I’m currently reprogramming my fibroblast cells using Simplicon RNA OKSG-cMyc TagRFP Kit and after 13 days, these colony/aggregates started to show up in all of my wells. I have 6 wells, 2 wells coated with geltrex the rest with matrigel (1:100 for all wells). I am not sure if these are really iPSC colonies or just the cells aggregated together to form a spheroid (which is odd cuz even when culturing normally they wouldn’t become like this). I’ll perform AP staining and check for RFP another 2 days before the next medium change, but it’s just that I’m quite nervous about this is all. All of the pictures are under 4x objective lens.
Hi, Ahmad Khairul Aizad Ahmad Khairi
I would guess that they are partially initialized cells; AP staining may look slightly positive, but many of the intrinsic undifferentiated marker groups would be negative, e.g. Oct4 and Nanog.
Since the introduced initialization factors seem to be functional, I would culture a portion of the cells in expanded culture and observe for the next two weeks to see if fully initialized iPS cell colonies arise.
On the other hand, excess cells would be cryopreserved.
If the protocol indicates the appearance of initialized colonies in 2 weeks, I would guess that the efficiency of transduction and expression level of the initialization factor group may have been low.
Sincerely yours
Hi,
In my opinion, some of them look very optimistic. I recommend you wait about 2 days. Then add ROCK inhibitor for 4 hours and try to transfer some colonies. Remember that after transfer, the ROCK inhibitor should still be in the medium for 24 hours. Colonies should be transferred gently, one colony at a time.
Good luck!
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