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!
I would start by checking the lines haven’t been mixed up, either by sequencing the samples, or, depending on the mutation, you could do a PCR using primers targeting the mutation site, which might be easier.
I would also double check all the other obvious things to make sure nothing else has changed – were the cells grown in a different incubator, on a different plate type, were you using a new reagent/has the lot of a reagent changed in a significant way, have any of the parameters in your analysis pipeline changed at all, did a software update to a new version in between experiments, etc.
Assuming that everything remains the same or at least not substantially changed, it may be that the difference isn’t as robust as you thought it was – especially if it can be lost to small changes like those above. I’m not sure optimising the differentiation would recover your result if the differentiation conditions you’re using currently were what produced the original difference. You should consider in your quality control checks not just contamination or SNPs in the iPSCs but also the quality of each differentiation though, and how much this changes. Were the original differentiations much better/different to this new one? Quantifying differentiation efficiency and comparing between them rather than just looking for a go/no go passing grade on the differentiation may give you more insight into whether something has gone wrong in these newer experiments.
The suggestion to try and find a different isogenic pair from a different genetic background is also a good one, always a nice way to confirm a phenotype is not line specific, though I recognise these aren’t always easy to find with rare fAD mutations. Introducing a point mutation into a healthy line could be another way to go here, to make your own isogenic pair without any other disease background.
Good luck! Do report back and let us know what happens.
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!
Alzheimer's Society
