NOTCH signaling specifies arterial-type definitive hemogenic endothelium from human pluripotent stem cells
DOI: 10.1038/s41467-018-04134-7
Authors: Gene I. Uenishi; Ho Sun Jung; Akhilesh Kumar; Mi Ae Park; Brandon K. Hadland; Ethan McLeod; Matthew Raymond; Oleg Moskvin; Catherine E. Zimmerman; Derek J. Theisen; Scott Swanson; Owen J. Tamplin; Leonard I. Zon; James A. Thomson; Irwin D. Bernstein; Igor I. Slukvin
Step 1: iPSC/ESC maintainance
day 0 ~ 1
culture “adherent” human PSC in “E8” for “1” days. For ECM, use “Collagen” at “0.005” “g/m2”. Medium should contain factors shown in the following table, and should be changed once in “1” days.
| concentration | unit | |
|---|---|---|
| Y27632 | 1.0e-5 | mol/l |
Step 2: Primitive streak 1
day 1 ~ 3
culture “adherent” human PSC in “unspecified medium” for “2” days. For ECM, use “Collagen” at “0.005” “g/m2”. Medium should contain factors shown in the following table, and should be changed once in “2” days.
| concentration | unit | |
|---|---|---|
| BMP4 | — | — |
| FGF | 5.0e-5 | g/l |
| Activin A | 1.5e-5 | g/l |
| LiCl | 0.002 | mol/l |
| Y27632 | 5.0e-7 | mol/l |
Step 3: Primitive streak 2
day 3 ~ 4 (duration missing in database; assumed 1 day)
culture “adherent” human PSC in “unspecified medium”. For ECM, use “Collagen” at “N/A” “N/A”.
Step 4: mesoderm 1
day 4 ~ 6
culture “adherent” human PSC in “unspecified medium” for “2” days. For ECM, use “Collagen” at “0.005” “g/m2”. Medium should contain factors shown in the following table, and should be changed once in “2” days.
| concentration | unit | |
|---|---|---|
| FGF | — | — |
| VEGF | 5.0e-5 | g/l |
| SB431542 | 2.5e-6 | mol/l |
Ideally, “mesoderm” should express “APLNR”, “PDGFRA” at the end of culture.
Step 5: mesoderm 2
day 6 ~ 7 (duration missing in database; assumed 1 day)
culture “adherent” human PSC in “unspecified medium”. For ECM, use “Collagen” at “N/A” “N/A”.
Ideally, “mesoderm” should express “KDR”, “PDGFRA”, “PECAM1” at the end of culture.
Step 6: endothelial 1
day 7 ~ 8
culture “adherent” human PSC in “unspecified medium” for “1” days. For ECM, use “Collagen” at “0.01” “g/m2”. Medium should contain factors shown in the following table, and should be changed once in “1” days.
| concentration | unit | |
|---|---|---|
| DAPT | 1.0e-5 | mol/l |
| DLL1-Fc | — | — |
| DMSO | 0.1 | % |
| FGF | — | — |
| VEGF | — | — |
| EGF | — | — |
| IGF1 | — | — |
| IGF-II | — | — |
| TPO | — | — |
| IL-6 | 5.0e-5 | g/l |
| SCF | 2.0e-5 | g/l |
| IL-3 | — | — |
| FLT3L | 1.0e-5 | g/l |
| Y27632 | 5.0e-6 | mol/l |
| JAG1-Fc | — | — |
Ideally, “EC” should express “CDH5”, “CD43”, “CD73”, “NOTCH1”, “DLL4”, “EFNB2”, “HEY2”, “SOX17”, “CXCR4”, “NR2F2”, “MYB”, “GATA2”, “HAND1”, “HES1”, “NOTCH4”, “JAG2” at the end of culture.
Step 7: endothelial 2
day 8 ~ 12
culture “adherent” human PSC in “unspecified medium” for “4” days. For ECM, use “Collagen” at “0.01” “g/m2”. Medium should contain factors shown in the following table, and should be changed once in “4” days.
| concentration | unit | |
|---|---|---|
| DAPT | 1.0e-5 | mol/l |
| DLL1-Fc | — | — |
| JAG1-Fc | — | — |
| DMSO | 0.1 | % |
| FGF | — | — |
| VEGF | — | — |
| EGF | — | — |
| IGF1 | — | — |
| IGF-II | — | — |
| TPO | — | — |
| IL-6 | 5.0e-5 | g/l |
| SCF | 2.0e-5 | g/l |
| IL-3 | — | — |
| FLT3L | 1.0e-5 | g/l |
Ideally, “EC” should express “HES1”, “NOTCH1”, “CD34” at the end of culture.