Isolation,Culture,andDifferentiationofProgenitorCells

Isolation, Culture, and Differentiation of Progenitor Cells from the Central Nervous System

Scott R. Hutton and Larysa H. Pevny¹

UNC Neuroscience Center, Department of Genetics, University of North Carolina, Chapel Hill, NC 27599, USA

¹Corresponding author (larysa_pevny@med.unc.edu )

INTRODUCTION

The ability to prospectively identify and characterize neural progenitor cells in vivo has been difficult due to a lack of cell-surface markers specific for these cell types. A widely used in vitro culture method, known as the Neurosphere Assay (NSA), has provided a means to retrospectively identify neural progenitor cells as well as to determine both their self-renewalcapacity and their ability to generate the three primary cell types of the nervous system: neurons, astrocytes, and oligodendrocytes. Today, combined with the establishment of multiple transgenic mouse strains expressing fluorescent markers and advances in cell isolation techniques such as fluorescence-activated cell sorting (FACS), the NSA provides a powerful system to prospectively elucidate neural progenitor characteristics and functions. Here we describe methods for the isolation, culture, and differentiation of neural progenitors from the developing mouse and adult cortex.

RELATED INFORMATION

This protocol was adapted from the method by Polleux and Ghosh (2002).

MATERIALS

Reagents

Dulbecco’s phosphate-buffered saline (PBS), 1X sterile (Sigma D8537)

 Enzyme solution for neurosphere assay (prewarmed)

 Heavy inhibitory solution (HI) (prewarmed)

Laminin stock solution (1 mg/mL) (Sigma)

Store stock solution in 0.1-mL aliquots at –20°C. Just before use (Step 14), prepare a 1:50 dilution by diluting 0.1 mL of stock solution in 5 mL of sterile 1X PBS supplemented with Ca⁺⁺and Mg⁺⁺ (Sigma D8662).

 Light inhibitory solution (LI) (prewarmed)

Mice (embryonic or adult)

 NEP basal medium

 NEP basal medium containing 2% horse serum (Invitrogen; heat-inactivated for 30 min at 60°C)

 NEP complete medium

 Neurobasal medium (PSG)

 Paraformaldehyde (PFA; Sigma), 4% in 1X PBS

Poly-D-lysine (Sigma)

Reconstitute in H₂O for a stock solution of 1 mg/mL and store in 1.0-mL aliquots.

 Trypsin-EDTA (Sigma) (optional; see Step 20)

Equipment

Centrifuge

Dishes, polystyrene (6- or 10-cm nontreated) (BD Falcon)

Dissection tools for removing mouse brain from skull

Forceps, sterilized (#5 pointed) (Fine Science Tools 11252-30)

Hemacytometer

Incubator preset to 37°C (humidified, 5% CO₂)

Knife, microsurgical (5-mm) (MSP/Surgical Specialties 7516)

Parafilm

Pipette (P200; P20 may be used in place of P200 in Step 20)

Plates, polystyrene, 96-well flat bottom, low cell binding (Corning 3474) (optional; see Step 21)

Razor blade, sterile

Scissors, sterilized microspring (8.5-cm) (Fine Science Tools 15009-08)

Slides, eight-well chamber (Nunc 177402)

Transfer pipettes, sterile disposable (Fisher)

Tubes, sterile conical (15- and 50-mL)

Tubes, sterile microcentrifuge (1.5-mL)

Water bath preset to 37°C

METHOD

Dissection

• 1. Carefully remove the brain from the skull of an embryonic or adult mouse and place in a clean dish containing ice-cold 1X PBS. 
  To dissect tissue from an early embryo (<E16) 
  

  • i. Separate the two hemispheres of the brain. Carefully separate the region of interest (e.g., dorsal telencephalon) using a microsurgical knife (Fig. 1). 
    
    

    View larger version (40K): [in this window] [in a new window]

    Figure 1. A sagittal view of a single cerebral hemisphere from an E12.5 mouse embryo, demonstrating the location of the dorsal telencephalon.

  • ii. Using fine-tipped forceps and a microsurgical knife, carefully remove the meninges from the tissue. 
    Meninges must be removed from the tissue, since they will not digest efficiently in enzyme solution. 
    

  To collect tissue from a late-stage embryo (>E16) or adult 
  

  • iii. Using a razor blade, cut a coronal slice of the brain containing the region of interest (lateral ventricle, hippocampus, etc.) (Fig. 2A). 
    
    

    View larger version (32K): [in this window] [in a new window]

    Figure 2. (A) Dorsal view of the adult mouse brain. The red bar indicates the location of the cut used to isolate periventricular tissue. (B) The resulting cross-section of tissue with lateral ventricles exposed. Periventricular tissue should be dissected where indicated by the red box.

  • iv. Carefully remove the tissue of interest using the forceps and microsurgical knife (Fig. 2B). 
    

• 2. Using a sterile transfer pipette, carefully transfer the tissue to a nontreated polystyrene dish containing cold 1X PBS. To speed up enzymatic digestion, cut the tissue into smaller pieces using the microspring scissors. 
  
  

• 3. Using a sterile transfer pipette, transfer the tissue to a 15-mL conical tube containing 10 mLof enzyme solution. Minimize the amount of PBS transferred with the sample. Incubate at 37°Cfor 20 min, carefully mixing every ~5 min. 
  Do not vortex. 
  
  

• 4. Add another 10 mL of enzyme solution. Incubate for 20 min at 37°C, mixing occasionally. 
  Incubation times may vary. The tissue is ready when it achieves a thick, viscous consistency. 
  
  

• 5. In a sterile hood, carefully remove the enzyme solution using a pipette, leaving the tissue at the bottom of the tube. 
  
  

• 6. Add 4.5 mL of LI solution to the tube. Carefully flick the tube, remove the solution, andrepeat with another 4.5 mL of LI solution. 
  Caution: The tissue will go into solution easily and should not be mixed with a pipette. 
  
  

• 7. Remove the LI solution, leaving the tissue at the bottom of the tube, and add 6 mL of HI solution. Incubate for 2 min at 37°C. Gently remove the HI solution. 
  
  

• 8. Add 5 mL of NEP basal medium, flick the tube, and remove the medium. 
  
  

• 9. Add 0.5-1.0 mL of NEP complete medium and triturate 10-20 times, until the tissue pieces are dissociated. 
  More medium may be required, depending upon the amount of tissue used. 
  
  

• 10. Count the cells using a hemacytometer and add the appropriate number to a nontreated polystyrene dish containing NEP complete medium. 
  Cell density should be 1 x 10⁶ cells per 6-cm plate, or 2 x 10⁶ cells per 10-cm plate. However, the plating density will vary between different age points and different brain regions. See Discussion. 
  
  

• 11. Incubate cells in a humidified 37°C incubator (+5% CO₂). Monitor the dishes daily for neurosphere formation (Fig. 3A). 
  
  

  View larger version (36K): [in this window] [in a new window]

  Figure 3. (A) Neurospheres derived from an E12.5 mouse dorsal telencephalon after 6 d in culture. (B) Neurosphere attachment to a poly-D-lysine/laminin-coated slide, 1 d after plating. (C) β-tubulin-III (red) and glial fibrillary acidic protein (GFAP) (green) labeling of neurosphere-derived neurons and astrocytes, respectively.

  Adult lateral ventricle neurospheres take ~1 wk to form, while embryonic neurospheres are observed after a few days. 
  
  

• 12. Once spheres have formed, replace medium every 3 d by transferring spheres to a 15-mL conical tube and letting them settle to the bottom by gravity at 37°C (centrifugation is not recommended). After the spheres have settled, remove the medium and replace with fresh NEP complete medium. Transfer spheres to a fresh dish.