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axolGEM iPSC-Derived Neural Stem Cells LRRK2 G2019S HOM

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Description

Human iPSC-Derived Neural Stem Cells that have been genetically edited using CRISPR-Cas9 technology to introduce the G2019S mutation (GGC>AGC) in the LRRK2 gene. This line is homozygous for the G2019S mutation so both alleles contain the mutation. Click on the product images to see the data and further details. 

The G2019S mutation in LRRK2 has been implicated in autosomal-dominant familial Parkinson's disease with late onset (Fonzo et al., 2006, Thaler et al., 2009). The G2019S mutation increases the kinase activity of LRRK2 causing increased autophosphorylation and substrate phosphorylation that may affect neuronal cell health in Parkinson's disease patients (West et al., 2005). 

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 Homozygous for the LRRK2 G2019S mutation (GGC>AGC)
Genetic modification Contains a puromycin resistance cassette (intronic)
Size ≥1.5 million cells
Kit components 1 vial of axolGEM Neural Stem Cells (≥1.5 million cells) and 1 bottle of Plating-XF Medium (30 mL)
Growth properties Adherent
Shipping conditions Dry ice
Storage conditions Liquid nitrogen

Frequently Asked Questions

Plate the cells on Readyset + Surebond (ax0052)

Always plate onto Surebond (ax0041) if you intend to detach the cells for continued expansion

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

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).

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.

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

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.

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.

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.

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.

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.  

At day 21, spontaneous synaptic activities should be detected, and day 35 synchronised burst firing should occur.

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.

We typically use PAX6, SOX2, Nestin, FOXG1, OTX, ASPM, N-cadherin and Ki67 to identify NSCs.

NeuN, TBR1, TUJ1, MAP2, GAD67, VGLUT1, Synaptophysin, CTIP2, CUX1 and BRN2 can be used to identify cerebral cortical neurons.

Technical Resources

References

  • Thaler A, Ash E, Gan-Or Z et al. The LRRK2 G2019S mutation as the cause of Parkinson's disease in Ashkenazi Jews. Journal of Neural Transmission (2009)
  • West AB, Moore DJ, Biskup S et al. Parkinson's disease-associated mutations in leucine-rich repeat kinase 2 augment kinase activity. Proc Natl Acad Sci USA (2005)
  • Di Fonzo A, Tassorelli C, De Mari M et al: Comprehensive analysis of the LRRK2 gene in sixty families with Parkinson's disease. Eur J Hum Genet (2006)