PSC 神经诱导培养基

神经诱导失败有几种可能的原因：

•高品质的hPSC细胞对于成功完成神经诱导至关重要。在神经诱导前去除已分化和部分分化的hPSC细胞。
•在铺板后续用于诱导的hPSC细胞之前，我们推荐用户对细胞进行计数，因为过低或过高的细胞汇合度都将减低诱导效率。诱导实验的推荐铺板密度为2–2.5 x 10E4个细胞/cm2。
•应铺板细胞团（而非单个细胞的悬液）来进行诱导。
•为了增加诱导效率，可在hPSC传代时使用10 µM ROCK抑制剂Y27632进行过夜处理，以避免细胞过多死亡。

我们不推荐使用Gibco StemPro NSC SFM扩增以Gibco PSCs神经诱导培养基（货号 A1647801）诱导出来的NSCs，因为使用这一培养基来扩增NSCs时，某些hPSCs细胞系来源的NSCs形态将发生改变。我们推荐使用神经元扩增培养基来扩增这些NSCs，如此处（https://tools.thermofisher.com/content/sfs/manuals/MAN0008031.pdf）所述。

我们将细胞培养在Gibco Essential 8 Flex培养基中长达15代，未发现细胞的后续分化潜能受到任何影响。进行拟胚体分化和使用Gibco PSC神经诱导培养基（货号A1647801），Gibco PSC心肌细胞分化试剂盒（货号A25042SA），Gibco PSC定形内胚层诱导试剂盒（货号A27654SA）等产品进行分化均证明这些细胞拥有三系分化潜能。

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.