Human Induced Pluripotent Stem Cell Derived Neuronal Cells Cultured on Chemically-Defined Hydrogels for Sensitive In Vitro Detection of Botulinum Neurotoxin

Abstract

Botulinum neurotoxin (BoNT) detection provides a useful model for validating cell-based neurotoxicity screening approaches, as sensitivity is dependent on functionally competent neurons and clear quantitative endpoints are available for correlating results to approved animal testing protocols. Here, human induced pluripotent stem cell (iPSC)-derived neuronal cells were cultured on chemically-defined poly(ethylene glycol) (PEG) hydrogels formed by “thiol-ene” photopolymerization and tested as a cell-based neurotoxicity assay by determining sensitivity to active BoNT/A1. BoNT/A1 sensitivity was comparable to the approved in vivo mouse bioassay for human iPSC-derived neurons and neural stem cells (iPSC-NSCs) cultured on PEG hydrogels or treated tissue culture polystyrene (TCP) surfaces. However, maximum sensitivity for BoNT detection was achieved two weeks earlier for iPSC-NSCs that were differentiated and matured on PEG hydrogels compared to TCP. Therefore, chemically-defined synthetic hydrogels offer benefits over standard platforms when optimizing culture conditions for cell-based screening and achieve sensitivities comparable to an approved animal testing protocol.

3D culture models of Alzheimer’s disease: a road map to a “cure-in-a-dish”

“…However, the use of heterogeneous Matrigel with under-defined and complex protein components, may cause variability for drug testing [70, 97]. Natural hydrogels based on simple extracellular matrix protein components may be potential alternatives for Matrigel [76, 98, 99]. Synthetic hydrogels generally provide much better matrix uniformity and reproducibility than natural hydrogels [94, 100–102].…”

Assessment of ELISA as endpoint in neuronal cell-based assay for BoNT detection using hiPSC derived neurons

“…While previous data has indicated greater sensitivity of iCell Neurons compared to HIP Neurons (Pellett, et al, 2015; Whitemarsh, et al, 2013), the HIP Neurons can be seeded at greater density resulting in higher total protein concentration in cell lysates. The protein concentrations of the cell lysates were determined to be 90.4 ±12.2 and 285 ± 36.7 μg / ml for iCell Neurons and HIP Neurons, respectively.…”

“…Jones, personal communication) can be used to detect intracellularly cleaved SNAP-25 in a sensitive hiPSC derived neurons cell model (Pellett, et al, 2015; Whitemarsh, et al, 2012), detection of cleaved SNAP-25 in cell lysates from hiPSC derived neurons exposed to serial dilutions of BoNT/A1was compared by ELISA and Western blot. While the Western blot data indicated dose-dependent SNAP-25 cleavage ranging from 100 % to 0 % cleavage as previously described (Whitemarsh, et al, 2012), the ELISA resulted in a very low and non-quantifiable signal (data not shown).…”

“…Therefore, ELISA based detection methods for the SNAP-25 cleavage product have been developed for quantitation of BoNT/A1 in various cell models (Fernandez-Salas, et al, 2012; Nuss, et al, 2010). However, this ELISA detection platform has not been developed for the highly sensitive and human specific hiPSC derived neuron cell models (Pellett, et al, 2015; Whitemarsh, et al, 2012a). Initial analyses of an ELISA based detection system in hiPSC derived neurons (iCell Neurons, Cellular Dynamics Inc.) and HIP Neurons (Globalstem) resulted in a low and non-quantifiable signal.…”

“…The neurons were matured for 18 days prior to use in the toxin assays. Primary mouse spinal cord cells were prepared as previously described (Pellett, et al, 2015; Whitemarsh, et al, 2013) and cultured for 18 days prior to use in a toxin assay. All cell types were incubated at 37°C in a humidified 5% CO 2 atmosphere.…”