3D vascular-spheres of human mesenchymal stem cells derived from arteries.

Mesenchymal Stem Cells (MSCs) are adult multipotent cells identified within several human tissues including arteries. Besides the fibroblast-like morphology, growth in adhesion and mesenchymal phenotype, MSCs have distinctive features as self-renewal, differentiation in multiple mesengenic lineages under appropriate stimuli and immunomodulatory properties. Another well-known MSC characteristic is the capacity to grow in a non-adherent condition as spheres. Here, we examined the attitude of MSC harvested from human vascular wall (hVW-MSCs) to form spheres when cultured in a 3D cell culture model; a phenotypic, molecular, morphological and metabolomic characterization of vascular-spheres was also provided. hVW-MSCs were enzymatically isolated from human femoral arteries and in vitro expanded in a 2D culture model. To generate vascular-spheres, ultralow attachment and hanging drops techniques were employed. Immunophenotype and pluripotent genes were respectively analyzed by immunohistochemistry and Real Time PCR; morphology and ultrastructural organization through histology and electron microscopy; cell viability and proliferation with LIVE/DEATH and ki-67 proliferation marker, respectively; metabolomic profile using liquid chromatography - mass spectrometry. In 2D cultures, hVW-MSC cells displayed an elongated morphology with mesenchymal antigens and multi lineage differentiation potential. In 3D cultures, hVW-MSCs spontaneously formed floating vascular-spheres with CD44+, CD105low, CD90low profile expressing SOX2, OCT4 and NANOG stemness genes. In histology, vascular-spheres were organized in multiple layers of elongated cells enclosing round cells. Specifically, the outer layer was composed of undifferentiated spindle cells with poor organelle content while the core was formed by rounded cells with a loosely arranged as observed in electron microscopy. Additional ultrastructural investigation of vascular-spheres highlighted the presence of a complex cell to cell communication system including nanotubular projections and multi-vesicular bodies enclosing exosomes. Furthermore, living cell were disposed in the outer layers, whereas dead ones in the core. Interestingly, hVW MSCs spheres acquired a quiescent status as documented by low the percentage of ki-67+ cells and slowed metabolism for the increased accumulation of intermediate metabolites. Taken together, these data proved that vascular-spheres of hVW-MSCs possess characteristics commonly found in stem niche as stemness, quiescence and reduced metabolic activity. This in vitro 3D model can be improved to deep the knowledge on hMSCs properties for future application in medical field.