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Description
Axol Human iPSC-Derived Neural Stem Cells
Axol Human iPSC-Derived Neural Stem Cells (NSCs) are derived from integration-free, induced pluripotent stem cells (iPSCs) under fully defined neural induction conditions. The NSCs express typical markers of cerebral cortical neural stem and progenitor cells such as PAX6, FOXG1 and nestin, and spontaneously form polarized neural tube-like rosette structures when plated as a monolayer in culture (see below). Additionally, Axol NSCs are capable of generating a spectrum of cerebral cortical excitatory and inhibitory neurons that are electrically active and have the ability to form functional synapses and circuits in vitro. After thawing and plating, the neural stem cells terminally differentiate to acquire mature electrophysiological properties, and form functional synaptic networks over a period of 40 ~ 50 days.
Axol NSCs are easy to differentiate to neurons or mixed neural cell types, following our protocols and using our tailored media and reagent bundles. A highly pure population of neurons can be generated from Axol NSCs following the synchronous differentiation protocol. Using our specialized coating reagents, neurons derived from Axol NSCs can be maintained in culture long-term (>1 year). NSCs are available from multiple donors to suit your research needs and have been characterized extensively.
Product Specification
Starting material | Fibroblasts |
Donor gender | Male |
Donor age at sampling | 74 yrs |
Karyotype | Normal |
Reprogramming method | Episomal vector |
Induction method | Monolayer & chemically defined medium |
Genetic modification | None |
Size | ≥1.5 million cells |
Growth properties | Adherent |
Shipping conditions | Dry ice |
Storage conditions | vapour phase 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)
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).
I am not sure what coating I should use for iPSC-derived neural stem cells?
We recommend SureBond-XF: Xeno-free coating reagent (Ax0053) in combination with Poly-D-Lysine (Sigma Aldrich, P7405)
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.
Can I use other coating reagents such as Poly-D-Lysine?
We only recommend using SureBond-XF (Ax0053) in combination with Poly-D-Lysine (Sigma Aldrich, P7405).
What are the APOE genotypes of the Alzheimers disease Neural Stem Cell products?
Please follow the link to download the PDF file with APOE genotype information: https://www.axolbio.com/web/binary/saveas?model=ir.attachment&field=datas&filename_field=name&id=92400&t=1556550049465
Can the iPSC-derived NSCs be expanded and frozen down again?
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.
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.
How long can I expand the NSCs before differentiation?
Axol do not recommend the expansion of NSCs prior to differentiation. For best results, thaw the frozen NSC and then begin the differentiation process 24 hours later.
How much medium and coating would I need for differentiating a vial (1.5 million cells) of NSCs to striatal neurons?
ReadySet: 12 mL SureBond: 90 uL Striatal Neuron Medium: 85 mL Below we laid out the calculations: Assuming you'd like to plate the cells at our recommended density - 35,000 cells per square centimetre, you would have 43 square centimetres (or 4.5 wells of a 6-well plate) of cells. Coating: For ReadySet coating, we recommend 250 uL per square centimetre. So, you'd need 11 mL of ReadySet per vial of cells (250 uL x 43 cm2) For SureBond top coating, we recommend adding 120 uL in 6 mL PBS; and then coat 100 uL of diluted SureBond per square centimetre. So, you'd need at least 86 uL of SureBond (in 4.3 mL of PBS) per vial of cells. Medium: Assuming you'd use 6-well plates to start with your experiments, each well would require 2 mL medium. For plating 1 vial of cells (19 mL total): Spin down: 10 mL Then, 2 mL for each well x 4.5 wells = 9 mL Medium A for 1 vial of the cells (40 mL total): Day 1 - complete medium change - 2 mL for each well x 4.5 wells = 9 mL Day 4, 7, 10, 13 and 16 - 2/3 medium change (1.33 mL) - 6.7 mL total for each well x 4.5 wells = 30.2 mL Medium B for 1 vial of the cells (31 mL total): Day 19, 22, 25, 28 and 31 - 2/3 medium change - 6.7 mL x 4.5 wells = 30.2 mL Hence, you'd need 81 mL of medium per vial of cells to differentiate and mature the cells to Day 33 for assaying.
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.
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
- Poulsen ET, Iannuzzi F, Rasmussen HF, et al., An aberrant phosphorylation of amyloid precursor protein tyrosine regulates its trafficking and the binding to the clathrin endocytic complex in neural stem cells of Alzheimer's disease patients. Journal of Frontiers in Molecular Neuroscience (2017)
- Zollo A, Allen Z, Rasmussen H, et al., Sortilin-Related Receptor Expression in Human Neural Stem Cells Derived from Alzheimer’s Disease Patients Carrying the APOE Epsilon 4 Allele. Neural Plasticity (2017)