VEGF, FGF2, and BMP4 regulate transitions of mesoderm to endothelium and blood cells in a human model of yolk sac hematopoiesis
DOI: 10.1016/j.exphem.2021.08.006
Authors: Freya F. Bruveris; Elizabeth S. Ng; Edouard G. Stanley; Andrew G. Elefanty
Step 1: iPSC/ESC maintainance
day 0 ~ 1 (duration missing in database; assumed 1 day)
culture “adherent” human ESC in “unspecified medium”. For ECM, use “Fibroblast” at “N/A” “N/A”.
Ideally, “ESC” should be obtained at the end of culture.
Step 2: mesoderm 1
day 1 ~ 3
culture “spheroid” human ESC in “APEL medium” for “2” days. Medium should contain factors shown in the following table, and should be changed once in “2” days.
| concentration | unit | |
|---|---|---|
| Activin A | 1.0e-5 | g/l |
| BMP4 | 2.0e-5 | g/l |
| VEGF | 3.0e-5 | g/l |
| SCF | 4.0e-5 | g/l |
Ideally, “mesoderm” should express “PDGFRA” at the end of culture.
Step 3: endothelial 1
day 3 ~ 12
culture “unspecified method” human mesoderm in “APEL medium” for “9” days. Medium should contain factors shown in the following table, and should be changed once in “9” days.
| concentration | unit | |
|---|---|---|
| methylcellulose | 1 | % |
| SCF | 1.0e-4 | g/l |
| EPO | 2000 | U/l |
| VEGF | 5.0e-5 | g/l |
| IL-3 | 5.0e-5 | g/l |
| IL-6 | 5.0e-5 | g/l |
| TPO | 5.0e-5 | g/l |
| BMP4 | 2.0e-5 | g/l |
| FGF | 1.0e-5 | g/l |
| SU5402 | 0.005 | mol/l |
| DMH-1 | 7.0e-4 | mol/l |
| Activin A | 1.0e-5 | g/l |
| SB431542 | 0.004 | mol/l |
| CHIR90021 | 0.003 | mol/l |
| IWR1 | 0.005 | mol/l |
| XAV939 | 0.005 | mol/l |
| SANT-1 | 2.5e-4 | mol/l |
| purmorphamine | 0.001 | mol/l |
| DAPT | 0.01 | mol/l |
Ideally, “EC” should express “SOX17”, “CD34”, “PECAM1”, “KDR”, “TEK” at the end of culture.