Have you guys began hiPSC differentiation in to cardiomyocytes?


#1

Have you guys began hiPSC differentiation in to cardiomyocytes?


#2

Yes! Our method at the Allen Institute for Cell Science was adapted from the small molecule protocol described in Lian et al., Nature Protocols, 2013, and has been optimized for the differentiation of cardiomyocytes in a 6-well format using the Allen Cell Collection, derived from the hiPSC WTC parental line released by the Conklin Laboratory at the J. David Gladstone Institute.

Check out our SOP: Cardiomyocyte differentiation methods v1.0.pdf for more details!


#3

From the Allen Cell Collection, MYL7-mEGFP cell line, Day 12 after differentiation


#4

You can also find our methods video where we set up hiPSCs for cardiac differentiation here:

https://www.allencell.org/instructional-videos-and-tutorials-for-cell-methods.html


#5

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?


#6

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!


#7

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!


#8

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