PSC Neural Induction Medium - FAQs

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.

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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Yes, neurospheres can be maintained in PSC Neural Expansion medium. The recipe for Neural Expansion medium is found in the Neural Induction Medium product manual.

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Antibodies that are not stored or handled properly may result in loss of staining quality and should be replaced. Note that Nestin should stain filaments in the cytoplasm whereas Pax6, Sox1, and Sox2 should stain the nucleus. Oct4 should stain the nucleus of pluripotent cells, but not NSCs.

If you are seeing extensive cell death and the NSCs are cryo-preserved under P4, we recommend treating with 5 µM ROCK Inhibitor Y27632 after plating.

Yes, NSCs induced by PSC Neural Induction Medium can be differentiated into motor neurons. We do not offer a validated protocol, but you can refer to the following publication or perform a literature search for additional methods:
http://rd.springer.com/article/10.1007/s12015-014-9541-0

We have tested the following PSC cell lines for the PSC Neural Induction Medium:
• H9 embryonic stem cells
• iPSC line created in-house
• Episomal iPSC cell line

We recommend using neural expansion medium with the following composition to expand NSCs generated using the PSC Neural Induction Medium: - 50:50 Neurobasal medium: Advanced DMEM/F12 plus PSC Neural Induction Supplement.

PSC lines can behave differently, and we recommend performing your own validation experiments if you want to use PSC Neural Induction Medium, or you do not observe good expansion using the Neurobasal and Advanced DMEM/F12 combination.

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It is not necessary or recommended to add ROCK inhibitor to PSC Neural Induction Medium when differentiating cells into neural stem cells. You would only add ROCK inhibitor to iPSC or ESC before initiation of neural induction and when expanding neural stem cells using Neural Expansion Medium.

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No, NSCs derived by PSC Neural Induction Medium cannot be differentiated into cells of the PNS. They can be differentiated into neurons and glial cells in the central nervous system. Sensory, sympathetic and parasympathetic neurons, as well as Schwann cells in the PNS are derived from precursor cells of the neural crest.

PSC Neural Induction Medium has not been tested and is not recommended for the conversion of mouse PSCs into NSCs.

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NSCs derived from some human PSC lines may have an increased proliferation rate. In this case, we recommend decreasing NSC plating density to 5x10^4 cells/cm2.

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We recommend adding ROCK inhibitor Y27632 into cell suspension to reach a final concentration of 5 µM if the passage number of NSCs is under P4. NSCs from some human PSC lines may be more sensitive to dissociation. For NSCs derived from those PSC lines, the overnight treatment with Y27632 after P4 decreases cell death after re-plating.

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To prevent extensive cell death during late stage neural induction, we recommend checking whether cells are over-confluent. If they are, change the PSC Neural Induction Medium every day with 5 mL per well of a 6-well plate.

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For PSCs cultured in Essential 8 Medium, cells may detach during neural induction. To prevent cell detachment, we recommend coating your culture plates with Vitronectin at 1µg/cm2 when splitting PSCs.

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We recommend that you discard the culture and begin the process again. You will want to select and maintain a high quality of PSCs before inducing neural differentiation.

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Due to variable parameters such as the confluence of PSCs, cell clump passaging, cell attachment and the property of different PSC lines, it may be difficult to determine the splitting ratio. To obtain the optimal starting density for neural induction with PSC Neural Induction Medium we recommend estimating the cell number of PSC clumps before plating. Instructions for estimating the cell number of PSC clumps can be found in the protocol (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0008031.pdf). PSC culture should be at 15-25% confluence on day 1 of PSC splitting (Day 0 of neural induction).

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Neural induction can be started with human PSCs cultured on 6-well plate or culture dishes. PSCs cultured in flasks are not recommended because it is difficult to remove non-neural differentiated colonies in flasks.

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PSC Neural Induction Medium has been tested on human PSCs cultured in both feeder-free and feeder-based conditions. To eliminate MEF contamination, human PSCs cultured in feeder-free conditions such as Essential 8 and StemPro hESC SFM are strongly recommended for starting neural induction.

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Yes, PSC Neural Induction Medium works for both human ESCs and iPSCs.

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Unfortunately, we cannot provide the exact composition of PSC Neural Induction Medium because it is proprietary information. The only information we can provide is that the medium contains BSA, small molecules, and products capable of inhibiting GSK and TGF-beta pathways.

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Yes. We have seen compatibility with the following differentiation kits provided by Thermo Fisher Scientific: PSC Cardiomyocyte Differentiation Kit (Cat. No. A2921201), PSC Definitive Endoderm Induction Kit (Cat. No. A3062601), PSC Neural Induction Medium (Cat. No. A1647801), and PSC Dopaminergic Neuron Differentiation Kit (Cat. No. A3147701).

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There are several possibilities causing the failure of neural induction:

- High quality of hPSC cells is critical to the success of neural induction. Remove differentiated and partially differentiated hPSC cells before neural induction.
- Before plating hPSCs for induction, cell counting is recommended because too low or too high cell confluency will reduce induction efficiency. The recommended plating density for induction is 2-2.5 x 10E4 cells/cm2.
- Cell clumps (and not a single cell suspension) should be plated for induction.
- To increase induction efficiency, overnight treatment with 10 µM ROCK Inhibitor Y27632 at the time hPSC splitting can be used to prevent extensive cell death.

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We do not recommend using Gibco StemPro NSC SFM to expand NSCc which are induced using Gibco PSC Neural Induction Medium (Cat. No. A1647801), because the morphology of NSCs induced from some lines of hPSCs will change if this medium is used to expand NSCs. We recommend using the Neural Expansion Medium to expand such NSCs, as stated here ( https://tools.thermofisher.com/content/sfs/manuals/MAN0008031.pdf).

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We have seen no effect on downstream differentiation potential for cells cultured in Essential 8 Flex Medium for up to 15 passages. Tri-lineage potential has been demonstrated from embryoid bodies as well as using Gibco PSC Neural Induction Medium (Cat. No. A1647801), Gibco PSC Cardiomyocyte Differentiation Kit (Cat. No. A25042SA) and Gibco PSC Definitive Endoderm Induction Kit (Cat. No. A27654SA).

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