Differentiation and characterization of human pluripotent stem cell-derived brain microvascular endothelial cells

DOI: 10.1016/j.ymeth.2015.10.016

Authors: Matthew J. Stebbins; Hannah K. Wilson; Scott G. Canfield; Tongcheng Qian; Sean P. Palecek; Eric V. Shusta

Step 1: iPSC/ESC maintainance

day 0 ~ 1

culture “adherent” human PSC in “mTeSR” for “1” days. For ECM, use “Matrigel” at “0.083” “g/l”. Medium should contain factors shown in the following table, and should be changed once in “1” days.

Step 2: Primitive streak 1

day 1 ~ 2

culture “adherent” human PSC in “DMEM/F12” for “1” days. For ECM, use “Matrigel” at “0.083” “g/l”. Medium should contain factors shown in the following table, and should be changed once in “1” days.

Ideally, “EC” should be obtained at the end of culture. Ideally, “NPC” should be obtained at the end of culture.

Step 3: endothelial 1

day 2 ~ 4

culture “adherent” human PSC in “hESFM” for “2” days. For ECM, use “Matrigel” at “0.083” “g/l”. Medium should contain factors shown in the following table, and should be changed once in “2” days.

Ideally, “EC” should express “Claudin-5”, “Occludin”, “ZO-1”, “GLUT-1”, “P-gp” at the end of culture.

Step 4: endothelial 2

day 4 ~ 5

culture “adherent” human PSC in “hESFM” for “1” days. For ECM, use “collagen IV/fibronectin” at “N/A” “N/A”. Medium should contain factors shown in the following table, and should be changed once in “1” days.

Ideally, “EC” should express “CDH5”, “PECAM1”, “Claudin-5”, “Occludin”, “ZO-1”, “GLUT-1”, “P-gp”, “BCRP”, “MRP1” at the end of culture.