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The embryonic endothelium is a known source of hematopoietic stem cells. Moreover, vessel-associated progenitors/stem cells with multilineage mesodermal differentiation potential, such as the 'embryonic mesoangioblasts', originate in vitro from the endothelium. Using a genetic lineage tracing approach, we show that early extra-embryonic endothelium generates, in a narrow time-window and prior to the hemogenic endothelium in the major embryonic arteries, hematopoietic cells that migrate to the embryo proper, and are subsequently found within the mesenchyme. A subpopulation of these cells, distinct from embryonic macrophages, co-expresses mesenchymal and hematopoietic markers. In addition, hemogenic endothelium-derived cells contribute to skeletal and smooth muscle, and to other mesodermal cells in vivo, and display features of embryonic mesoangioblasts in vitro. Therefore, we provide new insights on the distinctive characteristics of the extra-embryonic and embryonic hemogenic endothelium, and we identify the putative in vivo counterpart of embryonic mesoangioblasts, suggesting their identity and developmental ontogeny.

Mesoangioblasts have been characterized as a population of vessel-associated stem cells able to differentiate into several mesodermal cell types, including skeletal muscle. Here, we report that the paired box transcription factor Pax3 plays a crucial role in directing mouse mesoangioblasts toward skeletal myogenesis in vitro and in vivo. Mesoangioblasts isolated from the aorta of Pax3 null embryos are severely impaired in skeletal muscle differentiation, whereas most other differentiation programs are not affected by the absence of Pax3. Moreover, Pax3(-/-) null mesoangioblasts failed to rescue the myopathic phenotype of the alpha-sarcoglycan mutant mouse. In contrast, mesoangioblasts from Pax3 gain of function, Pax3(PAX3-FKHR/+), mice display enhanced myogenesis in vitro and are more efficient in regenerating new muscle fibers in this model of muscular dystrophy. These data demonstrate that Pax3 is required for the differentiation of mesoangioblast stem cells into skeletal muscle, in keeping with its role in orchestrating entry into the myogenic program.

Mesoangioblasts are stem cells capable of differentiating in various mesodermal tissues and are presently regarded as suitable candidates for cell therapy of muscle degenerative diseases, as well as myocardial infarction. The enhancement of their proliferation and survival after injection in vivo could greatly improve their ability to repopulate damaged tissues. In this study, we show that the bioactive sphingolipid sphingosine 1-phosphate (S1P) regulates critical functions of mesoangioblast cell biology. S1P evoked a full mitogenic response in mesoangioblasts, measured by labeled thymidine incorporation and cell counting. Moreover, S1P strongly counteracted the apoptotic process triggered by stimuli as diverse as serum deprivation, C2-ceramide treatment, or staurosporine treatment, as assessed by cell counting, as well as histone-associated fragments and caspase-3 activity determinations. S1P acts both as an intracellular messenger and through specific membrane receptors. Real-time polymerase chain reaction analysis revealed that mesoangioblasts express the S1P-specific receptor S1P3 and, to a minor extent, S1P1 and S1P2. By using S1P receptor subtype-specific agonists and antagonists, we found that the proliferative response to S1P was mediated mainly by S1P2. By contrast, the antiapoptotic effect did not implicate S1P receptors. These findings demonstrate an important role of S1P in mesoangioblast proliferation and survival and indicate that targeting modulation of S1P-dependent signaling pathways may be used to improve the efficiency of muscle repair by these cells. Disclosure of potential conflicts of interest is found at the end of this article.

Early activation of myogenesis in the somite depends on signals from surrounding tissues. Canonical beta-catenin dependent Wnt signalling preferentially activates Myf5. We now show, in explant experiments with presomitic mesoderm, that the expression of another myogenic determination factor, MyoD, depends on non-canonical Wnt signalling, probably emanating from the dorsal ectoderm. Inhibitors of PKC block MyoD expression, indicating that the intracellular Wnt pathway depends on this kinase. In the absence of Myf5 and Mrf4, this activation is only minorily affected and we identify Pax3 as the transcriptional mediator responsible for MyoD expression. When embryos expressing a constitutively active form of Pax3, PAX3-FKHR, are used for these studies in the presence of PKC inhibitors, MyoD expression is not affected, suggesting that Wnt signalling acts on the transcriptional activity of Pax3.

The molecular regulation of smooth muscle differentiation is currently far less well understood than that of striated muscle, in part because in this cell type, the differentiated state is plastic and reversible. In recent years, however, several molecules, the best characterized of which is myocardin, have been shown to be necessary and sufficient to promote at least partial smooth muscle differentiation. Indeed, mice deficient in myocardin have a severe reduction of smooth muscle tissue. However, possibly because of multiple embryological origins, which include mesenchyme, neural crest, and even endothelium, different types of smooth muscle cells differ in their expression of myocardin and of other potential regulatory molecules. Here, we will review recent work on the topic, focusing on the mesoangioblast, a recently described vessel-associated stem cell, whose differentiation into smooth muscle is dependent upon expression of msx2 and necdin, but not of myocardin.

Mesoangioblasts are vessel-derived stem cells that can be induced to differentiate into different cell types of the mesoderm such as muscle and bone. The gene expression profile of four clonal derived lines of mesoangioblasts was determined by DNA micro-array analysis: it was similar in the four lines but different from 10T1/2 embryonic fibroblasts, used as comparison. Many known genes expressed by mesoangioblasts belong to response pathways to developmental signalling molecules, such as Wnt or TGFbeta/BMP. Interestingly, mesoangioblasts express receptors of the TGFbeta/BMP family and several Smads and, accordingly, differentiate very efficiently into smooth muscle cells in response to TGFbeta and into osteoblasts in response to BMP. In addition, insulin signalling promotes adipogenic differentiation, possibly through the activation of IGF-R. Several Wnts and Frizzled, Dishevelled and Tcfs are expressed, suggesting the existence of an autocrine loop for proliferation and indeed, forced expression of Frzb-1 inhibits cell division. Mesoangioblasts also express many neuro-ectodermal genes and yet undergo only abortive neurogenesis, even after forced expression of neurogenin 1 or 2, MASH or NeuroD. Finally, mesoangioblasts express several pro-inflammatory genes, cytokines and cytokine receptors, which may explain their ability to be recruited by tissue inflammation. Our data define a unique phenotype for mesoangioblasts, explain several of their biological features and set the basis for future functional studies on the role of these cells in tissue histogenesis and repair.

In 2002 we published an article describing a population of vessel-associated progenitors that we termed mesoangioblasts (MABs). During the past decade evidence had accumulated that during muscle development and regeneration things may be more complex than a simple sequence of binary choices (e.g., dorsal vs. ventral somite). LacZ expressing fibroblasts could fuse with unlabelled myoblasts but not among themselves or with other cell types. Bone marrow derived, circulating progenitors were able to participate in muscle regeneration, though in very small percentage. Searching for the embryonic origin of these progenitors, we identified them as originating at least in part from the embryonic aorta and, at later stages, from the microvasculature of skeletal muscle. While continuing to investigate origin and fate of MABs, the fact that they could be expanded in vitro (also from human muscle) and cross the vessel wall, suggested a protocol for the cell therapy of muscular dystrophies. We tested this protocol in mice and dogs before proceeding to the first clinical trial on Duchenne Muscular Dystrophy patients that showed safety but minimal efficacy. In the last years, we have worked to overcome the problem of low engraftment and tried to understand their role as auxiliary myogenic progenitors during development and regeneration. [ABSTRACT FROM AUTHOR]

Different cells can contribute to repair following vascular injury by differentiating into smooth muscle (SM) cells; however the extracellular signals involved are presently poorly characterized. Mesoangioblasts are progenitor cells capable of differentiating into various mesoderm cell types including SM cells. In this study the biological action exerted by the pleiotropic sphingolipid sphingosine 1-phosphate (S1P) in human mesoangioblasts has been initially investigated by cDNA microarray analysis. Obtained data confirmed the anti-apoptotic action of this sphingolipid and identified for the first time a strong differentiating action toward SM cells. Quantitative mRNA and protein analysis corroborated the microarray results demonstrating enhanced expression of myogenic marker proteins and regulation of the expression of transcription factor GATA6 and its co-regulator, LMCD1. Importantly, GATA6 up-regulation induced by S1P was responsible for the enhanced expression of SM-specific contractile proteins. Moreover, by specific gene silencing experiments GATA6 was critical in the pro-differentiating activity of the cytokine TGFβ. Finally, the pharmacological inhibition of endogenous S1P formation in response to TGFβ abrogated GATA6 up-regulation, supporting the view that the S1P pathway plays a physiological role in mediating the pro-myogenic effect of TGFβ. This study individuates GATA6 as novel player in the complex transcriptional regulation of mesoangioblast differentiation into SM cells and highlights a role for S1P to favour vascular regeneration. [ABSTRACT FROM AUTHOR]