Hi All, I am starting the cardiac differentiation of my HiPSC lines using J.Wu lab's CDM3 based protocol. My cells remain healthy after CHIR99021 treatment of 6 uM and even 4uM for both 24 and 48 hr induction. However, I notice total cell death after 48 hrs with the addition of Wnt inhibitor Wnt -C59 (cells look fine at 24 hr after the addition of Wnt-C59). Has anyone experienced the same? I use 2uM of Wnt-C59 in CDM3 media
I've been troubleshooting similar issues recently, albeit using a completely different protocol. There would be instances of abrupt cell death or detachment from the plate mid-differentiation, and even after beating began in two procedures. This is apparently not unusual, however. In the 2015 paper by Burridge and Wu they indicate that there is intrinsic variability for this differentiation procedure, in contrast to the high reproducibility reported in the prior 2014 article (see excerpt below). With that said, my differentiation issues have recently been resolved after making adjustments to the seeding density and the time allowed for their expansion. These two factors ultimately affect the degree of confluence by D0. These factors are essential and, I've learned, may require optimization. Here is another recommendation from the Wu article that may give you an idea for how to resolve the issue (as I found out the hard way). Hope this helps.
"The major optimizable factors are the seeding density (1:12 to 1:20), the number of days of pluripotent growth (3 or 4 days), and the narrow range at which the CHIR99021 is effective in this system (5 to 7 μM). A simple experiment is to seed the cells at densities of 1:12, 1:15, and 1:20, treat them with 5 μM, 6 μM, or 7 μM of CHIR99021 during the day 0 to day 2 window, and assess for cell survival and epithelial to mesenchymal transition. We found only a minimal influence of modification of the later steps on differentiation."
"For cardiac differentiation, we have now used the protocol described here for >200 hiPS cell lines. The protocol is not 100% reproducible, and we have found that some lines go through periods of very successful differentiation followed by periods when they become refractory as they progress from passage 25 to beyond passage 90. Nevertheless, we have not found an hiPS cell line that is completely recalcitrant to differentiation. In 6-well plates, we have also commonly observed that some wells differentiate acceptably, and that others do not differentiate at all or are subject to total cell death. Taken together, these observations suggest that a controlling factor in differentiation efficiency is a combination of passage number, cell density, proliferation rate, and the microenvironment established in response to CHIR99021 and Wnt-C59 treatment."
Hi Walker Krepps,
Thanks for the suggestions, they are very helpful. May I ask which Wnt inhibitor did you use for your iPSC? Which one is better WntC59 or IWP4?
We've been using IWP4, although I tend to see IWP2 more commonly used across the spectrum of monolayer protocols. Referencing back to Burridge and Wu's 2014 paper, IWP2 displayed greater consistency across varying concentrations compared to IWP4 (see screencap from Supp. Methods). That would be what I'd opt for if I was looking for a sure bet. But, IWP4 has worked for us and I don't imagine there'd be much of a difference between the two. I've never used Wnt-C59 so I can't speak to that, but it looks like its similarly effective at lower concentrations.
Yes makes sense, I will try with IWP2 and see how it goes. Also, which media base do you recommend? RPMI/B27 or CDM3?
I am also suspecting if my CDM3 media itself is not very fresh and effective.
We use the standard RPMI/B27 (-insulin) for the first five days, then switch over to RPMI/B27 (+insulin) every 2-3 days afterwards. I agree with what you mentioned about media freshness. I try not to use anything over a month old but many papers suggest 2 weeks at most.
Makes sense! Thank you very much for your responses. I will try with RPMI/B27 too.
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