed that iPSCs-derived neurons (i.e., motor neurons, cortical neurons, dopaminergic neurons, and sensory neurons) express all the necessary receptors and substrates for BoNT intoxication, and are sensitive for detection of several BoNTs serotypes with different potencies, making iPSC-derivatives cells of choice for BoNT research [ 96 , 157 , 160 , 161 , 162 , 163 ]. Motor neurons derived from hiPSCs have proved to be highly sensitive to BoNT intoxication [ 157 , 160 , 161 ].…”