Hi Yanny, there is no straight forward way or a single cell marker to identify or confirm hMSC phenotype. To ascertain your cultured or isolated cells as hMSC you have to follow the International Society for Cellular Therapy's guideline which is that MSCs must express CD73, CD90, CD105 and lack the expression of CD34, CD45, CD14, CD11b, CD19 or MHC class II antigens. In addition they must show their tri-lineage differentiation potential. However, you also can use commercially available hMSC characterization kit from Millipore for immunophenotyping of your cultured MSC. Its a simple fluorescence immunocytochemistry technique. I used this kit for my thesis and publication. I am sending this paper. You can have a look if you wish. Good luck.

Article Mesenchymal stem cells promote alveolar epithelial cell woun...

This is the publication, in Cytotherapy (the Official Journal of the International Society for Cellular Therapy) which describes what Khondoker just explained: http://www.ncbi.nlm.nih.gov/pubmed/16923606

You can do an RNA-seq and compared to published data sets. That would also show if your culture has mutated, differentiated, or has deregulated expression of specific genes.

I have done differentiation of hMSCs and they successfully differentiated to osteocytes, chondrocytes and adipocytes. Just thinking what other method to prove that the cells are really hMSCs. Thank you for sharing everyone!

Whilst the paper quoted above is useful, it is a little out of date as it doesn't reflect our current understanding of the in-vivo identity of hMSC which we usually observe in-vitro. For example there are distinct subsets of hMSC that are CD34+ but lose their expression in culture.

As the hMSC we see in-vitro are a very heterogenous cell population, it makes surface marker expression difficult to interpret, however there are now distinct subsets of hMSC with distinct anatomical locations in-vivo that have been described.

http://www.ncbi.nlm.nih.gov/pubmed/18786417

http://www.ncbi.nlm.nih.gov/pubmed/23818229

For you to be able to call a single cell a hMSC then you need to show that it is multipotent (adipo, osteo and chondr) when expanded clonally.

I agree with ANDREI MOROZ, the paper of Dominici et al "Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement" explains you if you are isolating and culturing human mesenchymal stem cells.

A good overview and methodology can be found here:

Charbord, P. Mesenchymal stem cell characterization, Biomedical and Health Research , pp. 27-35 (2012)

Dear Yanny,

the best way of answering such question is to analyse the marker profile of your cells. MSCs have several specific markers ( CD90, CD105, CD73) that can be analysed with various techniques such as immunestaining, flow cytometry or by analysing their expression levels via RT-PCR. Classicaly there is also the need to prove that MSCs lack the markers typically present in the haematopoietic cell precursors, such as CD45, CD14,CD34, etc... As a good way of proceeding you should use the same sample cells used at the beginning of the three lineage differentiation (if you have stocks) and test their levels of such markers. Proving the exsistence of such markers with a double test (i.e. immunostaining and RT-PCR) together with the differentiation data will be good enough. However you have to take into the account wich source your MSC are from (fat, bone marrow, other), because there might be some other markers that are niche-specific and could further prove that your cells are MSC from a specific tissue derivation. All the best.

Indeed all of the above is correct.. meanwhile we can consider the paper by Domicini for the characterization.. At least in clinical trial applications to FDA or any other Health Ministry Organizations it is adequate ...

I agree that the paper of Dominici et al "Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement." establishes some standard basis on isolation and characterization of human mesenchymal stem cells in culture. However, this paper dates from 2006 and, as the authors predicted, these criteria more than likely need to be redefined, including further assays as those demonstrating clonogenesis and low immunogenicity. For instance, increasing body of data demonstrate that MSCs inhibit proliferation of allogeneic T cells and express low levels of major histocompatibility complex class I and II molecules.

Best regards.

If its MSC I completely agree to Stefania and Andrei. There are set guidelines for MSC characterisation which demands cells to be culture adherent, to be positive for cell surface markers like CD73, CD90 and CD105 and negative for markers like Cd14, Cd34 and CD45. MSc also requires to be differentiate into alteast osteogenic, adipogenic and chondrogenic lineages.

Similarly for characterisation of other cells a combination of cell surface, intracellular marker as well as functional tests can be used (eg. HSC -CD34+IVE, low Cd45 and ability to reconstitute the cellular components in lethally irradiated mice).

Thank you all for the suggestions. Really appreciate it. Can anyone send me the articles by Dominici et al, please?

Here we go!

Thank you Santiago Roura :-)

This may be contentious, but I do not believe it is possible yet to prove cells are adult MSCs. The marker profiles suggested will tell you if the cells are mesenchymal or hematopoietic in origin, but they will not tell you if they are stem cells. When you compare CD expression to CFU counts, the CFU counts are dramatically lower, suggesting the CD profile labels a population of cells of which only a small fraction are "stem" like. Data from multipotency assays are only really valid if clonal populations are used, otherwise how can you eliminate the possibility that your sample is part osteoprogenitors, part chondroprogenitors, and part adipoprogenitors but they are not necessarily the same cell. The lack of a real prospective assay for the identification of MSCs is the biggest problem in the field.

using differentiation markers for facs or immunostaining does not necessarily prove they are human. As in my experience the markers can and do cross react with other species. the panel configuration can indicate towards a species. However, conformation using human specific primers is probably a better option whereby the your primers can be made to be species specific (rt-pcr) . putting the facs/immuno panel plus the species specific primers will together increase the probability that the species is human. As for stem cells differentiation asays such as osteogenic/chondrogenic and adipogenic differentiation would be the key.