This protocol describes three in vitro assays that collectively assess the functions of human periventricular endothelial cells and their interaction with human GABAergic interneurons. Results from these assays will provide critical insight into the link between periventricular endothelial cells and brain disorders. These assays are simple, low-cost and allow measurement of cell migration in the range of centimeters, which is not achieved by other existing assays.
Defects in migration and distribution of GABAergic interneurons are associated with psychiatric disorders, such as autism, epilepsy, schizophrenia and depression. Therefore, it is critical to study the interaction of periventricular endothelial cells with GABAergic interneurons in human context to address the pathogenesis of these disorders. Begin by preparing human cells for the assay.
Allow human periventricular endothelial cells to reach 70 to 80%confluency then dissociate them according to the manuscript's directions and count them using the trypan blue exclusion method. To prepare human GABAergic interneurons, warm cell dissociation solution and an aliquot of neuronal medium at 37 degrees celsius for 10 minutes. Then aspirate the medium from each well containing cells and wash them with one millimeter of PBS per well.
Detach the cells by adding 0.5 milliliters of prewarmed dissociation solution per well and incubating them at 37 degrees celsius for five minutes. After the incubation, add one milliliter of neuronal medium per well and transfer the cell solution into a 15 milliliter conical tube, gently triturating to dissociate cell clumps. Centrifuge the cells at 380 times G for five minutes at room temperature, aspirate the supernatant and resuspend the cell pellet in one milliliter of neuronal medium.
Then count the live cells using trypan blue exclusion. Prepare control human endothelial cells by warming the cell dissociation solution and an aliquot of endothelial medium at 37 degrees celsius, 10 minutes prior to use. Aspirate medium from each well and wash them with one milliliter of PBS per well.
Detach the cells by adding 0.5 milliliters of the prewarmed dissociation solution to each well and incubating the plate at room temperature for five minutes. Then, add one milliliter of endothelial cell medium per well to neutralize the dissociation solution, and transfer the cells to a 15 milliliter conical tube. Centrifuge the cells at 200 times G for five minutes, aspirate the supernatant and resuspend the cells in one milliliter of endothelial cell medium.
Then use the trypan exclusion method to count the cells. Prepare one well culture insert by cutting three sides of one well of a two well silicone culture insert with a sterile blade. Remove the insert with sterile tweezers and place it in the center of a poly-L-ornithine and laminin-coated dish, then press along the edge of the insert to fix it to the surface.
Carefully turn the dish upside down to verify that the insert is firmly adhered, and mark the boundary of the insert compartment using a permanent black market with an ultra fine tip. Suspend human GABAergic interneurons in neuronal medium and human periventricular endothelial cells and endothelial cell medium at a concentration of 30, 000 per 70 microliters, then seed 70 microliters of the cell solution inside each one well culture insert. Add one milliliter of neuronal medium to the neuron dish to fill the area around the insert and prevent the coating from drying.
Similarly, add one milliliter of endothelial cell medium to the periventricular endothelial cell dish. Check the cells under a microscope to verify that they are not leaking from the insert compartment, then incubate them at 37 degrees celsius and 5%carbon dioxide for 24 hours. After the incubation, check the cells again under the microscope to verify that they have properly attached.
48 hours after seeding, gently remove the insert with sterile tweezers and check the cells under the microscope to verify that the cell layer remains undisturbed. Remove the medium from both the neuron and the periventricular endothelial cell dishes, and add one milliliter of fresh medium to each dish. Incubate the cells at 37 degrees celsius and 5%carbon dioxide for five days.
Then, remove the medium, fix the cells with 4%PFA for 10 minutes and wash them three times with PBS. To perform the co-culture assay, suspend 30, 000 GABAergic interneurons and 30, 000 human periventricular endothelial cells in 70 microliters of co-culture medium, then seed this cell solution inside a one well insert compartment. After 48 hours, remove the insert.
Incubate the co-culture at 37 degrees celsius and 5%carbon dioxide for five days. After the incubation, remove the medium, fix the cells with 4%PFA and wash them three times with PBS. To perform the chemo-attraction assay, place a three well culture insert in the center of a poly-L-ornithine and laminin-coated 35 millimeter dish, turn the dish upside down, and mark the boundary around the middle compartment of the insert using a permanent marker with an ultra fine tip.
Seed 30, 000 GABAergic interneurons in 70 microliters of neuronal medium in the middle compartment, then seed 10, 000 periventricular endothelial cells and 10, 000 control endothelial cells and 70 microliters of their respective medium in the two outer compartments. Add one milliliter of co-culture medium along the side of the dish to prevent the coating on the dish from drying. After 48 hours, remove the insert and incubate the cells at 37 degrees celsius and 5%carbon dioxide for 36 hours.
After the incubation, remove the medium, fix the cells and wash them three times with PBS. Long distance and co-culture migration assays were used to investigate the interactions between periventricular endothelial cells and GABAergic neurons. In the long distance migration assay, cells started out as a rectangular patch on day zero, but by 48 hours, they had migrated out into the cell-free space.
Immunocytochemical staining with Anti-Active Caspase-3 antibody, a marker of apoptosis, showed no apoptotic signal in the seeded neurons. In the co-culture migration assay, when interneurons were co-seeded with human periventricular endothelial cells, neurons traveled farther distances compared to when interneurons were seeded alone or when co-seeded with control endothelial cells. In the chemo-attraction assay, a significantly higher number of interneurons migrated towards periventricular endothelial cells compared to control endothelial cells, confirming that GABAergic interneurons respond selectively to chemo-attractive cues secreted by periventricular endothelial cells.
These assays can be modified using ligands, inhibitors or assay arenes to get mechanistic insights into interactions of human periventricular endothelial cells with human interneurons. They can also be used to study the interaction of human periventricular endothelial cells with other neural cells as reported by our group and others. When attempting this procedure, it is important to fix the insert firmly on the dish and keep it undisturbed throughout the assay, otherwise it may lead to cell leakage, cell detachment or misalignment of the insert with the drawn boundary.
Our work has indicated cell autonomous role of human periventricular endothelial cells in pathogenesis of psychiatric disorders like schizophrenia, epilepsy and autism. These assays will therefore allow assessment of diseased periventricular endothelial cells derived from patients with these disorders using the IPSC technology.
