Hi, I’m just starting the Cardiomyocyte differentiation using the Allen Institute protocol. Currently at day 8 of differentiation and I’m starting to see some isolated beating clusters. I noticed that in the protocol the expected result was that there will be uniform beating across the entire well.
Would you happen to know when this uniform beating usually occur? If I don’t see uniform beating by the designated day, does that mean my differentiation failed?
Hello,
We usually expect to see variation on when beating should begin between experiments, but around day 7 to day 9 you should see specific areas of the well beating. In our experience, by day 12 we usually see uniform beating across the well in experiments where the differentiation was optimal.
Please keep in mind that optimization of our protocol may be required to account for variation between cell lines. A good place to start would be varying the initial stem cell seeding density (we find that between 125k-250k cells/well in a 6wp is a good range to test).
We would suggest collecting your cells and running a flow experiment to determine the percent of cells that are troponin positive (cTnT+), which should give you a quantitative result on how successful your differentiation was.
Let me know if you have any more questions!
Hi, thank you for your response.
I ran the flow on these cells and got a very low cTnT + percentage (< 1%). I want to optimize the protocol further, do you suggest increasing or decreasing the CHIR concentration? Additionally, should I keep the IWP2 concentration at an equal ratio to the CHIR concentration?
Thanks!
Hello,
Sorry to hear your differentiations are not working.
Would you mind providing me with some more information on the cell line you are using? In particular, are you using one of our lines and does the line you are using have good stem cell morphology? (see Pictures of Healthy vs Unhealthy hiPSC colonies for examples). Could you post an example of how your stem cells look if they do not look similar to the pictures we have provided?
Although we do see variations in cTnT+ levels between our lines and from differentiation experiments, <1% cTnT+ is extremely low for a well with beating clusters and below the range we see even in subpar differentiations at day 12 using this protocol. Have you validated your flow protocol for staining and detecting cTnT in cardiomyocytes?
We have a video on our website showing our differentiation setups (https://youtu.be/cCVUrKGRINs). We have found that the timing of the media changes is important. Have you been changing the media from Chiron to IWP and IWP to B27- as close to 48hrs as possible?
If so, because your cTnT+ is so low my first suspicion would be that potentially something is off with one of the reagents. We lot test our B27 minus insulin. We have found that lot to lot variations can lead to unsuccessful differentiations. Have you tested multiple B27 minus insulin lots in a controlled experiment?
Thanks,
Haseeb
Hi Haseeb, thank you for the reply.
The line I’m using is a patient-derived iPSC line that we reprogrammed ourselves here in the lab. I didn’t use the same line that you are using. They have good morphology prior to differentiation (no cratering, no spontaneous differentiating regions) and are kept at 70-80% confluency prior to routine passaging.
I’m unsure if it’s the flow protocol or the differentiation protocol that’s the issue, because I saw beating clusters on the edges of the plate and there was no beating cells in the middle at all. I timed the media changes from Chiron supplemented media to IWP2 media as close to 48 hrs as possible, and I’ve used the flow protocol before to detect cTNT+ cells derived using a commercial differentiation kit and they were able to detect cTNT+ cells.
if it’s not too much trouble, could you share the flow protocol used by Allen Cell to evaluate differentiation efficiency?
Thank you!
Hi @hwidyastuti
We do have a protocol for troponin-T staining of fixed cells (fixed in 4%PFA/PBS for 10 min). The steps are as follows:
Flow Cytometry Prep
We use the BD FACS AriaIII for sorting. Please let us know if you have further questions! Happy to help.
Hi Haseeb,
Thank you for the protocol! I’ve had some success differentiating commercial iPSC lines using your protocol (>97% cTNT+). I’m currently working on a patient line and I’ve identified a seeding density at which they would produce beating clusters but the beatings are localized on the periphery. I suspect I would only get less than 50% cTNT+ cells.
What do you suggest I should do next? Should I increase the Chiron concentration or change the seeding density?
Thank you! I really appreciate all your help with this.
@hwidyastuti We haven’t worked with disease lines ourselves, which we know can be a bit more finicky. However, we will in the near future, and would love to know if you gave your suggestion a try and if it worked.
Thanks!
Hi everyone,
What is considered a successful differentiation based on cTTN+? Should it be above 90%? 75%?
Is there in general a correlation between %cTTN+ and homogeneity of beating cell in the well?
Hi Danuta! Hope you’re doing well! We use 70% cTnT+ to define “passing” wells in our cardiac differentiations, but the ideal threshold is typically determined by the intended application of the cells. Some want very pure populations, such as for single cell studies, whereas others want some non-cardiomyocytes to act in a supporting role (such as in certain tissue engineering approaches).
We do find there is some correlation between %cTnT+ and if we get nice, even sheets of beating cardios in the differentiation dish, but typically we can only roughly estimate the purity of cardios by looking at them. Usually we can judge if a well is >50% cTnT+ or <50% cTnT+ by visual inspection, but anything more specific than that is difficult to judge without flow cytometry.
Hope that helps and let me know if there’s anything else we can help with!
Becky
Hi Becky! As fait would have it, I’m too differentiating CM now. Good to still be able to count on your expertise!
We are using wild-type WTC-11 straight for Coriell. Usually we find only areas of beating CM, and the highest cTTN+ we got was 35% last week. I’m trying to figure out how to improve it.
We tried 400k/500k/600k in 6wp, because the protocol the team had optimized before. joined called for 500k. Based on this discussion here, I’m guessing we need to test lower densities too.
Besides densities, are there other things you’d suggest trying?
Thanks!!
That’s so exciting! Glad I can be of help!
For our differentiation method, we typically plate 150k-250k cells per well of a 6 well plate. If you’re using our differentiation method, I would also recommend lot testing your B27 without insulin, as well as trying different concentrations of Chiron and IWP2 (we adjust our concentrations every time we buy a new lot of these 2 reagents).
Hope that helps, and definitely happy to answer any more questions!
Becky
Hi,
I have found that incubating the plate with matrigel 0.337mg/ml for overnight for gel formation rather than one hour improved the time until the whole well of cells started beating. Previously with one hour matrigel gel formation, the well did not fully beat until 12-14 days. With overnight cells beat at day 7. thanks