Neuro Toxicity Testing

Applied StemCell Neuro Toxicity

Cell-based assays of drug toxicity are becoming crucial tools in current translational research and drug discovery. It is critical to learn as much as possible about new drug candidates, with high throughput human in vitro assays, before moving candidates into expensive animal models testing. Cell-based in vitro alternatives are a perfect fit for your overall strategy for neuro toxicity testing and represent a paradigm shift for toxicology and drug discovery efforts. Many current testing protocols involve expensive, time consuming and ethically debated in vivo animal models, Thus, drug discovery project teams are trying to minimize the use of animals for cost, time and ethical reasons. In addition, animal models are not suitable for screening large numbers of compounds efficiently and economically. ASC offers cell-based assays for toxicity testing in many types of biologically relevant human neuronal cells derived from induced pluripotent stem cells (iPSCs).

We offer different neuronal lineage cells for toxicity testing that are derived from our iPSCs, including dopaminergic neurons, astrocytes, oligodendrocytes, GABAergic neurons and mixed neurons (excitatory and inhibitory).

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 neural toxicity testing is the perfect solution:

  • Our testing is highly reproducible providing confidence in all data generated.
  • ASC neuronal cell lines are fully characterized by immunocytochemistry and whole genome profiling.
  • Possible endpoints include oxidative stress, DNA damage, apoptosis, neurite outgrowth.
  • Our iPSC-derived neural cells are cryopreserved and well characterized with little batch to batch variations.
  • 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 neuro toxicity testing are now considered effective, 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.
  • Our cell-based assays for toxicity testing are a very effective path to a better understanding of environmental toxicants as well.

Let us do your neuro toxicity 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

Parkinson's disease Modeling with Control and Engineered iPSCs and differentiated neuronal lineage cells

  • Control Lines: Well-characterized, integration-free control iPSC lines generated from male and female CD34+ cells (cord blood) using episomal vectors. These lines were also used for engineering isogenic lines with disease mutations.
  • Engineered isogenic lines: From parental control iPSCs with knockout mutations of genes associated with PD; and reporter lines with knock-in of lineage-specific reporters and in safe-harbor locus.

Genome Modified iPSC Lines

1. Neuronal Disease Specific Isogenic Knockout Lines: 

Isogenic Knock-out Lines

Associated Disease

Description

PARK2 -/-

PD

Biallelic KO

PARK7 -/-

PD

Biallelic KO

PINK1 -/-

PD

Biallelic KO

LRRK2 -/-

PD

Biallelic KO

Park2-/-; Park7-/-

PD

Biallelic KO

Park2-/-; Pink1-/-

PD

Biallelic KO

APOE -/-

Alzheimer's disease

Biallelic KO

SOD1 -/-

ALS

Biallelic KO

DICS1 -/-

Schizophrenia

Biallelic KO

CNTNAP2 -/-

Autism

Biallelic KO

BDNF -/-

CNS

Biallelic KO

2. Lineage-specific Reporter Gene Knock-in Lines:

Knock-in Lineage-specific Reporters

Description

MAP2-Nanoluc-Halotag KI

Neuron reporter

GFAP-Nanoluc-Halotag KI

Astrocyte reporter

MBP-Nanoluc-Halotag KI

Oligodendrocyte reporter

3. Safe Harbor Locus Reporter Gene Knock-in Lines:

Safe-harbor knock-in lines

Description

CAG-GFP, AAVS/Chr19

Ubiquitous reporter

DCXp-GFP

Neuron reporter

Generation of Isogenic Panels of Neurons and Glia from iPSCs using Neural Stem Cells (NSCs) as a Stable Intermediate

Genome edited iPSCs were differentiated into NSCs which were used as a stable intermediate for further directed differentiation into neurons and glia. This established an isogenic panel of neuronal lineage cells for comparitive drug screening across different cell types. Below is an example of characterization of a genome edited iPSC line (ASE-9405; APOE-/-)

The ASE-9405 iPSC line is engineered with a bi-allelic (homozygous) knockout for APOE gene (APOE-/-) that has been implicated in Alzheimer’s disease etiology. The parental iPSC is ASE-9109 which is an integration-free, normal karyotype iPSC derived from male, CD34+ cord blood cells. The APOE knockout line can be further differentiated into an isogenic panel of neurons and glia for disease modeling and drug/ toxicity screening applications.

1. Genotyping of APOE-/- bi-allelic KO clone F4: APOE KO

technical-genomedited-ipsc-apoe-ko-1

technical-genomedited-ipsc-apoe-ko-2

Figure 1. Sequence alignment between parental/ control iPSC line (ASE-9109; male) and APOE-/- clone. The homozygous knockout clone shows a 4 bp deletion in allele 1, a 3 bp deletion in allele 2, and a 4bp insertion in allele 2, as compared to wild type (WT) sequence in parental iPSC line.

2. Expression of Pluripotency Markers

technical-genomeedited-ipsc-apoe-ko-3

Figure 2. Expression of pluripotency markers in APOE-/- iPSC line. The homozygous knockout iPSC line, APOE-/-, expresses pluripotency markers OCT4, NANOG, TRA-1-60, and TRA-1-81, indicating pluripotency of the iPSC line after genome editing. Nucleus stained with DAPI (blue).

3. Expression of APOE in neural stem cells differentiated from APOE-/- iPSC-line

technical-genomeedited-ipsc-apoe-ko-4

Figure 3. Expression of APOE in neural stem cells (NSC) differentiated from master/ parental iPSC line (ASE-9109) and APOE-/- iPSC line (ASE-9405) was quantified using qPCR. The APOE mRNA was significantly downregulated in the APOE-knockout NSC as compared to expression in the parental/ control iPSC line. 

WT APOE-/- 
1022 -4

Figure 4. APOE Expression in APOE knockout NSCs by microarray. APOE expression in the knockout NSC line was significantly lower than the wild type NSC line.

4. Differentiation of APOE -/- iPSCs into Neurons and GFAP

technical-genomeedited-ipsc-apoe-ko-5

Figure 5. Differentiation of APOE-/- iPSCs into cortical neurons and astrocytes. The APOE-/- line can be differentiated into cortical neurons (GABA; green) and astrocytes (GFAP; red) to model Alzheimer's disease. Other markers: Neuronal marker (MAP@; blue) and nucleus staining (DAPI; blue)

 

Support Materials
References
  • Pei Y, Peng J, Behl M, Sipes NS, Shockley KR, Rao MS, Tice RR, Zeng X. Comparative neurotoxicity screening in human iPSC-derived neural stem cells, neurons and astrocytes. Brain research. 2016 May 1;1638:57-73.
  • Shaltouki A, Sivapatham R, Pei Y, Gerencser AA, Momčilović O, Rao MS, Zeng X. Mitochondrial alterations by PARKIN in dopaminergic neurons using PARK2 patient-specific and PARK2 knockout isogenic iPSC lines. Stem cell reports. 2015 May 12;4(5):847-59.
  • Efthymiou A, Shaltouki A, Steiner JP, Jha B, Heman-Ackah SM, Swistowski A, Zeng X, Rao MS, Malik N. Functional screening assays with neurons generated from pluripotent stem cell–derived neural stem cells. Journal of biomolecular screening. 2014;19(1):32-43.
  • Shaltouki, A., Peng, J., Liu, Q., Rao, MS., and Zeng, X. Efficient generation of astrocytes from human pluripotent stem cells in defined conditions. Stem Cells. 2013 May;31(5):941-52.
  • Swistowski, A, Peng, J, Liu, Q, Mali, P, Rao, MS, Cheng, L, and Zeng, X. Efficient generation of functional dopaminergic neurons from human induced pluripotent stem cells under defined conditions. Stem Cells. 2010 Oct;28(10):1893-904.
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