br Materials and methods br Results br Discussion

Materials and methods
Results
Discussion Strategies aiming at controlled divergence of ASCs to specific differentiated cell types essentially rely on prolonged culture in the presence of specific sets of growth factors and/or matrix proteins (Zuk et al., 2001). Specific culture conditions were reported to induce subpopulations of ASCs to express endothelial markers as PECAM1 (CD31) (Miranville et al., 2004), Tie2 (Konno et al., 2010), KDR and CD34 (Wosnitza et al., 2007; Konno et al., 2010). Reprogramming of fibroblasts to pluripotency, or direct conversion of fibroblasts to differentiated, functional cells, is based on transfer of specific sets of transcription factors (Takahashi & Yamanaka, 2006; Vierbuchen et al., 2010; Ieda et al., 2010). Human neonatal fibroblasts were transdifferentiated to endothelial cells, applying transduction of the induced pluripotent stem cell-factors Oct4 and Klf4 under inductive signaling conditions (Li et al., 2013). Here, we report on an approach that aims at controlled in vitro induction of endothelial features in ASCs by combining optimized culture conditions described by Konno et al. (2010) with direct reprogramming of the calcium sensing receptor using SOX18, an endothelial-specific transcription factor that plays a key role in endothelial differentiation and development of the vasculature (François et al., 2010). We could confirm that under the growth conditions applied, human ASCs show expression of the endothelial markers CDH5, PECAM1 and TIE2 at low levels (<0.4%) compared to primary endothelial cells. Additional transduction of ASCs with SOX18 tended to further increase expression of these genes, however, this increase did not reach significant levels. CLDN5, a gene encoding the endothelial-specific tight junction protein claudin 5 and earlier described by our group as a direct target gene of SOX18 in HUVEC (Fontijn et al., 2008), showed significant upregulation upon SOX18 transduction in ASCs. Final mRNA levels amounted to a fraction of the levels observed in HUVEC and the protein could not be detected using immunofluorescence. In contrast, we found that SOX18 transduction specifically induced high expression levels of CD34 and KDR. Apart from their function in vasculogenesis and angiogenesis, KDR and CD34 are considered markers that occur in endothelial differentiation as early as the hemangioblast-stage. Their appearance, upon SOX18 transduction, together with the vascular patterning genes MMP7, EFNB2, SEMA3G and CXCR4, shows that in ASCs, ectopic expression of SOX18 is sufficient to induce a subset of genes involved in endothelial differentiation and vasculogenesis. The limited expression of markers of mature endothelial cells indicates that for further endothelial differentiation additional transcriptional programming, complementary to SOX18, is required. Here, it should be noted that in our experiments, SOX18 was constitutively expressed from a viral promoter, rather than from the native promoter which is likely subject to regulatory mechanisms. In vivo, the function of SOX18 in endothelial differentiation and vascular development has proven difficult to dissect, due to the redundant function of members of the SOXF subfamily: SOX18, SOX17 and SOX7. Vascular anomalies observed in different animal models, including the naturally occurring ragged allelic mutants in mouse (Pennisi et al., 2000a; Downes et al., 2009), SOX18-null mouse (Pennisi et al., 2000b; François et al., 2008) and subsequent combined SoxF knock outs in mouse and zebra fish (Sakamoto et al., 2007; Herpers et al., 2008; Pendeville et al., 2008; Cermenati et al., 2008; Matsui et al., 2006; Sacilotto et al., 2013) point at the crucial roles of SOX18 in endothelial differentiation, arteriovenous specification, lymphangiogenesis and maintenance of vascular structural integrity. Young et al. (Young et al., 2006) studied the role of SOX18 in angiogenesis in an in vitro tube formation assay using endothelial cells. Overexpression of wild type SOX18 and the dominant negative SOX18 RaOp mutant resulted in gain and loss of tube formation, respectively. These studies reveal that correct spatiotemporal SOX18 expression is essential in several stages of vascular development.