A novel efficient strategy to generate liver sinusoidal endothelial cells from human pluripotent stem cells
DOI: 10.1038/s41598-024-64195-1
Authors: Shang-Ping Tian; Jian-Yun Ge; Yu-Mu Song; Xiao-Qing Yu; Wen-Hao Chen; Yu-Ying Chen; Di Ye; Yun-Wen Zheng
Step 1: iPSC/ESC maintainance
day 0 ~ 1 (duration missing in database; assumed 1 day)
culture “adherent” human PSC in “StemFit Basic04 medium”. For ECM, use “Matrix511-silk” at “5.0e-6” “l/m2”. Medium should contain factors shown in the following table.
| concentration | unit | |
|---|---|---|
| Y27632 | 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 “Matrix511-silk” at “5.0e-6” “l/m2”. Medium should contain factors shown in the following table, and should be changed once in “1” days.
| concentration | unit | |
|---|---|---|
| Ham’s F-12K | 50 | % |
| polyvinyl alcohol | 1 | g/l |
| N2 | 1 | % |
| B27 | 1 | % |
| MTG | 0.45 | mol/l |
| GlutaMAX | 1 | % |
| ITS | 0.1 | % |
| Activin A | 3.0e-5 | g/l |
| BMP4 | 4.0e-5 | g/l |
| CHIR90021 | 6.0e-6 | mol/l |
| FGF | 2.0e-5 | g/l |
Ideally, “PS” should be obtained at the end of culture.
Step 3: mesoderm 1
day 2 ~ 3
culture “adherent” human PS in “IMDM” for “1” days. For ECM, use “Matrix511-silk” at “5.0e-6” “l/m2”. Medium should contain factors shown in the following table, and should be changed once in “1” days.
| concentration | unit | |
|---|---|---|
| Ham’s F-12K | 50 | % |
| polyvinyl alcohol | 1 | g/l |
| N2 | 1 | % |
| B27 | 1 | % |
| MTG | 0.45 | mol/l |
| GlutaMAX | 1 | % |
| ITS | 0.1 | % |
| BMP4 | 4.0e-5 | g/l |
| GDC-0941 | 2.5e-6 | mol/l |
| forskolin | 1.0e-5 | mol/l |
| SB431542 | 6.0e-6 | mol/l |
| VEGF | 1.0e-4 | g/l |
| XAV939 | 1.0e-6 | mol/l |
| ascorbic acid-2-phosphate | 0.2 | g/l |
Ideally, “mesoderm” should be obtained at the end of culture.
Step 4: mesoderm 2
day 3 ~ 4
culture “adherent” human mesoderm in “IMDM” for “1” days. For ECM, use “Matrix511-silk” at “5.0e-6” “l/m2”. Medium should contain factors shown in the following table, and should be changed once in “1” days.
| concentration | unit | |
|---|---|---|
| Ham’s F-12K | 50 | % |
| polyvinyl alcohol | 1 | g/l |
| N2 | 1 | % |
| B27 | 1 | % |
| MTG | 0.45 | mol/l |
| GlutaMAX | 1 | % |
| ITS | 0.1 | % |
| SB431542 | 6.0e-6 | mol/l |
| DMH1 | 2.5e-7 | mol/l |
| DAPT | 1.0e-5 | mol/l |
| VEGF | 1.0e-4 | g/l |
| XAV939 | 1.0e-6 | mol/l |
| ascorbic acid-2-phosphate | 0.2 | g/l |
Ideally, “mesoderm” should be obtained at the end of culture.
Step 5: endothelial 1
day 4 ~ 5
culture “adherent” human mesoderm in “IMDM” for “1” days. For ECM, use “Matrix511-silk” at “5.0e-6” “l/m2”. Medium should contain factors shown in the following table, and should be changed once in “1” days.
| concentration | unit | |
|---|---|---|
| Ham’s F-12K | 50 | % |
| polyvinyl alcohol | 1 | g/l |
| N2 | 1 | % |
| B27 | 1 | % |
| MTG | 0.45 | mol/l |
| GlutaMAX | 1 | % |
| ITS | 0.1 | % |
| SB431542 | 6.0e-6 | mol/l |
| DAPT | 1.0e-5 | mol/l |
| PD0325901 | 5.0e-7 | mol/l |
| CHIR90021 | 1.0e-6 | mol/l |
| ascorbic acid-2-phosphate | 0.2 | g/l |
Ideally, “EPC” should express “KDR”, “PECAM1”, “CD34”, “CDH5” at the end of culture.
Step 6: endothelial 2
day 5 ~ 7
culture “adherent” human EPC in “EGM2” for “2” days. For ECM, use “Matrix511-silk” at “5.0e-6” “l/m2”. Medium should contain factors shown in the following table, and should be changed once in “2” days.
| concentration | unit | |
|---|---|---|
| StemPro | 50 | % |
| polyvinyl alcohol | 1 | g/l |
| SB431542 | 6.0e-6 | mol/l |
| FGF | 2.0e-5 | g/l |
| cAMP | 2.0e-4 | mol/l |
Ideally, “LSEC” should express “LYVE1”, “STAB2”, “FCGR2B”, “CD36”, “FLT4”, “PLVAP”, “c-MAF”, “CLEC1B”, “CLEC4M”, “CRHBP”, “DNASE1L3”, “EPAS1”, “FCN3”, “LIFR”, “NID1”, “OIT3” at the end of culture.