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axolGEM iPSC-Derived Neural Stem Cells LRRK2 G2019S HET

Please be aware that the correct volume of Neural Plating Medium ax0033 (not Xeno-Free) needed for your specific order volume of cells will be supplied FREE OF CHARGE.


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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 heterozygous for the G2019S 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 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 Heterozygous for the LRRK2 G2019S mutation (GGC>AGC)
Genetic modification Contains a puromycin resistance cassette (intronic)
Size ≥1.5 million cells
Growth properties Adherent
Shipping conditions Dry ice
Storage conditions Liquid nitrogen

Frequently Asked Questions

Plate the cells on Readyset + Surebond (ax0052)

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).

We recommend SureBond-XF: Xeno-free coating reagent (Ax0053) in combination with Poly-D-Lysine (Sigma Aldrich, P7405)

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.

We only recommend using SureBond-XF (Ax0053) in combination with Poly-D-Lysine (Sigma Aldrich, P7405).

We do not recommend expansion or re-freezing of the the NSCs. Axol cannot guarantee the viability of the iPSC-derived NSCs and functionality of the neurons derived after re-freezing or expansion.

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

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.  

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)