Differentiating Neural Stem Cells into Neurons and Glial Cells

Introduction

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)

Primary antibodies

Lineage markerAntigenConcentrationSubtypesReactivity*
NSCNestin1:1,000RabbitHu, Rt, Ms
 Sox21:200Mouse IgG2aHu
NeuronDcx1:400RabbitHu, Rt, Ms
 MAP21:200Mouse IgG1Hu, Rt, Ms
GlialA2B51:100Mouse IgMHu, Rt, Ms
 CD441:50Mouse IgGHu
OligodendrocyteGalC1:200Mouse IgGHu, Rt, Ms
AstrocyteGFAP1:200RabbitHu, Rt, Ms
*Hu = human, Rt = rat, Ms = mouse

Secondary antibodies

Ex/Em* (color)Alexa Fluor
2nd host2nd againstCat. No.Concentration
346/442 (Blue)350GoatMouse IgMA315521:1,000
  GoatMouse IgGA210491:1,000
  GoatRat IgGA210931:1,000
  GoatRabbit IgGA210681:1,000
  DonkeyGoat IgGA210811:1,000
495/519 (Green)488GoatMouse IgMA210421:1,000
  GoatMouse IgGA110291:1,000
  GoatRat IgMA212121:1,000
  GoatRat IgGA110061:1,000
  GoatGoat IgGA110341:1,000
  DonkeyMouse IgMA110551:1,000
590/617 (Red)594GoatMouse IgMA210441:1,000
  GoatMouse IgGA110291:1,000
  GoatRat IgMA212131:1,000
  GoatRat IgGA110071:1,000
  GoatRabbit IgGA110371:1,000
  DonkeyGoat IgGA110581:1,000
496, 536, 565/576
(Red)
NAGoatMouse IgMM315041:500
  GoatMouse IgGP8521:1,000
  GoatRabbit IgGP2771MP1:1,000
*Approximate excitation and emission maxima, in nm; NA = not applicable

Preparing media

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.
ComponentFinal concentrationAmount
KnockOut D-MEM/F-121X97 mL
GlutaMAX-I Supplement2 mM1 mL
bFGF (prepared as 100 μg/mL stock)20 ng/mL20 μL
EGF (prepared as 100 μg/mL stock)20 ng/mL20 μL
StemPro Neural Supplement2%2 mL

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.

ComponentFinal concentrationAmount
Neurobasal Medium1X97 mL
B-27 Serum-Free Supplement2%2 mL
GlutaMAX-I Supplement2 mM1 mL

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.

ComponentFinal concentrationAmount
D-MEM1X97 mL
N-2 Supplement1%1 mL
GlutaMAX-I Supplement2 mM1 mL
FBS1%1 mL

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.

ComponentFinal concentrationAmount
Neurobasal Medium1X97 mL
B-27 Serum-Free Supplement2%2 mL
GlutaMAX-I Supplement2 mM1 mL
T3*30 ng/mL0.1 mL

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

Preparing matrix

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.

Differentiating neural stem cells

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.

Characterizing NSCs and differentiated lineages by immunocytochemistry

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.

Expected results

A
B
    
C
D
Figure 1. Fluorescence images (20X) of Gibco hNSCs that have been cultured in StemPro NSC SFM for three passages, and then allowed to differentiate into neurons, oligodendrocytes, or astrocytes. Upon directed differentiation, cells start to lose the undifferentiated NSC marker, nestin, but stain positive for the differentiated cell type markers Dcx, GalC, and GFAP. Cells were stained for the undifferentiated NSC markers nestin (red) and SOX2 (green) prior to directed differentiation (Panel A). Cell were then differentiated into neurons and glial cells, and respectively stained for the neuronal marker Dcx (green) (Panel B), for the oligodendrocyte marker GalC (red) (Panel C), or for the astrocyte marker, GFAP (green) (Panel D). The nuclei were counterstained with DAPI (blue) in panels B–D.

Troubleshooting

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

Possible causeSolution
Culture medium contains bFGFRemove bFGF from culture medium
Cell density too high and endogeneous bFGF is preventing differentiationReduce cell density
Concentration of GlutaMAX-I Supplement is incorrectUse the GlutaMAX-I Supplement at a final concentration of 2 mM
Cells have been passaged too many timesObtain new Gibco human neural stem cells

References

LT154     17-Mar-2011