Hi we recently reprogrammed fibroblasts into iPSCs, the morphology is off but we are wondering why they are still staining with tra-1-60? We picked the colonies to see if the morphology would change once re-plated but they look the same. According to this paper https://www.nature.com/articles/nbt.1580 , fully reprogramed cells stain positive for tra-1-60 but ours do not look like iPSCs.
Your iPSCs have differentiated... the colonies are not tightly packed
The colonies could be a mixture of pluripotent stem cells and differentiated cells. Usually, when iPSC/ESC colonies are maintained longer in a single culture dish more than 10 days they tend to differentiate. I would suggest, using TrypLE and dissociating the colonies to single cells and seeding it again. From there you can pick colonies with proper iPSC colony morphology and expand them.
I do agree with some answers above since those iPSC samples look like undergo spontaneous differentiation. You could try the earlier suggestion using TrypLE to dissociate colonies to single cells prior to the re-seeding step. Another suggestion from me is, that you can try to use enzyme-free dissociation using ReleSR, which I think could be more effective to eliminate differentiated cells because only non-differentiated cells would be detached as aggregates. However, please pay attention to incubation time.
You can refer to this link for ReleSR and its procedure:
https://www.stemcell.com/relesr.html
Hopefully it helps your research, good luck!
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