DifferentiatingGlialPrecursorCellsintoAstrocytesandOligodendrocytes

實驗概要

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.

主要試劑

Neurobasal? Medium

D-MEM

GlutaMAX?-I

B-27? Serum-Free Supplement

N-2 Supplement

T3 (Liothyronine)

FBS

Geltrex? Reduced Growth Factor Basement Membrane Matrix

Laminin

Poly-L-Ornithine

Water, distilled

KnockOut? D-MEM/F-12

Dulbecco’s Phosphate-Buffered Saline (D-PBS) without Ca²  and Mg²  

Dulbecco’s Phosphate-Buffered Saline (D-PBS)

StemPro? NSC SFM

實驗材料

GIBCO? Rat Glial Precursor Cells

實驗步驟

1.        Preparing Matrix

1)        Matrix for Oligodendrocyte Differentiation

        i.              Prepare a 1:500 dilution of poly-L-ornithine in distilled water for a final concentration of 20 μg/mL.

       ii.              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).

    iii.              Incubate the culture vessel at 37°C in a humidified atmosphere of 5% CO₂ for at least 1 hour.

    iv.              Rinse the culture vessel once with distilled water.

      v.              Prepare a 1:1,000 dilution of laminin in distilled water for a final concentration of 1 μg/mL.

     vi.              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).

  vii.              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.

2)        Matrix for Astrocyte Differentiation

         i.              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.

       ii.              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.

     iii.              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).

     iv.              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.

       v.              Immediately before use, remove all Geltrex? solution,  wash once with D-PBS with calcium and magnesium, and replace it with  pre-warmed complete medium.

2.        Differentiation of GRPs

1)        Differentiation to Oligodendrocytes

         i.              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.

      ii.              Culture the cells for 2 days, then change the medium to Oligodendrocyte Differentiation Medium.

    iii.              Change the medium every 3-4 days.

2)        Differentiation to Astrocytes

        i.              Plate glial precursor cells on Geltrex? coated plate in StemPro? NSC SFM at a density of 2.5 × 10⁴ cells/cm².

      ii.              Culture the cells for 2 days, then change the medium to Astrocyte Differentiation Medium.

    iii.              Change the medium every 3-4 days.