Induction of Neural Stem Cells from Human Pluripotent Stem Cells Using Gibco® PSC Neural Induction Medium

Human pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), are excellent resources for studying cell fate specification, disease modeling and drug screening. The first important step in producing various neural cells from PSCs is the induction of PSCs to neural stem cells (NSCs). Conventional methods of NSC derivation from human PSCs involving embryoid body (EB) formation or co-cultures with stromal cell lines have several disadvantages including a time-consuming protocol and variability in the quality of resulting NSCs. We have developed a serum-free neural induction medium, which can convert human PSCs into NSCs in one week with high efficiency, but without the laborious processes of EB formation and mechanical NSC isolation.

Neural Induction Medium (for 500 mL)

1. To prepare 500 mL of complete Gibco® Neural Induction Medium, aseptically mix the following components:

• Neurobasal® Medium 490 mL
• Gibco® Neural Induction Supplement 10 mL

2. Complete Neural Induction Medium can be stored at 2–8°C in the dark for up to 2 weeks. Warm the Gibco® Neural Induction Medium in a 37°C water bath for 5–10 minutes before using. Do not warm the Neural Induction Medium in a 37°C water bath for longer than 10 minutes, as this may cause degradation of the medium.
Note: Gibco® Neural Induction Supplement can be thawed in a 37°C water bath for 5 minutes or at 2–8°C overnight, and then aliquoted and frozen at –5°C to –20°C to allow for preparation of smaller volumes of complete medium. Avoid repeated thawing and freezing of the Neural Induction Supplement.

Neural Expansion Medium (for 100 mL)

1. To prepare 100 mL of complete Neural Expansion Medium, aseptically mix the following components:

• Neurobasal® Medium 49 mL
• Advanced™ DMEM⁄F-12 49 mL
• GIBCO® Neural Induction Supplement 2 mL

2. Complete Neural Expansion Medium can be stored at 2–8°C in the dark for up to 2 weeks. Warm the Neural Expansion Medium in a 37°C water bath for 5–10 minutes before using.

ROCK Inhibitor Y27632 Solution (5 mM)

1. To prepare 5 mM ROCK inhibitor Y27632 solution, aseptically mix the following components:

• Y27632 1 mg
• Distilled water 0.625 mL

2. After dissolving, filter through a 0.22 μm filter, aliquot 20–50 μL into sterile tubes, and store at –5°C to –20°C in the dark for up to 1 year. Thawed Y27632 solution can be kept at 2–8°C for up to 1 week.
Note: The Molecular Weight of Y27632 is 320.26, which may vary between lots depending on the water content.

Coating Culture Vessels with Geltrex® LDEV-Free, hESC-Qualified Basement Membrane Matrix

1. Thaw a vial of Geltrex® LDEV-Free hESC-Qualified Reduced Growth Factor Basement Membrane Matrix at 2–8°C overnight.
Note: Thawed Geltrex® matrix can be aliquoted and frozen at –5°C to –20°C, or stored at 2–8°C for up to 2 weeks. Avoid repeated thawing and freezing.
2. To create a working solution, dilute the thawed Geltrex® matrix solution 1:100 with cold Neurobasal® Medium on ice.
3. Quickly cover the whole surface of each culture vessel with the Geltrex® matrix solution (refer to Table 1).
4. Incubate the culture vessels in a 37°C, 5% CO₂ incubator for 1 hour.
5. Culture vessels can now be used. Just prior to use, aspirate the diluted Geltrex® solution from the culture vessels. Cells can be plated directly onto the Geltrex® matrix-coated culture vessels without rinsing. Coated culture vessels can also be stored at 2–8°C for up to one week. When storing, seal culture vessels with Parafilm® laboratory film to prevent drying. Before using, warm up the coated culture vessels stored at 2–8°C at room temperature for 30 minutes.

Methods Quick Links

• Culture and Preparation of human PSCs for Neural Induction
• Neural Induction
• Harvesting and Expansion of P0 NSC
• Cryopreservation of NSCs
• Recovery of Cryopreserved NSCs
• Staining for Pluripotent (Oct4) and NSC (Nestin, Pax6, Sox1, and Sox2) Markers

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1. On day 7 of neural induction (Fig. 2C), NSCs (P0) are ready to be harvested and expanded.
2. Prepare Geltrex® matrix-coated vessels prior to next steps.
3. Aspirate the spent Neural Induction Medium with a Pasteur glass pipette from the 6-well plate(s) to be passaged.
4. Gently add 2 mL of DPBS without CaCl₂ and MgCl₂ into each well of a 6-well plate and aspirate the DPBS to rinse the cells.
Note: Add DPBS towards the wall of the well to avoid cell detachment.
5. Add 1 mL of pre-warmed StemPro® Accutase® Cell Dissociation Reagent to each well of 6-well plate and incubate for 5–8 minutes at 37°C until most of cells detach from the surface of the culture vessels.
6. Use a cell scraper to detach the cells off the surface of the plates.
7. Transfer the cell clumps using a 5-mL pipette and place into a 15 or 50-mL conical tube.
8. Add 1 mL of DPBS to each well of 6-well plate to collect residual cells and transfer the cell suspension to the conical tube.
9. Gently pipette the cell suspension up and down 3 times with a 5-mL or 10-mL pipette to break the cell clumps.
10. Pass cell suspension through a 100-μm strainer and centrifuge the cells at 300 × g for 4 minutes.
11. Aspirate the supernatant, re-suspend the cells with DPBS (3-5 mL of DPBS for all cells from 1 well of a 6-well plate) and centrifuge the cells at 300 × g for 4 minutes.
12. Aspirate the supernatant, re-suspend the cells with pre-warmed complete Neural Expansion Medium (e.g., 1 mL for all cells from 1 well of a 6-well plate).
13. Determine the cell concentration using your preferred method.
14. Dilute the cell suspension with pre-warmed complete Neural Expansion Medium to 2 × 10⁵–4 × 10⁵ cells/mL.
15. Add ROCK inhibitor Y27632 into cell suspension to a final concentration of 5 μM.
16. Aspirate the Geltrex® matrix solution from Geltrex® matrix-coated vessels and add the diluted cell suspension into each culture plate/dish to plate the cells at a density of 0.5 × 10⁵–1 × 10⁵ cells/cm² as shown in Table 2.

Note: Avoid splashing of the medium to the outsides of the well to avoid contamination.

18. After overnight incubation, change to complete Neural Expansion Medium to eliminate the ROCK inhibitor Y27632. Continue to exchange the Neural Expansion Medium without Y27632 every other day thereafter.
19. Usually, NSCs reach confluency on day 4–6 after plating (Fig. 3A, B). When NSCs reach confluency, they can be further expanded in complete Neural Expansion Medium. Expanded NSCs can be cryopreserved (see protocol below) or differentiated into specific neural cell types following the protocol of your choice.

Note: After dissociation of P0–P4 NSCs, the overnight treatment with the ROCK inhibitor Y27632 at a final concentration of 5 μM is required at the time of plating to prevent cell death for both expansion and differentiation into glial and neuronal cells (Fig. 3C).

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1. Make sure your NSC have been passaged to at least P1 before cryopreservation.
2. When NSCs reach confluency, warm the appropriate amount of StemPro® Accutase® Reagent in a 37°C water bath according to Table 3.

1. Prepare Geltrex® matrix-coated dishes as previously described.
2. Wear eye protection as cryotubes stored in the liquid phase of liquid nitrogen may accidentally explode when warmed.
3. Wear ultra low temperature cryo-gloves. Remove cryotubes of NSCs from the liquid nitrogen storage tank using metal forceps.
4. Immerse the vial in a 37°C water bath without submerging the cap. Swirl the vial gently.
5. When only an ice crystal remains, remove the vial from the water bath.
6. Spray the outside of the vial with 70% ethanol and place it in the cell culture hood
7. Pipette the cells gently into a sterile 15-mL conical tube using a 1-mL pipette.
8. Add 1 mL of DPBS into the vial to collect the resident cells. Transfer the DPBS from the vial and drop-wise add it to the 15-mL conical tube. While adding, gently move the tube back and forth to mix the NSCs. This reduces osmotic shock to cells.
9. Centrifuge the cells at 300 × g for 5 minutes and aspirate the supernatant.
10. Resuspend the cell pellet in DPBS. Centrifuge at 300 × g for 5 minutes and aspirate the supernatant.
11. Re-suspend the cell pellet in the appropriate amount (e.g., 1 mL for all NSCs from 1 vial) of pre-warmed Neural Expansion Medium.
12. Determine the cell concentration using your preferred method.
13. Dilute the cell suspension with pre-warmed Neural Expansion Medium into a solution containing 2 × 10⁵–4 × 10⁵ cells/mL.

Note: If the NSCs were cryo-preserved before passage 4, addition of the ROCK inhibitor Y27632 solution at a final concentration of 5 μM into the cell suspension is required to prevent cell death.

14. Aspirate the Geltrex® matrix solution from the Geltrex® matrix-coated vessels and add the appropriate amount of diluted cell suspension into each culture vessel to plate cells at the density of 0.5 × 10⁵–1 × 10⁵ cells/cm2 (see Table 4).

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The table below lists some potential problems and solutions that may help you troubleshoot your neural induction experiments.

LT177             Publication Number MAN0008031    Rev 1