Haematopoietic stem and progenitor cell heterogeneity is inherited from the embryonic endothelium
DOI: 10.1038/s41556-023-01187-9
Authors: Joey J. Ghersi; Gabriel Baldissera; Jared Hintzen; Stephanie A. Luff; Siyuan Cheng; Ivan Fan Xia; Christopher M. Sturgeon; Stefania Nicoli
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.
Step 2: Primitive streak 1
day 1 ~ 4
culture “spheroid” human ESC in “unspecified medium” for “3” days. Medium should contain factors shown in the following table, and should be changed once in “3” days.
| concentration | unit | |
|---|---|---|
| BMP4 | 1.0e-5 | g/l |
| FGF | 5.0e-6 | g/l |
| Activin A | 1.0e-6 | g/l |
| SB431542 | 6.0e-6 | mol/l |
| CHIR90021 | 3.0e-6 | mol/l |
| IWP2 | 3.0e-6 | mol/l |
| l-glutamine | 0.002 | mol/l |
| Ascorbic Acid | 0.001 | mol/l |
| MTG | 4.0e-4 | mol/l |
| Transferrin | 0.15 | g/l |
Step 3: mesoderm 1
day 4 ~ 5 (duration missing in database; assumed 1 day)
culture “spheroid” human PS in “StemPro”. Medium should contain factors shown in the following table.
| concentration | unit | |
|---|---|---|
| CHIR90021 | 3.0e-6 | mol/l |
| l-glutamine | 0.002 | mol/l |
| Ascorbic Acid | 0.001 | mol/l |
| MTG | 4.0e-4 | mol/l |
| Transferrin | 0.15 | g/l |
| FGF | 5.0e-6 | g/l |
| VEGF | 1.5e-5 | g/l |
Ideally, “mesoderm” should express “KDR”, “HOXA” at the end of culture.
Step 4: endothelial 1
day 5 ~ 10
culture “spheroid” human mesoderm in “StemPro” for “5” days. Medium should contain factors shown in the following table, and should be changed once in “5” days.
| concentration | unit | |
|---|---|---|
| antagomir-128 | 2.0e-7 | mol/l |
| l-glutamine | 0.002 | mol/l |
| Ascorbic Acid | 0.001 | mol/l |
| MTG | 4.0e-4 | mol/l |
| Transferrin | 0.15 | g/l |
| FGF | 5.0e-6 | g/l |
| VEGF | 1.5e-5 | g/l |
| IL-6 | 1.0e-5 | g/l |
| IGF1 | 2.5e-5 | g/l |
| IL-11 | 5.0e-6 | g/l |
| SCF | 5.0e-5 | g/l |
| EPO | 2000 | U/l |
Ideally, “EC” should express “HOXA”, “CD34”, “CD43” at the end of culture.
Step 5: endothelial 2
day 10 ~ 19
culture “adherent” human EC in “StemPro” for “9” 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 “9” days.
| concentration | unit | |
|---|---|---|
| antagomir-128 | 2.0e-7 | mol/l |
| l-glutamine | 0.002 | mol/l |
| Ascorbic Acid | 0.001 | mol/l |
| MTG | 4.0e-4 | mol/l |
| Transferrin | 0.15 | g/l |
| TPO | 3.0e-5 | g/l |
| IL-3 | 3.0e-5 | g/l |
| SCF | 1.0e-4 | g/l |
| IL-6 | 1.0e-5 | g/l |
| IL-11 | 5.0e-6 | g/l |
| IGF1 | 2.5e-5 | g/l |
| EPO | 2000 | U/l |
| VEGF | 5.0e-6 | g/l |
| FGF | 5.0e-6 | g/l |
| BMP4 | 1.0e-5 | g/l |
| FLT3L | 1.0e-5 | g/l |
| SHH | 2.0e-5 | g/l |
Ideally, “HSPC” should express “CD34”, “PTPRC” at the end of culture.