Human iPSC-Derived Neural Stem Cells (Male)

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).

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

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.

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.

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.

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.