Mesenchymal stem cells – definition and assays. Part II – Assay-based definition discussion

by Alexey Bersenev on December 6, 2010 · 2 comments

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Because defining of true stem cell characteristics MSC in vivo, such as self-renewal, appeared to be difficult task, there is an ongoing debate about their identity as stem cells. Today I’m going to cite some reviews, highlighting the reasons for such debate and challenging current MSC definition.

Exerpt from the review:

Despite the terminology routinely applied, whether MSCs qualify as “stem cells” remains a legitimate question.

Because the experimental proof whether MSCs or even their subsets fulfill the stem cell definition is still lacking on a single cell level, the term “stem cell” seems to be inappropriate.

There is no problem with identification of MSC in vitro. MSC identity currently based on well established immunophenotyping, morphology, tri-lineage differentation capability (oste-, chondro-, adipo-), clonogenicity and colony formation. In vivo transplantation assays is the main current problem in MSC identification. The field is lacking strong data supporting engraftment, differentiation, multipotentiality and self-renewal in vivo.

Paolo Bianco and co-authors wrote about divergence in vitro and in vivo assays for MSC identification:

… the wide- spread use of in vitro assays in lieu of the defining in vivo assays is one of the most important sources of confusion, or at least controversy and disagreement, as to the nature, identity, and potency of MSCs…

In brief, stringency of assays is a must to claim any kind of potency, and in vitro assays are not stringent enough. Therefore, whereas a bulk of evidence widely reproduced in multiple laboratories demonstrates the genuine skeletogenic potential of bone marrow-derived stromal progenitors, evidence in support of the idea of a virtually ubiquitous, broadly multipotent postnatal progenitor of mesodermal and nonmesodermal derivatives is not equally as strong, as it is largely based on nonstringent assays. Importantly, when in vitro and in vivo assays are conducted on the same test cell population, it is obvious that the results yielded by the two kinds of assays may or may not converge (Krebsbach et al., 1999; Satomura et al., 2000). Hence, it is impossible to predict in vivo bone formation on the basis of in vitro mineralization, in vivo adipogenesis on the basis of in vitro adipogenesis, and so on.

About lack of data supporting MSC ability to self-renew in vivo:

Can human MSCs be serially transplanted, like murine HSCs? The answer is a definite maybe. However, one fundamental fact, again, prevents overly strict comparisons or equations between the two systems. Purified murine HSCs are neither cultured, nor expanded, nor in any way induced to proliferate ex vivo before transplantation, whereas human MSCs are. Hence, unless one can provide a rigorous assessment of self-renewal over a defined number of population doublings, it cannot be stated whether MSCs are shorter-lived (less extensively self-renewing) than murine HSCs. Serial transplantation may not necessarily be required to claim self- renewal in general, beyond the boundaries of hematopoiesis.

However, evidence for self-renewal of bone marrow skeletal progenitors, or for any other entity alluded to as MSCs, has long been missing, and was actually left as one of the major unsolved questions in the original work of Friedenstein. As a result, the question has remained open whether bone marrow-derived MSCs can indeed be seen as stem cells, or should rather be called simply “mesenchymal stromal cells”, in the lack of evidence of self-renewal as a defining feature of “stemness”.

Discussion about necessity of testing MSC self-renewal in serial transplantation assays was echoed in Ulrich’s Lindner review:

Based on this conservative and strict definition of “true” stem cells, it is currently not possible to confirm that MSCs fulfill these properties. First, an at least time limited or lifelong self-renewal capacity of human MSCs could not be proven. Moreover, the inductive in vitro differentiation capacity of MSCs after application of induction media is not comparable to the hematopoietic repopulation which takes place in NOD/ SCID mice after transplantation of ˜true” HSCs.

Thus, some of the inherent biological properties of the HSC concept are not necessarily transferable to other adult stem cells.

Thus, considering self-renewal and multipotency for defining characteristics of adult stem cells requires the development of an in vivo assay based on the same strict principles as it is used for defining HSCs. But it should be adapted to the specific biology of the cell type under study.

Another matter of debate is multipotentiality of MSC in vivo, noted by Darwin Prockop:

The debate on whether MSCs can be called stem cells in part revolves around the question of whether they can be differentiated into nonmesenchymal cells.

Prockop about resolving some controversies in the field:

How can we resolve the confusion generated by the use of different preparations of MSCs and MSC-like cells? More detailed comparisons of the cells in the same laboratories will be of some help. The most critical question is probably which cell preparations will be the most effective for therapy of specific diseases. This question probably cannot be answered until reproducible in vivo models are developed that can become assays for the cells that are as effective as the marrow-ablated mouse is for assays of hematopoietic stem cells.

So, can we used a term “mesenchymal stem cell” based only on in vitro assays? Would be “skeletal stem cell” better fit for MSC identity? Should we used a term “stem” in definition of MSC at all? What do you think?

Finally, what can we learn from such discussions? I’d conclude by quoting Paolo Bianco:

Nonetheless, the term has gained such global usage that it would perhaps be futile to suggest replacing it with another that would better adhere to the known biology of the system. Debating nomenclature always conveys a flavor of pedantry. However, debating misconceptions that accompany the popular use of any term may correct flawed experimental approaches based on mistaken assumptions, may trigger experimental advances, and may ultimately promote better understanding. The term MSC is indeed questioned, and questionable, because it conveys assumptions that were neither included in the original concept of nonhematopoietic stem cells in the BM nor supported by direct experimental evidence.

Also read: Mesenchymal stem cells – definition and assays. Part I: A brief history of the term

{ 2 comments… read them below or add one }

Vlad December 6, 2010 at 8:24 am

Alex – what is your opinion about this article? –

The authors demonstrated that – “Nestin+ MSCs contain all the bone-marrow colony-forming-unit fibroblastic activity and can be propagated as non-adherent ‘mesenspheres’ that can self-renew and expand in serial transplantations“.


Dr. Bersenev December 6, 2010 at 8:37 am

Thanks for linking to this paper. This is great article! One of the best that I’ve read this year. One of the best in 2010 according Faculty of 1000 evaluations –
This study is so good that I’d love to write separate post about it. Here in “Mesencymal stem cells – definition and assays” series (part III or IV) and on Hematopoiesis.

I guess the reviews that I cited here were written before this study was published, so all of them said: “Lack of evidence for self-renewal in vivo”. I think, even we have got one good study testing MSC self-renewal, assay was not reproduced yet in other labs. Also, Nestin+ is not commonly currently used marker for mouse MSC. Well, maybe things will change after this study.


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