Hello Everyone
I tried to differentiate Embryoid bodies (EBs) from mouse Embryonic Stem cells (E14tg2a) following the protocol I attached. However i failed during the process because despite cells aggregate and accumulate they don't differentiate and form an Embryoid body. I already tried with a larger amount of cells (1000 cells per Embryoid body) in the same medium and they formed correctly but when are treated with Activin A (5 ng/ml and 50 ng/ml) mesoderm isn't induced (what is my principal purpose) so i decided to try with a lowest amount of cells and according to my literature research 250 cells is a the lowest amount feasible to differentiate into an embryoid body. Any suggestions will be well received.
Hello
links here will help you
Good Luck
https://www.ncbi.nlm.nih.gov/pubmed/19686051
https://www.thermofisher.com/sa/en/home/references/protocols/cell-culture/stem-cell-protocols/ipsc-protocols/formation-embryoid-bodies-mouse-embryonic-stem-cells.html
http://onlinelibrary.wiley.com/doi/10.1002/9780470151808.sc01d03s4/abstract
http://onlinelibrary.wiley.com/doi/10.1634/stemcells.2006-0523/pdf
Hi Tamara,
This is a great method for generating consistent embryoid bodies. The APEL medium in the paper is defined and serum free and can be purchased from Stem Cell Technologies.
I usually aggregate between 3000 - 5000 cells/EB for the method for the best results. I work on blood cell formation from human ES cells and the best mesoderm inducing cytokine combination I use is BMP4 (10-20ng/mL), Act A (10-20ng/mL), VEGF (30-40ng/mL) and SCF (40ng/mL).
Hope that helps!
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Hello,
I think that this protocol should be little modificated.
1. Use accutase instead of trypsin.
2. Use RevitaCell Supplement (10X) (Thermo Scientific) to increase the EB viability in SFM (handing drop methods).
3. Change medium in to the mesoderm inductin medium at day 3 post EB forming.
4. Forming EB from 500-1000 cell per single EB.
Good Luck
I follow the protocol from this paper.
good luck
Article In vitro differentiation of retinal ganglion-like cells from...
Thank you to all of you for your suggestions. I will adjust my protocol to some of them.
Hey Tamara!
Out of personal experience in the lab and how hard it is to get good stem cell cluster, we have developed a plate for easy aggregation. Check out this video:
https://drive.google.com/open?id=0B1v-W0EVfIIEUFRnN3FMU3pZMmM
If you like, I can send you a test plate, it might help with your protocol!
Attached a little flyer about the plate.
By the way: this is how many clusters from hanging drops can be produced in our plate.
https://drive.google.com/open?id=0B1v-W0EVfIIEQ1RCUWNWcklEMU0
Regards,
Patrick
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