Generating Brain Endothelial Cells from Induced Pluripotent Stem Cells
Transcription
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.
