I need to induce mesenchymal-epithelial transition. Kind of urgently. I have already tried "good old times 5-Azacytidine" - did not work. I have already tried BMP7 - several times, several concentrations, several methods (with, w/o serum); nothing seems to work. Does anyone have other suggestions besides miRNA (been there, dont want to go there again if not necessary)? Would it help to just knockdown E-Cad? Or something like that? I am also happy to visit you with my cells in your lab to try things out and share a publication with you because this is the last bit which is missing. And I start to get devastated. Thanks everyone.
Dear Teresa,
interesting problem there.
Can you give more infos on which kind of mesenchymal cells are you trying to switch and towards which kind of epithelial ones (if this is a differentiation experiment)?
Let me know
Giampiero
Thanks Giampiero for answering that quickly.
Basically I am trying to switch fibrotic fibroblasts into a more epithelial state to force it to produce my protein of interest since I believe (or know by experiments) that type 2 EMT is responsible for the supression of it. That is why I used BMP7 - there is this massive study going on to stop tissue fibrosis - but it made it worse since BMP7 is also known to induce SNAI1, a typical mesenchymal marker (well, all the MET papers do not tell you about that information). I just want a reduction of mesenchymal markers and an induction of epithelial markers.The more the better.
As "simple" as that... Haha.
Teresa
Are you working with a clonal population of cells derived from a single cell or a mixed population of cells containing multiple different cell types? Utilizing a mixed population will have a serious impact on your studies.
I have followed the EMT literature since it was first proposed in the kidney. In renal and liver fibrosis, EMT does not actually occur, as recent accurate cell lineage tracing have essentially disproved the EMT hypothesis.
I am not sure I understand your question. You wish to drive fibroblast to an epithelial state, i.e. the reverse of EMT? MET happens in development, but has this ever been done in an in vitro system? I can't recall ever seeing a paper that promotes fibroblast differentiation into an epithelial cell.
I agree with Gregory Dressler because we tried to recreate this research using adult-derived genomically-labeled pluripotent (and totipotent) stem cells in mixed cultures with unlabeled lineage-committed progenitor cells in vitro. It did not happen. In all instances examined the only cells that formed the EMT were already epithelial-committed progenitor cells. We concluded that the mixed cell system lead to the erroneous results reported.
Thanks so much for the information... Well, I will try my luck with direct inhibition of mesenchymal markers since I think that it would be great for my work to show that reversal of in vitro EMT is inducing my protein of interest again even if this is very very artificial but it would certainly prove the point.
Thanks again.
Have a lovely day. And a lot of success in research. Well, I can certainly need it. :)
Teresa
Dear Teresa Otto, you are welcome. If there is anything that I could help you with, all you need do is ask. Professor Young
We have good experience with CHIR99021 and SB431542 inducing MET in human foreskin fibroblasts.
Hy guys, just picked this conversation up. I actually had the same interest as Teresa. Heiko, do you have any publication or protocol that I could read regarding your chemical induced MET?
Thanks.
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