In contrast to cMSCs, only little information exists regarding the features of the primary precursors, nMSCs, that give rise to these plastic-adherent cells (Fig. 2). Various groups have attempted thus far to purify MSCs by using different strategies. Indeed, using the monoclonal antibody Stro-1 (recognizing an as-yet-unknown antigen), Simmons et al. identified nMSCs as CD34+ cells and found them on the abluminal face of the vessels (Table 1) [47, 48]. Our group used the monoclonal antibody anti-CD49a molecule to enrich these cells and confirmed their stem cell phenotype in human beings and in rodents (CD133+ and Sca-1+, respectively) [49, 50]. CD49a is the α1 subunit of the VLA-1 integrin, a collagen IV and laminin receptor and was primarily described as an early marker of VSM cells in organogenesis . Recently, our group demonstrated that nMSCs could also be enriched within the CD200+ BM fraction . CD200 is a member of the immunoglobulin superfamily and is expressed by various cell types, including myeloid cells, endothelium, ovarian cells, trophoblasts and neurons. Native MSCs are also enriched within the low affinity nerve growth factor (LNGF-R or CD271+ BM fraction . As Stro-1 antigen and CD49a, the CD271 molecule is a reliable marker of nMSCs. In a seminal work, using immunohistochemistry, the LNGF-R was shown to be confined to a subset of BM stromal cells, the reticular cells, with dendrites irradiated toward haematopoietic cells and localized at the abluminal side of sinus. The reticular cells have been described to form a system of lacunae where haematopoietic cells are organized . These CD271+ cells were also positive for alkaline phosphatase, vimentin, CD13 but negative for CD34, CD45 and CD68. Interestingly, CD271+ stromal cells originate from vessels and ingrow within BM . Additional markers for the prospective isolation of nMSCs have been described recently, such as the neural glanglioside GD2 and the embryonic membrane molecule SSEA4 [56, 57]. Unfortunately, for the two latter markers no data exist on the enrichment factor obtained after selection, which does not allow for comparison with previous works. However, in contrast to cells from the GD2+, SSEA4+ or CD200+ fraction, CD49a+ nMSCs and CD271+ nMSCs express at a low level the pan-leucocyte marker CD45, which rapidly disappears when cells are cultured . This discrepancy needs further clarification to verify whether nMSCs do actually express CD45 at a low level, similarly to BM haematopoietic stem cells, or whether this is simply due to an experimental artefact. Finally, the CD146 molecule (an immunoglobulin protein also named MUC18 or S-endo) has been convincingly proposed as a marker of multipotent nMSCs . Immunohistochemical studies ascribed a sub-endothelial localization to CD146+ cells in the BM and, consistent with this finding, CD146+ nMSCs expressed multiple mural cell-specific molecules such as NG2, αSM-actin and calponin 1 and 3. In contrast to CD271+ or CD49a+ cells, CD146+ cells were CD45–. In addition, CD146+ cells were found to express very few osteogenic molecules but acquired these additional markers when induced to differentiate into osteoblasts or when transplanted in SCID mice with hydroxyapatite/βtricalciumphosphate (HA/βTCP) particles used as a scaffold. Interestingly, when CD146+ cells were transplanted, they could self-renew since they could generate multipotent CD146+ perivascular cells in serial transplantations. In contrast, cells in which CD146 was down-regulated (i.e. upon culture with the basic FGF), or silenced, no self-renewal was observed. Therefore, this is the first time that multipotential nMSCs have been defined as a specific population of perivascular cells with self-renewal capacities (Fig. 2). This location can explain the possibilities to obtain CFU-Fs from several tissues such as skeletal muscle, pancreas, adipose tissue, brain, spleen, liver, lung and thymus . Recently, Crisan et al. demonstrated that all nMSCs were of pericytic origin, but not all pericytes were nMSCs. Interestingly, sorted CD146+ perivascular cells, from CD34– and CD45– fractions (to exclude both endothelial and haematopoietic cells) of muscle and non-muscle tissues, displayed myogenic potential in vitro and in vivo. Regardless of their tissue origin, sorted pericytes subsequently cultured in cMSC conditions displayed markers of cMSCs (i.e. positive for CD44, CD73, CD90, CD105, CD166 and SSEA4 but negative for CD45, CD34, CD31 and CD144) and could also differentiate into chondrocytes, adipocytes and osteocytes. Nevertheless, the CD146 molecule was also shown to be expressed by other regenerative cells that have strong importance in bone healing: the EPCs . Therefore, investigating the role of the CD146 molecule in these settings would be of interest.
In addition to these recent reports, the perivascular origin of nMSCs is supported by other observations as well. In this context bona fide pericytes were previously shown to be multipotent cells because they were able to differentiate into osteoblasts, chondroblasts and adipocytes [62, 63]. Furthermore, clearly defined smooth muscle cells (αSM-actin+, caldesmon+ and myosin heavy chain+) from bovine aortic media have been described to undergo osteoblastic and chondroblastic differentiation when cultured appropriately . These observations could explain the calcification of vessels during atherosclerosis. The mechanisms of such vascular calcification seem to resemble those taking place during bone formation, because BMP2 and its target genes Runx2, osteocalcin and osteopontin were observed within atherosclerotic lesions [65, 66]. Moreover, inflammatory cytokines in aorta induce BMP2, which then promotes signalling through a muscle segment homeobox homolog (Msx2)/Wnt pathway leading to increased alkaline phosphatase activity and osteogenic differentiation . Interestingly, Msx2 was shown to be a regulatory factor for VSM differentiation in one hand and osteoblastic cell differentiation on the other hand [68, 69]. This latter finding suggests that whatever their tissue origin, nMSCs can retain molecular programs to generate either smooth muscle cells or osteo-chondroblastic cells, and such fine-regulation mechanisms can be disturbed in lesion and pathologic situations. This finding fits well with the description of mural mesodermal progenitors capable of generating either skeletogenic (osteoblasts, chondroblasts and adipocytes) or myogenic cells . The decision to give rise to one or the other differentiation pathway depends on molecular environmental influences.
The pericytic identity of nMSCs further suggests that nMSCs may reach the tissue from invading vessels during early events of bone formation. This process was shown to occur through the CD166 molecules which were found to be highly expressed by nMSCs from the perichondrium in the foetus. Activated leucocyte cell adhesion molecule (ALCAM), or CD166, is a membrane molecule that we and others found to be important for haematopoiesis-supporting stromal cells [71, 72]. Purified ALCAM+ cells could support haematopoiesis, osteoclastogenesis, and angiogenesis. Interestingly, in vitro inhibition of homophilic (ALCAM/ALCAM) and heterophilic (ALCAM/CD6) ALCAM-mediated adhesion prevented the blood vessel invasion into cartilage. It is likely that such an effect may be associated with ALCAM involvement in endothelial cell development probably through immature EPCs . In addition, Arai et al. first characterized nMSCs as cells capable of generating endochondral formation in the human foetus .
In contrast, when adult fracture healing is concerned, the origin of nMSCs, contributing to bone regeneration is still controversial since these cells are known to reside in a number of surrounding tissues, such as the periosteum, BM, synovium and trabecular bone [18, 74–77]. Native MSCs were also thought to be derived from surrounding skeletal muscle. This suggestion is a real possibility because MSCs might be obtained from multipotential myoblastic cells such as the well-known C2C12 murine cell line . However, during secondary fracture healing, the primary source of nMSCs giving rise to the callus is thought to be periosteum, notably because (i) callus development after fracture is strongly disturbed when the periosteum is removed and (ii) the periosteum produces BMPs during early events following fracture [79, 80]. Furthermore, to date convincing data support the presence of functional nMSCs within the periosteum that have strong proliferative and osteogenic capabilities in vitro and in vivo. Here also, vascularization is a crucial event for the initiation and propagation of the bone formation deriving from the periosteum. We can therefore suppose that periosteal nMSCs are located in the vessels and are induced to proliferate and differentiate into osteoblasts after fracture after vascular ingrowth into developing callus (Fig. 3).
To date, no data exist on the strict purification of nMSCs (i.e. one selected cell generating one multipotent CFU-F). Furthermore, heterogeneity is known to exist between CFU-Fs in terms of multipotentiality, and no observational tests exist to discern differences among them [18, 82, 83]. Finally, crucial questions remain to be elucidated: (i) are nMSCs or cMSCs true multipotent stem cells or are they more committed progenitors? (ii) In a pool of enriched nMSCs or cMSCs, does a hierarchy exist as that found in haematopoiesis (i.e. stem cells versus progenitors versus precursors versus mature cells)? Therefore, the identity of nMSCs remains obscure, and their characterization is undoubtedly crucial for understanding bone biology and its abnormalities (Table 1). Finally, since cMSCs derive from nMSCs, it is important that bone-reconstituting studies comparing both populations should also be performed, to prospectively evaluate their clinical outcome.