Neurotoxicity Testing

Applied StemCell Neuro Toxicity

Cell-based in vitro assays of drug toxicity are becoming crucial tools to screen new drug candidates before moving to expensive animal models testing. They are inexpensive, efficient, and ethically compatible preliminary screening alternatives to using animal models. Human iPSC-derived neuronal cells have been shown to be more physiologically relevant neuronal models than immortalized cell lines, can improve drug discovery and safety assessment, and reduce late stage drug attrition by providing a reliable drug testing platform.

We do it All! Applied StemCell (ASC) offers a one-stop-solution for neurological compound screening: generating iPSCs (healthy and diseased patient samples), disease modeling via gene editing for drug target discovery and efficacy testing, differentiation to cell lineage(s) of choice, cell line model characterization, as well as, a comprehensive cell-based test battery for drug efficacy, neurotoxicity and target discovery that are regulatory compliant. 

Generation of iPSCs Lines

Control iPSCs
Engineered iPSCs to model disease
Engineered reporter lines
Patient-specific iPSC lines 

CRISPR/Cas9 Mutation Correction in iPSCs

 

Differentiaton to Neural Cells

Neural Stem Cells (NSC)
Neurons (dopaminergic, cortical)
  Astrocytes
Oligodendrocytes

 

Disease Modeling & Drug Screening

Neurotoxicology assays
Neuroprotection screening
CNS drug efficacy testing
Screening for new drug targets


We provide stage-specific phenotype assays in our neurotoxicity/ drug screening platform:

  • Dose-response curve for neurotoxicity and drug efficacy
  • Screening in multiple cell types: iPSCs, NSCs, neural cells
  • Cellular Morphology
  • Synaptogenesis, network formation using neuronal co-cultures

Screening

Types of Assays

Estimated Timeline

Cytotoxicity & Cell Viability Assays

MTT/ MTS cell proliferation assay

LDH, Necrosis and Apoptosis assays

Luciferase (bioluminescence) expression

cAMP level measurement

4-6 weeks

Mitochondrial Toxicity Testing

Enzyme activity

Volume fraction detection

2-4 weeks

Functional Assays

Calcium influx/ imaging

Electrophysiology: Multielectrode array (MEA) analysis and Patch clamp recording

8-12 weeks

Quantitative Gene Expression

qPCR

RNA-seq using NGS (next generation sequencing)

2-8 weeks

Morphology

Neurite growth assay

Biomarker screening

2-4 weeks

Custom Assays

iPSC generation; characterization; gene editing; differentiation

Custom assay development

Based on project requirements

We also offer various neuronal lineage cells (Dopaminergic neurons, GABAergic/mixed neurons, astrocytes and oligodendrocytes, and neuronal co-cultures) for drug screening that are derived from:

  1. Parental/ Control iPSCs derived from multiple donors and tissue samples (cord blood; PBMCs; fibroblasts)
  2. Patient-derived iPSC lines (ALS8; Parkinson’s disease)
  3. Neuronal disease-specific isogenic knockout lines (Parkinson’s disease)
  4. Reporter iPSC lines (neural lineage-specific reporter iPSCs, iPSCs with reporter inserted at a safe harbor locus)

Custom cells: We also generate iPSCs and genome edit iPSCs.for specialized projects. Please inquire for details.

It is important to test a variety of cell types because no one-test can be used to examine all aspects of the human nervous system. Our cell-based assay for neurotoxicity testing is the perfect solution:

  • Our testing is highly reproducible providing confidence in all data generated.
  • The differentiated neuronal cells are fully characterized by immunocytochemistry and whole genome profiling.
  • iPSC models can be patient-specific and can be differentiated into many neural cell types helping to fulfill the promise of personalized medicine.
  • In vitro cell-based assays for neurotoxicity testing are now considered effective and regulatory compliant, by key governmental agencies, to assess risk.
  • A cell-based toxicity testing assay allows for prioritization of decisions to be made to move the most promising compounds into appropriate animal studies quickly.

Let us do your neurotoxicity testing with our consistent and reliable source of neuronal cell types. Contact us to discuss the cell-based assays we have for testing toxicity and the endpoints that make sense for your drug discovery project.

Technical Details

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.

TECHNICAL-neurotox-GFAP-MAP2-luciferase

B.

TECHNICAL-neurotox-MTT-assay

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.

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.

Ordering

2 Items

per page