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

Introduction

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.

Required Materials

  • Gibco® PSC Neural Induction Medium (consists of Neurobasal® Medium and GIBCO® Neural Induction Supplement, 50X (Cat. no. A1647801)
  • Advanced™ DMEM⁄F-12 (Cat. no. 12634) (needed for NSC expansion)
  • Geltrex® LDEV-Free hESC-qualified Reduced Growth Factor Basement Membrane Matrix (Cat. no. A1413301 or A1413302)
  • Distilled water (Cat. no. 15230)
  • Rock Inhibitor Y27632 (Sigma-Aldrich, Cat. no. Y0503)
  • DPBS without CaCl2 and MgCl2 (Cat. no. 14190)
  • StemPro® Accutase® Cell Dissociation Reagent (Cat. no. A11105)
  • Dimethyl sulfoxide (DMSO) (Cat. no. D12345)
  • Cell scraper (Fisher Scientific, Cat. no. 08-771-1A)
  • 15-mL and 50-mL sterile polypropylene conical tubes
  • 6-well culture plates (Fisher Scientific, Cat. no. 08-772-1B)
  • Pasteur glass pipettes
  • 2, 5 and 10-mL sterile pipettes
  • 100-μm strainer (Fisher Scientific, Cat. no. 08-771-19)
  • Nalgene® Mr. Frosty® Freezing Container (Fisher Scientific, Cat. no. 15-350-50)
  • 37°C humidified cell culture incubator with 5% CO2
  • Liquid nitrogen storage
  • Centrifuge
  • 37°C water bath

* For In Vitro Diagnostic use.

Immunocytochemistry reagents:

  • Molecular Probes® Human Neural Stem Cell Immunocytochemistry Kit (Cat. no. A24354) NOTE: this complete immunocytochemistry kit contains primary antibodies for four common NSC markers (Nestin, PAX6, SOX1, and SOX2), a matching set of Alexa Fluor®-labeled secondary antibodies, a nuclear DNA stain (DAPI), and all of the buffers necessary for performing the staining protocol.
  • OCT4 Rabbit Monoclonal Antibody (Cat. no. A13998)
  • Cover glass, 24 × 50 mm (Fisher Scientific, Cat. no. 22-050-232)

TOP

Ordering Information

Sku Name Size Price Qty
A1647801 PSC Neural Induction Medium 500 mL USD 395.00
12634010 Advanced DMEM/F-12 500 mL USD 41.60
A1413301 Geltrex™ LDEV-Free, hESC-Qualified, Reduced Growth Factor Basement Membrane Matrix 1 mL USD 44.50
15230204 Distilled Water 10 x 500 mL USD 111.00
14190136 DPBS, no calcium, no magnesium 1,000 mL USD 29.40
A1110501 StemPro® Accutase® Cell Dissociation Reagent 100 mL USD 37.60
D12345 DMSO, Anhydrous 10 x 3 mL USD 68.50
A24354 Human Neural Stem Cell Immunocytochemistry Kit 20 tests USD 695.00

Preparation of Media and Materials

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.

 

Culture vessel Surface area (cm2) Volume of diluted Geltrex® matrix (mL)
4-well chamber slide 1.8 cm2/well 0.3 mL/chamber
6-well plate 9.6 cm2/well 1.0 mL/well
35-mm dish 11.8 cm2 1.0 mL
60-mm dish 20 cm2 2 mL
100-mm dish 60 cm2 5 mL

 

Table 1 Volume of Geltrex® matrix solution required

 

Methods

Culture and Preparation of human PSCs for Neural Induction

1. Start with high quality human PSCs (with minimal or no differentiated colonies) cultured in feeder-free conditions such as in Essential 8™ Medium on Vitronectin or Geltrex® substrate or in StemPro® hESC SFM on Geltrex® matrix. For PSC culturing protocols, visit www.lifetechnologies.com/protocols.

Notes:

a. The quality of the PSCs (with minimal or no differentiated colonies) is critical for efficient neural induction. Remove any differentiated and partially differentiated colonies before passaging PSCs.
b. Human PSCs cultured on mouse embryonic fibroblasts can also be used for neural induction.

2. When PSCs reach ~70–80% confluency, split them into 6-well culture plates following the PSC subculturing protocol (see www.lifetechnologies.com/protocols).
3. After generating a PSC cell suspension, transfer a portion of the cell suspension to a 15-mL conical tube (e.g., 1 mL of 6 mL PSC suspension prepared from one well of a 6-well plate) to estimate the total cell number of the PSC cell suspension.
4. Centrifuge the 15-mL conical tube with the cells at 200 × g for 3 minutes and aspirate supernatant.
5. Add 1 mL of pre-warmed StemPro® Accutase® cell dissociation reagent into the 15-mL conical tube with the cells and incubate for 5 minutes at 37°C.
6. Gently pipette the cells up and down with a 1 mL pipette 5 times to dissociate the cells into a single cell suspension.
7. Determine the total cell number using your preferred method.
8. Add 2.5 mL PSC culture medium into each well of coated 6-well plates.
9. Gently shake the conical tube containing the PSC cell suspension and plate the PSCs into each well at 2.5 × 105–3 × 105 PSCs per well. For example, add 0.25–0.3 mL of PSC suspension into each well if the PSC suspension is 1 × 106 cells/mL.
10. Move the plates in several quick back-and-forth and side-to-side motions to disperse the cells across the surface and place them gently in a CO2 incubator.

Notes:

a. The split ratio varies depending on the confluence of PSCs before splitting and variability in PSC lines. Neural induction starts on day 1 of PSC splitting (about 24 hours after passaging). The starting density of PSCs should be about 10–20% confluency. When passaging PSCs, cells should be plated as small clumps and not as a single cell suspension. Avoid plating PSC as single cells as that can lead to increased cell death.
b. You may treat the cells overnight with 10 μM of ROCK inhibitor Y27632 by adding it into PSC medium at the time of splitting to prevent cell death.

TOP

Neural Induction

1. The workflow of NSC derivation is shown in Fig. 1 A. On day 1 of PSC splitting (10–20% confluency, Fig. 1 B), aspirate the spent medium to remove non-attached cells, and add 2.5 mL pre-warmed complete Gibco® Neural Induction Medium into each well of 6-well plate. Return the plates into the incubator.

neural induction

 

Figure 1. A: Workflow of NSC derivation from PSCs. B: Human PSCs cultured in feeder-free condition on day 1 of splitting with 10–20% confluency. C: Cells under neural differentiation on day 2 of neural induction. D–G: Representative images to show non-neural differentiation (Arrows) on day 2 of induction which is due to the poor quality of starter PSCs if used during neural induction.

2. On day 2 of neural induction, the morphology of cell colonies should be uniform (Fig. 1 C).

Notes:

a. If the quality of PSCs is good before neural induction, the morphology of the cells should appear as in the image (Fig. 1 C).
b. If the quality of starter PSC is poor, the cultures will be dominated by a number of non-neural colonies as shown in images (Fig. 1 D–G). In this case, there are two options
i. If there are few areas in dish exhibiting this morphology, cultures can be continued after removing such non-neural differentiated colonies. To remove non-neural differentiated colonies, mark all non-neural differentiation colonies with a microscopy marker. Tilt the plate and remove all unwanted colonies at the upper half of the well by pointing a Pasteur glass pipette to marked colonies to aspirate cells off. Turn the plate 180 degrees, and repeat. Perform this step one well at a time to prevent cells from becoming too stressed without medium
ii. If there are a large number of such non-neural colonies, it is recommended to discard the cultures and start with high quality PSCs.

3. On day 2 (about 48 hours after switching to Gibco® Neural Induction Medium), change medium by aspirating spent medium from each well. Add 2.5 mL of pre-warmed complete Neural Induction Medium per well.
4. On day 4 of neural induction, cells will be reaching confluency (Fig. 2 A). Mark all colonies if any non-neural differentiation is noticed, and remove such unwanted colonies with a Pasteur glass pipette. Aspirate the spent medium from each well. Add 5 mL of pre-warmed complete Neural Induction Medium per well.
5. On day 6 of neural induction, cells should be at near maximal confluence (Fig. 2 B). Remove any non-neural differentiated cells and add 5 mL of complete Neural Induction Medium into each well.
Note: Due to high cell density from day 4 onwards in the culture, doubling the volume of Neural Induction Medium is very critical for cell nutrition. Also, minimal cell death should be observed from day 4 to 7 after neural induction. If the color of cells turns brownish with many floating cells during day 4 to 7 of neural induction, it indicates that the starting density of PSCs was too high. In this case, change Neural Induction Medium every day with 5 mL per well.

morphology of cells 

Enlarge

Figure 2. A: The morphology of cells on Day 4, B: Day 6, and C: Day 7 of neural induction. D: The morphology of cells on day 7 of culture by omitting key components in neural induction supplement. *Flat non-neural cells.

TOP

Harvesting and Expansion of P0 NSC

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.

 


Culture vessel
Surface area (cm2) Volume of cell suspension (mL)
4-well chamber slide1.8 cm2/well0.46 mL/chamber
6-well plate9.6 cm2/well2.5 mL/well
35-mm dish11.8 cm23 mL
60-mm dish20 cm25 mL
100-mm dish60 cm215 mL

 

Table 2 Volume of cell suspension required

 

17. Move the culture vessels in several quick back-and-forth and side-to-side motions to disperse the cells across the surface and place them gently in the incubator.

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).

 PO NSC

Enlarge

 

Figure 3. A: P0 NSCs plated at a density of 1 × 105 cells/cm2 on day 1 of plating with Y27632 treatment. B: NSCs reach confluence on day 5 of plating. C: Without Y27632 treatment, intensive cell death takes place on day 1 of NSC plating.
TOP

Cryopreservation of NSCs

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.


Culture vessel
Surface area (cm2) Volume of StemPro® Accutase® reagent (mL)
6-well plate9.6 cm2/well1 mL/well
35-mm dish11.8 cm21 mL
60-mm dish20 cm22 mL
100-mm dish60 cm25 mL

 


Table 3 Volume of StemPro® Accutase® reagent required

 

3. Aspirate the spent medium and add pre-warmed StemPro® Accutase® Reagent solution into culture vessels according to Table 3.
4. Incubate for 3–5 minutes at 37°C until all cells detach from the surface of culture vessels.
5. Transfer the cells into a 15 or 50-mL conical tube using a 5-mL pipette.
6. Add the appropriate amount of DPBS into each well of the vessel (e.g., 1 mL for 1 well of a 6-well plate) to collect the residual cells, and add the cell suspension to the tube.
7. Triturate the cell suspension 3 times with a 5-mL or 10-mL pipette to break the cell clumps.
8. Centrifuge the cells at 300 × g for 4 minutes and aspirate the supernatant.
9. Re-suspend the cells with the appropriate amount of DPBS and centrifuge the cells at 300 × g for 4 minutes.
10. Aspirate the supernatant and re-suspend the cells with the appropriate amount of Neural Expansion Medium.
11. Determine the cell concentration using your preferred method.
12. Dilute the cell suspension with Neural Expansion Medium to 2 × 106–4 × 106 cells/mL.
13. Add the same volume of Neural Expansion Medium containing 20% DMSO.
14. Allocate 1 mL cell suspension into each cryotube and freeze cells at –80°C overnight in Nalgene® Mr. Frosty® Freezing Containers with isopropanol.
15. Transfer the cells into a liquid nitrogen tank next day for long term storage.  

TOP

Recovery of Cryopreserved NSCs

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).


Culture vessel
Surface area (cm2) Volume of cell suspension (mL)
4-well chamber slide 1.8 cm2/well 0.46 mL/chamber
6-well plate 9.6 cm2/well 2.5 mL/well
60-mm dish 20 cm2 5 mL
100-mm dish 60 cm2 15 mL
T25 Flask 25 cm2 6.5 mL
T75 Flask 75 cm2 19.5 mL

 

Table 3 Volume of StemPro® Accutase® reagent required

 

15. Move the culture vessels in several quick back-and-forth and side-to-side motions to disperse the cells across the surface and place the vessels gently in the incubator.

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

16. On day 2 of cell plating, exchange the Neural Expansion Medium. Continue to exchange the medium every other day until the NSCs reach confluency and are ready for further expansion.

Note: If NSCs were under P4, the overnight treatment with the ROCK inhibitor Y27632 is required at the time of NSC plating to prevent cell death. At day 1 after NSC plating, replace the medium with complete Neural Expansion Medium without Y27632 to eliminate Y27632 from the culture.

TOP

Staining for Pluripotent (Oct4) and NSC (Nestin, Pax6, Sox1, and Sox2) Markers

Note: We recommend using the Molecular Probes® Human Neural Stem Cell Immunocytochemistry Kit (Cat. no. A24354). This complete immunocytochemistry kit contains primary antibodies for four common NSC markers (Nestin, Pax6, Sox1, and Sox2), a matching set of Alexa Fluor®-labeled secondary antibodies, a nuclear DNA stain (DAPI), and all of the buffers necessary for performing the staining protocol. In addition, pluripotency marker Oct4 (Cat. no. A13998 is compatible with the reagents in the NSC immuncytochemistry kit) can be used as a negative control marker for NSCs.

1. Plate the dissociated NSCs at a density of 1 × 105–3 × 105 cells/cm2 into 4-well chamber slides

Note: For P0-P4 NSCs, treatment with 5 μM ROCK inhibitor Y27632 is essential to prevent cell death.

2. At day 1 following NSC plating, remove the spent medium and add 0.4 mL of Fixative Solution (4% formaldehyde in DPBS) per well and incubate for 15 minutes at room temperature.

Note: Add solutions toward the wall of the well to avoid cell detachment.

Note: Fixed cells can be stored in 0.4 mL 1X DPBS(Wash Buffer)/per well, wrapped to avoid evaporation, at 2–8°C for up to to 2 weeks.

3. Remove the Fixative Solution and, add 0.4 mL of Permeabilization Solution (0.5% Triton® X-100 in DPBS) into each well and incubate for 15 minutes at room temperature.

4. Remove the Permeabilization Solution and add 0.4 mL of Block Solution (3% BSA in DPBS) into each well and incubate for 30 minutes – 1 hour at room temperature.

5. Remove the Blocking Solution, and add 0.2 – 0.4 mL of primary antibody against pluripotent NSC markers (see Table 4 below for options), diluted 1:50 – 1:100 in Blocking Solution, into each well. Incubate at room temperature for 3 hours or  2–8°C overnight.

Table 4. Recommended antibody staining options.

Color options: 

  

Green1 (e.g., FITC filter)

 

Orange2 (e.g., Cy®3 / TRITC filter)

or Red3 (e.g., Texas Red® filter)

Antibody combination # 1: Nestin + Sox2

Primary antibody

anti-Nestin (host: mouse)

anti-Sox2 (host: rabbit)

Secondary antibody

Alexa Fluor®488 donkey anti-mouse

Alexa Fluor®555 donkey anti-rabbit

or

Alexa Fluor®594 donkey anti-rabbit

Antibody combination # 2: Sox1 + Pax6 or Oct4

Primary antibody

anti-Sox1 (host: goat)

anti-Pax6 or anti-Oct4 (host: rabbit)

Secondary antibody

Alexa Fluor®488 donkey anti-goat

Alexa Fluor®555 donkey anti-rabbit

or

Alexa Fluor®594 donkey anti-rabbit

1Ex/Em 495/519 nm (green)

2Ex/Em 555/565 nm (orange)

3Ex/Em 590/617 nm (red)

6. Rinse the cells 3 times with 0.4 mL of 1X DPBS (Wash Buffer) per well, with 1-2 minutes per rinse at room temperature.

7. Remove the Wash Buffer, add 0.2 mL of the appropriate secondary antibody (see Table 4), diluted 1:250-1:500 in Blocking Solution, into each well. Incubate for 1 hour at room temperature.

8. Rinse the cells 3 times with 0.4 mL of 1X DPBS (Wash Buffer) per well, with 1-2 minutes per rinse at room temperature.

Note: If desired, stain the nuclear DNA of the cells in each well by adding 1-2 drops/mL of NucBlue® Fixed Cell Stain (DAPI) into the last wash step.

9. Image the cells immediately. Cells can also be stored at 4OC, wrapped to avoid evaporation, in the dark for up to 1 week.

Note: Alternatively, remove the Wash Buffer, apply a suitable quantity (about 1 drop) of ProLong® Gold Antifade Reagent to each well, cover with a coverslip, and air dry the slide in the dark overnight before imaging.

 

TOP

stained_with_DAPI_(blue)
Antibody combination # 1: Nestin+ Sox2 with additional DAPI (nuclear DNA) staining.

Antibody combination # 2: Sox1 + Pax6 with additional DAPI (nuclear DNA) staining.

 

Figure 4. Neural stem cells derived from an iPSC line using Gibco® PSC Neural Induction Medium (Cat. no.  A1647801) were stained for NSC markers Nestin and Sox2 (antibody combination #1) or Sox1 and Pax6 (antibody combination #2) and nuclear DNA (DAPI) using the Neural Stem Cell Immunocytochemistry Kit (Life Tech. # 24354). Please note that not all NSC will stain positive for PAX6, this is normal and to be expected (i.e., only NSCs that possess forebrain neuron fate commitment will stain positive). In our experience ~15-50% of NSCs generated using Gibco® PSC Neural Induction Medium stain positive for Pax6.

Troubleshooting

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

Question/Problem Solution
Does Gibco® Neural Induction Medium work for both human ESCs and iPSCs? Yes, Gibco® Neural Induction Medium works for both human ESCs and iPSCs.
Does neural induction medium work for human PSCs cultured on feeders (mouse embryonic fibroblasts, MEF) and feeder-free conditions? Neural induction medium has been tested on human PSCs cultured in both feeder-free and feeder-based conditions. To eliminate MEF contamination, we strongly recommend starting neural induction by using human PSCs cultured in feeder-free conditions such as Essential 8™ Medium.
Which format of cultures should I use for neural induction? Neural induction can be started with human PSCs cultured on 6-well plate or culture dishes. We do not recommend starting neural induction by using PSCs cultured in flasks because it is difficult to remove non-neural differentiated colonies from PSCs in flasks.
The starting density of human PSCs is too low or too high. 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. By following the protocol to estimate cell number of PSC clumps before plating, PSC should be 10–20% confluent on day 1 of PSC plating (i.e., Day 0 of neural induction).
Too many colonies with non-neural differentiation morphology in culture plate. Select and maintain high quality of PSCs before starting neural induction.
Cell detachment during neural induction. For PSCs cultured in Essential 8™ Medium, cells may detach during neural induction. To prevent cell detachment, coat culture plate with 10 μg/mL of Vitronectin when splitting PSCs.
Extensive cell death during the late stage of neural induction. Check whether cells are over-confluent. If so, change Gibco® Neural Induction Medium every day using 5 mL per well of a 6-well plate.
Extensive cell death after plating dissociated NSCs for expansion and differentiation. Check whether ROCK inhibitor Y27632 is added into cell suspension at final concentration of 5 μM if NSCs are 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.
NSC reach confluence in 2–3 days after passaging NSCs derived from some human PSC lines may have an increased proliferation rate. In this case, decrease NSC plating density to 5 × 104 cells/cm2.
Extensive cell death after thawing and plating cryopreserved NSCs. Check whether NSCs are treated with the ROCK inhibitor Y27632 after plating if NSCs are cryopreserved under P4.
Abnormal staining pattern of NSCs stained with antibodies against neural markers. Antibodies which 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.

FAQ's

1. Can Gibco® PSC Neural Induction Medium convert mouse PSCs into NSCs?
Gibco® PSC Neural Induction Medium has not been tested in mice and it is not recommended for the conversion of mouse PSCs into NSCs.
2. Can NSCs derived by Gibco® PSC Neural Induction Medium be differentiated into cells in the PNS?
No. NSCs derived by Gibco® PSC Neural Induction Medium can be differentiated into neurons and glial cells in the CNS. Sensory, sympathetic and parasympathetic neurons, as well as Schwann cells in the PNS are derived from precursor cells of the neural crest.

TOP

LT177             Publication Number MAN0008031    Rev 1