Medio de inducción neuronal de células madre pluripotentes (PSC)

PSC Neural Induction Medium is a medium to convert hPSCs into NSCs. At day 7 of neural induction, dissociate P0 NSCs with Accutase. The following recommendations can be use for differentiation of neurons or neurospheres: For differentiation of neurons: Use PSC Neural Induction Medium to convert hPSCs into NSCs. At day 7 of neural induction, dissociate P0 NSCs with Accutase. If dissociated NSCs are plated on a Geltrex-coated culture vessel in neural expansion medium, NSCs will grow as a mono-layer. For NSC expansion, it is necessary to treat NSCs with 5 µM ROCK inhibitor Y27632 at the time of plating to prevent cell death if NSCs are under P4. For differentiation of neurospheres: Use PSC Neural Induction Medium to convert hPSCs into NSCs. At day 7 of neural induction, dissociate P0 NSCs with Accutase. If dissociated NSCs are plated into a non-coated flask in neural expansion medium, dissociated NSCs will re-aggregate into small spheres. NSCs in each sphere will proliferate and form big neurospheres. We have not expanded NSCs in neurosphere format in-house. You can test expanding NSCs in neurospheres with and without ROCK inhibitor Y27632 to determine which is optimal for your workflow. Neural stem cells in adherent or neurosphere conditions can be differentiated into neurons using the appropriate protocol.

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Studies have not been performed using flow cytometry or qPCR to detect NKX2.1 expression in NSCs induced by Neural Induction Medium.

Yes, this is normal. SOX2 is expressed by both hPSCs and hPSC-derived NSCs, so you will see its expression in the induced NSCs from iPSCs.

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NSCs induced using the PSC Neural Induction Medium can be differentiated on glass slides/coverslips, but the glass should be cleaned well before coating. We recommend the following cleaning protocol:
1. Treat glass cover slips with1M HCl at RT for 2-3 h on a shaker.
2. Rinse with tap water 5 times, and then double distilled water for 2 times
3. Store cleaned cover slips in 70% ethanol.
4. Dry cover slips by transferring cover slips into culture plate with sterile forceps before poly-L-ornithine and laminin coating.

Neurospheres can be plated on laminin coated culture plates for neuron differentiation. The issue is that it is difficult to control the plating density of neurospheres. Alternatively, neurospheres can be dissociated into single cells and plate single cell suspension at a certain density such as 1-5 x 10^4 cells/cm2 onto laminin coated plates for neuron differentiation. For general neuron differentiation, Neurobasal+B27+N2 can be used. Growth factors such as BDNF and/or GDNF can be added into medium for improving survival of differentiating NSCs.

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