Dear Solutions Lab,
I’m in the second year of my PhD using iPSC-derived neurons to study a familial Alzheimer’s mutation. We have an isogenic control line, but in my last three differentiations the control neurons have shown a similar phenotype to the mutation line. Earlier experiments showed a clear difference.
I’ve checked for contamination and both lines passed our usual quality checks. My supervisor thinks I should optimise the differentiation, but I’m worried about spending months changing conditions just to recover the result we expected.
How would you work out whether this is a problem with the cells, variability in the differentiation, or evidence that the original finding isn’t as robust as we thought?
Thank you for taking a look.
Hello,
Thank you for your question. It’s understandable to feel concerned when a difference you previously observed disappears, especially when you’re deciding where to invest your time next. You’re right to want to understand what has changed before adjusting conditions to recover the result you expected.
My first step would be to confirm the identity of both lines by sequencing the mutation site. You mention that both lines passed your usual quality checks, but it isn’t clear whether these included checking that the mutant line carries the mutation and that the isogenic control does not. Mix-ups or mislabelling can happen during extended culture, so this is worth ruling out first.
I would also go back to early-passage frozen stocks of both lines, ideally from the stocks used when you first observed the difference. Repeating the differentiation with these would help establish whether the phenotype has changed during continued culture.
Finally, if other iPSC lines carrying the same mutation are available in a different genetic background, I would try to obtain them. Testing whether you see the same effect in an independent background would help you assess how robust the finding is and whether it might be specific to your original pair of lines.
Those would be my starting points before changing the differentiation protocol. Your instinct to question the result, rather than simply try to reproduce what you expected, is a good one.
I hope these suggestions help you work out your next steps. Best of luck with the experiments and the rest of your PhD!
