Studies using freshly isolated cells from the stromal vascul

Studies using freshly isolated KY 02111 manufacturer from the stromal vascular fraction (SVF) of sWAT characterized ASCs by their plastic adherence in culture, colony-forming capacity and the expression pattern of distinct cell surface markers (Mitchell et al., 2006). These cells can promote angiogenesis (Rehman et al., 2004) and can be used as autologous ASCs for regenerative therapy (Bourin et al., 2013). It has however not been shown by rigorous assays that ASCs are capable of differentiating outside of their lineage into functional phenotypes without extensive molecular engineering, or extreme chemical treatment (like with BMP, which will temporarily turn any fibroblastic cell into an osteoblast-like cell). The ASC population is broadly defined by the International Society for Cellular Therapy as a subset of cells positive for the mesenchymal stem/stromal cell (MSC) markers CD90 and CD105 (Mitchell et al., 2006), and the stem/progenitor cell marker CD34 (Sengenès et al., 2005; Sidney et al., 2014), lacking the expression of the endothelial marker CD31, summarized as CD34+/CD90+/CD105+/CD31− (Bourin et al., 2013). There is, however, no consensus about the exact pattern of markers being expressed by ASCs. No definitive singular marker to precisely define ASCs in human adipose tissues is known and although certain MSC markers are recommended, none are specific for a stem cell of any type. They are expressed on virtually all fibroblastic cells that are not stem cells based on rigorous assays (Bianco et al., 2008). According to the current model, in human adipose tissue, vessel-associated stromal cells are organized in two layers, perivascular in an inner ring and more peripheral in an outer ring (Zimmerlin et al., 2013). ASCs reside in the outer adventitial layer encircling small vessels; the spatial organization of ASCs in the vascular stroma of adipose tissues is however not precisely understood. Delta-like protein 1/preadipocyte factor 1 DLK1(PREF1) is an paternally expressed imprinted gene that encodes a transmembrane protein containing six epidermal growth factor-like repeats at the extracellular domain, a juxtamembrane region with a TNF-alpha-converting enzyme (TACE)-mediated cleavage site, a transmembrane domain and a short intracellular region (Smas and Sul, 1993; Laborda et al., 1993). Upon cleavage by the TACE protease, it can be released as a soluble protein (Sul, 2009). DLK1 is a crucial regulator of ASC homeostasis in WAT. Evidence was presented that DLK1 negatively regulates proliferation (Mortensen et al., 2012) and terminal differentiation of adipocyte progenitors into adipocytes and other mesenchymal lineages (Wang and Sul, 2009). Studies on mouse models confirm these inhibitory effects of DLK1. Animals with increased levels of DLK1 show less total body weight and a decrease in fat mass, whereas DLK1-null mice increases fat mass (Villena et al., 2008; Abdallah et al., 2007; Lee et al., 2003; Mortensen et al., 2012). DLK1 is implicated in stem/progenitor cell biology during embryonic development and in several organs of adults (Floridon et al., 2000; Sul, 2009; Abdallah and Kassem, 2012; Andersen et al., 2009b, 2013). In adult mouse fat tissues, adipocyte progenitors express DLK1 and its main role is most likely to regulate clonal expansion and terminal adipogenesis (Sul, 2009; Traustadottir et al., 2013). There is also evidence for a role of DLK1 in vascular endothelium of adipose tissue (Andersen et al., 2009a). We recently showed that DLK1+/CD105+/CD90+/CD34+/CD31− ASCs constitute a major population in early passage isolates from the SVF of human sWAT and that DLK1 regulates adipogenesis in these cells (Mitterberger et al., 2012). The aim of the present communication was to further characterize DLK1+ ASCs in human WAT.
Materials and methods
Results
Discussion Renewal of adipose tissue from progenitor cells is important for its homeostasis throughout life. DLK1-positive adipose precursors among others are required for adipose tissue expansion in adult mice Hudak et al. (2014). The mechanisms regulating expansion of the adipogenic lineage and terminal adipogenesis are however little understood, especially in human adipose tissues. In the present study, we demonstrated for the first time that the SVF of human sWAT contains three different cell populations defined by DLK1 and CD34. Only cs-DLK1−/cs-CD34+/CD90+/CD105/α-SMA+/CD45−/CD31− cells showed high proliferative and adipogenic capacity. Cs-DLK1+/cs-CD34− and cs-DLK1+/cs-CD34 cells, which bear cell-surface-tethered DLK1, consistently co-expressed α-SMA and CD31 and had a mixed hematopoietic and mesenchymal phenotype, could neither undergo proliferation nor terminal adipogenesis. While previous studies underscore that DLK1 restricts adipose tissue size by inhibiting clonal expansion and terminal adipogenesis of ASCs (Sul, 2009; Traustadottir et al., 2013), our data suggest that cell surface expression of DLK1 plays a role in the regulation of clonal expansion and terminal adipogenesis of human ASCs. Further work is necessary to elucidate the role of intracellular DLK1.