Feeder-free generation and characterization of endocardial and cardiac valve cells from human pluripotent stem cells

DOI: 10.1016/j.isci.2023.108599

Authors: Clifford Z. Liu; Aditi Prasad; Bharati Jadhav; Yu Liu; Mingxia Gu; Andrew J. Sharp; Bruce D. Gelb

Step 1: iPSC/ESC maintainance

day 0 ~ 1

culture “adherent” human PSC in “mTeSR Plus” for “1” days. For ECM, use “Matrigel” at “6.6” “%”. Medium should contain factors shown in the following table, and should be changed once in “1” days.

Step 2: mesoderm 1

day 1 ~ 3

culture “adherent” human PSC in “RPMI 1640” for “2” days. For ECM, use “Matrigel” at “6.6” “%”. Medium should contain factors shown in the following table, and should be changed once in “2” days.

Ideally, “ME” should express “CD13”, “CD56” at the end of culture.

Step 3: endothelial 1

day 3 ~ 5

culture “adherent” human ME in “RPMI 1640” for “2” days. For ECM, use “Matrigel” at “6.6” “%”. Medium should contain factors shown in the following table, and should be changed once in “2” days.

Ideally, “endocardial” should express “PECAM1”, “PECAM1”, “CD34”, “CDH5”, “NKX2.5”, “GATA4”, “GATA5”, “ISL1”, “HAPLN1”, “NPR3”, “NRG1”, “MEIS2”, “TMEM100”, “NFATc1”, “HAND2”, “TBX20”, “TK1”, “CYTL1”, “TMEM108”, “SGK1”, “TOP2A”, “UBE2C”, “CDC20” at the end of culture.

Step 4: endothelial 2

day 5 ~ 7

culture “adherent” human ME in “RPMI 1640” for “2” days. For ECM, use “Matrigel” at “6.6” “%”. Medium should contain factors shown in the following table, and should be changed once in “2” days.

Ideally, “EC” should express “CXCR4”, “ESM1”, “ANGPT2”, “FLT1”, “NOTCH4”, “SOX17”, “CDH5”, “TIE1”, “FLT4”, “CD34” at the end of culture.