Hi there, I differentiate cardiomyocytes from iPS cells. However, my differentiation is not consistent. Often I can achieve beating cardiomyocytes on day 8-9, but sometimes there is no beating at all. As well, after 8-9 day of differentiation spontaneous beating can decay. I use GiWi protocol for differentiation and start my differentiation when cells reach 80-90% confluence. But I have some doubts on initial cell seeding density: some protocols mention that seeding density should be 100000 cells/cm2, but it is a huge number and I'll get full confluence on day 2 after seeding. Can somebody give me advice or share a protocol that U use?
Dear Alex Mitriashkin ,
Here are a few recommendations which will hopefully help you to get reproducible cardiomyocyte differentiation (CM diff):
1. It is very important that the iPSC line that you are using is stable and not differentiating randomly on its own. A simple way to test for this is to check for pluripotency markers. (If you have done so already then maybe you can try to troubleshoot in the next steps).
2. The iPSC line "should" be maintained consistently and not allowed to be too confluent before splitting. Meaning they should be split in the same ratio every time and within the same no. of days. For example splitting the iPSCs in ratio 1:6 or a specific cell density every 5 days. If the splitting ratios or the days in which cells are split are not consistent then the CM diff will not work well.
3. In my experience the "seeding density" is extremely crucial for inducing good and reproducible CM diff and this is cell line specific. So I would recommend you to optimize the seeding density for your iPSC line if the above 2 options don't help you out.
Do you use medium containing B27 supplement and still observe reduction in beating frequency or areas? Then the reason behind the decay of spontaneous beating is mostly due to the fact that other non-CM cell types emerging from the differentiation protocol proliferate rapidly and cover up all the CM regions. In such a case you will hardly see any spontaneous contractions. My first guess would be that you need to optimize cell density for your specific iPSC line (if you are following the 2 points mentioned above for the maintenance of your iPSCs).
Hope this will help you out.
Hi Alex,
It is very normal to have large variations of iPSC differentiation into cardiomyocytes and other cell types. You can do your best to standardize your method to minimize variations between experiments. One of the most important points in differentiation efficiency is initial cell quality and density. Make sure you split cells evenly so that you will not have any overlapping and spontaneously differentiating iPSCs. I would recommend using cell density as a measure instead of cell counting so that you won’t have to dissociate your iPSCs into single cells because single cells tend to not grow and differentiate. I usually use cell density of 50-80% to start differentiation, but this somehow depends on iPSC line. Also, start differentiation between Day2 and Day3 of splitting cells, which should be at least 24 hours after you remove ROCK inhibitor (cells should have enough time to recover from ROCKi). Hope these would help improving your differentiation!
Dear Shambhabi Chatterjee ,
Thank you for your advice. I have checked my cells on pluripotency markers - they expressed well. For differentiation I use medium containing B27 -insulin and later B27 full
I'll follow your advice on iPS maintenance and optimization of seeding density.
As well, I have a question on purification: do you use lactation for it? Do you use DMEM without glucose + lactate?
Thank you!
Hi Emre Bektik ,
Thank you for your advice. You are right - I observe that differentiation efficiency is better when I start it on day 2-3. Good advice to try not to dissociate cells to single cells.
Thank you a lot!
Hi Jaba Tkemaladze
Yes, I saw this protocol, but don't think that it would be more robust than simple protocols by Wu or Kamp. But I'll try it next time. Thank you!
Dear Alex Mitriashkin
It is great that your iPSC are in good quality.
For differentiation part, the medium descriptions that you shared sounds good as well. The most important factor in the diff medium is to use GLUTAMAX instead of L-Glu. This made a huge difference in terms of quality and efficiency of my CM differentiations. I always use the medium only up to14 days.
Yes, I also perform metabolic selection to purify cells. I use RPMI without glucose and supplemented with lactate. It also contains L-ascorbic acid 2-phosphate and recombinant human albumin. Let me know if you need any further information.
Hope everything will go smoothly for the troubleshooting.
In particular, iPSCs derived from cardiomyocytes tend to differentiate more efficiently into cardiomyocytes, forming more mature cells compared to iPSCs derived from other tissues. Thus, history of the iPSC-derived cardiomyocytes needs to be taken into account when comparing cell phenotypes or gene expression profiles. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4447149/
Check this method used here https://www.google.com/search?q=cardiomyocytes+differentiation+from+iPSC%3F&client=ucweb-b&channel=sb
Also check https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6354100/
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