This is the first protocol allowing to differentiate human-induced pluripotent stem cells into brain microvascular endothelial cells with good barrier properties, mature tight junctions, and expression of endothelial cell adhesion molecules mediating immune cell migration into the central novice system. EECM-BMEC-like cells allow to study immune cell migration across the blood brain barrier with blood brain barrier models derived from the patients in a fully autologous fashion. Differentiating human-induced pluripotent stem cells from MS patients into EECM-BMEC-like cells has allowed us to identify intrinsic barrier defects in these in vitro models of the MS patients.
This cultural method will therefore allow us to identify the molecular pathways underlying these impaired barrier properties in the future. These methods can be applied to a broader spectrum of diseases where in serum morphology and immune cell infiltration into the CNS are critical for these pathogenesis, for example, stroke or neuroinfection. When trying this technique for the first time, use the same amount of cell materials.
The seeding density is the key to success of this protocol and needs to be adjusted depending on the clones used. Demonstrating the procedure will be Kinya Matsuo, assistant professor from my laboratory. On day five of the endothelial progenitor cell culturing aspirate the medium from the wells and add one milliliter of dissociation reagent to each well.
Incubate the plate for six to eight minutes at 37 degrees celsius. Using a micropipette dissociate and singularize the cells. Then, pass the cell suspension through a 40 micrometer cell strainer to filter into a 50 milliliter tube containing 10 milliliters of DMEM/F-12 10 medium.
To stop the digestion reaction, add DM DMEM/F-12 10 medium up to the 50 milliliter mark. Thoroughly pipette and reserve 10 microliters for counting the cells. Centrifuge the 50 milliliter tube at 200 g for five minutes at 20 to 25 degrees celsius to pellet the cells.
After centrifugation, remove the supernatant and resuspend the cell pellet with 10 milliliters of DMEM/F-12 10 medium. Transfer the cell suspension into a fresh 15 milliliter tube. Centrifuge at 200 g for five minutes at 20 to 25 degrees celsius to pellet the cells again.
Then resuspend the cell pellet into flow buffer one. Next, add the FCR blocking reagent at a one to 100 ratio and incubate for five minutes. Then add the one to 200 ratio diluted fluorescein isothiocyanate or FITC labeled CD31 antibody.
Incubate the suspension for 30 minutes in the dark at a temperature between 20 and 25 degrees celsius. At the end of the incubation, add 10 milliliters of flow buffer one to the sample and reserve 10 microliters of the suspension for flow cytometry analysis to determine the fraction of CD31 positive cells. Centrifuge the cells at 200 g for five minutes at 20 to 25 degrees celsius.
After centrifugation resuspend the cells with flow buffer one solution. Next, add five microliters of the FITC selection cocktail per 100 microliters of the cell suspension. Mix the solution thoroughly by pipetting and incubate in the dark for 15 minutes at a temperature between 20 and 25 degrees celsius.
Next, add five microliters of magnetic nanoparticles per 100 microliters of cell suspension. Pipette the mixture well and incubate in the dark for 10 minutes at 20 to 25 degrees celsius. Transfer the cell suspension to a five milliliter flow cytometry tube and add flow buffer one to get a total volume of 2.5 milliliters.
Then place the flow cytometry tube in the magnet for five minutes. In a continuous motion, invert the magnet and decant the cell suspension containing FITC CD31 antibody unlabeled cells. Maintain the magnet and tube in the inverted position for two to three seconds and then remove the remaining liquid.
Aspirate any droplets on the tube edge before returning the tube to an upright position. Retrieve the flow cytometry tube from the magnet and add 2.5 milliliters of flow buffer one to wash the remaining CD31 positive cells. Gently pipette the cells up and down two or three times to resuspend them.
Then place the flow tube into the magnet for five minutes. Repeat the washing at least four times to remove the FITC CD31 unlabeled cells from the tube. Remove the flow tube from the magnet and resuspend the purified CD31 positive cells with the desired amount of a suitable medium.
Reserve two 10 microliter aliquots of the suspension, one for cell counting and the second to carry out flow cytometry analysis to assess the purity of CD31 positive cells in post-magnetic activated cell sorting samples. Remove the HECSR medium from the six well plates containing endothelial and non-endothelial cell populations. Then add one milliliter of dissociation reagent to each well.
Carefully observe the cell morphology under a microscope. When the endothelial cells appear bright and round, usually within two to five minutes, detach them by tapping the edge of the plate. The non-endothelial cells remain attached to the plate.
Using a micropipette, carefully collect the detached endothelial cells without disturbing the non-endothelial cells and transfer them to a 15 milliliter or 50 milliliter centrifuge tube containing four milliliters of DMEM/F-12 10 per milliliter of dissociation reagent. Then add two milliliters of HECSR medium to the wells containing the remaining attached non-endothelial cells to establish the smooth muscle like cells or SMLCs and place the plate in the incubator. Transfer the endothelial cell suspension into a centrifuge tube.
Mix thoroughly and reserve 10 microliters of the suspension for cell counting. Centrifuge the remaining cells at 200 g for five minutes at 20 to 25 degrees celsius. Then add two milliliters of HECSR medium.
Next, remove the collagen for solution from a previously prepared collagen coded six well plate and add two milliliters of the endothelial cell suspension to each well. Then, incubate the plate at 37 degrees celsius in 5%carbon dioxide. Immunofluorescent staining of EECM-BMEC-like cell junctional molecules in including claudin-5, occludin and VE-cadherin was used to assess cell morphology and the presence of continuous and mature junctions.
Stimulation of the EECM-BMEC-like cells seeded on chamber slides with pro-inflammatory cytokines such as tumor necrosis factor alpha and interferon gamma diluted in SMLC derived conditioned medium upregulated the expression of adhesion molecules such as ICAM-1 and VCAM-1. Representative images of smooth muscle cell markers including alpha smooth muscle actin, calponin, and smooth muscle protein 22 alpha are shown here. Stimulation of the EECM-BMEC-like cells with pro-inflammatory cytokines like tumor necrosis factor alpha and interferon gamma upregulated the cell surface expression of several adhesion molecules including ICAM-1, VCAM-1, and P-selectin.
Further, stimulation with inflammatory cytokines also upregulated the expression of endothelial adhesion molecules and promoted the increased number of immune cells that adhered to EECM-BMEC-like cell monolayers EECM-BMEC-like cells are promising too for in-depth understanding of pathophysiological mechanisms at the blood-brain barrier level, and as a valuable tool to develop new therapeutic target for blood-brain barrier stabilization.
