Morphologically, my cells remain tightly packed in round colonies for about 30 passages, but are they still pluripotent?
Probably functional characterization assay would be more appropriate to confirm pluripotency, in addition to use of specific markers.
This depends on whether you are working with human or mouse cells. If it is mouse cells you are using you would test the cells potency by generating mice from them much the same as Yamanaka did when they first developed iPCS (PMID:16904174). However, this would be expensive and time consuming. You might instead test their expression of common stem-cell markers e.g. nanog, oct4 to confirm that the cells are stem-cell like. In addition, if you can direct them to differentiate into various lineages, e.g. endoderm, mesoderm, ectoderm, that would confirm their overall ability to make the basics of an embryo.
You should check various markers (for human pluripotent cells, Oct4/Nanog/SSEA4, etc.) + check whether the cells can differentiate into all three "derms", e.g.: mesoderm, endoderm and ectoderm direction. In this latter case again, check various markers of being expressed in endoderm-/ectoderm-/mesoderm-derived cells.
You must perform those above in order to prove your cells are pluripotent, regardless of their origin (iPSCs or others).
Good luck!
Thanks everybody veery much for the answers!!
Still, my cells are positive for Nanog, but when I am trying to differentiate them with TGF, both control and TGF treated cells express mesoderm markers as brachyury, Nkx2.5 and Mef2c.
Any ideas?
If the cells are pluripotent it should be capable to differentiate in osteo, adipo, chondrocytes when are culture in specific media. In this paper you find the protocol for testing the differentiation potential of stem cells. You also can test the proliferation, and expression of different markers (for ES- oct 4, nanog; for adult stem cells (MSC): CD90, CD73, CD29, phosphatase alcaline etc )
Article Integration Properties of Wharton's Jelly-derived Novel Mese...
You can also check if they are pluripotent by performing an alkaline phosphatase live stain. Akaline phosphatase is a phenotypic marker of undifferentiated embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). While AP is expressed in most cell types, its expression is highly elevated in pluripotent sstem cells.
If your cells are of human you may find useful the information of this paper:
http://www.ncbi.nlm.nih.gov/pubmed/16923606
Many thanks!
Forget to say that my cells are mouse stem cells (CGR8).
Till which passage number do you usually use your cells?
For mouse cells, there is really only one gold standard for pluripotency, making a chimera and showing that your cells contribute to all lineages. If you are unable to do this, then look for Oct4 expression which should be strong, nuclear, and in 90% or more of the cells.
A simple method is to use the embryoid body formation assay, and check for the various embryonic germ layer markers.
This is not an simple task. The gold standard is to make a chimeric mouse and see that your cells contribute to all tissues in the body including germ line transmission. For this, your ES cells should be genetically tagged, such as with EGFP or LacZ. Also, karyotyping can be very informative. We find that ES cells with greater than 85% of a normal karyotype (40 chromosomes) consistently give good germ line transmission.
The easiest way is to check the expression of some pluripotent makers such as SSEA-4, SOX2 and the transcription factors Oct-3/4 and Nanog. Good luck!
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