Hi everyone,
I've been differentiating cardiomyocytes from iPSCs using stem cell technologies' ventricular cardiomyocyte differentiation kit. However, I've recently encountered a significant reduction in differentiation efficiency. I initiate differentiation 2-3 days after seeding, once the cells reach 80-90% confluency. I always use fresh media and supplements. I also tried differentiating cardiomyocytes from two different cell lines, but the outcome was similar. I noticed an increase in the non-CM population, cells appear degenerated around D6-D8 of differentiation and the cells don't start beating as expected. Can anyone give me advice on the subject? Any suggestions you can share would be greatly appreciated.
Thank you
Hi Hazal,
Which substrate are you using when differentiating? I am not sure what could cause the sudden reduction in performance that you mention, if the protocol has previously worked well. Anything that might have changed in terms of reagents or cell conditions?
If you haven't been using full-length laminins yet, I highly recommend testing to see if they would solve the issues you mention - based on publications and feedback from other researchers. Laminin-521 and laminin-221 help you get consistently over >80% efficiency and faster differentiation (beating at day 9-10). See e.g. Lynn Yap et al. 2019 Cell Reports.
I hope your cells start beating again very soon!
Hi Hazal,
Unfortunately, not all hiPSC lines are characterized by 100% differentiation efficiency. Differentiation efficiency depends on: the cell line; different lines from a given patient may be characterized by different differentiation efficiency.
hiPSC at higher passages may accumulate mutations in genes important for differentiation and/or lose pluripotency and consequently lose differentiation potential. Therefore, it is worth performing karyotyping and/or STR and analysis of pluripotency gene expression.
Sometimes it also happens that the same protocol runs with different differentiation efficiency. Sometimes it is simply worth performing a cell enrichment procedure to obtain a more pure population.
Good luck!
Hi Hazal,
This thing delayed my PhD graduation and forced me to change the project. The answer is simple, but yet poorly studied — your refill media pH. I found it out just before pivoting to another field in 2021, thus haven't publish any paper. However, there is a paper from 2024 that describes this: https://pubmed.ncbi.nlm.nih.gov/39178847/
In my case it was that while media was in a fridge its pH gets higher (~7.8) even when it was HEPES media. And this pH kick while media change was enough to stop differentiation to CMs and shift to CFs. Not sure what is the critical timepoint for it, but most probably it is 4-6 day of differentiation.
To bypass this I used a trick warming media not in water bath, but in a petri dish in incubator for 10-15 minutes before changing media. To equilibrate pH.
Cheers,
Alex
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