High sensitivity of iPSC-derived motor neurons to the human-relevant Botulinum Neurotoxin serotypes E and F

Botulinum neurotoxins (BoNTs) are the most potent biological toxins known and widely used as biopharmaceuticals. Yet pharmaceutical potency testing still relies largely on the mouse bioassay, raising ethical concerns and conflicting with 3 R principles. Sensitive, serotype‑independent human cell‑based alternatives are particularly needed for the emerging serotypes BoNT/E and BoNT/F, for which no validated in vitro potency assay currently has been reported. In this study, human iPSC‑derived motor neurons were generated, cryopreserved as motor neuron progenitors, matured to day 30, and exposed to serial dilutions of BoNT/E or BoNT/F. Toxin activity was quantified via Western blot detection of SNAP25 (BoNT/E) using a full-length antibody, whereas for VAMP2 (BoNT/F), both full-length and neoepitope-specific antibodies were used to quantify substrate cleavage. IC₅₀ values were calculated by non‑linear regression and converted to LD₅₀/mL equivalents for comparison with mouse bioassay data. Measured IC₅₀ values were 0.769 pM for BoNT/E and 0.802 pM (full‑length) or 7.645 pM (neoepitope) for BoNT/F, corresponding to 4.54 LD₅₀/mL for BoNT/E and 2.46 LD₅₀/mL (full‑length) for BoNT/F, closely matching mouse bioassay potencies. Combining these data with prior results for BoNT/A and BoNT/B demonstrates a potency order: BoNT/A ≫ BoNT/B ≈ BoNT/F ≈ BoNT/E. These results confirm human iPSC‑derived motor neurons as a sensitive, physiologically relevant model capable of detecting four medically important BoNT serotypes. While Western blotting provides robust determination of potency, this cellular model well suited for adaptation to serotype-independent high‑throughput formats, paving the way for animal‑free BoNT potency testing. • iPSC-derived motor neurons detect BoNT/E and BoNT/F with sub-picomolar sensitivity • IC 50 values closely match mouse bioassay potencies for both serotypes tested • Human motor neurons show conserved potency ranking: BoNT/A > BoNT/B ≈ BoNT/F ≈ BoNT/E • Platform suitable for animal-free, serotype-independent BoNT potency testing