I am trying to obtain iPS cells through feeder and virus free condition. My cells stained with Oct4 and Tra-1-60 but they didn't make a colony. Their shape doesn't seem as a fibroblast but also not completely circular. I observed significantly MET on my cells and some cells have got still shape of MET. What is your suggestion? What do you think about it?
You need more Characterization (AFP staining; in-vitro and in-vivo differentiation to Endo Meso and Ecto lineage, Endo.....),
The Oct4 and Tra-1-60 positive cells do make colonies. If not, they are not true colonies. Please continue the method by which you reprogram the cells for more days to get colonies with clear boundaries.
Two markers are not sufficient to make the claim. You should wait until you have colonies, then do clonal selection of colonies and expand. Then do PCR for more markers (myc, KLF, SSEAs, etc) in addition to more staining as well (such as alkaline phosphatase). You then need to do something like transplantation of your cells into SCID mice and characterize teratoma formation to demonstrate that they have the potential to differentiate into all 3 germ layers before you can actually claim your reprogrammed cells are indeed iPS.
Don't forget karyotype analysis for any evident chromosomal abnormalities!
What type of somatic cells you are using? They probably expressed Oct-4 (or pseudo Oct-4) and Tra-1-60 before being reprogrammed. Don't you think they are just partially reprogrammed?
They came from woman liposaction. So I dont think so they were partially reprogrammed. But i will ask again to hospitol for their real source.They stained only with oct4 not with a Tra-1-60 but after treatment they started to stained also with tra1-60.
Thanks a lot for each suggestions.
What method of reprogramming are you using? Is the OCT4 expression endogenous or exogenous(ie what you put in)? The cells may not have fully established their own pluripotency network yet. We have experienced the same when sorting mRNA reprogrammed cells with Tra-1-60. Further transfections might be needed as was our case. Also, SSEA3 is a better marker, Tra-1-60 is less sensitive.
Hello, in our experience, regardless of the starting somatic cell, reprogramming method or media system used, a reprogrammed cell must meet a panel of charactristic before it can be called a pluripotent stem cell and be banked and used for downstream applications. Pluripotent stem cells (either hESC or iPSC) must form colonies, proliferate indefintely, express self renewal markers (SSEA4, Tra-160, Oct4, Nanog, AP) and demonstrate tri-lineage potential when allowed to differentiate spontaneously staining positive for AFP(endoderm), beta-3 Tubulin (ectoderm) and SMA (mesoderm). In addition, the clones selected must also show normal karyotype and demonstrate the correct transcritome signature. Cells that are partially reprogrammed may only show some charcateristics, but not the full panel of charcaterization. If you donot get colonies at 21-30 days post reprogramming initiation, you will not have any true iPSC. I would try again and look for all the afore mentioned markers. Good Luck and much success wished to you.
I agree with colleagues above. Oct4 and Tra-1-60 staining is not enough in this case. Best would be to select 4-5 markers to define the cell population. OCT4 also forms a trimeric complex with SOX2 and DNA to control the expression of genes involved with embryonic development, such as FGF4 (PMID: 10801796), UTF1 (PMID: 10409735), or even NANOG (PMID: 15743839).
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