Murine VSEL cells were first described as a CD45−Lin−Sca-1+ population in adult murine bone marrow (mBM) that showed high transcript levels of pluripotency genes, a nuclear localization of both Oct 4 and Nanog proteins, a demethylated Oct4 promotor, an overall euchromatic nuclear organization, a quiescent status that is maintained by a unique methylation pattern of certain imprinting genes, and an in vitro differentiation potential into neuronal, pancreatic, and cardiomyocyte-like cells representative of ectoderm, endoderm, and mesoderm, respectively (2, 11). The putative pluripotency of murine VSEL cells remains based on these preliminary findings, there having been in the meantime no demonstration of teratoma formation, tissue contribution following blastocyst injection or multilineage differential at a clonal level. Thus, the gold standards of pluripotent stem cell function have still not been demonstrated, and the mere detection of pluripotency-associated markers at mRNA and protein level must be regarded as an insufficient basis from which to claim pluripotency, especially as the assays used to detect the markers are prone to artifacts such as false-positives (7, 8, 12). While the case for pluripotency and quiescence of VSEL cells has not been convincingly made, murine VSEL cells have indeed been shown to have proliferation capacity and differentiation potential in vitro (2, 13). Furthermore, murine VSEL cells are able to form bone tissue in vivo, and may contribute to heart regeneration (14, 15), although the same cells failed in hematopoietic reconstitution assays and in teratoma formation assays (2, 11). Most recently, a population similar to murine VSEL cells has been identified in rat bone marrow and has been shown both to expand and differentiate in vitro and to contribute to heart regeneration in vivo (16). These data are not definitive proof of a specific adult stem cell function and are not compatible with the pluripotent and quiescent status claimed for murine VSEL, but they would be consistent with a bone marrow VSEL population containing mesenchymal stem or progenitor cells with in vivo regenerative potential.
Interestingly, supporting evidence in this respect has been provided by an independent Japanese group with the identification of a mesenchymal precursor overlapping with the CD45−Lin−Sca-1+ VSEL cell population (Fig. 1) (17). The CD45−Ter119−Sca-1+PDGFR-α+ (PαS) cells give rise to multipotent mesenchymal stromal cells in vitro, and are able to engraft in bone marrow when transplanted i.v. into irradiated mice. Furthermore, it was shown that at least some of the PαS cells originate from the neural crest and can give rise to neuronal cell types in vitro, suggesting that there may be a mixed mesodermal/ectodermal potential in the PαS population (18). Consistent with this, it has been shown that Nestin, a marker previously associated with neuronal stem cells, specifies the entire mesenchymal potential of the CD45− bone marrow population (19, 20). The fact that mesenchymal stem cells with very similar properties have been independently isolated from PαS cells and from CD45−Nestin+ cells suggests a degree of overlap also between these populations.
Figure 1. Heterogeneity of the murine VSEL cell population. The gating of VSEL cells shows that ∼60% of the VSEL cells express PDGF-Rα, thus resembling the PαS phenotype. A: Gating of Lin− mBM cells (CD5−, CD45R−, CD11b−, Ly-6G/C−, 7-4−, Ter-119−; lineage cell depletion kit, Miltenyi Biotec). B: Lin− cells: Gating of CD45-Sca-1+ VSEL cells. C: Lin−CD45+ cells: gating of c-kit+Sca-1+ hematopoietic stem and progenitor cells (KSL). D: Expression of PDGF-Rα in KSL cells and VSEL cells. Further experimental details are provided as Supporting Information.
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In summary, these examples demonstrate: (1) that mBM VSEL cells do contain multipotent mesenchymal stem cells, (2) that the mBM VSEL population is heterogeneous and currently insufficiently characterized, and (3) that an extra-mesenchymal differentiation potential of the VSEL population (into the neuronal lineage in this case) can be explained without invoking a pluripotent stem cell. The set of recently identified markers that allow a prospective isolation of mesenchymal stem cells from mBM should also help to clarify the true nature of the murine VSEL population.