Differentiating Human-Induced Pluripotent Stem Cells into Brain Microvascular Endothelial Cells
Transcription
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.
