This article describes a protocol for differentiating induced pluripotent stem cells (iPSCs) into brain endothelial cells. The process involves the use of rho-kinase inhibitors and growth factors to maintain undifferentiated states and promote cell survival, followed by a series of media changes to induce differentiation.
Take an induced pluripotent stem cell, or iPSC, culture in maintenance media containing rho-kinase inhibitors.
Growth factors keep cells undifferentiated, while rho-kinase inhibitors enhance cell survival.
Replace the media with fresh maintenance media, removing rho-kinase inhibitors. Incubate, promoting colony formation.
Regularly replace with unconditioned media, which provides a controlled environment, triggering iPSC differentiation into neural progenitors and then brain endothelial cells, followed by cell proliferation.
Replace with endothelial cell media containing retinoic acid and fibroblast growth factors. Incubate.
Fibroblast growth factors facilitate cell proliferation, while retinoic acid promotes brain endothelial cell maturation.
Add proteolytic enzymes to detach the cells. Collect the cells and centrifuge. Discard the supernatant.
Resuspend the cells in endothelial media and seed them onto a multi-well membrane insert coated with collagen and fibronectin.
The well contains endothelial media. Incubate for selective brain endothelial cell adhesion.
Replace the media with non-supplemented endothelial media for further experiments.
Resuspend 7.5 x 105 cells in 12 milliliters of stem cell maintenance medium and 10 micromolar ROCK inhibitor. Aspirate matrix from a T75 flask and transfer the cell suspension to the flask. Shake the flask to distribute the cells, and place it in the incubator.
Refresh the media every day for the next two days to remove ROCK inhibitor, and support the growth of stem cell colonies. On the third day, begin differentiation by changing the media to an unconditioned medium.
Change the medium daily for the next five days. Six days after starting differentiation, selectively expand the endothelial cell population by switching to endothelial cell or EC medium with 20 nanograms per milliliter bFGF and 10 micromolar retinoic acid. Incubate the cells for two days.
Prepare collagen IV and Fibronectin solution according to manuscript directions, and use it to coat cell culture plates. Aspirate the EC medium from the cells, and add 12 milliliters of cell dissociation reagent. Incubate the flask at 37 degrees Celsius until 90% of the cells have detached.
During the incubation time, remove the coating solution from the previously prepared plates, and let them dry in a sterile hood. Once the cells have detached, use a 10-milliliter pipette to create a single-cell suspension.
Transfer the cells to a 50-milliliter tube, and dilute them with an equal volume of human endothelial serum-free free medium. Then, count the cells with a hemocytometer. Pellet the cells at 1,500 g for 10 minutes. Then resuspend them in freshly prepared EC medium with bFGF and retinoic acid for a concentration of 2 million cells per milliliter.
Add 500 microliters of the cell suspension to the top of a 12-well insert and 1.5 milliliters of medium to the bottom. Distribute the cells evenly across the insert, and incubate the plate at 37 degrees Celsius and 5% carbon dioxide. On the next day, change the media to EC without bFGF or retinoic acid.
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