We are trying to differentiate the Cardiomyocytes from the human iPSC. We could do 6 differentiation sets properly but suddenly our cardiomyocyte differentiation got stoped we think the only change that we made in our protocol was the change in the splitting ratio of the iPSC. Does the iPSC colony number(Confluency) play such a critical role in the differentiation efficiency?
Thanks a lot Yohan. For sharing your valuable experience and suggestion.And another problem we are facing that instead of Cardiomyocytes we are getting many Fibroblast like cells.
ok . Thanks Yohan. Hi, Holger actually we are not using the protocol from the above mentioned labs but from Minami's lab iCEMS. The way we are is actually by formation of EB bodies in floating culture and then the induction of cardiomyocyte lineage..
Oh. Ok. Actuallyy we are using the Mef feeders for iPSC culture and we are using the Wnt signaling modulating compounds like XAV. I will look by trying the other method that you suggested. Thanks a lot for valuable suggestion.
Hi Ravindra, I totally agreed with Holger's advise. You better to use Matrigel instead of MEFs and allow the cells become confluent before starting the cardiac differentiation.
Hi Ravindra,
When using the feeder-dependent system, I have found it is best to passage them to Matrigel (diluted 1:60) for a passage or two before reseeding for diffferentiation. With regards to the density, the time of induction is critical and must be optimized per iPS cell line.
Best of luck!
Marcela
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