how do you confirm pluripotency after editing hiPSCs?

[CrisprWizard]

What is your preferred technique for verifying that hiPSCs remain pluripotent after editing? Do you use functional assays such as embryoid body formation, marker expression, or another method? I'd be interested in knowing what has worked best for you.

 

[AverageBrody]

What is your preferred technique for verifying that hiPSCs remain pluripotent after editing? Do you use functional assays such as embryoid body formation, marker expression, or another method? I'd be interested in knowing what has worked best for you.

honestly, I've had the best luck with a combination. Immunostaining for pluripotency markers is a quick visual check. But honestly, if you're doing any downstream differentiation, the ultimate proof is their ability to form functional derivatives of all three germ layers in vitro (e.g., via EB differentiation). Just relying on markers can sometimes pull the wool over your eyes if you're not careful.

 

[Crispy Fry]

What is your preferred technique for verifying that hiPSCs remain pluripotent after editing? Do you use functional assays such as embryoid body formation, marker expression, or another method? I'd be interested in knowing what has worked best for you.

I suggest stick with marker expression OCT4, SOX2, NANOG since those are quick and reliable. If you're feeling a little skeptical, you can maybe add EB just to double check the functionality. Agree with @AverageBrody on that.

 

[GenomeJockey]

Great advice! I completely agree, while marker staining is a quick way to get some initial insights, nothing really compares to EB formation when it comes to solid evidence of functionality. Do you typically culture EBs in suspension, or do you plate them after a while? I'm interested to hear what's been the most effective for your process.