Hi Daniel,

If I understand the question correctly you would like to know about the differentiation potential of your cells already known/assumed to be MSCs? In this case there are papers discussing the heterogeneity of the MSCs - different subpopulations and their potential to divide or differentiate. We recently published a paper, which might be partially answering your question - we follow longitudinally how MSCs lose their potential and ability to divide:

"An in vitro expansion score for tissue-engineering applications with human bone marrow-derived mesenchymal stem cells. " (also can be downloaded on my page) and show that out of 84 senescence related genes only two correlate with differentiation qualities. Hope this answer is of some help.

Hi Jivko. Thanks for the feedback, and I'll take a look at your publication. I agree that one can look at differentiation efficiency using end-point assays as a measure of stem cell function. However, what we'd like to do is have a faster read-out of the stem cell phenotype that can be measured prior to differentiation in order to assess whether changes in the culturing conditions promote multipotent lineage potential.

Your response nicely hits on one of the challenging issues here, namely the interplay between proliferative potential and stemness. Proliferative potential generally falls off with either senescence (high passage) or in the presence of differentiation cocktails. So many gene expression studies looking at either of these variables are likely to identify markers of cell proliferation. However, to what degree can we casually associate them with maintainence of the stem cell phenotype? I think in some ways this association can be somewhat misleading. We've found that you can maintain hMSCs in suspension culture for up to two weeks (conditions that cause them to arrest proliferation), yet upon replating, their differentiation potential is as good or better than before (PMID 22331826).

While I agree that proliferative markers and stemness are both anti-correlated with senescence or passage number, I'm not sure that we can meaningfully conclude there is a functional correlation between proliferative markers and stemness (at least in a general sense). What do you think?

Hi Daniel,

"While I agree that proliferative markers and stemness are both anti-correlated with senescence or passage number, I'm not sure that we can meaningfully conclude there is a functional correlation between proliferative markers and stemness (at least in a general sense)."

Quite right - we have seen that too. Passage number is not the only point to look at - there is much more going on, so we tried to decouple it and look at other markers. The potential from what we have seen is neither linked to the age of the donor (great news for the old folks, who will need autologous MSCs most)nor to telomere length. Also a lot of markers are only there but don't give out if the cell will pass the "quality control" or not. It is more an exception than a rule but there are samples where cells divide great yet with the time lose some of their potentials, and for these occasions we need to use a battery of tests. Exactly as using the markers "no hair and no teeth" will not reliable to tell a baby from an old man.

Hi Daniel,

You have a very nice problem to look at. Thought I have not really studied stemness genes in MSCs but in my recent work I found decrease in known stemness markers (Nanog and UTF1) by microarray analysis [PMID 23383279].

@Seppe- none of the iPS or ESC factors are expressed in the hMSCs I've worked with.

@Neeraj- thanks for the reference. Have you been able to demonstrate Nanog expression at the protein level. The locus is devoid of transcriptional activity (PolII occupancy, histone acetylation) in ChIP-Seq data, and though we can occassionally pick up Nanog transcripts by qPCR, I don't think Nanog is functionally expressed in adult hMSCs.

@Rodrigo- do you know whether CD146 expression decreases as a function of differentiation in hMSCs (such as in the bone and fat lineages)? Yes, I agree, pericyte markers should be enriched in hMSCs. Never tested these by qPCR, but it is worth a shot!

CD73, CD90, CD105 are few Human mesenchymal stem cell markers.

hi Daniel....well i have not checked any at protein level

@Neeraj- it would be critical to check at the protein level.

There was an eye-popping study published in Mol Cell, "Oct4 and Nanog directly regulate Dnmt1 to maintain self-renewal and undifferentiated state in mesenchymal stem cells." PMID 22795133. This study claims to find Nanog and Oct4 expressed in hMSCs. We use BM-MSCs from Lonza and have never been able to detect expresion of these proteins. Perhaps, MSC source matters for detecting these factors, but even if Oct4 or Nanog is present in a subset of hMSCs, I think the bulk of evidence suggests that these are NOT required to maintain the stemness phenotype in hMSCs.

Would be very interested to hear about any independent confirmation of the results reported in the aforementioned paper, especially with convincing Western blot data to demonstrate expression of these factors at the protein level!!! This is fairly important to future research in this field.

@Seppe- have you been able to find these factors in hMSCs? By what method?

Dear Daniel,

You may want to look at some other MSCs from other labs or even different sources of MSCs maybe WJ-MSCs and Adipose tissue MSCs.These may express some pluripotent,self-renewal or stemness markers.Microarray data suggests BM-MSCs express over 40 stem cell markers(May or may not correspond to stemness).

I do not not have any experience myself regarding this but i can relate to this question by suggesting to look at cells from other labs or sources because we do see some variability amongst different lots of MSCs we produce and even within passages.You may want to try some cells maybe from Darwin Prockop lab.

I do not remember well but i think some of my colleagues told me they saw oct4 and Nanog gene expression.I'll get back to you regarding this.

In some cases i hear Oct4 and Nanog is cytoplasmic and not nuclear altering/functioning in signalling stemness.Some scientists even report that cytoplasmic nanog positive MSCs promote tumor progression.http://www.ncbi.nlm.nih.gov/pubmed/22683467

@Charan- thanks for your feedback, but I disagree that self-renewal markers in hMSCs are related to pluripotency or iPS-reprogramming factors. There certainly seems to be a growing debate about the role of Oct4 and Nanog in somatic cells, but the most compelling data is that Oct4 and Nanog are restricted to iPS and ESCs.

The stem cell field has been led astray too many times by claims of pluripotency in somatic stem cells (for example, the MAPC controversy from the Verfaillie lab), and I think extreme caution is warranted in weighing these types of claims. Even if such expression occurred in rare populations of hMSC, I think it is quite clear that these markers are not strictly required for tri-potent lineage (osteo/adipo/chondro) differentiation, and therefore are insufficient to account for the MSC phenotype. Instead, it seems probable that some other set of transcription factors comprises the minimal set of genes necessary to confer this cellular phenotype.

@Daniel-Does a necessity lie in studying the stemness/stemness related markers in MSCs?I suppose stemness in MSCs would not matter to a great extent since MSCs are now well accepted to act in a paracrine manner rather than engrafting and differentiating.It could mostly be like a 'hit and run mechanism'.I am curious why you would want to track the stemness related markers unless you want to modify the MSCs with small molecules or vector based reprogramming.

I completely agree with Daniel mere expression of the marker on the somatic doesn't mean that cells are pluripotent.

And to nail down the set of markers for extremely heterogeneous population like MSCs is really difficult.

Adult human mesenchymal stem cells (hMSCs) possess multilineage differentiation potential, and differentiated hMSCs have recently been shown to have the ability to transdifferentiate into other lineages. However, the molecular signature of hMSCs is not well-known, and the mechanisms regulating their self-renewal, differentiation, and transdifferentiation are not completely understood.

Now I'm engaged to investigate this genes in mesenchymal stem cells of different tissue origin: Pou5f1, Nanog, KLF4, ERAS, GDF3, DPPA2, SALL4, TCL1, ZFP42, UTF1, BMI1, SOX1, SOX2, SOX10, SOX15 and SOX17. also proteomics can be very important for identification of MSCs molecular signature and comparative them with together and other lineages.