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. …

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. …

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 conce…

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 hiP…

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…

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 In round bottom 96 well plate, plate 2 wells per sample, 80-100ul cell suspension/well. Spin down at 200g, 3min, 25C (radius=15.1) Flick off…

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 perip…

@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 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-card…

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…

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Cardiomyocyte Differentiation Issue

hiPSC Culture Discussion

dsastre February 15, 2022, 8:16pm 9

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?

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Allen Cell Discussion Forum

Cardiomyocyte Differentiation Issue

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