We have been culturing Mouse D3 ES cells for some time, and quite routinely been differentiating them to beating cardiomyocytes. Recently after the five day culturing post hanging drop, we have observed no beating. I've double checked all the reagents and made everything fresh but no change. I'm hoping its not the cells, i have not got many stocks left and we desperately need them. Any suggestions welcome.
cheers
Dear Liaque
Its better to characterize the ES cells for pluripotency markers like SOX2, Oct4 and Nanong using flowcytometry. I also had the same problem with mouse ES cells and I found that the old cryovials do not differentiate efficiently compared to recently frozen ones. I didn't had the markers for characterization so after six month I ordered a new vial of D3 ES cells and they differentiated well using same protocol. One more thing you can check is the expression of cardiac markers ones cells are differentiated and also try to differentiate the cells in the presence of 100uM ascorbic acid which is acardiogenic compound. It might will tell you whether cells can still differentiate or not.
Send me protocol I will double check with mine one.
Cheers
Wasay
Thanks Wasay, its very unusual everything look spot on. Exactly the same as when you was doing them, we refer to your thesis quite often. Embryonic bodies look fine with the typical dark area in the centre even when we plate them they attach perfectly and the cells grow out just as anticipated but no beating!?? We even tried using a range of cell number, with no avail???
Hey Wasay,
Good news they are beating again. We took an early passage 3 from LN2 which worked. I am not 100% satisfied as the number of focci are just a few in each well, but satisfactory as the beats are very strong and clear. Thanks for all your advice on your visit.
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