Differentiating Neural Stem Cells into Neurons and Glial Cells

The protocols in this section describe the steps involved in differentiating neural stem cells (NSC) to neurons, astrocytes, and oligodendrocyte lineages in vitro. NSCs are self-renewing multipotent stem cells that can be proliferated in vitro in supportive culture systems such as Gibco StemPro NSC SFM and can further be differentiated into downstream lineages. The protocols described are primarily optimized with NSCs derived from human embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC). Some optimization in terms of reagent concentration and duration of in vitro differentiation is expected for NSCs from other species such as rat or mouse, as well as with NSCs derived from patient-specific iPSCs.

Required materials (for all steps and sub-protocols)

Cells

• Gibco Human Neural Stem Cells (H9 hESC-Derived) (Cat. No. N7800)

Reagents

• Gibco KnockOut D-MEM/F-12 (Cat. No. 12660)
• Dulbecco’s Modified Eagle Medium (D-MEM) (Cat. No. 11995)
• Dulbecco’s Phosphate-Buffered Saline (D-PBS) (Cat. No. 14040)
• StemPro NSC SFM (Cat. No. A10509-01)
• N-2 Supplement (Cat. No. 17502)
• Gibco B-27 Serum-Free Supplement (Cat. No. 17504)
• Gibco Neurobasal Medium (Cat. No. 21103)
• Antibiotic-Antimycotic solution (Cat. No. 15240)
• Fetal Bovine Serum, ES Cell-Qualified FBS (Cat. No. 16141)
• Gibco GlutaMAX-I (Cat. No. 35050)
• FGF-basic (AA 10–155), Recombinant Human (bFGF) (Cat. No. PHG0024)
• EGF, Recombinant Human (Cat. No. PHG0314)
• Gibco CELLstart CTS (Cat. No. A10142-01)
• Gibco Geltrex Reduced Growth Factor Basement Membrane Matrix (Cat. No. 12760)
• Poly-L-Ornithine (Sigma, Cat. No. P3655)
• Laminin (Cat. No. 23017)
• Dibutyryl cAMP (Sigma, Cat. No. D0627)
• T3 (Sigma, Cat. No. D6397)
• EM grade paraformaldehyde (Electron Microscopy Services, Cat. No. 19208)
• Invitrogen ProLong Gold antifade reagent (Cat. No. P36930)

StemPro NSC SFM complete medium

StemPro NSC SFM complete medium consists of KnockOut D-MEM/F-12 with Gibco StemPro Neural Supplement, EGF, bFGF, and GlutaMAX-I. Complete medium is stable for 4 weeks when stored in the dark at 2–8°C.

To prepare 100 mL of complete medium:

1. Reconstitute bFGF and EGF with 0.1% BSA solution (in KnockOut D-MEM/F-12) at a concentration of 100 μg/mL. You will need 20 μL of each per 100 mL of complete medium. Freeze unused portions in aliquots.
2. Mix the following components under aseptic conditions. For larger volumes, increase the component amounts proportionally: If desired, add 1 mL of Antibiotic-Antimycotic solution per 100 mL of complete medium.

Note: You may observe a white precipitate when thawing StemPro Neural Supplement; this precipitate will disappear when the supplement is completely thawed or dissolved.

Neural differentiation medium

Neural differentiation medium requires supplementation of Neurobasal medium with B-27 Serum-Free Supplement and GlutaMAX-I. Neural differentiation medium is stable for 2 weeks when stored in the dark at 2–8°C.

To prepare 100 mL of neural differentiation medium, aseptically mix the following components. For larger volumes, increase the component amounts proportionally. If desired, add 1 mL of Antibiotic-Antimycotic solution per 100 mL of medium.

If faster differentiation is desired, add dibutyryl cAMP (Sigma, Cat. No. 0627) to a final concentration of 0.5 mM at day 7 for a duration of 3 days, as indicated in the differentiation protocols.

Astrocyte differentiation medium

Astrocyte differentiation medium requires supplementation of D-MEM with N-2, GlutaMAX-I, and FBS. Astrocyte differentiation medium is stable for 4 weeks when stored in the dark at 2–8°C.

To prepare 100 mL of astrocyte differentiation medium, aseptically mix the following components. For larger volumes, increase the component amounts proportionally. If desired, add 1 mL of Antibiotic-Antimycotic solution per 100 mL of medium.

Oligodendrocyte differentiation medium

Oligodendrocyte differentiation medium requires supplementation of Neurobasal medium with B-27, GlutaMAX-I, and T3. Oligodendrocyte differentiation medium is stable for 2 weeks when stored in the dark at 2–8°C.

To prepare 100 mL of oligodendrocyte differentiation medium, aseptically mix the following components. For larger volumes, increase the component amounts proportionally. If desired, add 1 mL of Antibiotic-Antimycotic solution per 100 mL of medium.

* You can prepare a 30 μg/mL T3 stock solution (1,000X) in distilled water. Filter sterilize the T3 stock solution.

Coating culture vessels with CELLstart substrate

1. Dilute CELLStart CTS 1:100 in D-PBS with calcium and magnesium (i.e., 50 μL of CELLStart CTS into 5 mL of D-PBS).
2. Coat the surface of the culture vessel with the working solution of CELLStart CTS (14 mL for T-75, 7 mL for T-25, 3.5 mL for 60-mm dish, 2 mL for 35-mm dish).
3. Incubate the culture vessel at 37°C in a humidified atmosphere of 5% CO₂ in air for 1 hour.
4. Remove the vessel from the incubator and store it until use. Immediately before use, remove all CELLStart CTS solution and replace it with complete StemPro NSC SFM.

Note: You may coat the plates in advance and store them at 4°C, wrapped tightly with Thermo Scientific™ Parafilm™, for up to 2 weeks. Do not remove CELLStart CTS solution until just prior to use. Make sure the plates do not dry out.

Coating culture vesselswith Geltrex matrix

1. Thaw the Geltrex matrix bottle at 4°C overnight to prevent polymerization. The next day, dilute Geltrex matrix 1:2 with D-MEM/F-12 at 4°C to make 100X stock solution, using an ice bucket to keep the bottles cold. Quickly prepare 0.5-mL aliquots in 50-mL conical tubes (pre-chilled on ice), and store the tubes at –20°C.
2. Thaw 1 tube of Geltrex matrix (0.5 mL, aliquoted as above) slowly at 4°C, and add 49.5 mL of cold D-MEM/F-12 (1:100 dilution). Mix gently.
3. Cover the whole surface of each culture plate with the Geltrex matrix solution (1.5 mL for a 35-mm dish, 3 mL for 60-mm dish, 5 mL for a T-25 culture flask).
4. Seal each dish with Parafilm to prevent drying, and incubate 1 hour at room temperature in a laminar flow hood.
5. Immediately before use, remove all Geltrex matrix solution, wash once with D-PBS with calcium and magnesium, and replace pre-warmed complete medium.

Note: You may store the Geltrex matrix–treated dish at 4°C, wrapped tightly with Parafilm, for up to 1 month. Do not remove Geltrex matrix solution until just prior to use.

Coating culture vessels with poly-L-ornithine and laminin

1. Dissolve poly-L-ornithine in cell culture-grade distilled water to make 10 mg/mL stock solution (500X). Aliquot the solution and store it at –20°C until use.
2. Thaw the laminin slowly at 2–8°C and prepare 10 μg/mL working solution in cell culture-grade distilled water. Aliquot the working solution into polypropylene tubes, and store the tubes at –20°C until use. Avoid repeated freeze/thaw cycles. 
   Note: Laminin may form a gel if thawed too rapidly.
3. Dilute the poly-L-ornithine stock solution 1:500 in cell culture-grade distilled water to make 20 μg/mL working solution.
4. Coat the surface of the culture vessel (with or without cover slips) with the poly‑L‑ornithine working solution (14 mL for T-75, 7 mL for T-25, 3.5 mL for 60-mm dish, 2 mL for 35-mm dish).
5. Incubate the culture vessel overnight at 4°C or for 1 hour at 37°C.
6. Rinse the culture vessel twice with sterile water.
7. Coat the surface of the culture vessel (with or without cover slips) with the laminin working solution (14 mL for T-75, 7 mL for T-25, 3.5 mL for 60-mm dish, 2 mL for 35‑mm dish).
8. Incubate the culture vessel overnight at 4°C or for 2 hours at 37°C.
9. Rinse the culture vessel with D-PBS without calcium or magnesium, and store the vessel covered with D-PBS until use. Immediately before use, remove all D-PBS and replace it with complete StemPro NSC SFM.
   Note: You may coat the plates in advance and store them at room temperature, wrapped tightly with Parafilm, for up to 1 week. Do not remove D-PBS until just prior to use. Make sure the plates do not dry out.

Neural stem cells (NSCs) will proliferate as progenitors a few times even after the complete growth medium is replaced with the appropriate differentiation medium. If the cells reach 90% confluency, it might be necessary to split the cells at a 1:2 ratio. However, do not split the cells once they reach day 9–10 of differentiation when they can get damaged during the passaging process.

Differentiation into neurons

1. Plate neural stem cells on a polyornithine and laminin-coated culture dish in complete StemPro NSC SFM at
   2.5 –5  × 10⁴ cells/cm².
2. After 2 days, change the medium to neural differentiation medium. Change the spent medium every 3–4 days.
3. If expedited differentiation is desired, add 0.5 mM of dibutyryl cAMP (Sigma, Cat. No. D0627) to the differentiation medium daily starting at day 7 of differentiation for 3 days.

IMPORTANT!   Do not expose cells to air at any time after they have differentiated into neurons.

Differentiation into astrocytes

1. Plate the NSCs on a Geltrex matrix–coated culture dish in complete StemPro NSC SFM at 2.5 × 10⁴ cells/cm².
2. After 2 days, change medium to astrocyte differentiation medium. Change the spent medium every 3–4 days.

Differentiation into oligodendrocytes

1. Plate the NSCs on a polyornithine and laminin-coated culture dish in complete StemPro NSC SFM at
   2.5 –5 × 10⁴ cells/cm².
2. After 2 days, change the medium to oligodendrocyte differentiation medium. Change the spent medium every 3–4 days.

Preparing paraformaldehyde fixing solution

20% paraformaldehyde (PFA) stock solution

1. Add PBS to 20 g of EM grade paraformaldehyde (Electron Microscopy Services, Cat. No. 19208), and bring the volume up to 100 mL.
2. Add 0.25 mL of 10 N NaOH and heat the solution at 60°C using a magnetic stirrer until the solution is completely dissolved.
3. Filter the solution through a 0.22-μm filter, and cool on ice. Make sure the pH is 7.5–8.0.
4. Aliquot 2 mL in 15-mL tubes, freeze the tubes on dry ice, and store them at –20°C.

4% PFA for fixing

1. Add 8 mL of PBS into each 15-mL tube containing 2 mL of 20% PFA, and thaw each tube in a 37°C water bath.
2. Once the solution has dissolved, the tubes cool on ice.

Fixing cells

1. Remove culture medium and gently rinse the cells once with D-PBS, without dislodging the cells.
2. Fix the cells with 4% fresh Paraformaldehyde Fixing Solution (PFA) at room temperature for 15 minutes.
3. Rinse 3X with D-PBS containing Ca²⁺ and Mg²⁺.
4. Check for the presence of cells after fixing.
5. Proceed to staining, described below. You may store slides for up to 3–4 weeks in D-PBS at 4°C before staining. Do not allow slides to dry.
   

Staining cells

1. Incubate cells for 30–60 minutes in blocking buffer (5% serum of the secondary antibody host species, 1% BSA, 0.1% Thermo Scientific™ Triton™-X in D-PBS with Ca²⁺ and Mg²⁺).
   Note: If you are using a surface antigen such as GalC, omit Triton-X from the blocking buffer.
2. Remove the blocking buffer and incubate the cells overnight at 4°C with primary antibody diluted in 5% serum. Ensure that the cell surfaces are covered uniformly with the antibody solution.
3. Wash the cells 3X for 5 minutes with D-PBS containing Ca²⁺ and Mg²⁺ (if using a slide, use a staining dish with a magnetic stirrer).
4. Incubate the cells with fluorescence-labeled secondary antibody (5% serum in D-PBS with Ca²⁺ and Mg²⁺) in the dark at 37°C for 30–45 minutes.
5. Wash the cells 3X with D-PBS containing Ca²⁺ and Mg²⁺, and in the last wash, counter stain the cells with DAPI solution (3 ng/mL) for 5–10 minutes, and rinse with D-PBS.
6. If desired, mount using 3 drops of ProLong Gold antifade reagent per slide and seal with the cover slip. You may store the slides in the dark at 4°C.

The table below lists some causes and solutions to help you troubleshoot your potential differentiation problems.

1. Trujillo CA, Schwindt TT, Martins AH, et al. (2009) Novel perspectives of neural stem cell differentiation: from neurotransmitters to therapeutics. Cytometry A 75:38–53.
2. Elkabetz Y, Studer L (2008) Human ESC-derived neural rosettes and neural stem cell progression. Cold Spring Harb Symp Quant Biol 73:377–387.
3. Denham M, Dottori M (2009) Signals involved in neural differentiation of human embryonic stem cells. Neurosignal 17:234–241.

LT154     17-Mar-2011