• Neurotoxicity Screening

Neurotoxicity Screening

Cell-based in vitro assays for drug toxicity are becoming crucial tools to screen new drug candidates because they are inexpensive, efficient, and ethically compatible preliminary screening alternatives to the more expensive animal testing models.

Applied StemCell offers a one-stop shopping solution for neurological compound screening. Starting with the engineering of iPSCs from healthy and diseased patient samples; developing disease models via gene editing for drug target discovery and efficacy testing; differentiation into cell lineage(s); cell line model characterization; and ending with a comprehensive panel of cell-based tests for drug efficacy, neurotoxicity, and target discovery that are regulatory compliant.

Application Notes

Drug Screening for Neurological Disorders using iPSC-derived Neuronal Lineage Cells

Using iPSC-differentiated Neural Lineage Cells for Determining Cell Viability in Neurotoxicity Assays

A.

Graphs of cell viability of astrocytes and neurons during drug toxicity testing

Figure 1A. Luciferase activity (bioluminescence) was used to detect the cell viability of astrocytes and neurons derived from neural reporter iPSC lines (GFAP-Nanoluc/Halotag, ASE-9501; green and MAP2/Nanoluc-Halotag, ASE-9500; blue) when exposed to 100 µM of neurotoxin 1 and 2. Neurotoxin 1 reduced luciferase activity in astrocytes by ~ 80% and in neurons by ~40%. Neurotoxin 2 reduced luciferase activity in astrocytes by ~90% and in neurons by ~70%. Luciferase activity was measured (as % of control; DMSO-treated cells) to determine the extent of cytotoxicity of the compounds. These results were similar to toxicity measured by the MTT assay.

B.

Graphs of cell viability of astrocytes and neurons after exposure to neurotoxins during drug toxicity testing

Figure 1B. Cell viability of astrocytes and neurons after exposure to neurotoxins. Astrocytes and neurons derived from a control iPSC line (ASE-9109) were exposed to concentrations of 1, 10 and 100 μM concentrations of two neurotoxins, 1 and 2. Ce ll viability was evaluated using MTT assay (MTT tetrazolium salt) and cell survival was expressed as % of absorbance of viable cells normalized to control (DMSO-treated cells). The 1 µM concentration of both neurotoxins was not significantly cytotoxic in both cell types while there was mild cytotoxicity observed at the 10µM concentrations of the neurotoxins. The 100 µM concentration of both neurotoxins was significantly cytotoxic and resulted in ~80% reduction in cell viability in astrocytes and neurons. The results of this assay were similar to the expression of luciferase in astrocytes and neurons derived from a neuronal-reporter iPSC line (Figure 1A.).  

 

iPSC-differentiated Neurons Can be Used for Screening Potential Neuroprotective Compounds

iPSCs and derived neurons can be used for testing neuroprotective effects of compoinds using MTT asasy.

Table 1. Drugs that were neuroprotective in iPSC and derived neuronal cells, and used for human clinical trials

Neurotransmitter/ MAO Inhibitors:

Rasagiline, selegiline, nicotine, topiramate, amantadine, zonisamide, taurine

Antioxidant/ Mitochondrial Stabilizers:

Resveratrol, N-acetyl cysteine, lipoic acid, epigallocatechin gallate, creatine

Anti-inflammatories:

Rolipram, indomethacin, 7-nitroindazole, 3-aminobenzamide, phenanthridone

Table 2. Drug that were not neuroprotective in iPSC-based models but were neuroprotective in conventional cell lines and animal models:

Neurotransmitter/ MAO Inhibitors:

Donepezil, caffeine, theophylline, pergolide, apomorphine, riluzole, pramipexole

Antioxidant/ Mitochondrial Stabilizers:

Ascorbic acid, coenzyme Q10, uric acid, folic acid, ropinirole

Anti-inflammatories:

Minocycline, estradiol, clioquinol, plicamycin

Dopaminergic (DA) neurons derived from control iPSC lines were used to screen neurological compounds that have been shown to be neuroprotective in rodent and cell line models (Table 1 and 2). These derived-primary DA neurons were grown in 96-well plates and pretreated with one of each of the selected compounds. The cells were then exposed to either rotenone or MPP+ that are commonly used in dopamine toxicity models and cell viability was determined using the MTT assay. Only 18 out of the compounds (Table 1) were found to be neuroprotective in these iPSC-derived DA neurons, and these 18 compounds have been used in human Parkinson's disease clinical trials to test for neuroprotection. The rest of the compounds (Table 2) have either not been used in clinical trials or were not neuroprotective. This highlights the importance of using iPSC-derived neurons for large-scale screening neurological drugs before evaluating their efficacy in animal and human studies.

Technical Details

Cytotoxicity & Cell Viability Assays
Our assays evaluate MTT/ MTS cell proliferation; LDH, necrosis and apoptosis; Luciferase expression; and cAMP levels. The assays take 4-6 weeks.

Mitochondrial Toxicity Testing
Our mitochondrial tests evaluate enzyme activity and volume fraction. These assays take 2-4 weeks to complete.

Functional Assays
Our assays are Calcium influx/imaging; Electrophysiology - Multielectrode array (MEA) analysis and Patch clamp recording. The assays take 8-12 weeks.

Quantitative Gene Expression
We offer qPCR and RNA-sequencing using NGS (next generation sequencing) technology. These assays take 2-8 weeks to complete.

Morphology
We offer the following assays to evaluate cellular morphology: Neurite growth assay and Biomarker screening. These assays take 2-4 weeks.

Custom Assays
Custom assays include iPSC generation; characterization; gene editing; differentiation; custom assay development. Time frame is project dependent.

Have Questions?

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