Generation and characterization of cardiac valve endothelial-like cells from human pluripotent stem cells
DOI: 10.1038/s42003-021-02571-7
Authors: LinXi Cheng; MingHui Xie; WeiHua Qiao; Yu Song; YanYong Zhang; YingChao Geng; WeiLin Xu; Lin Wang; Zheng Wang; Kai Huang; NianGuo Dong; YuHua Sun
Step 1: iPSC/ESC maintainance
day 0 ~ 1 (duration missing in database; assumed 1 day)
culture “adherent” human PSC in “mTeSR”. For ECM, use “Matrigel” at “N/A” “N/A”. Medium should contain factors shown in the following table.
| concentration | unit | |
|---|---|---|
| Y27632 | 1.0e-7 | mol/l |
Step 2: Primitive streak 1
day 1 ~ 2
culture “adherent” human PSC in “Essential 6 medium” 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.
| concentration | unit | |
|---|---|---|
| BMP4 | 1.0e-4 | g/l |
| WNT3a | 2.5e-5 | g/l |
| CHIR90021 | 6.0e-7 | mol/l |
Ideally, “PS” should express “TBXT” at the end of culture.
Step 3: mesoderm 1
day 2 ~ 3
culture “adherent” human PS in “Essential 6 medium” 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.
| concentration | unit | |
|---|---|---|
| BMP4 | 5.0e-5 | g/l |
| FGF | 2.0e-5 | g/l |
Ideally, “CPC” should express “ISL1”, “KDR” at the end of culture.
Step 4: endothelial 1
day 3 ~ 4
culture “adherent” human CPC in “Essential 6 medium” for “1” days. Medium should contain factors shown in the following table, and should be changed once in “1” days.
| concentration | unit | |
|---|---|---|
| VEGF | 1.0e-4 | g/l |
| BMP4 | 1.0e-5 | g/l |
| TGFb1 | 1.0e-5 | g/l |
Ideally, “EC” should express “NPR3”, “CDH11”, “EDN1”, “GATA5”, “MEF2C”, “SOX17”, “THSD1”, “EMCN”, “TGFb2”, “NOTCH4”, “DLL3”, “DLL4”, “JAG1”, “PECAM1”, “VE-cad”, “GATA4”, “KDR”, “VWF”, “ENG”, “NOTCH1”, “ATF3”, “NFATc1” at the end of culture.
Step 5: endothelial 2
day 4 ~ 5
culture “adherent” human CPC in “Essential 6 medium” for “1” days. Medium should contain factors shown in the following table, and should be changed once in “1” days.
| concentration | unit | |
|---|---|---|
| VEGF | 1.0e-4 | g/l |
| BMP4 | 1.0e-5 | g/l |
| TGFb1 | 1.0e-5 | g/l |
Ideally, “EC” should express “NFATc1”, “SMAD6”, “HAND2”, “PROX1”, “HEY2”, “TBX2”, “NOTCH1”, “NOTCH3”, “DLL4”, “NTRK2”, “VE-cad”, “NFKB1”, “DKK2”, “DKK3”, “ID1”, “ID2”, “CCND1”, “WNT2”, “GATA5”, “BMP4”, “ATF3”, “CXCL12”, “LDB2”, “MGP”, “CDH13”, “KLF2”, “KLF4”, “KLF9”, “KLF11”, “EGR1”, “CXCL2”, “JUN”, “FOS”, “FOSB”, “GATA2”, “THBD”, “MSX1”, “SELE”, “IGFBP7”, “CAV1”, “ITM2A”, “GJA5”, “PALMD”, “TSPAN8”, “ISLR”, “HMCN1”, “SELP”, “CDH11”, “NFATc5” at the end of culture.