WP2 - The Identity of Marrow MSCs in vivo

The identity of in vivo cells initiating MSC cultures (in vivo MSCs) remains poorly understood. From the pioneering work of Alexander Friedenstein we all know that these cells are rare in the bone marrow (BM) and that they are non-hematopoietic and adherent to plastic. However, the sole adherence technique does not sufficiently enrich for in vivo MSCs, since the adherent cell fraction also contains mature and immature cells of the hematopoietic lineage. Therefore several laboratories across the world are now actively working on the development of more selective MSC purification methods. These are primarily based on the surface antigen expression in MSCs; however some investigators also use methods based on unique biophysical or biochemical properties of these cells. The panel of MSC-selective markers is rapidly growing (Stro-1, CD49d, CD271, CD73, CD105, GD2, CD200, SSEA-4, CD146 and others), but a consensus opinion as to which marker is the most useful for the in vivo MSC purification is still lacking.

Interestingly, despite the fact that different purification methods have been used by independent investigators, some common features of in vivo MSCs have now begun to emerge. It appears that in vivo MSCs have a unique stromal morphology characterized by long cytoplasmic extensions reminiscent of dendrites, which are likely to facilitate an organization of individual cells into a larger cellular network (Figure 2).

Figure 2. A single in vivo MSC purified using a negative depletion with CD45 and CD235a antibodies and subsequently stained with Stro-1/Alexa-488 labeled secondary antibody. Note long cytoplasmic projections extending from the main body of the cell.

 

A similar stromal network has been commonly described on sections of marrow biopsies and is called a reticular cell network by histologists (Figure 2). These marrow reticular cells and in vivo MSCs share a panel of common markers (such as CD271, VCAM-1/CD106, CD10 and alkaline phosphatase) and it is reasonable to suggest that in vivo MSCs are somehow concealed within this network. Reticular cells are known to produce an extracellular matrix in marrow cavities; they strengthen marrow sinusoids, develop into adipocytes or osteoblasts and support hematopoietic cell maturation. The future challenge will therefore be to design new techniques capable of dissecting these potentially different subsets of marrow reticular cells and uncover the true identity of the most primitive MSCs.

Figure 3. Stromal reticular cells detected on a bone marrow trephine biopsy section using CD10 antibody. Note their long cytoplasmic projections similar to that of in vivo MSCs.

 

It is well-known that MSCs don't perpetually self-renew in standard culture. In fact the proliferative potential of MSC's tends to decline with accrued cell divisions, whilst a risk of potentially harmful mutations increases. The knowledge of in vivo MSC biology is therefore critical for the development of better MSC expansion protocols, potentially yielding safer products for therapy. Workpackage 2 (WP2) of Purstem is designed to address some of these important issues. We are looking at developing better methods to purify marrow reticular cells and to study their heterogeneity. We aim to generate MSC cultures from highly-purified in vivo MSCs and to monitor particular changes that occur in standard and optimized culture conditions. The molecular signature of MSCs, before and at different stages of cultivation, is studied using a number of modern genomics approaches, with a particular emphasis on the preservation of bone-forming potency of MSCs. From a Regenerative Medicine perspective, we believe that this knowledge will contribute to the establishment of new manufacturing practices and safety standards based on optimally-cultured BM MSCs that most closely resemble MSCs resident in vivo.