CD32 captures committed haemogenic endothelial cells during human embryonic development
DOI: 10.1038/s41556-024-01403-0
Authors: Rebecca Scarfò; Lauren N. Randolph; Monah Abou Alezz; Mahassen El Khoury; Amélie Gersch; Zhong-Yin Li; Stephanie A. Luff; Andrea Tavosanis; Giulia Ferrari Ramondo; Sara Valsoni; Sara Cascione; Emma Didelon; Laura Passerini; Giada Amodio; Chiara Brandas; Anna Villa; Silvia Gregori; Ivan Merelli; Jean-Noël Freund; Christopher M. Sturgeon; Manuela Tavian; Andrea Ditadi
Step 1: iPSC/ESC maintainance
day 0 ~ 1 (duration missing in database; assumed 1 day)
culture “adherent” human ESC in “DMEM/F12”. Medium should contain factors shown in the following table.
| concentration | unit | |
|---|---|---|
| KSR | 25 | % |
| PSA | 1 | % |
| Glutamine | 0.002 | mol/l |
| 2-mercaptoethanol | 0.1 | % |
| NEAA | 0.7 | % |
| ciprofloxacin | 0.001 | g/l |
| FGF | 2.0e-5 | g/l |
Ideally, “ESC” should be obtained at the end of culture.
Step 2: mesoderm 1
day 1 ~ 2 (duration missing in database; assumed 1 day)
culture “spheroid” human ESC in “IMDM”. Medium should contain factors shown in the following table.
| concentration | unit | |
|---|---|---|
| Ham’s F12 | 25 | % |
| BSA | 0.005 | % |
| B27 | — | — |
| N2 | — | — |
| PSA | 1 | % |
| ciprofloxacin | 0.001 | 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: mesoderm 2
day 2 ~ 3 (duration missing in database; assumed 1 day)
culture “spheroid” human ESC in “IMDM”. Medium should contain factors shown in the following table.
| concentration | unit | |
|---|---|---|
| CHIR90021 | 3.0e-6 | mol/l |
Step 4: endothelial 1
day 3 ~ 8
culture “spheroid” human ESC in “StemPro” for “5” days. Medium should contain factors shown in the following table, and should be changed once in “5” days.
| concentration | unit | |
|---|---|---|
| BMP4 | 1.0e-5 | g/l |
| SB431542 | 6.0e-6 | mol/l |
| PSA | 1 | % |
| 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 |
| IL6 | 1.0e-5 | g/l |
| IGF1 | 2.5e-5 | g/l |
| IL11 | 5.0e-6 | g/l |
| SCF | 5.0e-5 | g/l |
| EPO | 2000 | U/l |
| LDN193189 | 2.5e-7 | mol/l |
Ideally, “EC” should express “CD34”, “ACE”, “HOXA” at the end of culture. Ideally, “HEC” should express “RUNX1”, “GFI1”, “MYCN”, “RAB27B”, “HOXA9”, “HOXA10”, “FCGR2B”, “KCNK17”, “H19”, “SPN”, “MYB”, “CD32”, “HES1” at the end of culture. Ideally, “AEC” should express “CXCR4”, “DLL4”, “HEY1”, “HEY2”, “SOX17”, “GJA5” at the end of culture.
Step 5: endothelial 2
day 8 ~ 9
culture “adherent” human ESC and human ESC in “StemPro” for “1” days. “ESC” should be “downregulated” for “FCGR2B”. For ECM, use “Matrigel” at “N/A” “N/A”. Medium should contain factors shown in the following table, and should be changed once in “1” days.
| concentration | unit | |
|---|---|---|
| L-685,458 | 1.0e-5 | mol/l |
| DMSO | — | — |
| Y27632 | 1.0e-5 | mol/l |
| Glutamine | 1 | % |
| Ascorbic Acid | 0.05 | g/l |
| Transferrin | 0.15 | g/l |
| MTG | 4.0e-4 | mol/l |
| TPO | 3.0e-5 | g/l |
| VEGF | 1.0e-5 | g/l |
| FGF | 5.0e-6 | g/l |
| IL3 | 3.0e-5 | g/l |
| SCF | — | — |
| IGF1 | 5.0e-5 | g/l |
| IL6 | 1.0e-5 | g/l |
| IL11 | 5.0e-6 | g/l |
| EPO | 4000 | U/l |
Ideally, “HC” should express “PTPRC”, “CD34”, “RUNX1C” at the end of culture. Ideally, “NK” should express “PTPRC”, “CD56” at the end of culture. Ideally, “TBXT” should be obtained at the end of culture.