Lamin A/C-dependent chromatin architecture safeguards naïve pluripotency to prevent aberrant cardiovascular cell fate and function
DOI: 10.1038/s41467-022-34366-7
Authors: Yinuo Wang; Adel Elsherbiny; Linda Kessler; Julio Cordero; Haojie Shi; Heike Serke; Olga Lityagina; Felix A. Trogisch; Mona Malek Mohammadi; Ibrahim El-Battrawy; Johannes Backs; Thomas Wieland; Joerg Heineke; Gergana Dobreva
Step 1: iPSC/ESC maintainance
day 0 ~ 2
culture “adherent” mouse ESC in “DMEM/F12” for “2” days. For ECM, use “Gelatin” at “0.1” “%”. Medium should contain factors shown in the following table, and should be changed once in “2” days.
| concentration | unit | |
|---|---|---|
| LIF | 2.0e+6 | U/l |
| BMP4 | 1.0e-5 | g/l |
Step 2: Primitive streak 1
day 2 ~ 4
culture “spheroid” mouse 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 | |
|---|---|---|
| Ham’s F-12 | 25 | % |
Step 3: Primitive streak 2
day 4 ~ 5 (duration missing in database; assumed 1 day)
culture “spheroid” mouse ESC in “IMDM”. Medium should contain factors shown in the following table.
| concentration | unit | |
|---|---|---|
| Ham’s F-12 | 25 | % |
| Activin A | 5.0e-6 | g/l |
| VEGF | 5.0e-6 | g/l |
| BMP4 | 1.0e-7 | g/l |
Step 4: mesoderm 1
day 5 ~ 6 (duration missing in database; assumed 1 day)
culture “spheroid” mouse ESC in “DMEM”. Medium should contain factors shown in the following table.
| concentration | unit | |
|---|---|---|
| FBS | 15 | % |
| Glutamine | 0.002 | mol/l |
| 2-mercaptoethanol | 1.0e-4 | mol/l |
| NEAA | 1.0e-4 | mol/l |
| sodium pyruvate | 0.001 | mol/l |
| D-glucose | 4.5 | g/l |
| PSA | 1 | % |
Ideally, “mesoderm” should express “EOMES”, “MESP1” at the end of culture.
Step 5: mesoderm 2
day 6 ~ 7 (duration missing in database; assumed 1 day)
culture “adherent” mouse ESC in “StemPro-34 medium”. For ECM, use “Gelatin” at “0.1” “%”. Medium should contain factors shown in the following table.
| concentration | unit | |
|---|---|---|
| Glutamine | 0.002 | mol/l |
| Ascorbic Acid | 0.05 | g/l |
| VEGF | 5.0e-6 | g/l |
| FGF | 1.0e-5 | g/l |
| FGF10 | 2.5e-5 | g/l |
Ideally, “CP” should express “Nkx2-5” at the end of culture.
Step 6: endothelial 1
day 7 ~ 8 (duration missing in database; assumed 1 day)
culture “adherent” mouse ESC in “unspecified medium”. “ESC” should be “downregulated” for “Lmna”, “upregulated” for “GATA4”. For ECM, use “Gelatin” at “0.1” “%”.
Ideally, “EC” should express “PECAM1”, “KDR” at the end of culture.
Step 7: endothelial 2
day 8 ~ 9 (duration missing in database; assumed 1 day)
culture “adherent” mouse ESC in “unspecified medium”. For ECM, use “Gelatin” at “0.1” “%”.
Ideally, “EC” should express “PECAM1”, “KDR” at the end of culture.