Stepwise differentiation and functional characterization of human induced pluripotent stem cell-derived choroidal endothelial cells

DOI: 10.1186/s13287-020-01903-4

Authors: Kelly Mulfaul; Joseph C. Giacalone; Andrew P. Voigt; Megan J. Riker; Dalyz Ochoa; Ian C. Han; Edwin M. Stone; Robert F. Mullins; Budd A. Tucker

Step 1: iPSC/ESC maintainance

day 0 ~ 1

culture “adherent” human PSC in “mTeSR” for “1” days. For ECM, use “Matrigel” at “N/A” “N/A”. 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 ~ 3

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

Step 4: endothelial 1

day 6 ~ 9

culture “adherent” human PSC in “endothelial cell growth medium” for “3” days. For ECM, use “Matrigel” 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 5: endothelial 2

day 9 ~ 11

culture “adherent” human PSC in “endothelial cell growth medium” for “2” days. For ECM, use “Matrigel” at “N/A” “N/A”. Medium should contain factors shown in the following table, and should be changed once in “2” days.

Ideally, “EC” should express “PECAM1”, “CA4”, “RGCC”, “CDH5” at the end of culture.