In Vitro Differentiation of Gata2 and Ly6a Reporter Embryonic Stem Cells Corresponds to In Vivo Waves of Hematopoietic Cell Generation
DOI: 10.1016/j.stemcr.2017.11.018
Authors: Mari-Liis Kauts; Carmen Rodriguez-Seoane; Polynikis Kaimakis; Sandra C. Mendes; Xabier Cortés-Lavaud; Undine Hill; Elaine Dzierzak
Step 1: iPSC/ESC maintainance
day 0 ~ 1 (duration missing in database; assumed 1 day)
culture “adherent” mouse ESC in “DMEM”. Medium should contain factors shown in the following table.
| concentration | unit | |
|---|---|---|
| FBS | 15 | % |
| GlutaMAX | 0.002 | mol/l |
| Na-pyruvate | 0.001 | mol/l |
| PSA | 1 | % |
| 2-mercaptoethanol | 5.0e-5 | mol/l |
| NEAA | 1.0e-4 | mol/l |
| LIF | 1.0e+6 | U/l |
Ideally, “ESC” should be obtained at the end of culture.
Step 2: Primitive streak 1
day 1 ~ 2 (duration missing in database; assumed 1 day)
culture “spheroid” mouse ESC in “IMDM”. Medium should contain factors shown in the following table.
| concentration | unit | |
|---|---|---|
| FCS | 15 | % |
| PSA | 1 | % |
| GlutaMAX | 0.002 | mol/l |
| Ascorbic Acid | 0.05 | g/l |
| MTG | 4.0e-4 | mol/l |
| Transferrin | 0.3 | g/l |
| proteome-free hybridoma medium | 5 | % |
| BMP4 | 5.0e-6 | g/l |
Ideally, “PS” should express “TBXT” at the end of culture.
Step 3: mesoderm 1
day 2 ~ 3 (duration missing in database; assumed 1 day)
culture “spheroid” mouse ESC in “IMDM”. Medium should contain factors shown in the following table.
| concentration | unit | |
|---|---|---|
| FCS | 15 | % |
| PSA | 1 | % |
| GlutaMAX | 0.002 | mol/l |
| Ascorbic Acid | 0.05 | g/l |
| MTG | 4.0e-4 | mol/l |
| Transferrin | 0.3 | g/l |
| proteome-free hybridoma medium | 5 | % |
Ideally, “ME” should express “KDR” at the end of culture.