Efficient generation of liver sinusoidal endothelial-like cells secreting coagulation factor VIII from human induced pluripotent stem cells

DOI: 10.1016/j.omtm.2024.101355

Authors: Seiji Mitani; Chihiro Hosoda; Yu Onodera; Yoko Takabayashi; Asuka Sakata; Midori Shima; Kohei Tatsumi

Step 1: iPSC/ESC maintainance

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

culture “adherent” human PSC in “StemFit AK02N”. For ECM, use “Laminin” at “N/A” “N/A”.

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

Step 2: Primitive streak 1

day 1 ~ 2

culture “adherent” human PSC in “RPMI” 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, “PS” should express “TBXT”, “MIXL1” at the end of culture.

Step 3: Primitive streak 2

day 2 ~ 3

culture “adherent” human PSC in “RPMI” 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.

Step 4: mesoderm 1

day 3 ~ 4

culture “adherent” human PSC in “RPMI” 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, “mesoderm” should express “KDR”, “FOXF1”, “HAND1”, “HAND2”, “POU5F1”, “NANOG” at the end of culture.

Step 5: endothelial 1

day 4 ~ 7

culture “adherent” human PSC in “RPMI” 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 express “CD34”, “PECAM1”, “NRP2”, “EPHB4”, “CXCR4”, “EFNB2” at the end of culture.

Step 6: endothelial 2

day 7 ~ 9

culture “adherent” human PSC in “EGM2” 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 “FCGR2B”, “LYVE1”, “F8”, “CD36”, “MRC1”, “CLEC1B”, “CD14”, “PECAM1”, “GATA4”, “MAF”, “MCFD2”, “LMAN1” at the end of culture.