Overall, this report provides a transcriptional map of immunomodulatory MSCs through global RNA profiling and bioinformatics-based comparative analysis with several other mouse tissues and cell types. By analyzing arrays from the same platform (Affymetrix 430) we were able to perform statistical analyses combining results across different laboratories. Thus, we overcame the limitation of comparisons across different platforms, generally regarded as problematic from a biological perspective because the measurements may represent different physical quantities . Conversely, the approach used in this study offers a more precise assessment of the true degree of differential expression and helps refine the list of candidate genes warranting biological validation.
By applying principal component and hierarchical clustering analyses, we identified a distinctive gene expression profile for MSCs. MSC gene expression profile appeared in fact to be characteristic and substantially homogeneous. One of the most significant findings of this analysis was the nearly perfect sorting of tissues by germinal layer of origin utilizing an unsupervised classification. As expected, being MEFs derived from MSCs, their expression profile was highly similar. Furthermore, previous studies have shown that cultured MEFs maintain some of the multipotency of MSCs . Surprisingly, MSCs and DCs displayed a similar profile as well, a finding further confirmed by qRT-PCR analysis. We speculate this could be due to the fact that both MSCs and DCs are involved in the determination of bystander cell fate, respectively HSCs and T cells, by cell-cell contact-dependent mechanisms.
When focusing on the list of differentially expressed genes we found that many of them had been reported in previous transcriptional studies of MSCs, thus validating the sensitivity and reproducibility of our approach [18, 23]. Among MSC specific genes, several proteins belonging to the Wnt signaling pathway or Wnt-regulated genes (Sfrp1, Dvl2, Axin1, Tcf3, Rarg, Cspg2, Efnb2) were detected. Wnt proteins have been previously reported to promote myogenesis , inhibit chondrogenesis  and adipogenesis  and to have a dual effect on osteogenesis . Some of the most abundantly expressed genes are also inhibitors (Sfrp1, Sfrp2, Dkk3) or act downstream (Thbs1, Thbs2, Cyr61, Sema3a, Sfrp2) of the Wnt signaling pathway . Among the differentially expressed genes we were also able to find most recently described molecules involved in stem cell renewal such as Dkk3  and Serpinf1 (PEDF) . Altogether, these results are consistent with the involvement of Wnt signaling in mesenchymal lineage specification.
We also used GO-based functional classification to characterize both differentially expressed and MSC specific genes. This analysis confirmed our primary observation that differentially expressed transcripts were significantly enriched in molecules present in the extracellular space and involved in cell adhesion process. On the other hand, MSC specific genes were mainly nucleoplasm-located transcription factors involved in development. Importantly, the observation that these transcription factors are expressed at low levels is consistent with most recent findings about the biology of ESCs. These cells have been recently shown to express low levels of transcription factors by means of the polycomb group proteins and by specific chromatin modification patterns thus keeping them poised for activation during development . It can be speculated that a similar mechanism may be working in further committed stem cells such as MSCs.
The combined transcriptional and comparative analyses presented here allowed us to identify several secreted molecules that may be critical in determining cell functions. Several of these molecules have been previously reported to play a pivotal role in the constitution of the HSC niche synapse  or as being expressed by hematopoiesis-supportive stromal cells  [See Additional file 4]. For example, angiopoietin-1 (Angpt1) is considered one of the major regulators of blood vessel stability and, in contrast with angiopoietin-2 (Angpt2), it seems to exert a potent anti-inflammatory effect . Moreover, Angpt1 and its receptor endothelial-specific receptor tyrosine kinase(Tek) are known key components of the HSC niche, namely of the cell-cell signaling between osteoblasts and HSCs. Furthermore, Angpt1 is likely to be one of the most important molecules in the induction of the "quiescent" state of the HSCs, by enhancing their tight adhesion to osteoblasts, inhibiting cell division, arresting cells in the G0 phase of the cell cycle and promoting cell survival under myelosuppressive stress .
Osteopontin (Spp1) is a highly acidic phosphoprotein with pleiotropic effects, including regulation of inflammation, cell adhesion and angiogenesis . In addition, it has recently been demonstrated to be a pivotal molecule in limiting the size of the HSC pool . Similarly, it has also been shown that the thrombin-cleaved form of Spp1 promotes the quiescence of HSCs by exerting a profound suppression of proliferation of HSCs without inducing apoptosis . Interestingly, Spp1-deficient mice developed a milder form of EAE, and activated T cells from these animals produced less IFNγ and more IL-10 than their wild type counterparts consistently with the role of a pro-inflammatory molecule . Although an elevated Spp1 expression in therapeutic MSCs is in apparent contradiction with its previously reported proinflammatory role, its pleiotropism and ubiquitousness suggest that it may interact with different receptors at different times and through different regulatory networks, thus preventing a straightforward characterization of its biological functions.
Thrombospondin (Thbs)-1 and -2 are extracellular matrix proteins that share several effects on cell growth, adhesion, survival and differentiation. In particular, Thbs1, secreted by DCs, has been demonstrated to inhibit both TCR signaling and T cell proliferation, mainly acting through the CD47 receptor . In addition, Thbs1 has also been described as part of the HSC niche . Both Thbs1 and, to a lesser extent, Thbs2 have been studied as inhibitors of endothelial growth . On the other hand, Thbs2 has been described as an autocrine inhibitor of proliferation secreted by MSCs which acts through cell-cycle inhibition without the induction of apoptosis .
Fibronectin-1 (Fn1) is another major component of the bone marrow stroma. The interaction of Fn1 with β1-integrins, VLA-5 in particular, has been shown to be a negative regulator of HSC proliferation by preventing them from entering the cell cycle .
Galectin-1 (Lgals1) is an endogenous lectin characterized by its affinity for beta-galactosides which has been demonstrated to exert an immunosuppressive effect on T cells through both apoptotic and non-apoptotic mechanisms . This molecule has been shown to be expressed by hematopoietic-related tissues  and to inhibit HSC growth particularly at high concentrations . Most recently, Lgals1 has been shown to play a role within the NSC niche . We were able to confirm MSC expression of this molecule both by ELISA and immunocytochemistry assays.
Finally, semaphorins are molecules that were originally identified as mediators of axon guidance but have also been described as part of the immunological synapse . Semaphorin 3A (Sema3a), in particular, has also been recently demonstrated to mediate T cell proliferation inhibition by arresting T cells in the G0/G1 phase of the cell cycle .