Hi everyone,
So these are images of IPSCs 2 days post passaging/splitting. I noticed that the overall morphology of colonies are not compact or round and without distinct border. However, at higher magnification, i can still see cellular morphology of typical IPS cell with scnat cytoplasm and prominent nucleoli
Any help is much appreciated!
Hello - (caveat - I am more familiar with ESC cells, rather than iPSCs; and your culture method choice will influence morphology subtly too), but these do look like they are differentiating (or at least stressed). The main comparison I'm looking at is the edge cells compared to the middle cells in the same aggregate - these look much more elongated and less immature than the center cells which is indicative of differentiation. However, you mention these are 2d-post-split, so it could just be stressed cells from splitting. The key here is if they round back out and make more compact&dense aggregates by ~4d post split (with likely some small areas of differentiation). If they do, then I would recomend adjusting your split technique to be a bit gentler to avoid this issue in the future - as stressed cells are much more likely to spontaneously differentiate (and depending on your downstream experiments, also less likely to behave properly there).
the plating density is too high. you need to allow cells to form the colonies 1st, and then judge about the shape, select best colonies manually (the progeny of each individual original clone) and then expand. iPSCs stabilize with more passages in stem cell media. Right now (as shown in your photo) one cannot say much about the potential of cells to form nice round colonies (which we all prefer to have in hESC culture).
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