This article outlines a protocol for differentiating human-induced pluripotent stem cells (iPSCs) into brain microvascular endothelial cells (BMECs). The process involves several steps, including the use of specific growth factors and media to promote cell differentiation and adhesion.
Begin with a multi-well plate containing human-induced pluripotent stem cells, or iPSCs, in a medium with a Rho-kinase inhibitor, which promotes cell survival.
Replace the medium with an essential medium containing nutrients and growth factors.
Incubate to allow the proliferation and differentiation of iPSCs into precursor cells.
Replace this medium with a differentiation medium supplemented with fibroblast growth factors and retinoic acid, then incubate.
The growth factors facilitate the differentiation of precursor cells into microvascular endothelial cells, while retinoic acid promotes the synthesis of tight junction proteins.
Wash and treat the cells with an enzyme-EDTA solution to detach the cells, creating a single-cell suspension.
Centrifuge to collect the cells and remove the supernatant. Then, resuspend the cells in a fresh differentiation medium.
Seed this cell suspension into a multi-well plate coated with collagen and adhesion proteins.
Incubate to allow cell adhesion, forming tight junctions between the cells, resembling brain microvascular endothelial cells.
To induce BMEC differentiation from human iPSCs, wash the confluent iPSC culture with DPBS one time before incubating the cells with the appropriate volume of enzymatic EDTA for approximately five minutes at 37 degrees Celsius.
When a single-cell suspension has been obtained, collect the cells by centrifugation, and resuspend the pellet in an appropriate volume of stem cell medium, supplemented with 10 micromolar ROCK inhibitor for counting.
Dilute the cells to a 1.56 times 10 to the fourth cells per cubic centimeter concentration, and seed 2 milliliters of cells into individual wells of a 6-well flat-bottom. After 24 hours in the cell culture incubator, replace the supernatant in each well with E6 medium and return the plate to the incubator for four more days.
To purify the iPSC-derived BMECs, coat the sub-culturing plates with the appropriate volume of a freshly prepared collagen IV and Fibronectin solution per well, and incubate the plates for a minimum of two hours at 37 degrees Celsius.
On day 6 of differentiation, collect the cells by centrifugation, and resuspend the pellets in the appropriate volume of fresh human endothelial serum-free medium supplemented with B27, basic fibroblast growth factor, and retinoic acid. Plate the cells into each well of a collagen- and Fibronectin-coated 24-well flat-bottom plate.
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