Could anyone give me some suggestions on the differentiation medium for in vitro differentiation of mouse (not human) embryonic stem cell into cardiomyocyte?
I have read different protocols and I decided to follow the protocol on Jove http://www.jove.com/video/825. Embryoid body will be formed through the hanging drop method and in the well of 96-well ultralow attachment plate. After the formation of embryoid body, they will be transferred into gelatin-coated plates with the differentiation medium. However, the Jove protocol did not mention the component of the differentiation medium.
The classic Hanging-Drop method suggested the mESC differentiation medium as follow:
390 ml IMDM (with glutamine and HEPES; Invitrogen) supplemented with:
100 ml heat-inactivated fetal bovine serum (FBS; Invitrogen)
5 ml penicillin (10,000 U/ml)/streptomycin (10,000 μg/ml); Invitrogen
5 ml MEM nonessential amino acids (100×; Invitrogen)
0.5 ml β-mercaptoethanol (0.7 mM; Sigma-Aldrich)
However, I also read other protocol using RPMI as the base. Some protocol also included F12, B27 or BMP4. Is it important to include these components into the medium as well?
I am concerning the concentration of serum as well. There are up to 20% serum in the classic protocol but I read paper suggesting low serum conditions promote cardiomyocyte differentiation. They use only 1% and some even use 0.2% serum.
It is my first time to differentiation embryonic stem cell. I would be very grateful if anyone can give me some suggestions on it.
Hello. For the differenciation into cardiomyocytes I also use the hanging drop method.
Day 0: hanging drop on the lied of a bacterial plate (sleeve opened under the hood), 20 µL each drop with 500 cells (25000 cells/mL). Don't forget to add PBS on the bottom of your plate to avoid evaporation.
Day 2: harvest the drops (you should be able to see small EBs already), and put them in a new bacterial plate filled with differentiation media (I don't use low addherent plates... this method works perfectly ;))
Leave your cells in the incubator until Day 5.
Day5: Harvest your cells (it helps to make circles with your plate in order to collect all the EBs in the center of the plate). And pour them in a gelatin coated culture dish.
Here, add Retinoic acid to the media in your plate with a final concentration of 10^-8.
Day 6: change the media (still with RA)
Change the media every two days. You should start to see strong beatings around Day 8.
For the media I personnally use a 20% FBS media without any antibiotics (and obviously no LIF), NEAA, NaPyr, Bmercapto. My protocol works to assess the ability of differenciation (I tried it so far on three different cell lines: R1, G4 and CGR8). I have to admit that I haven't really look more deeply into the addition of F12 B27 or BMP4. However, I know that you can use them to achieve more quickly the "beating state" of your cells, but as they are quite expensive, I never used them.
Hope this helps. Send me a message if you have any other questions.
Dear Fleur Bourdelais,
Thank you for your great tips! I will add retinoic acid in my media.
So can I confirm that your differentiation media is only IMDM with 20% FBS (with retinoic acid since day 5) without any other component?
And what is the percentage of cardiomyocyte you can obtain generally?
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