Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons

1. Culture and Storage of Human Periventricular Endothelial Cells

1. Maintain human periventricular endothelial cells on basement membrane matrix-coated (see Table of Materials) 6-well plates in periventricular endothelial cell medium (E6 medium containing 50 ng/mL VEGF-A, 100 ng/mL FGF2 and 5 µM GABA) at 37 °C and 5% CO₂. Change medium every alternate day.
2. Thaw basement membrane matrix in 4 °C, and make a 1:100 solution by diluting it in cold DMEM/F12 medium. Coat each well of a 6-well plate with 1 mL of matrix solution. Incubate plates at 37 °C for at least 1 h before use.
3. Allow human periventricular endothelial cells to reach a confluency of 80%-90%. Aspirate medium from the well. Wash the wells once with 1 mL of sterile 1x PBS per well.
4. Detach cells by adding 1 mL of cell dissociation solution (see Table of Materials) per well. Incubate at 37 °C for 5 min. After 5 min, add 1 mL of periventricular endothelial cell medium. Transfer the cell solution into a 15 mL conical tube.
   NOTE: We use Accutase for cell dissociation here, as opposed to TrypLE in sections 3 and 4.
5. Centrifuge cells at 500 x g for 5 min at room temperature, aspirate the supernatant and resuspend the cell pellet in 1 mL of periventricular endothelial cell medium.
6. Count live cells using the trypan blue exclusion method. Seed cells in fresh matrix-coated plates at a density of 1.2 x 10⁵ cells/cm². Incubate at 37 °C and 5% CO₂.
7. Store human periventricular endothelial cells by cryopreserving in freezing medium (90% periventricular endothelial cell medium and 10% DMSO).
   1. Dissociate and collect cells following steps 1.3 and 1.4 above. Count cells in the solution by the trypan blue exclusion method.
   2. Centrifuge cells at 500 x g for 5 min at room temperature. Aspirate the supernatant and resuspend the cell pellet at 5 x 10⁶ cells/ mL of freezing medium.
   3. Dispense 1 mL of freezing medium plus cells per cryovial. Place the vials in isopropanol-filled chamber and cool overnight in -80 °C at 1 °C/min. Transfer vials to a liquid nitrogen tank on the next day for long term storage.

2. Preparation of Human Periventricular Endothelial Cells for Assay

1. Allow human periventricular endothelial cells to reach 70%-80% confluency.
2. Dissociate cells following steps 1.3 to 1.5 as described above. Count cells using the trypan blue exclusion method.

3. Preparation of Human GABAergic Interneurons for Assay

NOTE: Human induced pluripotent stem cell (iPSC)-derived GABAergic interneurons and the neuronal medium were commercially purchased (see Table of Materials). The neurons are generated by differentiating a human fibroblast-derived iPSC line following a protocol developed by the manufacturer. The cells were thawed and cultured according to manufacturer's protocol.

1. Thaw human GABAergic interneurons and culture them in 12-well plate for two weeks to a confluency of 70%-80%.
2. On the day of the assay, warm cell dissociation solution (see Table of Materials) and an aliquot of neuronal medium at 37 °C for 10 min before use.
3. Aspirate medium from each well containing the cells. Wash cells with 1 mL of sterile 1x PBS per well.
4. Detach cells by adding 0.5 mL of pre-warmed dissociation solution per well and incubate at 37 °C for 5 min. Add 1 mL of neuronal medium per well. Transfer cell solution into a 15 mL conical tube. Gently triturate to dissociate cell clumps.
5. Centrifuge cells at 380 x g for 5 min at room temperature, aspirate the supernatant and resuspend the cell pellet in 1 mL of neuronal medium. Count live cells using the trypan blue exclusion method.

4. Preparation of Control Human Endothelial Cells for Assay

NOTE: Control human iPSC-derived endothelial cells and endothelial cell medium were commercially purchased (Table of Materials). These endothelial cells are generated by differentiating a human fibroblast-derived iPSC line to endothelial fate following a protocol developed by the manufacturer. The cells were thawed and cultured on Fibronectin substrate according to manufacturer's protocol. Fibronectin-coated plates were prepared following manufacturer's protocol.

1. Thaw control human endothelial cells and culture them in 6-well plate to a confluency of 80%-90%.
2. On the day of the assay, warm cell dissociation solution (see Table of Materials) and an aliquot of endothelial medium at 37 °C for 10 min before use.
3. Aspirate the medium from each well containing the cells. Wash cells with 1 mL of sterile 1x PBS per well.
4. Detach cells by adding 0.5 mL of pre-warmed dissociation solution per well. Incubate at room temperature for 5 min. Add 1 mL of endothelial cell medium per well to neutralize the dissociation solution. Transfer cell solution into a 15 mL conical tube.
5. Centrifuge cells at 200 x g for 5 min at room temperature. Aspirate supernatant and resuspend the cell pellet in 1 mL of endothelial cell medium. Count live cells using the trypan blue exclusion method.

5. Preparation of One-well Culture Inserts

1. Thaw 1 mg/mL laminin solution at room temperature or overnight at 4 °C.
2. Coat an appropriate number of 35 mm dishes with 0.01% poly-L-ornithine solution (1 mL per dish). Incubate the dishes in room temperature for at least 1 h.
3. Dilute 1 mg/mL laminin solution 1:300 in sterile water to a final concentration of 3.3 µg/mL immediately before use.
4. Completely aspirate poly-L-ornithine from each dish. Rinse each dish thoroughly 3x with sterile water and aspirate completely to avoid poly-L-ornithine-induced cell toxicity.
5. Add 1 mL of 3.3 µg/mL laminin solution to each dish and incubate at 37 °C overnight or at least 1 h. Remove laminin solution from the dish immediately before use.
   NOTE: Alternatively, store the laminin-containing dishes in 4 °C. Equilibrate the dishes in a 37 °C cell culture incubator before use.
6. Cut three sides of one well of a two-well silicone culture insert (Figure 1A) using a sterile blade to generate a one-well insert (Figure 1B).
   NOTE: Keep the two-well insert firmly attached to the surface of the original packaging while cutting to ensure a smooth cut and protect the adhesiveness of the insert.
7. Aspirate laminin solution from the dishes.
   NOTE: Do not wash the dishes with sterile PBS or water after laminin incubation. Wet surfaces will prevent tight adhesion of the culture insert.
8. Remove one-well insert with sterile tweezers and place it in the center of the poly-L-ornithine/laminin coated dish. Press along the edges of the insert to fix it to the surface of the dish.
9. Carefully turn the dish upside down to verify that the insert is firmly adhered.
10. Keep the dish upside down and mark the boundary of the insert compartment using a permanent black marker with an ultra-fine tip (Figure 1C).

6. Long-distance Migration Assay

1. Suspend human GABAergic interneurons at a concentration of 3 x 10⁴ cells/70 µL of neuronal medium. Seed 70 µL of cell solution inside each one-well culture insert.
   NOTE: The seeding density of interneurons is as per manufacturer's recommendation.
2. Suspend human periventricular endothelial cells at a concentration of 3 x 10⁴ cells/70 µL of periventricular endothelial cell medium. Seed 70 µL of cell solution inside each one-well culture insert.
   NOTE: The number of human periventricular endothelial cells seeded at a 1:1 ratio with the number of seeded neurons.
3. Add 1 mL of neuronal medium in neuron dish to fill the area around the insert and prevent the coating from drying. Similarly, add 1 mL of periventricular endothelial cell medium in periventricular endothelial cell dish.
   NOTE: Add medium slowly along the edge of the dish so that the insert is not disturbed.
4. Check under a microscope to verify that cells are not leaking from the insert compartment.
5. Incubate cells for 24 h at 37 °C and 5% CO₂. After 24 h incubation, check under microscope to verify that cells have attached properly and there is no overnight leak.
6. After 48 h of seeding, gently remove the insert using a sterile tweezer. Check under the microscope to verify that the cell layer remains undisturbed (day 0).
7. Remove medium from the neuron dish and add 1 mL of fresh neuronal medium. Similarly, remove medium from the periventricular endothelial cell dish and add 1 mL of fresh periventricular endothelial cell medium.
   NOTE: Set aside a required number of dishes and fix with 4% PFA for day 0 images.
8. Incubate cells for 5 days at 37 °C and 5% CO₂. After 5 days, remove medium, fix cells with 4% PFA for 10 min, and wash 3x with 1x PBS.
9. Stain neurons with anti-human β-Tubulin or anti-human MAP2 antibody, and endothelial cells with anti-human CD31 antibody. At the end of immunostaining, add 1 mL of antifade mounting medium to each dish.

7. Co-culture Migration Assay

1. Co-suspend 3 x 10⁴ GABAergic interneurons and 3 x 10⁴ human periventricular endothelial cells in 70 µL of co-culture medium (50% periventricular maintenance medium without GABA and 50% neuronal medium). Seed this cell solution inside the one-well insert compartment. Prepare an appropriate number of such assay dishes.
   NOTE: GABA was not added in the co-culture medium to exclude the effect of exogenous GABA on migration.
2. Check under a microscope to verify that cells are not leaking from the insert compartment.
3. Slowly add 1 mL of co-culture medium along the side of the dish to prevent the coating from drying.
   NOTE: Add medium slowly along the edge of the dish so that the insert is not disturbed.
4. As a first control, seed 3 x 10⁴ human GABAergic interneurons only in 70 µL of co-culture medium per one-well insert. Prepare an appropriate number of such dishes.
5. As a second control, co-seed 3 x 10⁴ GABAergic human interneurons with 3 x 10⁴ control human endothelial cells in 70 µL of co-culture medium per one-well insert. Prepare an appropriate number of dishes.
6. Incubate the dishes for 24 h at 37 °C and 5% CO₂. After 24 h incubation, check under a microscope to verify that cells have attached properly and there is no leak.
7. After 48 h of seeding, gently remove the insert using a sterile tweezer. Check under the microscope to verify that the cell layer is not disturbed (day 0).
8. Remove medium and add 1 mL of fresh co-culture medium.
   NOTE: Set aside an appropriate number of dishes for acquiring day 0 images.
9. Incubate cells for 5 days at 37 °C and 5% CO₂.
10. After 5 days, remove medium, fix cells with 4% PFA for 10 min, and wash 3x with 1x PBS.
11. Stain with anti-human β-Tubulin or anti-human MAP2 antibody to label the neurons. At the end of immunostaining, add 1 mL of antifade mounting medium to each dish.

8. Chemo-attraction Assay

1. Place a three-well culture insert in the center of a poly-L-ornithine/laminin coated 35 mm dish using sterile tweezers.
2. Turn the dish upside down. Mark the boundary around the middle compartment of the insert using a permanent black marker with ultra-fine tip.
3. Seed 3 x 10⁴ human GABAergic interneurons in the middle compartment in 70 µL of neuronal medium (Figure 3A).
4. Seed 10⁴ human periventricular endothelial cells in 70 µL of periventricular endothelial cell medium and 10⁴ control endothelial cells in 70 µL of control endothelial cell medium in the two outer compartments respectively (Figure 3A).
5. Add 1 mL of co-culture medium (50% periventricular maintenance medium without GABA and 50% neuronal medium) along the side of the dish to prevent the coating on the dish from drying.
6. Check under microscope to verify that cells are not leaking from the insert compartment.
7. Incubate cells for 24 h at 37 °C and 5% CO₂. After 24 h incubation, check under a microscope to verify that cells have attached properly and there is no leak.
8. After 48 h of seeding, gently remove the insert using a sterile tweezer. Check under the microscope to verify that the cell layer is not disturbed (day 0).
9. Remove medium and add 1 mL of fresh co-culture medium.
   NOTE: Set aside required number of dishes for day 0 images.
10. Incubate cells for 36 h at 37 °C and 5% CO₂. After 36 h, aspirate medium, fix cells with 4% PFA for 10 min, and wash 3x with 1x PBS.
11. Stain human GABAergic interneurons with anti-human β-Tubulin or anti-human MAP2 antibody. At the end of the staining procedure, add 1 mL of antifade mounting medium in each dish.

9. Imaging and Data Analysis

1. Place the immuno-stained assay dish under a microscope at 4X magnification.
2. Keep one long-edge of the rectangular boundary (made in step 5.10 above) in the field of view. Take images of cells in the cell-free space adjacent to that boundary. Acquire images along the right long-edge and the left long-edge of the rectangular boundary (Figure 2B).
   NOTE: Cells positioned diagonally with respect to the rectangle are not considered due to ambiguity in selecting the short- or long-edge as starting mark. Also, numbers of cells migrating across the short-edge are often significantly fewer (possibly due to lesser number of starting cells along the short-edge) and are not considered.
3. Open the images in ImageJ. Calculate distance between each cell and the boundary mark (Figure 2D) using ImageJ.
4. To assess migration in terms of cell numbers, set a specific distance from the boundary in the acquired image in ImageJ. Count the number of cells that are present within this distance. Calculate average number, standard deviation, and statistical significance using appropriate software.