Differentiating Neural Stem Cells into Neurons and Glial Cells

实验概要

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 selfrenewing multipotent stem cells that can be  proliferated in vitro in supportive culture systems such as 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.

主要试剂

KnockOut™ D-MEM/F-12

Dulbecco’s Modified Eagle Medium (D-MEM)

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

StemPro® NSC SFM

N-2 Supplement

B-27® Serum-Free Supplement

Neurobasal® Medium

Antibiotic-Antimycotic solution

Fetal Bovine Serum, ES Cell-Qualified FBS

GlutaMAX™-I

FGF-basic (AA 10–155), Recombinant Human (bFGF)

EGF, Recombinant Human

CELLstart™ CTS™

Geltrex™ Reduced Growth Factor Basement Membrane Matrix

Poly-L-Ornithine

Laminin

Dibutyryl cAMP

T3

EM grade paraformaldehyde

ProLong® Gold antifade reagent 

实验材料

GIBCO® Human Neural Stem Cells (H9 hESC-Derived)

实验步骤

1. Preparing Matrix

1) Coating Culture Vessels with CELLstart™

       a. 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).

        b. 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).

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

        d. 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.

2) Coating Culture Vessels with Geltrex™

       a. Thaw  the Geltrex™ bottle at 4°C overnight to prevent polymerization. The  next day, dilute Geltrex™ 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.

        b. Thaw 1 tube of Geltrex™ (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.

        c. 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).

       d. Seal each dish with Parafilm® to prevent drying, and incubate 1 hour at room temperature in a laminar flow hood.

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

3) Coating Culture Vessels with Poly-L-Ornithine and Laminin

       a.  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.

        b. 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.

        c. Dilute the poly-L-ornithine stock solution 1:500 in cell  culture-grade distilled water to make 20 μg/mL working solution.

        d. 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).

       e. Incubate the culture vessel overnight at 4°C or for 1 hour at 37°C.

       f. Rinse the culture vessel twice with sterile water.

        g. 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).

       h. Incubate the culture vessel overnight at 4°C or for 2 hours at 37°C.

         i. 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.

2. Differentiating Neural Stem Cells

1) Differentiation into Neurons

       a. Plate neural stem cells on a polyornithine and laminin-coated culture dish in complete StemPro® NSC SFM at 2.5 × 10⁴–5 × 10⁴ cells/cm².

       b. After 2 days, change the medium to neural differentiation medium. Change the spent medium every 3–4 days.

        c. 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.

2) Differentiation into Astrocytes

       a. Plate the NSCs on a Geltrex™-coated culture dish in complete StemPro® NSC SFM at 2.5 × 10⁴ cells/cm².

       b. After 2 days, change medium to astrocyte differentiation medium. Change the spent medium every 3–4 days.

3) Differentiation into Oligodendrocytes

       a. Plate the NSCs on a polyornithine and laminin-coated culture dish in complete StemPro® NSC SFM at 2.5 × 10⁴–5 × 10⁴ cells/cm².

       b. After 2 days, change the medium to oligodendrocyte differentiation medium. Change the spent medium every 3–4 days.

3. Characterizing NSCs and Differentiated Lineages by Immunocytochemistry

1) Preparing Paraformaldehyde Fixing Solution

       a. Add  PBS to 20 g of EM grade paraformaldehyde (Electron Microscopy Services,  Cat. no. 19208), and bring the volume up to 100 mL.

        b. 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.

       c. Filter the solution through a 0.22-μm filter, and cool on ice. Make sure the pH is 7.5–8.0.

       d. Aliquot 2 mL in 15-mL tubes, freeze the tubes on dry ice, and store them at –20°C.

2) Fixing Cells

       a. Remove culture medium and gently rinse the cells once with D-PBS, without dislodging the cells.

       b. Fix the cells with 4% fresh Paraformaldehyde Fixing Solution (PFA) at room temperature for 15 minutes.

       c. Rinse 3X with D-PBS containing Ca² and Mg² .

       d. Check for the presence of cells after fixing.

        e. 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.

3) Staining Cells

       a.  Incubate cells for 30–60 minutes in blocking buffer (5% serum of the  secondary antibody host species, 1% BSA, 0.1% 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.

        b. 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.

       c. 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).

       d. 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.

       e. 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.

        f. 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.