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