Vascular endothelial cells derived from transgene-free pig induced pluripotent stem cells for vascular tissue engineering
DOI: 10.1016/j.actbio.2024.12.033
Authors: Luke Batty; Jinkyu Park; Lingfeng Qin; Muhammad Riaz; Yuyao Lin; Zhen Xu; Xuefei Gao; Xin Li; Colleen Lopez; Wei Zhang; Marie Hoareau; Meghan E. Fallon; Yan Huang; Hangqi Luo; Jiesi Luo; Séverine Ménoret; Peining Li; Zhenting Jiang; Peter Smith; David H. Sachs; George Tellides; Ignacio Anegon; Jordan S. Pober; Pentao Liu; Yibing Qyang
Step 1: iPSC/ESC maintainance
day 0 ~ 1 (duration missing in database; assumed 1 day)
culture “adherent” pig PSC in “DMEM/F12”. For ECM, use “Gelatin” at “N/A” “N/A”. Medium should contain factors shown in the following table.
| concentration | unit | |
|---|---|---|
| N2 | — | — |
| B27 | — | — |
| l-glutamine | 0.002 | mol/l |
| NEAA | 1 | % |
| pen/strep | 1 | % |
| 2-mercaptoethanol | 1.0e-4 | mol/l |
| CHIR90021 | 2.0e-7 | mol/l |
| WH-4-023 | 3.0e-7 | mol/l |
| XAV939 | 2.5e-6 | mol/l |
| Ascorbic Acid | 0.065 | g/l |
| LIF | 1.0e-5 | g/l |
| Activin A | 2.0e-5 | g/l |
| FBS | 0.3 | % |
| FGF | 2.0e-6 | g/l |
Ideally, “PSC” should express “POU5F1”, “NANOG”, “SSEA-4”, “alkaline phosphatase” at the end of culture.
Step 2: mesoderm 1
day 1 ~ 5
culture “adherent” pig PSC in “N2B27 medium” for “4” days. For ECM, use “Gelatin” at “N/A” “N/A”. Medium should contain factors shown in the following table, and should be changed once in “4” days.
| concentration | unit | |
|---|---|---|
| N2 | — | — |
| B27 | — | — |
| l-glutamine | 0.002 | mol/l |
| CHIR90021 | 6.0e-6 | mol/l |
| BMP4 | 2.5e-5 | g/l |
Ideally, “mesoderm” should be obtained at the end of culture.
Step 3: endothelial 1
day 5 ~ 7
culture “adherent” pig mesoderm in “StemPro” for “2” days. For ECM, use “Gelatin” at “N/A” “N/A”. Medium should contain factors shown in the following table, and should be changed once in “2” days.
| concentration | unit | |
|---|---|---|
| l-glutamine | 0.002 | mol/l |
| VEGF | 2.0e-4 | g/l |
| forskolin | 2.0e-6 | mol/l |
Ideally, “EC” should express “PECAM1”, “NOS3”, “VWF” at the end of culture.
Step 4: endothelial 2
day 7 ~ 9
culture “adherent” pig EC in “EGM2” for “2” days. For ECM, use “Gelatin” at “N/A” “N/A”. Medium should contain factors shown in the following table, and should be changed once in “2” days.
| concentration | unit | |
|---|---|---|
| FBS | 10 | % |
| VEGF | 5.0e-5 | g/l |
Ideally, “EC” should express “PECAM1”, “NOS3”, “VWF”, “CDH5”, “ICAM1” at the end of culture.