PMC

Detection of BoNT/B, C, and E activity in hiPSC-derived neurons. Seven-day cultures of hiPSC-derived neurons and RSC cells were exposed to serial dilutions of BoNT/B (A), C (B), and E (C) for 48 h in parallel. Cell lysates were analyzed for the respective SNARE target protein cleavage by Western blot, and data from three Western blots were quantified by densitometry and data plots were generated.

Comparison of BoNT/A1 sensitivity of hiPSC-derived neurons plated on different substrates. At 2 weeks after plating, the cells were exposed to the indicated concentrations of BoNT/A1 and cell lysates were analyzed for SNAP-25 cleavage by Western blot. Data from three Western blots were quantified by densitometry, and data plots were generated. There were no statistically significant differences in sensitivity of neurons grown on the different substrates. The substrates are shown in the figure legend on the right.

Western blot and densitometry data of antibody protection assay in hiPSC-derived neurons. Serial dilutions of antibody and toxin were preincubated for 1 h prior to exposure to neurons, and cell lysates were analyzed for SNAP-25 cleavage by Western blot. The data from three Western blots were quantified by densitometry and average, and SDs are shown in the graph. The amounts of antibody used are indicated. (A) hiPSC-derived neurons were exposed to 1.5 U of toxin and antibody for 24 h. (B) hiPSC-derived neurons were exposed to 55 U of toxin-antibody mixture in cell stimulation medium for 5 min, the mixture was removed, cells were washed twice, and incubated for 24 h.

A) BoNT/A1 sensitivity of hiPSC-derived neurons and RSC cells. hiPSC-derived neurons were plated 4 and 7 days prior to toxin exposure, and RSC cells were cultured for 2 weeks prior to toxin exposure. All cells were exposed to the indicated toxin dilutions for 48 h, and cell lysates were analyzed for SNAP-25 cleavage by Western blot. (B) Time dependence of detecting BoNT/A1 activity in hiPSC-derived neurons. The hiPSC-derived neurons were exposed to serial BoNT/A1 dilutions for 6, 16, 24, and 48 h. The assay time is shown in the figure legend on the right. Data from three Western blots were quantified by densitometry, and data plots and EC₅₀ values were generated. The maturation time and EC₅₀ values are shown in the figure legend below.

BoNT receptor expression in hiPSC-derived neurons. (A) hiPSC-derived neurons were plated on PLO-laminin(BD)–coated six-well plates at a density of 125,000 cells/cm². Images were acquired at 1, 3, 8, and 14 days after plating at ×200 magnification via an Olympus IX81 microscope. (B) hiPSC-derived neurons were cultured for 4, 7, 14, and 21 days and assayed via Western blot for expression levels of BoNT receptors. A representative Western blot of four replicates is shown. (C) hiPSC-derived neurons were cultured for 5, 10, 15, and 20 days and then used to perform qPCR assays. Adult human brain RNA was used as a comparison sample (STX [syntaxin], SV2 [synaptic vesicle protein], and SYT [synaptotagmin]).

Activity-dependent BoNT/A1 uptake by neurons. (A) hiPSC-derived neurons and RSC cells were exposed to 55 or 275 U of BoNT/A1 in cell stimulation medium for 1, 5, 10, and 15 min, followed by toxin removal and 24 h incubation. Cell lysates were analyzed by Western blot for SNAP-25 cleavage. A representative Western blot of four replicates is shown. (B) hiPSC-derived neurons were exposed to 55 U of BoNT/A1 in both neuronal and cell stimulation medium for 1, 5, and 10 min. Cell lysates were analyzed by Western blot for SNAP-25 cleavage. A representative Western blot of four replicates is shown. (C) Neurons were exposed to 1.7–55 U of BoNT/A1 for 5 min in cell stimulation medium, washed twice with neuronal medium, and incubated for 24 h. Data from four Western blots were quantified by densitometry, and data plots were generated. (D) BoNT/A1 uptake rate of hiPSC-derived neurons compared to RSC cells. The cells were exposed to 82 U of BoNT/A1 for up to 10 h in parallel in either neuronal medium (NM) or cell stimulation medium (CSM). Cell lysates were analyzed for SNAP-25 cleavage by Western blot, and data from three Western blots were quantified by densitometry and data plots were generated. The cell and medium types are shown in the figure legend on the right.