Human induced pluripotent stem cells (hiPSC) hold great promise for providing 
various differentiated cell models for in vitro toxigenicity testing. For 
Clostridium botulinum neurotoxin (BoNT) detection and mechanistic studies, 
several cell models currently exist, but none examine toxin function with 
species-specific relevance while exhibiting high sensitivity. The most sensitive 
cell models to date are mouse or rat primary cells and neurons derived from 
mouse embryonic stem cells, both of which require significant technical 
expertise for culture preparation. This study describes for the first time the 
use of hiPSC-derived neurons for BoNT detection. The neurons used in this study 
were differentiated and cryopreserved by Cellular Dynamics International 
(Madison, WI) and consist of an almost pure pan-neuronal population of 
predominantly gamma aminoisobutyric acidergic and glutamatergic neurons. Western 
blot and quantitative PCR data show that these neurons express all the necessary 
receptors and substrates for BoNT intoxication. BoNT/A intoxication studies 
demonstrate that the hiPSC-derived neurons reproducibly and quantitatively 
detect biologically active BoNT/A with high sensitivity (EC(50) ∼0.3 U). 
Additionally, the quantitative detection of BoNT serotypes B, C, E, and BoNT/A 
complex was demonstrated, and BoNT/A specificity was confirmed through antibody 
protection studies. A direct comparison of BoNT detection using primary rat 
spinal cord cells and hiPSC-derived neurons showed equal or increased 
sensitivity, a steeper dose-response curve and a more complete SNARE protein 
target cleavage for hiPSC-derived neurons. In summary, these data suggest that 
neurons derived from hiPSCs provide an ideal and highly sensitive platform for 
BoNT potency determination, neutralizing antibody detection and for mechanistic 
studies.