Human-Relevant Sensitivity of iPSC-Derived Human Motor Neurons to BoNT/A1 and B1

Abstract

The application of botulinum neurotoxins (BoNTs) for medical treatments necessitates a potency quantification of these lethal bacterial toxins, resulting in the use of a large number of test animals. Available alternative methods are limited in their relevance, as they are based on rodent cells or neuroblastoma cell lines or applicable for single toxin serotypes only. Here, human motor neurons (MNs), which are the physiological target of BoNTs, were generated from induced pluripotent stem cells (iPSCs) and compared to the neuroblastoma cell line SiMa, which is often used in cell-based assays for BoNT potency determination. In comparison with the mouse bioassay, human MNs exhibit a superior sensitivity to the BoNT serotypes A1 and B1 at levels that are reflective of human sensitivity. SiMa cells were able to detect BoNT/A1, but with much lower sensitivity than human MNs and appear unsuitable to detect any BoNT/B1 activity. The MNs used for these experiments were generated according to three differentiation protocols, which resulted in distinct sensitivity levels. Molecular parameters such as receptor protein concentration and electrical activity of the MNs were analyzed, but are not predictive for BoNT sensitivity. These results show that human MNs from several sources should be considered in BoNT testing and that human MNs are a physiologically relevant model, which could be used to optimize current BoNT potency testing.

Botulinum Neurotoxin A4 Has a 1000-Fold Reduced Potency Due to Three Single Amino Acid Alterations in the Protein Receptor Binding Domain

“…Measuring SNAP-25 cleavage as an outcome for BoNT/A sensitivity ( Table 1 ), human motor neurons showed an ~8000-fold differential sensitivity between BoNT/A1 and BoNT/A4, while rat spinal cord cells showed an ~2000-fold differential sensitivity between BoNT/A1 and BoNT/A4. This assay-specific difference may be due to the more sensitive human motor neurons cells, which were ~14-fold more sensitive to BoNT/A1 than rat spinal cord cells as also previously observed [ 33 , 34 , 35 , 36 ].…”

Recent Developments in Botulinum Neurotoxins Detection

“…In comparison with the mouse bioassay, human MNs exhibit a superior sensitivity to BoNT toxinotypes A1 (0.003–0.5 pM) and B1. However, SiMa cells exhibited much lower sensitivity (5.5 pM) than human MNs, and appeared unsuitable to detect any BoNT/B1 activity [ 98 , 99 ].…”

Validation of a Novel Double Control Quantitative Copy Number PCR Method to Quantify Off-Target Transgene Integration after CRISPR-Induced DNA Modification

“…Cell-based alternative methods have been established but must be improved to enhance sensitivity [3]. To this end, the coding sequence of a reporter luciferase (Gaussia luciferase, GLuc) used in a recently developed neuroblastoma cell-based functional assay [4][5][6] was incorporated into a genetic safe harbor of induced pluripotent stem cells (iPSCs), which can be differentiated into highly sensitive motor neurons [7] by the CRISPR/Cas9 method.…”