Electron Microscopy. At the service of vascular stem cells research.

In the last few years, we and other few groups have built a solid scientific background aimed at demonstrating the existence of resident stem cells in adult large and medium-sized human arteries. These cells are engaged in the regulation of normal arterial wall homeostasis but under stressful circumstances they are also supposed to contribute to arterial wall disease. In our experience, electron microscopy (EM) has been of great help in solving several critical issues that have arisen in the execution of research; further EM was invaluable in making critical decisions for choosing the best method for subsequent study of proteins and transcripts. In this study, we report some of the situations where we feel that EM plays a major role in vascular stem cell research. Characterization of the primary cell isolate. There are numerous methods used for the isolation of cells from the arterial wall. The more selective make use of magnetic beads coated with specific antibodies directed against surface molecules selectively expressed by the cell population of interest. In the case of CD34 positive populations EM has allowed us to verify how the cells isolated by this method are homogenous. For other investigation purposes, methods of isolation making use of enzymes are more appropriate; in this case, it is often necessary grow cells in suspension or in adhesion to plastic. Before establishing of culture and during the early passages (1-3), EM shows that cell isolates are composed of several cytotypes which are then lost in the course of the next passages. The selection of, for example, mesenchymal stem cells (MSC) that is achieved by cell adhesion to plastic, is never absolute and this raises doubts about the true differentiation potential of these cells. Characterization of resident vascular wall-mesenchymal cells (vw-MSC). The MSC are considered necessary to keep the architecture of the stem cell niche and to maintain in good health more embryonic-like stem cells and progenitors. MSC are currently identified by using a panel of monoclonal antibodies against surface molecules, i.e., CD44, CD90, CD105, CD166. EM allows to reveal the subcellular morphology of the MSC that is mesenchymal in nature and to assume, for comparison with the normal cell architecture of the arterial wall, the putative anatomical location of the niche stem. Our studies have thus allowed us to focus attention on the adventitia and in particular on the vasa vasorum compartment. Identification of the stem cell niche. The vasa vasorum constitute a branched vascular system that has the function of oxygenate and nourish the arterial wall. The vasa vasorum can be distinguished on the basis of their size in first order vasa vasorum, with a diameter greater than 100 micrometers, and second order vasa vasorum, with a diameter of less than 100 micrometers. These classes of vasa vasorum have a similar cell composition with endothelial cells and pericytes. By means of immunohistochemistry and monoclonal antibodies against nestin, an intermediate filament originally seen in brain stem cells, and its promoter WT1, we have observed that some vasa vasorum cells intensely co-express these two molecules; these vasa vasorum are collected in hot spots placed within a particular organoid structure that aggregates nerva vasorum, arterioles, venules and less than 50 micrometers-sized vasa vasorum. Using EM, we observed prominent perivascular collections of spindle cells with extremely thin and long cytoplasmic projections which establish functional contacts between structures belonging to different morphological and functional compartments. These cells have the potential to correspond to the MSC that we isolate from the arterial wall. This organoid adventitia area may represent the putative vascular stem cell niche. Establishing the differentiating potential of MSC. To exploit the differentiating potential and plasticity of MSC classical histochemical methods, i.e. oil red O, von Kossa and Alcian-PAS staining, are frequently employed. In our opinion EM is the gold standard to validate adipogenic and osteo-chondrogenic differentiation protocols. Features of early subcellular mesengenic differentiation can be derived from the past knowledge that pathologists have accumulated studying poorly differentiated sarcomas.