Engineering the haemogenic niche mitigates endogenous inhibitory signals and controls pluripotent stem cell-derived blood emergence
DOI: 10.1038/ncomms15380
Authors: Nafees Rahman; Patrick M. Brauer; Lilian Ho; Tatiana Usenko; Mukul Tewary; Juan Carlos Zúñiga-Pflücker; Peter W. Zandstra
Step 1: iPSC/ESC maintainance
day 0 ~ 1
culture “adherent” human ESC in “DMEM/F12” for “1” days. “ESC” should be “unspecified regulation” for “RUNX1C”. For ECM, use “Fibroblast” at “N/A” “N/A”. Medium should contain factors shown in the following table, and should be changed once in “1” days.
| concentration | unit | |
|---|---|---|
| KSR | 19 | % |
| PSA | 0.5 | % |
| NEAA | 1 | % |
| 2-mercaptoethanol | 1 | % |
| FGF | 1.0e-5 | g/l |
Step 2: mesoderm 1
day 1 ~ 4
culture “spheroid” unknown species cell and human ESC and human PSC in “StemPro” for “3” days. “ESC” should be “unspecified regulation” for “RUNX1C”. Medium should contain factors shown in the following table, and should be changed once in “3” days.
| concentration | unit | |
|---|---|---|
| SB431542 | 6.0e-6 | mol/l |
| CHIR90021 | 3.0e-6 | mol/l |
| StemPro34 supplements (ascorbic acid) | 0.05 | g/l |
| Glutamine | 1 | % |
| PSA | 1 | % |
| MTG | 4.0e-4 | mol/l |
| Transferrin | 0.15 | g/l |
| Y27632 | 0.1 | % |
| BMP4 | 4.0e-5 | g/l |
| VEGF | 5.0e-5 | g/l |
| SCF | 4.0e-5 | g/l |
| FGF | 5.0e-6 | g/l |
Step 3: endothelial 1
day 4 ~ 7
culture “spheroid” unknown species cell and human ESC and human PSC in “StemPro” for “3” days. “ESC” should be “unspecified regulation” for “RUNX1C”. Medium should contain factors shown in the following table, and should be changed once in “3” days.
| concentration | unit | |
|---|---|---|
| StemPro34 supplements (ascorbic acid) | 0.05 | g/l |
| Glutamine | 1 | % |
| PSA | 1 | % |
| MTG | 4.0e-4 | mol/l |
| Transferrin | 0.15 | g/l |
| BMP4 | 4.0e-5 | g/l |
| VEGF | 5.0e-5 | g/l |
| SCF | 4.0e-5 | g/l |
| FGF | 5.0e-6 | g/l |
Ideally, “EC” should express “CD34”, “VECAD”, “VWF” at the end of culture.