Human definitive haemogenic endothelium and arterial vascular endothelium represent distinct lineages
DOI: 10.1038/ncb3161
Authors: Andrea Ditadi; Christopher M. Sturgeon; Joanna Tober; Geneve Awong; Marion Kennedy; Amanda D. Yzaguirre; Lisa Azzola; Elizabeth S. Ng; Edouard G. Stanley; Deborah L. French; Xin Cheng; Paul Gadue; Nancy A. Speck; Andrew G. Elefanty; Gordon Keller
Step 1: iPSC/ESC maintainance
day 0 ~ 1
culture “adherent” human ESC in “unspecified medium” for “1” days. For ECM, use “Matrigel” at “N/A” “N/A”. Medium should be changed once in “1” days.
Ideally, “ESC” should be obtained at the end of culture.
Step 2: Primitive streak 1
day 1 ~ 3
culture “spheroid” human ESC in “StemPro” for “2” days. Medium should contain factors shown in the following table, and should be changed once in “2” days.
| concentration | unit | |
|---|---|---|
| PSA | 1.0e-5 | g/l |
| Glutamine | 0.002 | mol/l |
| Ascorbic Acid | 0.001 | mol/l |
| MTG | 4.0e-4 | mol/l |
| Transferrin | 0.15 | g/l |
| BMP4 | 1.0e-5 | g/l |
| FGF | 5.0e-6 | g/l |
Step 3: Primitive streak 2
day 3 ~ 4
culture “spheroid” human ESC in “StemPro” for “1” days. Medium should contain factors shown in the following table, and should be changed once in “1” days.
| concentration | unit | |
|---|---|---|
| BMP4 | 1.0e-5 | g/l |
| FGF | 5.0e-6 | g/l |
| CHIR | 3.0e-6 | mol/l |
| IWP2 | 3.0e-6 | mol/l |
| Activin A | 1.0e-6 | g/l |
Step 4: mesoderm 1
day 4 ~ 6
culture “spheroid” human ESC and human ESC in “StemPro” for “2” days. “ESC” should be “upregulated” for “NOTCH1”. Medium should contain factors shown in the following table, and should be changed once in “2” days.
| concentration | unit | |
|---|---|---|
| L-685,458 | 1.0e-5 | mol/l |
| DMSO | — | — |
| 4-OHT | 1.0e-6 | mol/l |
| PD0325901 | 1.0e-6 | mol/l |
| LY294002 | 1.0e-5 | mol/l |
| PSA | 1.0e-5 | g/l |
| Glutamine | 0.002 | mol/l |
| Ascorbic Acid | 0.001 | mol/l |
| MTG | 4.0e-4 | mol/l |
| Transferrin | 0.15 | g/l |
| VEGF | 1.5e-5 | g/l |
| FGF | 5.0e-6 | g/l |
| IL-6 | 1.0e-5 | g/l |
| IL-11 | 5.0e-6 | g/l |
Step 5: mesoderm 2
day 6 ~ 9
culture “spheroid” unknown species cell in “StemPro” for “3” days. Medium should contain factors shown in the following table, and should be changed once in “3” days.
| concentration | unit | |
|---|---|---|
| PSA | 1.0e-5 | g/l |
| Glutamine | 0.002 | mol/l |
| Ascorbic Acid | 0.001 | mol/l |
| MTG | 4.0e-4 | mol/l |
| Transferrin | 0.15 | g/l |
| VEGF | 1.5e-5 | g/l |
| FGF | 5.0e-6 | g/l |
| IL-6 | 1.0e-5 | g/l |
| IL-11 | 5.0e-6 | g/l |
| SCF | 5.0e-5 | g/l |
| IGF1 | 2.5e-5 | g/l |
| EPO | 2000 | U/l |
Step 6: endothelial 1
day 9 ~ 10
culture “adherent” human EC in “unspecified medium” for “1” days. Medium should contain factors shown in the following table, and should be changed once in “1” days.
| concentration | unit | |
|---|---|---|
| L-685,458 | — | — |
| DMSO | — | — |
Ideally, “EC” should be obtained at the end of culture.
Step 7: endothelial 2
day 10 ~ 17
culture “adherent” human EC in “unspecified medium” for “7” days. For ECM, use “Matrigel” at “N/A” “N/A”. Medium should contain factors shown in the following table, and should be changed once in “7” days.
| concentration | unit | |
|---|---|---|
| L-685,458 | — | — |
| DMSO | — | — |
Ideally, “EC” should express “CDH5”, “PTPRC”, “cKIT”, “RUNX1C”, “MYB”, “TAL1”, “EFNB2”, “DLL4”, “NR2F2” at the end of culture.