How do you prove your MSCs are really MSCs?

Differentiation, markers, what is the gold standard in your opinion?

In my opinion, the 2006 ISCT position statement is the gold standard for defining what is and is not a human MSC.

http://www.ncbi.nlm.nih.gov/pubmed/16923606
Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop Dj, Horwitz E. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006;8(4):315-7.

That paper lists three basic criteria that I think are essential:
1) plastic adherence and fibroblastic morphology
2) express CD105, CD73 and CD90, and lack expression of CD45, CD34, CD14 or CD11b, CD79alpha or CD19 and HLA-DR
3) in vitro differentiation to bone, fat, and cartilage.

Both the surface markers and differentiation capacity should always be characterized for each new batch, or else you're just doing sloppy science. I also like to monitor the colony-forming efficiency and forward/side scatter of the cells to give a basic indication of their proliferative potential and size/granularity.

Hi Bonnie,

I appreciate that it is still the gold standard but practices have moved a long way in four years. Do you not think there should be a limit on passage number (given that an individual passage can be defined). Different papers have come out with different unique markers for 'MSCS', all of which seem to be different cell populations. I think now is the time to determine and characterise new populations of cells under the MSC umbrella. Do you not agree?

Certainly there are lots of interesting ways of separating subpopulations of the cells, including markers like cd146, stro-1, etc. I was just trying to give an answer that would satisfy the basic question "I have some cells, can I call them MSCs?"

And anyway, I'm not quite convinced that any of these techniques or markers actually isolate the optimal cells for all applications. Consequently, I'm inclined to avoid 'throwing the baby out with the bathwater', so to speak. In most cases for basic research I'd rather work with the broadest population of MSCs (even if this means they have more contaminating differentiated cells) in order to ensure I'm not discarding the cells that turn out to be important for whatever application I'm interested in.

Regarding passage number, it's definitely critical, but I'm not sure there should be a universal limit for passage number unless there's also a universal guideline for seeding and splitting densities. I think what really matters is keeping to a reasonable number of population doublings and avoiding letting the cells become too confluent.

What's a reasonable number in your opinion? There is talk of passage 10 cells proliferating like crazy and losing their chemotactic ability.

There is the danger of throwing out the best cells by purifying further, but do you not think in the future we will find homogenous populations within this that can be used for specific therapies?

As for the answer on how to be sure what are MSCs, there is no good answer. Especially after excellent papers suggesting that fibroblasts can differentiate into all three lineages, adhere to the plastic surface and express markers designated as MSC markers. In addition, they showed similar immunomodulatory capacity and the mechanisms by which this modulation is mediated. We tried FSP1 marker in order to differ these two populations using dental MSCs (which, by the way, can go over 20 passages), but without larger success. As MSCs and fibroblasts belong to the same differentiation lineage, there should be some markers that could label earlier stadiums (CD271 for example), but there is lot to be done in that field.

As the discussion is on and there's a long way to get down to defining the true MSCs, any group using the so-called "MSCs" should define as completely as possible the methodology they used such as isolation method, passage interval, no. of passages, seeding and splitting density, medium conditions, characterisation methods, till some concensus is built in the coming time by bodies like ISCT.

But do you not feel that the time has come for researchers to be more specific. Definitions for MSCs as they are may be good enough for MSCs but do you not feel that groups should start characterising the cells they are using and give them each individuals MSC subpopulation names so that people understand what cells they are reading about when they go through publications. ie. MSC-1, MSC-2 etc?

Well certainly.But if each group starts to characterising the cells don't you think there would be a number of MSC subpopulations. I think that in vivo studies to trace the generation of mesenchymal lineage should be taken up, so that those identified cells can set the standard criteria for designating a population of cells as MSCs or not.

What are you thoughts on markers such as CD271. I was at the 4th Uk Mesenchymal Stem cell meeting yesterday and this seemed to be very popular?

CD271 could be a promising marker but the problem is its fast membranous down-regulation in culture conditions. So it could be used for the ex vivo isolation of MSCs.

Does anyone have an opinion on the isolated cells that are not adherent in culture? I know that the MSCs are typically isolated based on their adherent quality, but some people think that selecting for this may eliminate important stem cell populations from fresh isolations (before they're cultured, or just at the beginning of culturing them).