I am trying to reprogram human and murine cell lines using the 4 times transfection protocol. After the 4th transfection, I plated the cells on a layer of inactivated MEFs. A week has passed since the last transfection and my cells are confluent, but I don't see any colonies/iPSC-like cells yet. How should I passage them? Normally how long will it take for the spheres to appear?
I am new to this field and would appreciate sincerely your kind advice. Thanks a million.
Hi Rachel. The time table is diffferent if you are reprogramming human or mice cells. In general, in human the iPSC appears around 10 days and are ready to pick around 20 days. In mouse the protocols is faster, you see around 5/7 days and you pick around 15 days. Of course depend on your protocol, in my case I use Sendai Virus. A very crucial point is the passage to Mef. So remember to use always rock inhibitor and a delicated trypsine. At this point I have never seen a colony but you can appreciate a general different morphology of your cells. Good luck!
Hi Clara, so for mouse the colonies normally appear 5-7 days after being plated on inactivated MEFs you mean? And do you ever need to split them, after you plate the transduced cells on MEFs and before colonies appear?
The protocol I am passed does not require ROCK inhibitor for reprogramming human cells, will human iPSCs still be generated without it? I know when reprogramming mouse cells LIF is a must but I am not very sure about the human ones.
For mouse you don't have need to pass on inactivated Mef (but it is better to use pre-coated gelatine plate) after 5 days from transfection the iPSC appears on your plate. LIF is necessary to maintain the undifferentiated state of stem cells, so you must change the medium every day with fresh LIF. For human, in general after 10 days from transfection the colonies appears. The protocol that I use (but now there are many protocol in literature) suggests to pass cells after 7 days from transfection, so at this point you don't see the real colonies but the morphology of cells is different. Many protocols don't specify the use of Rock but trust me that it is necessary to reduce the death of cells and to have a good efficiency of reprogramming. I hope to make myself clear.
Hi Clara! Thanks, that's very clear. Indeed there are so many protocols out there that it can be quite confusing for new starters like myself. Thanks for sharing your experience.
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