This is the first time I studied induced pluripotent stem cell. I used a non-viral vector conjugated with a plasmid (encoding :Oct4, Sox2, Klf4 v-Myc and GFP) to transfect L929 Fibroblast cell line.
Here is how they look after transfection. I am concerned about the cells whether they are differentiated or not. Could you please help me about the issue?
Did my L929 fibroblast cells differantiate into IPCS ?
image F is promising , it seems that your cells are transforming but you have to wait till they transform into proper colony to decide
Too soon to know, but the flat stuff on the periphery looks more promising than that 3D clump. What media are you using?
We use DMEM-F12+ %10 FBS+ 1X GlutaMax+ 0.1 mM MEM nonessential aminoacids + 100 mM b-mercaptoethanol+ 10 ng/ml bFGF.
You dont use FBS with this experiment, you should use knockout serum with knock out medium plus those additives you have
Is the serum you have mentioned is Gibco KnockOut Serum Replacement ?
Could you please suggest me a brand name that you prefer for your own experiments?
Actually, in most reprogramming methods, you DO use FBS containing media for a period up to a week while the cells are more in their fibroblast-like state. Subsequently you change to a reprogramming media.
For lentiviral and Sendai based reprogramming, I use E7 media.
I'm a bit concerned about reprogramming of an immortalized line. I've never tried it nor ever have read anything about it, but would assume that the chromosomal abnormalities would interfere with iPSc function. Of course I suppose it depends on what your intended use is for these cells.
Thank you for all your answers. Here is how my cells look like 20th day. What do you think?
I think you need to start over with the right media sequence. Are you still using the same media?
At this stage a reprogrammed colony would at least begin show a distinct border, it would be flat, and individual cells with a high nucleus to cytoplasm ratio would be visible.
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