Hello to all, recently I used the hanging drop methods to obtain murine embryonic bodies. Do the embryonic stem cells need to be selected? If not, will the fibroblast effect the formation of the embryonic bodies? Besides, does the passage number effect its differentiation?
Hi,
Embryonic stem cells should express pluripotency marker (OCT4, SOX2, NANOG) befor embryoid bodies forming. MEF (after mitomycin C or radial inactivation) produce importent for mESC growth factor. Conditioned medium (with growth factor produced by MEF) can by used as medium for embryoid bodies development (without LIF and bFGF).
mESC can be cultured up to 50 passage. During culture mESC can accumulate mutations, which can have consequence in changes their differentation potency.
Hi Lin,
It not clear for me, I guess that you use co-culture system for your embryonic stem cells . If it is , you have to prepare ESCs from feeder cells via pre-plating.
As you know, for EBs formation, there are some points:
First of all, the amount of cells per drop, in addition the volume of each drop and duration are very important in differentiation process. In my opinion, the passage number is effective in differentiation,
Hi,
When mESC passage exceed 35, whether the formed EB can be differentiated into the cardiogenesis or not?
Yes. The cardiomyocytes can be obtained from mESC older than 35 passage.
The older mESC will effect the potency of EBs?
No. After 10 days in embryoid bodies growing in special media you should see betting cardiomyocytes.
https://www.youtube.com/watch?v=VdqqcD3wqzk
Hi Kejun,
Your EBs should still form nicely but one would expect 'contaminating' EBs to have an effect on the subsequent differentiation of your mESCs.
If you are worried about the fibroblasts you can try to deplete them, seed your plates for 10-20 minutes, tap them to dislodge your ESCs and replate those. Fibroblasts tend to adhere much faster than ESCs allowing you to deplete them this way. There are other methods (but more costly), e.g. miltenyi has magnetic beads to remove fibroblasts.
Will passage number affect differentiation: I would say definitely yes. In my experience EBs from a late passage mESC actually formed beating cardiomyocytes easier. This is likely because of my culture conditions, the FBS used etc resulting the skewing of my mESCs toward mesoderm differentiation.