I am trying to reprogram MEF (Mouse Embryonic Fibroblasts) into iPSCs. During the process, cells are shrinking, clumping, dying and detaching from the surface (non-coated) as you can see in two different figures taken at two different resolutions. I have no previous experience in iPSCs generation so can anyone please confirm by looking at morphology that either my cells are being reprogrammed or not? Is detaching from the surface is common during reprogramming? AP-staining might be the best way to identify iPSCs but I don't have that in my lab yet so is there any way to confirm by morphology? What should I do with the clumped and detached cells?
Experiment details: I didn't coat the surface with Matrigel or Gelatin. These cells are not grown on feeder layer. This detachment happened once I changed the medium from normal to KO DMEM/F12 after 5 days of transfection with reprogramming factors.
Many thanks in advance!
Hello Nasir.
I agree with Justyna's view.
Your MEF concentration appears to be far high when you start to plate them. All different forms of tissue arrays may be caused by predominant differential cell population. It is the best practice to mix single cell suspension completely after MEF preparation, followed by aliquoting for storage.
Once you thawed a vial, use an appropriate concentration according to protocol. You should really have everything ready. Otherwise, do not waste time to try.
Best wishes.
Dear Justyna,
Thank you for confirming. Can you please guide me to get iPSCs by transfecting MEF with non-replicating and non-integrating plasmid containing reprogramming factors? Should I use coated wells along with some small molecules like ascorbic acid and sodium butyrate? Your guidance will be really helpful for me. Thank you!
Dear Prof. Sang Ho Lee,
Thank you for pointing out one of my mistakes. Actually, I started first transfection at 70% confluency but MEFs grow exponentially which leads to over-confluency very soon (even before 2nd transfection). Next time I will start at 30-40% confluency (although I am still afraid that it will become overconfluent very soon again). Can you please guide me to achieve iPSCs using non-replicating and non-integrating plasmid containing reprogramming factors? Should I use coated wells along with some small molecules like ascorbic acid and/or sodium butyrate? Your guidance will be really helpful for me as I have no past experience or senior around me. Thank you!
Hello Nasir.
I suppose that you have read all the papers concerning many ideas. However, it would be a good idea to stick to one of them to try to follow exactly whether you can execute it properly and obtain iPSCs according to the procedure provided by the article. By achieving that, you would get a full confidence in every aspects of iPSCs or ESCs that are most demanding cell culture in the field. Then you could apply what is in mind to generate iPSCs in the most plausible way.
However, if you combine everything together in an effort for generation of iPSCs, it would not work properly because the reported articles showing specific ways of iPSC generation adopted different somatic cells and different culture conditions as well.
Familiarize yourself to iPSCs or ESCs on the basis of your intended direction first. Your supervisor will be glad to discuss everything mentioned above.
Best wishes.
Thank you so much Prof. Sang Ho Lee. I will follow your suggestions and will update once I get some success.
I am surprised that no one has pointed out the obvious for anyone who has done reprogramming...
First, yes, you should have coated the plates with matrigel.
Second, and most importantly, no those are not iPSCs but the photo on the bottom left clearly shows cells that are in the early stages of reprogramming. Some of them quite likely will continue on to become iPSCs.
Finally, a pet peeve: Embryonic fibroblasts do not exist. MEFs are Murine Fetal Fibroblasts.
Since I found this question 4 months late, what happened with the culture? Did you get any iPSCs from it or from subsequent ones?
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