Differentiating Glial Precursor Cells into Astrocytes and Oligodendrocytes

Glial precursor cells (GPCs), also known as glial restricted progenitors (GRP) or oligodendrocyte progenitor cells (OPCs), are cells that have the potential to differentiate into oligodendrocytes or astrocytes. The GPC population is derived from tissue or is generated from pluripotent cells by differentiation, which is induced by exogenously applied factors. Here we described a culture system that can be adjusted to favor differentiation into either astrocytes or oligodendrocytes.

Required Materials 

Cells

• GIBCO Rat Glial Precursor Cells (Cat. no. N7746-100)

Media and Reagents

• Neurobasal Medium (Cat. no. 21103-049)
• D-MEM (Cat. no. 11995)
• GlutaMAX-I (Cat. no. 35050)
• B-27 Serum-Free Supplement (Cat. no. 17504-044)
• N-2 Supplement (Cat. no. 17502-048)
• T3 (Liothyronine) (Sigma, Cat. no. T6397)
• FBS (Cat. no. 16000)
• Geltrex Reduced Growth Factor Basement Membrane Matrix (Cat. no. 12760)
• Laminin (Cat. no. 23017)
• Poly-L-Ornithine (Sigma, Cat. no. P4957)
• Water, distilled (Cat. no. 15230)
• KnockOut D-MEM/F-12 (Cat. no. 12660)
• Dulbecco’s Phosphate-Buffered Saline (D-PBS) without Ca2+ and Mg2+ (Cat. no. 14190)
• Dulbecco’s Phosphate-Buffered Saline (D-PBS) (Cat. no. 14040)
• StemPro NSC SFM (Cat. no. A10509-01)

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

To prepare 100 mL of complete medium, mix the following components under aseptic conditions. For larger volumes, increase the component amounts proportionally.

Preparing Astrocyte Differentiation Medium

Astrocyte differentiation medium uses D-MEM medium supplemented with N-2, GlutaMAX-I, and FBS. Complete medium is stable for 2 weeks when stored in the dark at 2-8°C.

Matrix for Oligodendrocyte Differentiation

1. Prepare a 1:500 dilution of poly-L-ornithine in distilled water for a final concentration of 20 μg/mL.
2. Add 2 mL of 20 μg/mL poly-L-ornithine solution to a 35-mm dish (0.5 mL for 4-well plate or slide, 0.25 mL for 8-well slide).
3. Incubate the culture vessel at 37°C in a humidified atmosphere of 5% CO₂ for at least 1 hour.
4. Rinse the culture vessel once with distilled water.
5. Prepare a 1:1,000 dilution of laminin in distilled water for a final concentration of 1 μg/mL.
6. Add 2 mL of 1 μg/mL laminin solution to a 35-mm dish (0.5 mL for 4-well plate or slide, 0.25 mL for 8-well slide).
7. Incubate the culture vessel at 37°C in a humidified atmosphere of 5% CO₂ for at least 1 hour. Store it at 4°C until use.   

Note: You may coat the plates in advance and store them at 2-8°C, wrapped tightly with Parafilm, for up to 4 weeks.

Matrix for Astrocyte Differentiation

1. Thaw the Geltrex bottle at 4°C overnight to prevent polymerization. The next day, prepare a 50X stock solution of Geltrex by diluting to a final concentration of 10 mg/mL with D-MEM/F-12. Use an ice bucket to keep the bottle at 4°C. Quickly prepare 1 mL aliquots in 50-mL conical tubes (pre-chilled on ice), and store the tubes at –20°C.
2. Thaw one tube of Geltrex (1 mL aliquot described above) slowly at 4°C, and add 49 mL of cold D-MEM/F-12. Mix gently.
3. Cover the whole surface of each culture plate with the Geltrex 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. Store at 4°C until use.
5. Immediately before use, remove all Geltrex solution, wash once with D-PBS with calcium and magnesium, and replace it with pre-warmed complete medium.

Note: You may store the Geltrex-treated dish at 2-8°C, wrapped tightly with Parafilm, for up to 1 month. Ensure that plates do not dry out. Do not remove the Geltrex solution until just prior to use.

Differentiation to Oligodendrocytes

1. Plate glial precursor cells on poly-L-ornithine and laminin coated plate in StemPro NSC SFM at a density of 2.5 × 10⁴-5 × 10⁴ cells/cm².
2. Culture the cells for 2 days, then change the medium to Oligodendrocyte Differentiation Medium.
3. Change the medium every 3-4 days.

Differentiation to Astrocytes

1. Plate glial precursor cells on Geltrex coated plate in StemPro NSC SFM at a density of 2.5 × 10⁴ cells/cm².
2. Culture the cells for 2 days, then change th emedium to Astrocyte Differentiation Medium.
3. Change the medium every 3-4 days.