Efficient differentiation and purification of human induced pluripotent stem cell-derived endothelial progenitor cells and expansion with the use of inhibitors of ROCK, TGF-β, and GSK3β

DOI: 10.1016/j.heliyon.2020.e03493

Authors: Hiromasa Aoki; Misaki Yamashita; Tadahiro Hashita; Koichi Ogami; Shinichi Hoshino; Takahiro Iwao; Tamihide Matsunaga

Step 1: iPSC/ESC maintainance

day 0 ~ 1

culture “adherent” human PSC in “Essential 8 Flex medium” for “1” days. For ECM, use “VTN-N” at “0.01” “g/m2”. Medium should contain factors shown in the following table, and should be changed once in “1” days.

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

Step 2: Primitive streak 1

day 1 ~ 2

culture “adherent” human PSC in “DMEM/F12” for “1” days. For ECM, use “VTN-N” at “0.01” “g/m2”. Medium should contain factors shown in the following table, and should be changed once in “1” days.

Step 3: mesoderm 1

day 2 ~ 3

culture “adherent” human PSC in “DMEM/F12” for “1” days. For ECM, use “VTN-N” at “0.01” “g/m2”. Medium should contain factors shown in the following table, and should be changed once in “1” days.

Step 4: endothelial 1

day 3 ~ 4

culture “adherent” human PSC in “DMEM/F12” for “1” days. For ECM, use “VTN-N” at “0.01” “g/m2”. Medium should contain factors shown in the following table, and should be changed once in “1” days.

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

Step 5: endothelial 2

day 4 ~ 5

culture “adherent” human PSC in “HE-SFM” for “1” days. For ECM, use “porcine skin gelatin” at “0.1” “%”. Medium should contain factors shown in the following table, and should be changed once in “1” days.

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