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Description
Human iPSC-Derived Neural Stem Cells that have been genetically edited using CRISPR-Cas9 technology to introduce the P301L mutation (CCG>CTG) into the MAPT gene. This line is heterozygous for the P301L mutation where one allele contains the mutation and the other allele is wild type at the locus. Click on the product images to see the data and further details.
The P301L mutation MAPT has been implicated in frontotemporal dementia and parkinsonism (Dumanchin et al., 1998). The mutation affects only the 4R isoforms of MAPT since the exon containing the mutation is spliced out of 3R isoforms (Hutton et al., 1998). Aggregated MAPT/tau protein in affected patients consisted mainly of the 4R isoform (Hutton et al., 1998). The P301L mutation promotes aggregation of MAPT/tau protein into ordered paired helical filaments and beta sheet formation in vitro (von Bergen et al., 2001).
Product Specification
| Starting material | Dermal fibroblast |
| Donor gender | Female |
| Donor age at sampling | 64 yrs |
| Karyotype | Normal |
| Reprogramming method | Episomal vector |
| Induction method | Monolayer & chemically defined medium |
| Genetic modification | Heterozygous for the MAPT P301L mutation (CCG>CTG) |
| Genetic modification | Contains a puromycin resistance cassette (intronic) |
| Size | ≥1.5 million cells |
| Kit components | 1 vial of Neural Stem Cells (≥1.5 million cells) and 1 bottle of Neural Plating-XF Medium (30 mL) |
| Growth properties | Adherent |
| Shipping conditions | Dry ice |
| Storage conditions | Liquid nitrogen |
Frequently Asked Questions
I want to carry out imaging/confocal microscopy, how should I plate the cells?
Plate the cells on Readyset + Surebond (ax0052)
I want to passage and expand the iPSC-derived neural stem cells, what coating shall I use?
Always plate onto Surebond (ax0041) if you intend to detach the cells for continued expansion
I am not sure what coating I should use for iPSC-derived neural stem cells?
Surebond-XF - Xeno-free coating reagent needed for endpoint assays on plastic, this is the easiest method of coating, with no wash steps. Surebond + Readyset - Coating reagent needed for endpoint assays on glass or 96-well plates Surebond- Coating reagent needed when passaging of NSCs is required
For cortical neurogenesis, when do I switch from the Neural Differentiation-XF medium to Neural Maintenance Medium-XF?
Please switch to the Maintenance Medium-XF after the suggested period of Neural differentiation medium-XF treatment (see page 12 and 15 of the Human iPSC-Derived Neural Stem Cell Master Protocol below).
A sheet of neurons is peeling from the corners , is this normal and can I stop it?
Please make sure you change medium gently and avoid adding the medium from one side of the wells throughout the 5-6 weeks of culture. If the cells start to peel from the corners, it can be repaired by adding Surebond (ax0041) into your standard feeding media. Usually, we use 120 uL Surebond in 12 mL medium for a few days until the layer re-attach. This can be applied no matter what coating has been used.
When using the XF medium system for iPSC-derived NSCs, what seeding densities should I use?
Please follow the links below: Sync. differentiation: https://www.axolbio.com/web/binary/saveas?model=ir.attachment&field=datas&filename_field=name&id=77214&t=1556543155516 Spontaneous differentiation: https://www.axolbio.com/web/binary/saveas?model=ir.attachment&field=datas&filename_field=name&id=77213&t=1556543179728
Can I use other coating reagents such as Poly-D-Lysine?
We do not recommend using coating reagents whcih we have not tested with our cells. They might result in poor quality cultures and low adherence. Our SureBond, SureBond-XF and SureBond+ReadySet have been optimized to complement our cells and provide consistent results.
Can the iPSC-derived NSCs be expanded and frozen down again?
We do not recommend re-freeze the NSCs. Axol cannot guarantee the viability of the iPSC-derived NSCs and functionality of the neurons derived after re-freezing.
How long can the iPSC-derived NSCs be expanded for?
A long expansion period will increase the number of glial cells in your final population. We recommend conducting fewer passaging steps (< 3 passages) over a shorter period of time in order to reduce the glial cell population.
What is the composition of pure neuronal population in terms of cell types?
It is possible to achieve a 90% pure population of cerebral cortical neurons after terminal differentiation using Neural Differentiation-XF Medium (System B). Repeated expansion of the NSCs will increase the glial population and conversely decrease the neuronal population.
What is the ratio of cortical deep layer neurons (Tbr1 & Ctip2 positive) and upper layer neurons (CUX1 & BRN2 positive) after Axol iPSC-derived NSC differentiation?
The ratio of deep to upper layer neurons will change with the number of days in culture. After 2 weeks in Neural Maintenance-XF Medium, approx. 60% of neurons express deep layer markers but this will decrease with length of time in culture. We would recommend spontaneous differentiation for over 40 days to see a large percentage of upper layer neurons.
When would I expect to see synaptic activity?
At day 21, spontaneous synaptic activities should be detected, and day 35 synchronised burst firing should occur.
Will I be able to detect long-term potentiation and long-term depression?
Yes, Axol iPSC-Derived Cortical Neurons when co-cultured with astrocytes have been shown to respond to high frequency stimulation resulting in a change in spike frequency presenting as a depression of potentiation of network transmission.
What markers are commonly used to detect the Cortical NSCs?
We typically use PAX6, SOX2, Nestin, FOXG1, OTX, ASPM, N-cadherin and Ki67 to identify NSCs.
What markers are commonly used to detect neurons?
NeuN, TBR1, TUJ1, MAP2, GAD67, VGLUT1, Synaptophysin, CTIP2, CUX1 and BRN2 can be used to identify cerebral cortical neurons.
Technical Resources
References
- Dumanchin C, Camuzat A, Campion D et al. Segregation of a missense mutation in the microtubule-associated protein tau gene with familial frontotemporal dementia and parkinsonism. Human Molecular Genetics (1998)
- Hutton M, Lendon CL, Rizzu P et al. Association of missense and 5'-splice-site mutations in tau with the inherited dementia FTDP-17. Nature (1998)
- von Bergen M, Barghorn S, Li L et al. Mutations of tau protein in frontotemporal dementia promote aggregation of paired helical filaments by enhancing local beta-structure. The Journal of Biological Chemistry (2001)