Efficient and Reproducible Differentiation Protocol for Pluripotent Stem Cells into Functional Endothelial Cells: Unveiling the Path to Vascular Regeneration
DOI: 10.1016/j.arcmed.2024.103142
Authors: Manizheh Azhdari; Axel zur Hausen; Nasser Aghdami; Mohamadreza Baghaban-Eslaminejad
Step 1: iPSC/ESC maintainance
day 0 ~ 1 (duration missing in database; assumed 1 day)
culture “adherent” human PSC in “DMEM/F12”. For ECM, use “Matrigel” at “N/A” “N/A”. Medium should contain factors shown in the following table.
| concentration | unit | |
|---|---|---|
| Glutamine | 0.002 | mol/l |
| NEAA | 1 | % |
| 2-mercaptoethanol | 1.0e-4 | mol/l |
| KSR | 10 | % |
| PSA | 100000 | IU/l |
| ITS | — | — |
| streptomycin | 100 | g/l |
| FGF | 1.0e-4 | g/l |
Ideally, “PSC” should be obtained at the end of culture.
Step 2: mesoderm 1
day 1 ~ 6
culture “adherent” human PSC in “alpha-MEM” for “5” days. For ECM, use “Collagen” at “N/A” “N/A”. Medium should contain factors shown in the following table, and should be changed once in “5” days.
| concentration | unit | |
|---|---|---|
| FBS | 10 | % |
| PSA | 1 | % |
| NEAA | 1 | % |
Ideally, “mesoderm” should express “KDR” at the end of culture.
Step 3: endothelial 1
day 6 ~ 7 (duration missing in database; assumed 1 day)
culture “adherent” human mesoderm in “EGM2”. For ECM, use “Collagen” at “0.08” “g/m2”. Medium should contain factors shown in the following table.
| concentration | unit | |
|---|---|---|
| VEGF | — | — |
| FGF | — | — |
Ideally, “EC” should express “PECAM1”, “CDH5”, “MCAM”, “VWF”, “CDH5”, “EphB2”, “EphB4” at the end of culture.