I tried cardiomyocyte differentiation several times with B27-insulin but it didn't work
I used 6 micromolar CHIR for wnt activator on day 0 (for 24h)
and 2 micromolar XAV939 as a wnt inhibitor on day 3 (for 48h)
The first confluency for induction is 90-95%
But I have not gotten beating on day7-10 yet.
I was wondering if anyone could help me.
I am also facing with that kind of problems. Have you tried optimization of iPSCs confluency ?
Simge kelekçi I have tried twice so far and in these experiments, the iPSCs confluency was 90-100%
which confluency do you recommend?
another thing that I doubted about is the pluripotency od iPSCs that I am working on
I want to measure the Nanog and OCT3/4 before differentiation
Fatemeh Etezadi I hope you have solved your diff problem, since it's been a while. In general, for any experiment, you don't want to let the iPSC reach 100% confluence before starting an experiment. The level of spontaneous differentiation in confluent cultures compromises their pluripotency and downstream success of your experiment.
Danuta Sastre
Simge kelekçi
I could differentiate hiPSCs to CMs with 94.6% cTnT+ after 14 days of differentiation and 99.3% cTnT+ after 50 days of differentiation.
I optimized the cell number for first seeding, CHIR99021 concentration, WNT inhibitor, and timeline of differentiation.
The procedure was optimized based on Lian and et al, 2012 protocol, but we should optimize our protocol based on the material that we used.
Anyway, I got a good result with flowing information.
1. 1*105 hiPSC/cm2
2. 4 days for preculture to hiPSCs to get over confluent.
3. Use differentiation medium (B-27 supplemented-RPMI minus insulin)
4. Add 12 micromolar CHIR on day 5 (Day 0 of differentiation)
5. Remove the CHIR exactly after 24 h
6. Add 2 micromolar XAV939 on day 7 (Day 3 of differentiation)
7. Remove XAV after 48 h
8. Change medium every other day
9. Add insulin on day 10 (B-27 supplemented-RPMI plus insulin) (Day 7 of differentiation)! you can use B-27 supplemented-RPMI minus insulin and add insulin manually.
10. You can see beating within days 7 to 14 of differentiation.
The hiPSCs line that I used was the 253G1 cell line.
I hope this experience be useful for you.
Happy new year.
Fatemeh.
Thanks so much for your detailed answer, Fatemah! This is definitely helpful and I'll pass along to my lab.
Danuta Sastre
Great, please let me know if you have any further questions.
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