Human iPSC-Derived Neural Stem Cells – Epilepsy Patient
iPSC-Derived Neural Stem Cells for Epilepsy Studies
Using our expertise in reprogramming and neural induction, Axol has differentiated neural stem cells from iPSCs derived from a patient that was clinically diagnosed with epilepsy. Our blog discusses the need for human iPSC-derived neural cell models from healthy and patient donors to help further our understanding of neuronal networks and identify effective treatments for conditions such as epilepsy.
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.
|Starting material||Dermal fibroblast|
|Donor age at sampling||5 months|
|Donor age of onset||in utero|
|Reprogramming method||Episomal vector|
|Induction method||Monolayer & chemically defined medium|
|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)|
|Cell type||iPSC-derived neural stem cells|
|Disease information||The patient presented with prenatal (3-4 weeks before delivery) tremors in utero. After birth, the patient exhibited bradycardia, abnormal EEG (central apneas present) and earlymyoclonic encephalopathy. The patient’s epilepsy was characterized further by: noted erratic seizures (1 to 5 per day), poor oral and motor skills, inability to follow with gaze and the brainstem auditory evoked response (BAER) revealed only waveforms I and III suggestive of upper pontine dysfunction. The donor was treated with phenobarbital and Klonopin.|
|Shipping conditions||Dry ice|
|Storage conditions||Liquid nitrogen|
Do you have any 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.