Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE

All procedures involving animals are performed according to the principles of laboratory animal care approved by the UK Home Office under the animals (scientific procedures) act 1986 (PPL No. 80/2457 to SP).

1. Derivation of Somatic Neural Stem/Progenitor Cells (NPCs) from the Subventricular Zone (SVZ) of the Brain of Adult Mice

1. Preparation of dissection instruments and media
   NB: These two solutions must be prepared 1-2 hr before dissections instruments are ready and prewarmed at 37 ºC at least 20-30 min before use (it helps to dilute papain and optimizes enzyme activity).
   
   1. Autoclave one straight sharp scissor, one small surgical scissor and two small curved serrated forceps.
   2. ‘Digestion’ solution: Prepare two separate 50 ml tubes with:
      Solution #1: 25 ml Earle’s Balanced Salt Solution (EBSS) + 10 mg ethylenediaminetetraacetic acid (EDTA) + 10 mg L-cysteine; and
      Solution #2: 25 ml EBSS + 50 mg papain.
   3. Prepare ‘Complete Growth Medium’ (CGM): complete mouse basal medium with mouse proliferation supplements, heparin 0.002%, basic fibroblast growth factor (bFGF; 10 ng/ml), epidermal growth factor (EGF; 20 ng/ml), and antibiotics (Pen/Strep).
2. Brain dissection
   NB: The removal of the temporal bones can easily damage the tissue because of their sharpness. To avoid this, firmly secure the bones with the forceps and pull out until the entire bone has been removed.
   
   1. For every preparation of NPCs, cull by cervical dislocation n= 5-7, 4-8 week old C57BL/6 mice.
   2. Cleanse with 70% ethanol (in water) the hair of the culled mice and cut behind the ears with the straight sharp scissor to remove the head;
   3. Make a midline incision using a small surgical scissor to cut the skin over the skull. Using the forceps, fold up the two patches of skin to expose the skull.
   4. With the small surgical scissor perform two little cuts at the base of the skull and remove the bones under the pons/medulla (ventral skull). Proceed by pulling out the temporal bones. With the same scissor perform a cut all along the skull, starting from the base to the bulbs, without damaging the surface of the brain. Also, perform a cut between the frontal bones and the eyes, to facilitate the removal of the parietal bones. With two forceps start removing the skull by pulling each of the parietal bones towards the outside. While pulling, pay attention to the meninges, which can damage the brain when the bones are being removed.
   5. Once the skull has been removed, slide the forceps between the brain and the skull to completely detach the meninges left. Gently lift up the brain from the skull. Cut the optic nerves to release the brain and finally collect the brain in a tube with cold phosphate buffered saline (PBS).
   6. Keep the tube on ice and repeat the same procedure for all the mice.
3. Dissection of the SVZ, neurosphere formation and maintenance assays
   Average number of cells x dilution factor x 10⁴
   
   Where 10⁴ represents the conversion of the total volume of the hematocytometer chamber (total volume = 0.1 mm³ -> 10-4 cm³ -> 10-4 ml). When considering the dilution factor, both the one done with CGM and with the vital stain have to be taken into consideration. As an example, if 160 cells have been counted in the 4 corner squares, the final density (cells/ml) of the cell suspension will be:
   
   160/4 x 5 (dilution with CGM) x 2 (dilution with vital stain) x 10⁴
   
   1. Turn on a dissection hood equipped with a dissecting microscope.
   2. Clean all the surfaces with 70% ethanol (in water), and spray with an anti Mycoplasma solution to lower the risk of contamination.
   3. Cover the bottom of a beaker with sterile gauze (to preserve sharpened instruments) and fill it with 70% ethanol (in water). Soak in the beaker two pairs of forceps, one pair of micro scissors and a surgical blade.
   4. Place the whole brain on the brain-slicer matrix.
   5. Using two razor blades perform a coronal sectioning 2 mm from the anterior pole of the brain, excluding the optic tracts, and 3 mm posterior to the previous cut; and place the tissue on a clean Petri dish.
   6. Under the dissecting microscope isolate the SVZ tissue and cut into 1 mm³ pieces using iridectomy scissors. Repeat for all the brains.
      1. Mix well the two solutions (section 1.1.2) above and filter through a 0.22 μm filter immediately after the brains are dissected and the SVZs are ready to be digested.
   7. Transfer the sections into 30 ml of ‘Digestion’ solution, gently resuspend with a10 ml pipette and incubate 30 min at 37 °C in 5% CO₂. Every 15 min gently shake the tube 2-3x.
   8. Centrifuge at 300 x g for 10 min.
   9. Remove the supernatant and resuspend the pellet in 200 μl of CGM by pipetting first with a P1000 and then with a P200 pipette (10-20x each), until obtaining a single cell suspension.
   10. Take the suspension up to 5 ml with CGM and transfer to a T25 cm² flask.
   11. After 5-7 days in vitro (DIV) neurospheres will form. Collect the suspension into a 15 ml tube and centrifuge 10 min at 150 x g.
   12. Remove the supernatant and resuspend the pellet into 200 μl of CGM.
       Dissociate the neurospheres mechanically by passaging the cells 100-150x with a P200 pipette, until obtaining a single cell suspension and take it up to 1 ml with CGM.
   13. Dilute the cell suspension, e.g. 10 μl of cell suspension in 40 μl of CGM to obtain a 1:5 dilution, and mix well. Mix 10 μl of the dilute cell suspension with 10 μl of a vital stain and mix well.
   14. Fill a hemocytometer counting chamber with 10 μl of a 1:1 cell suspension:vital stain solution. Separately count the number of live (white) and dead (blue) cells in the 4 corner squares. To determine the number of cells/ml, apply the following calculation:
   15. Resuspend 2 x 10⁵ cells in 5 ml of CGM and transfer to a T25 cm² flask;
   16. Repeat sections 1.3.11-1.3.12 every 4-5 DIV. From the second passage of expansion onwards, assess the cellular viability by vital stain exclusion and start building up a continuous growth curve by plating NPCs at clonal density (8,000 cells/cm2), as described²¹. Keep the cell numbers and repeat the procedure every 4-5 DIV. To generate a continuous growth curve, proceed as follows:
       NB: To have a good cell preparation, after 4-5 DIV spheres should have reached a diameter of 150-200 μm. To have a good estimate of the cell density, it is important to find an optimal dilution factor in order to have well distributed, nonoverlapping cells throughout the hematocytometer. Ideally, there should be between 50-200 total cells. When counting, only the cells comprised in the square without touching the borders have to be considered. Also, the viability of the cells is a crucial factor to have a healthy preparation. Importantly, it should be greater of 90%. A linear growth curve is another indicator of a healthy cell preparation.
       
       1. Count the number of live and dead cells (e.g. 1.3 x 10⁶).
       2. Define the growth rate dividing the number of live cells by the number of plated cells (e.g. 1.3 x 10⁶/2 x 10⁵= 6.5).
       3. Calculate the total number of cells by multiplying the growth rate for the total number of cells present at the previous time point (at the beginning= 2 x 10⁵).
       4. Report the mean ± standard deviation to build a linear trend line.
   17. Starting from passage 6, mechanical dissociation is replaced by enzymatic dissociation. After centrifugation at 150 x g for 10 min, remove the supernatant, resuspend the pellet in 200 μl of cell aggregate dissociation solution, resuspend 7-8x with a P200 and incubate 10 min at 37 °C, 5% CO_2.
   18. Resuspend 7-8x with a P200 to obtain a single cell suspension and add 800 μl of CGM. Count the cells (both live and dead) and establish their viability. Resuspend 2 x 10⁵ cells in 5 ml of CGM and transfer to a T25 cm² flask.
4. Viral transduction of NPCs for in vivo cell tracking
   NB: To verify the efficiency of viral transduction, build a parallel growth curve with transduced and nontransduced NPCs. In addition, the clonal efficiency, that is the absolute number of neurosphere forming cells present within a cell preparation or a cell cycle analysis by DNA content (with propidium iodide, PI), may be used as further indications of the cell status. If the percentage of infected cells should not be good, the protocol of viral transduction can be repeated a second time with the same population of cells. Once the level of infection is satisfactory and the growth curve is similar to that obtained with wild type cells, transduced NPCs can be expanded and/or transplanted.
   
   1. Harvest the neurospheres and obtain a single cell suspension. Plate the cells at high density (1.5 x 10⁶ cells in a T75 cm² flask) in 10 ml CGM.
   2. After 12 hr add 3 x 10⁶ T.U./ml of a third generation lentiviral vector pRRLsin. PPT-hCMV engineered with the E. coli-derived β-galactosidase (lacZ) or with green fluorescent protein (GFP).
   3. 48 hr later, harvest the cells, centrifuge at 300 x g for 10 min and replate the cells at a 1:1 ratio.
   4. After 3 passages in vitro verify the efficiency of the infection. Flow cytometry is commonly used to test infection efficacy, and the satisfactory level of infection is commonly accepted to be in the range of 75-95% of positive cells. Check again the efficiency of the infection after 3 further passages of expansion to verify the maintenance of the marker expression.
5. Neurosphere freezing
   1. Harvest the neurospheres from a T25 cm² flask, centrifuge at 300 x g for 10 min, and discard the supernatant.
   2. Resuspend the pellet with 1 ml of freezing medium (CGM with 10% dimethyl sulfoxide (DMSO).
   3. Place the cryovials at -80 °C within a freezing container.
   4. After at least 24 hr move the cells to a cryobox and store at -80 °C for months, or bring to liquid nitrogen (N₂) for longer storage.
6. Neurosphere thawing
   NB: To avoid the toxic effects of prolonged contact of NPCs with DMSO, the thawing process must be as fast as possible.
   
   1. Remove the cryovial from the -80 °C/N₂ and keep it on dry ice.
   2. Quickly thaw the vial on a water bath, until almost all the cell suspension is defrosted and only a small piece of iced suspension remains.
   3. Resuspend the cell suspension with 5 ml of prewarmed fresh CGM.
   4. Centrifuge at 300 x g for 10 min.
   5. Remove the supernatant, resuspend gently with 5 ml of fresh CGM and plate the cell suspension in a clean, untreated T25 cm² flask.
7. 1.7) NPC characterization
   NB: The last wash PBS can be replaced with 0.05% sodium azide-PBS to prevent fungal growth or contaminations, and coverslides are stored at 4 °C for 2-3 weeks.
   
   NB: To stain for intracellular markers add a permeabilizing agent to PBS in the blocking solution. If the source of the primary antibody is goat, bovine serum albumin (BSA) or serum of other than goat should be used.
   
   NB: To stain for intracellular markers use a permeabilizing agent in the primary antibody solution.
   
   1. Place a 13 mm glass coverslide on the bottom of a 24 well plate. Add 150 μl of coating solution on the top of each coverslide to create a small drop. Incubate for at least 30 min at 37 °C, 5% CO_2.
   2. Harvest the neurospheres and dissociate to obtain a single cell suspension.
   3. Count live cells and dilute to obtain 80,000 cells/35 μl. Remove the excess of coating solution from the coverslide by gentle aspiration, and plate the 35 μl of cell suspension.
   4. Incubate 25 min at 37 °C, 5% CO₂. Quickly check at the contrast phase microscope if the cells have started to adhere.
   5. Prepare the differentiation medium by mixing basal mouse medium with the appropriate supplements (see Table 1) and antibiotics (Pen/Strep).
   6. Add 400 μl of differentiation medium and incubate at 37 °C, 5% CO_2.
   7. After 3 DIV, change half of the medium with fresh differentiation medium.
   8. After 6 DIV, remove the medium and wash once with PBS. Under a chemical hood remove the PBS and add 300 μl of prewarmed 4% paraformaldehyde (PFA) 4% sucrose. Incubate for 5 min at room temperature (RT). Remove the PFA and wash 3x with PBS.
   9. To proceed with immunocytochemistry, remove the PBS and block with PBS 10% normal goat serum (NGS) (blocking solution), and incubate 1 hr at RT.
   10. Remove the blocking solution and add the desired primary antibody diluted with PBS 1% NGS. Incubate at 4 °C O/N or, alternatively, 120 min at RT.
   11. After the incubation, wash 2x with PBS. Incubate with the appropriate secondary antibody diluted with PBS 1%NGS. Incubate 60 min at RT.
   12. Wash 2x with PBS and counter-stain the nuclei with 4′,6-diamidino-2-phenylindole (DAPI) diluted 1:20,000 with PBS permeabilizing agent for 3 min at RT in the dark. Wash twice with PBS and once with distilled water.
   13. Put a drop of mounting medium on a glass tissue slide and with forceps mount the coverslide with the cells facing the mounting medium. Gently press the coverslide to squeeze out the excess of mounting medium. Leave the coverslide in the dark at RT until the mounting medium is dried and store at 4 °C.

2. Myelin Oligodendrocyte Glycoprotein (MOG)-induced Experimental Autoimmunity in C57Bl/6 Mice

1. Preparation of emulsion
   1. On a glass beaker prepare an emulsion composed of Incomplete Freund’s adjuvant containing 4 mg/ml of Mycobacterium tuberculosis [otherwise defined as Complete Freund’s Adjuvant (CFA)] and 200 μg of MOG (peptide 35-55), considering that the total amount of emulsion per mouse will be 300 μl.
      NB: Appropriate controls of MOG-immunized mice in CFA are mice immunized with CFA only. The expected variance of disease onset in MOG-immunized mice is 11.3 (±1.8) dpi.
      
      NB: When calculating the total volume of emulsion needed, consider twice as much volume as the number of mice to immunize. Glassware should be preferred to plasticware to minimize the waste of part of the emulsion.
      
      1. With a glass syringe holding a 19 G needle blend for about 30 min, always keeping the emulsion on ice.
      2. To verify if the emulsion is ready, drop a small amount of emulsion on water. The emulsion is ready to be injected only if the drop remains as it is and does not disperse.
      3. Immunize the control group of mice with CFA only. Treat experimental mice (MOG immunized) with MOG in CFA. The expected variance of disease onset in MOG immunized mice is 11.3 (±1.8) dpi.
   2. Transfer the 19 G needle to a 1 ml plastic syringe and aspirate the emulsion. Avoid bubbles, change the needle with a 25 G and leave the syringe on ice. Syringes filled with the emulsion are ready to be used and should be kept on ice for a couple of hr.
2. Immunization
   1. Place the mice (6-8 weeks old, female) into a warming box and wait for about 10 min to allow the tail vein to dilate.
   2. Transfer one mouse from the warming box to a mouse restrainer. Close the restrainer to avoid any movement of the mouse.
   3. Fill a 1 ml insulin syringe with 100 µl of pertussis toxin (5 μg/ml) avoiding making bubbles. Slowly inject the solution in the tail vein. Remove the needle and press for a few seconds with a piece of paper to plug the bleeding.
   4. Place the mouse back in the cage and repeat the same procedure for all the mice.
   5. Anesthetize a mouse with continuous isoflurane inhalation (1-2%, 2 L/min) and inject 100 µl of the emulsion subcutaneously at the level of the two flanks and the base of the tail (300 μl emulsion/mouse). Slowly remove the syringe, avoiding the leakage of the emulsion.
   6. Mark the mouse with an ear puncher, place the mice in the cage and check until full recovery. Repeat for all the mice.
   7. After 48 hr (2 days post immunization, dpi) repeat section 2.2.3.
3. Behavioral analysis of EAE mice
   1. Starting from 5 dpi, weigh the mice daily and monitor their locomotor performances by using the scale scoring system described in Table 1.

3. Injection of Neural Stem/Progenitor Cells into the Tail Vein (i.v.)

1. Cell preparation
   1. Harvest neurospheres by centrifugation at 300 x g for 10 min.
   2. Remove the supernatant and add 200 μl of cell aggregate dissociation solution. Incubate 10 min at 37 °C, 5% CO_2.
   3. Gently resuspend 10-12x (avoiding bubbles) with a P200 until obtaining a single cell suspension and take to 800 μl with basal medium without Ca²⁺ and Mg²⁺.
   4. Count the cells by using a vital stain to assay cell viability and dilute the suspension with basal medium without Ca²⁺ and Mg²⁺ to obtain a final density of 10⁶ cells/150 μl. Keep the cell suspension on ice until use.
2. NPC i.v. injection
   NB: The ideal dissociation into single cell suspension and the absence of air bubbles are two key aspects to consider to reduce the risk of either clumps/aggregates vs emboli formation which might result in vein occlusion and consequent death.
   
   1. At the peak of the disease (16-18 dpi), weigh and score the mice. Distribute the mice according to their score in order to have homogeneous groups.
   2. Place the mice into a warming box and wait for about 10 min to allow the tail vein to dilate.
   3. Transfer one mouse from the warming box to a mouse restrainer. Close the restrainer to avoid any movement of the mouse.
   4. Fill a 1 ml insulin syringe with 150 μl of cell suspension and make sure to remove all the bubbles. Slowly inject the solution into the tail vein. Remove the needle and press for a few seconds with a piece of paper to plug the bleeding (Figures 6A and 6B).
   5. Place the mouse in the cage and check until recovery. Repeat the same procedure for all the mice.

4. Injection of Neural Stem/Progenitor Cells into the Cisterna Magna (i.c.v)

1. Cell preparation
   1. Harvest NPCs by centrifuging at 300 x g for 10 min.
   2. Remove the supernatant and add 200 μl of cell aggregate dissociation solution. Incubate 10 min at 37 °C, 5% CO_2.
   3. Gently resuspend 10-12x with a P200 until obtaining a single cell suspension and take to 800 μl with basal medium without Ca²⁺ and Mg²⁺.
   4. Count the cells and dilute the suspension with basal medium without Ca²⁺ and Mg²⁺ to obtain the desired cell density. Keep the cell suspension on ice until use.
2. 4.2) NPC i.c.v. injection
   1. At the peak of the disease (16-18 dpi), weigh and score the mice. Distribute the mice according to their score in order to have homogeneous groups.
   2. Place the mouse on a stereotaxic apparatus under continuous isoflurane inhalation (1-2%, 2 L/min). Secure the head of the mouse with the ear bars. Then adjust both the mouth and nose pieces. Make sure the head of the mouse is flat and fixed.
   3. Swab the head of the mouse with Povidone-iodine (PVP-I) and make an incision to cut the skin in the posterior portion of the head to expose the skull.
   4. Using a micromanipulator, insert the tip of a microliter syringe into the cleft between the occiput and the atlas vertebra through the intact muscles and ligaments in the midline at the back of the neck. The bent part of the needle is kept in close contact with the internal surface of the occiput for the entire length, as described²².
   5. Slowly insert the needle and wait a few sec. The operator is meant to learn to recognize the feeling of the needle being “hooked” to the mouse skull, prior to start injecting the cell preparation. Slowly inject the volume of cells (within 3-5 min). Leave the needle in place for additional few seconds after the injection and remove slowly the syringe.
   6. Stitch up the skin and place the mouse into a recovery cage until full recovery.

5. Tissue Processing

1. Deeply anaesthetize the mice with a mixture of 0.5 ml ketamine (100 mg/ml), 0.25 ml xylazine (23.32 mg/ml) and 4.25 ml sterile water and transcardically perfuse, washing with saline-EDTA and fixing with 4% PFA. Remove both the vertebral columns and brains and postfix in 4% PFA for 12 hr at 4 °C. Wash the tissue in PBS, remove the spinal cords from the bones and leave the tissue at least 48 hr in 30% sucrose (in PBS) at 4 °C. Embed the tissues in optimal cutting temperature (OCT) compound and snap freeze with liquid nitrogen, as described².
2. Use a microtome to cut the tissues in 10-12 μm thick slices.
3. Alternatively, embed fresh tissue in agarose and cut tissue using a vibratome 50-80 μm thick slices.
4. In both the cases, incubate O/N at 37 °C in 5-bromo-4-chloro-3-indolyl-β D-galactopyranoside (X-gal) solution to detect nuclear β-galactosidase activity.
5. Recut sections containing β-gal+ cells into 5 µm slices and double stain using antiglial fibrillary acidic protein (GFAP) for astrocytes (50 μg/ml), antineuronal nuclear antigen (NeuN) for neurons (1μg/ml) and antiNestin for undifferentiated neural cells (10 μg/ml).
6. Proceed as described in sections 1.7.11 and 1.7.12. For multiple stainings make sure to use secondary antibodies conjugated with different fluorophores.
7. For tissues containing GFP labeled cells proceed as explained in steps 5.1-5.3 and proceed with the multiple stainings.
8. Add some drops of mounting medium to the slides and cover with a tissue coverslip.
9. For sections stained with biotin conjugated antibodies prepare a solution of avidin biotin complex (ABC solution, see Table 2) and leave in agitation for 45 min.
10. Incubate the slices 5 min with 1% H₂O₂ to block the activity of endogenous peroxidase.
11. Wash twice with PBS.
12. Incubate with ABC solution for 1hr at RT.
13. Wash twice with PBS.
14. Incubate with 3,3′-diaminobenzidine solution and 0.3% H₂O₂. Monitor the reaction under a microscope and wash with PBS as soon as the slice start getting brown (usually around 5-10 min).
15. Wash twice with PBS and proceed as described in step 5.7.

Comprehensive list of materials and reagents is shown in Table 2.