Protocol for efficient generation of human artery and vein endothelial cells from pluripotent stem cells
DOI: 10.1016/j.xpro.2024.103494
Authors: Kyle M. Loh; Sherry Li Zheng; Kevin J. Liu; Qingqing Yin; Zhainib A. Amir-Ugokwe; Sawan K. Jha; Yue Qi; Vanessa K. Wazny; Alana T. Nguyen; Angela Chen; Faith-Masong Njunkeng; Christine Cheung; Edda Spiekerkoetter; Kristy Red-Horse; Lay Teng Ang
Step 1: iPSC/ESC maintainance
day 0 ~ 1
culture “adherent” human PSC in “mTeSR Plus” for “1” days. For ECM, use “Geltrex” at “1” “%”. Medium should contain factors shown in the following table, and should be changed once in “1” days.
| concentration | unit | |
|---|---|---|
| PSA | 1 | % |
| Thiazovivin | 1.0e-6 | mol/l |
Ideally, “PSC” should be obtained at the end of culture.
Step 2: Primitive streak 1
day 1 ~ 2
culture “adherent” human PSC in “IMDM” for “1” days. For ECM, use “Geltrex” at “1” “%”. Medium should contain factors shown in the following table, and should be changed once in “1” days.
| concentration | unit | |
|---|---|---|
| Ham’s F12 | 50 | % |
| Chemically defined lipid concentrate | — | — |
| MTG | — | — |
| insulin | 7.0e-4 | g/l |
| Transferrin | — | — |
| polyvinyl alcohol | — | — |
| PSA | — | — |
| Activin A | — | — |
| BMP4 | — | — |
| CHIR90021 | — | — |
| FGF | — | — |
Ideally, “PS” should be obtained at the end of culture.
Step 3: Primitive streak 2
day 2 ~ 3 (duration missing in database; assumed 1 day)
culture “adherent” human PSC in “unspecified medium”. For ECM, use “Geltrex” at “1” “%”.
Step 4: mesoderm 1
day 3 ~ 4
culture “adherent” human PSC in “IMDM” for “1” days. For ECM, use “Geltrex” at “1” “%”. Medium should contain factors shown in the following table, and should be changed once in “1” days.
| concentration | unit | |
|---|---|---|
| Ham’s F12 | 50 | % |
| Chemically defined lipid concentrate | — | — |
| MTG | — | — |
| insulin | 7.0e-4 | g/l |
| Transferrin | — | — |
| polyvinyl alcohol | — | — |
| PSA | — | — |
| BMP4 | — | — |
| VEGF | — | — |
| Forskolin | — | — |
| XAV939 | — | — |
| SB505124 | — | — |
| GDC-0941 | — | — |
| Ascorbic Acid | — | — |
Ideally, “mesoderm” should be obtained at the end of culture.
Step 5: mesoderm 2
day 4 ~ 5 (duration missing in database; assumed 1 day)
culture “adherent” human PSC in “unspecified medium”. For ECM, use “Geltrex” at “1” “%”.
Step 6: endothelial 1
day 5 ~ 6
culture “adherent” human PSC in “IMDM” for “1” days. For ECM, use “Geltrex” at “1” “%”. Medium should contain factors shown in the following table, and should be changed once in “1” days.
| concentration | unit | |
|---|---|---|
| Ham’s F12 | 50 | % |
| Chemically defined lipid concentrate | — | — |
| MTG | — | — |
| insulin | 7.0e-4 | g/l |
| Transferrin | — | — |
| polyvinyl alcohol | — | — |
| PSA | — | — |
| VEGF | — | — |
| XAV939 | — | — |
| DMH1 | — | — |
| SB505124 | — | — |
| RO4929097 | — | — |
| Ascorbic Acid | — | — |
| GDC-0941 | — | — |
Ideally, “EC” should express “CDH5”, “PECAM1”, “SOX17”, “CXCR4”, “DLL4” at the end of culture.
Step 7: endothelial 2
day 6 ~ 7
culture “adherent” human PSC in “IMDM” for “1” days. For ECM, use “Geltrex” at “1” “%”. Medium should contain factors shown in the following table, and should be changed once in “1” days.
| concentration | unit | |
|---|---|---|
| Ham’s F12 | 50 | % |
| Chemically defined lipid concentrate | — | — |
| MTG | — | — |
| insulin | 7.0e-4 | g/l |
| Transferrin | — | — |
| polyvinyl alcohol | — | — |
| PSA | — | — |
| CHIR90021 | — | — |
| PD0325901 | — | — |
| SB505124 | — | — |
| RO4929097 | — | — |
| Ascorbic Acid | — | — |
Ideally, “EC” should express “CDH5”, “PECAM1”, “NR2F2”, “APLNR”, “NT5E”, “FLRT2” at the end of culture.