Preparation of iPS cell-derived CD31+ endothelial cells using three-dimensional suspension culture

DOI: 10.1016/j.reth.2018.06.004

Authors: Shinako Masuda; Katsuhisa Matsuura; Tatsuya Shimizu

Step 1: iPSC/ESC maintainance

day 0 ~ 1 (duration missing in database; assumed 1 day)

culture “adherent” human PSC in “Primate ES Cell Medium”. Medium should contain factors shown in the following table.

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

Step 2: Primitive streak 1

day 1 ~ 3

culture “spheroid” human PSC in “mTeSR” for “2” days. Medium should contain factors shown in the following table, and should be changed once in “2” days.

Step 3: Primitive streak 2

day 3 ~ 6

culture “spheroid” human PSC in “StemPro” for “3” days. Medium should contain factors shown in the following table, and should be changed once in “3” days.

Step 4: mesoderm 1

day 6 ~ 8

culture “spheroid” human PSC in “StemPro” for “2” days. Medium should contain factors shown in the following table, and should be changed once in “2” days.

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

Step 5: endothelial 1

day 8 ~ 11

culture “adherent” human mesoderm in “unspecified medium” for “3” days. For ECM, use “Collagen IV” at “N/A” “N/A”. Medium should contain factors shown in the following table, and should be changed once in “3” days.

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

Step 6: endothelial 2

day 11 ~ 14

culture “adherent” human EC in “unspecified medium” for “3” days. For ECM, use “Collagen IV” at “N/A” “N/A”. Medium should contain factors shown in the following table, and should be changed once in “3” days.

Ideally, “EC” should express “PECAM1”, “CDH5”, “VWF”, “KDR” at the end of culture.