I am attempting to perform cardiomyocyte differentiation following a fairly standard GiWi protocol: Grow iPSCs to 90% confluent, initiate differentiation by switching to RPMI 1640 with B-27 (minus insulin) and CHIR99021. However, 1 day after switching to RPMI, I am seeing most, if not all, of my cells die, even with very low CHIR concentrations (
Hi Joseph Philip Licata
I'm sorry to hear about the difficulties you're experiencing in your differentiation protocol. Cell death during differentiation protocols can be frustrating and it's often due to a combination of factors. Here are a few suggestions that might help you troubleshoot your protocol. Media Change Shock: Rapid changes in culture conditions, such as switching from a stem cell medium to a differentiation medium, can cause stress to the cells and lead to cell death. You may want to consider a gradual transition into the differentiation medium. This could be accomplished by adding increasing amounts of RPMI 1640 with B-27 (minus insulin) to the stem cell medium over a couple of days .Cell Density: Cell density at the start of the differentiation process is very important for successful cardiomyocyte differentiation. You mentioned that you're initiating differentiation when the iPSCs reach 90% confluence. While this is a good starting point, the optimal confluence can vary depending on the specific cell line. You might need to optimize the starting cell density for your specific iPSC line. CHIR99021 Concentration: CHIR99021 is a potent GSK3 inhibitor used to initiate mesoderm differentiation, which is the first step towards cardiomyocyte differentiation. However, its optimal concentration can vary depending on the specific iPSC line and the culture conditions. Even though you've already tried lower concentrations, you might want to test a range of concentrations to find the optimal one for your cells. Quality of Reagents: Always ensure the quality of all your reagents. For instance, the quality of the B-27 supplement (minus insulin) can greatly affect the differentiation efficiency. If it's expired or has been improperly stored, it could lead to cell death. Also, ensure that the Matrigel matrix is not expired and has been stored and handled correctly. Cell Health Prior to Differentiation: The health of the iPSCs before you start differentiation can greatly impact the success of your differentiation protocol. Stressed, over-confluent, or unhealthy iPSCs will not differentiate efficiently and might die when subjected to the stress of differentiation. Always ensure your iPSCs are healthy and happy before starting the differentiation process. Use of ROCK Inhibitor: You mentioned that you are using 10µM Y27632 for the first 24 hours after seeding. Although this is a common practice to enhance cell survival following dissociation, prolonged exposure to Y27632 can impair iPSC differentiation. You might want to experiment with removing the Y27632 earlier or reducing its concentration.
Remember that differentiation protocols often require some optimization depending on the specifics of your iPSC line and culture conditions. I hope that these suggestions can help you troubleshoot your protocol. Good luck with your experiments!
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