Medial HOXA genes demarcate haematopoietic stem cell fate during human development
DOI: 10.1038/ncb3354
Authors: Diana R. Dou; Vincenzo Calvanese; Maria I. Sierra; Andrew T. Nguyen; Arazin Minasian; Pamela Saarikoski; Rajkumar Sasidharan; Christina M. Ramirez; Jerome A. Zack; Gay M. Crooks; Zoran Galic; Hanna K. A. Mikkola
Step 1: iPSC/ESC maintainance
day 0 ~ 1 (duration missing in database; assumed 1 day)
culture “adherent” human ESC in “DMEM/F12”. For ECM, use “MEF feeder” at “N/A” “N/A”. Medium should contain factors shown in the following table.
| concentration | unit | |
|---|---|---|
| KSR | 20 | % |
| PSA | 50000 | U/l |
| Glutamine | 0.001 | mol/l |
| NEAA | 0.5 | % |
| FGF | 1.0e-5 | g/l |
| 2-mercaptoethanol | 1.1e-4 | mol/l |
Step 2: mesoderm 1
day 1 ~ 3
culture “spheroid” unknown species cell and human ESC in “IMDM” for “2” days. Medium should contain factors shown in the following table, and should be changed once in “2” days.
| concentration | unit | |
|---|---|---|
| BMP4 | 1.0e-5 | g/l |
| FLT3L | 5.0e-5 | g/l |
| SCF | 3.0e-4 | g/l |
| FBS | 15 | % |
| NEAA | 1 | % |
| 2-mercaptoethanol | 3.0e-7 | mol/l |
| PSA | 100000 | U/l |
| Glutamine | 0.001 | mol/l |
Ideally, “dummy” should express “CD34”, “THY1”, “CD43” at the end of culture.
Step 3: endothelial 1
day 3 ~ 5
culture “adherent” unknown species cell and human ESC in “alpha-MEM” for “2” days. Medium should contain factors shown in the following table, and should be changed once in “2” days.
| concentration | unit | |
|---|---|---|
| TPO | 2.5e-5 | g/l |
| SCF | 2.5e-5 | g/l |
| FLT3L | 2.5e-5 | g/l |
| ATRA | 1.0e-6 | mol/l |
| AM580 | 2.0e-7 | mol/l |
| FBS | 20 | % |
| PSA | 100000 | U/l |
| Glutamine | 0.001 | mol/l |
| DMSO | 0.004 | % |
Ideally, “dummy” should express “CD34”, “CD38”, “THY1”, “PTPRC”, “GPI-80” at the end of culture. Ideally, “HSPC” should express “HOXA3”, “HOXA5”, “HOXA6”, “HOXA7”, “HOXA9” at the end of culture. Ideally, “HSPC” should express “RARB”, “RARG”, “RARA” at the end of culture. Ideally, “HSPC” should express “CDH5”, “NOS3”, “SOX17”, “DLL4”, “HEY2”, “ROBO4”, “EMCN”, “PROCR”, “PBX1”, “MECOM”, “EVI1”, “ERG”, “GFI1”, “GATA3”, “HLF” at the end of culture.