Several published studies on this topic

Several published studies on this topic provide insights into the relationship between the two cell types but there is still a limited knowledge concerning the proliferation behavior of cells in coculture. For a detailed analysis on cell proliferation in coculture, immunomagnetic beads coupled with an anti-CD31 antibody were used to separate both cell types. This technique allowed confirming that both cell types were proliferating, although with significantly different growth rates, being it higher for MSCs. This difference in cell proliferation could explain the different percentage of both cell types in coculture (Fig. 2). Moreover, MSCs growing in coculture (CoMSC) showed a clearly higher cell growth rate as compared with MSCs in monolayer. On one hand, this result points towards the potential of HUVECs to stimulate MSCs proliferation. On the other hand, MSCs could curtail ECs capability for expansion due to their high proliferative capacity in coculture. Notwithstanding that HUVECs in monoculture presented a higher growth rate than in coculture, CoHUVECs proliferated for a longer period than HUVECs alone. This ruled out the possibility that CoHUVECs may be dying since, in fact, there was an increases in cell number. Noteworthy, in monoculture when endothelial cells are maintained at confluence for an extended period of time, they become tightly packed but show no tendency to overlap or overgrow. Then, when cell density becomes to high, cells start to detach and die (Marin et al., 2001). Another parameter that could influence cell proliferation was the extracellular matrix produced, since an increase on one of its major components was detected (upregulation of collagen type I). In parallel, a study was perform to validate that the significant increase in CoMSC proliferation was indeed related with HUVECs presence, and not with differences in terms of cell seeding densities (CoMSCs cell seeding density was 1500 cells/cm2 instead of 3000 cells/cm2 as the MSC monoculture). For that purpose MSCs were seeded alone at 1500 cells/cm2, which allow comparing with CoMSC of the same real “age” (having gone through the same cell divisions) during the 21days of culture (Fig. 2C). Although MSCs at lower cell density presented a higher proliferation rate in the first weak, in the following two weeks CoMSCs continued to exhibit the highest proliferation (Fig. 2C), corroborating the potential of HUVECs to stimulate MSCs proliferation. It is well established that the cytoskeleton plays important roles in cell morphology, adhesion, growth and signaling. The dapt secretase network, one of the three components of the cytoskeleton, is of critical importance in the determination of the mechanical properties of living cells. In this study, it was shown that the actin cytoskeleton changes from an apparently well-organized structure, with long fine fibers running in parallel along the cell axis in MSCs monocultures, to a more random arrangement of the actin cytoskeleton in cocultures (Fig. 3A). Yourek and colleagues observed this type of cytoskeleton changes upon osteogenic differentiation (Yourek et al., 2007). They further stated that this reorganization could be related with the natural reaction of the actin cytoskeleton in bone cells to the shear stress that occurs in vivo during bone modeling/remodeling. Grellier et al. (2009a) also observed a similar cell rearrangement in osteoprogenitor cells (HOPs) cocultured with ECs (Grellier et al., 2009a). They further observed HUVECs migration along HOPs and suggested that the direct contact between the two cell types could stimulate the release of chemotactic factors that, in turn, would stimulate HUVEC migration. To support the hypothesis that cytoskeleton changes of CoMSC could be related with their osteogenic differentiation in the presence of endothelial cells, further studies were made. Consequently, ALP cytochemistry and activity, and also gene expression analyzes of osteogenic markers, were performed. ALP activity is considered an early osteogenic marker since its expression increases from the beginning of cell differentiation and increases throughout extracellular matrix maturation (Stein et al., 1990). As shown in Fig. 3B there was an increased in the number of ALP stained cells in coculture comparing to monoculture even after only 3days, suggesting a commitment of CoMSCs into the osteogenic lineage. The ALP activity profiles and gene expression were assessed and both support the previous data (Figs. 3C and 4B). The gene profile of type I collagen, a protein of the extracellular matrix and the main organic component of bone tissue, was also higher in CoMSC (Fig. 4A). In order to further analyze osteogenic commitment of CoMSC, gene expression of two other early markers of osteogenic differentiation, Runx2 and bone sialoprotein (BSP), was evaluated by real time quantitative RT-PCR assay. Runx2, is an earlier transcription factor proven essential for commitment to osteoblatogenesis. BSP is a major non-collagenous extracellular matrix protein in bone and promotes the initial formation of mineral crystals (Ganss et al., 1999; Zhang et al., 2009). Both genes were significantly upregulated, which further confirms the differentiation of CoMSC along the osteoblastic lineage (Fig. 4C). Other gene that was investigated was osteocalcin (OCN) that is a noncollagenous calcium-binding bone protein and is considered a late-stage osteogenic differentiation marker (Ilmer et al., 2009). OCN expression did not increase and, even after 21days, there was no upregulation. Overall, the genetic profile obtained points towards an osteogenic commitment of MSCs culture in the presence of ECs, but cells were not fully mature.