White adipocyte progenitor cells were identified

White adipocyte progenitor cells were identified in vivo (Rodeheffer et al., 2008), and evidence provided by several studies indicates that these cells are responsible for regenerating adipocyte numbers in fat tissues (Bertrand et al., 1978; Gesta et al., 2007; Hauner et al., 1989; Hausman and Hausman, 2006; Rodeheffer et al., 2008; Zuk et al., 2002). In addition to adipocyte hypertrophy, an increased proliferation and adipogenic differentiation of ASC contributes to the adipose tissue growth that is characteristic for obesity (Drolet et al., 2008; Gesta et al., 2007; Tchoukalova et al., 2010). ASC reside most likely mainly at perivascular sites in adipose tissues (Crossno et al., 2006; Tang et al., 2008; Zimmerlin et al., 2010). The number of intermediate stages between adipose progenitors and a mature adipocytes is uncertain but once committed, ASC can enter an adipocyte differentiation program (adipogenesis) to acquire their specific functions as adipocytes (Cristancho and Lazar, 2011; Farmer, 2006; Rosen and MacDougald, 2006). Adipogenesis in human ASC involves growth arrest, early and terminal differentiation, including morphological changes, lipid accumulation and the expression of fat cell specific genes, for example fatty BB-94 binding protein-4 (FABP4) and adipokines, such as leptin and adiponectin. The stages of adipocyte differentiation are orchestrated by a transcriptional cascade involving adipogenic key factors, such as the nuclear receptor peroxisome proliferator-activated receptor-γ2 (PPARγ2) and members of the CCAAT/enhancer-binding protein (C/EBP) family (Farmer, 2006; Rosen and MacDougald, 2006). Adipose-derived stromal cells (ASC) are most frequently isolated by collagenase digestion of white adipose tissue, yielding a stromal vascular fraction (SVF) which contains a mixture of heterogeneous cell types that are further purified by differential centrifugation, plating and plastic adherence (Hauner et al., 1989; Mitchell et al., 2006; Zuk et al., 2002). It has been shown that a single adherent cell derived from the SVF can be expanded generating a daughter cell population with multipotent differentiation capacity, defined as an adult stem cell (Guilak et al., 2006; Mitchell et al., 2006). As ASC possess no unique morphological characteristics great efforts are made to characterize this cell type by specific marker proteins. Although in many studies specific pattern of cellular surface markers were assigned to human ASC comprising the combination CD90+/CD105+/CD34+/CD31− (Gimble et al., 2011; Maumus et al., 2008; McIntosh et al., 2006; Mitchell et al., 2006; Schaffler and Buchler, 2007; Sengenes et al., 2005, 2007; Zimmerlin et al., 2010), there is no complete consensus on the antigen expression pattern that will precisely define human ASC. Moreover, evidence was presented that the immunophenotype of ASC can change during expansion in cell culture, likely influenced by adherence to plastic culture dishes, and proliferation in medium with high concentrations of fetal bovine serum (FBS), that contains variable and undefined factors influencing proliferation, differentiation and survival of adult stem cells (McIntosh et al., 2006; Mitchell et al., 2006; Parker et al., 2007). Delta-like protein 1/preadipocyte factor-1 (DLK1/PREF1), which was originally identified as an inhibitor of adipogenesis in the murine preadipocyte cell line NIH 3T3-L1 (Smas and Sul, 1993), is used as preadipocyte marker (Rodeheffer et al., 2008; Smas and Sul, 1993; Tang et al., 2008; Tseng et al., 2005; Wang et al., 2008). Similar to Notch, DLK1(PREF1) is an epidermal growth factor repeat containing transmembrane protein that is proteolytically converted at the extracellular domain to generate a physiologically active soluble form that inhibits adipocyte differentiation (Sul, 2009). Adipocyte progenitors are the sole cell type expressing DLK1(PREF1) in mouse adipose tissues. DLK1(PREF1) knockout mice show accelerated fat deposition, whereas mice overexpressing DLK1(PREF1) in adipose tissue exhibit decreased fat mass (Gesta et al., 2007; Lee et al., 2003; Moon et al., 2002; Sul, 2009; Villena et al., 2008). DLK1(PREF1) was shown to play a role in the regulation of cell commitment and differentiation in murine multipotent mesenchymal cells regulating fat mass and bone mass formation in vivo (Abdallah et al., 2007a, 2011; Moon et al., 2002; Villena et al., 2008; Wang and Sul, 2009). DLK1(PREF1) can also stimulate bone resorption in mice (Abdallah et al., 2011). DLK1(PREF1) suppresses C/EBPβ and C/EBPδ gene expression in mouse preadipocytes which leads to prevention of adipogenesis (Wang and Sul, 2009). Moreover, DLK1(PREF1) can direct murine multipotent mesenchymal cells to the chondrogenic lineage but it inhibits differentiation into osteoblasts as well as chondrocytes (Chen et al., 2011; Harkness et al., 2009; Wang and Sul, 2009). DLK1(PREF1) was also shown to be expressed in human bone marrow-derived (BM) mesenchymal stem cells (MSC), but at relatively low levels (Abdallah et al., 2004, 2007b, 2011; Jing et al., 2009). It keeps these cells in an undifferentiated state and regulates their differentiation into osteoblasts or adipocytes, at least in part by influencing their microenvironment composition (Abdallah et al., 2007a, 2007b, 2011; Jing et al., 2009). Low expression of DLK1(PREF1) was also found in human cord blood (CB) MSC correlating with a strong adipogenic differentiation potential (Kluth et al., 2010). Moreover, DLK1(PREF1) overexpression inhibits adipogenesis as well as osteogenesis in CB MSC (Kluth et al., 2010). Together these data suggest that DLK1(PREF1) is an important regulator of mesenchymal stem cell differentiation. Thus DLK1(PREF1) could be a useful marker to characterize human ASC, however, the DLK1(PREF1) status of these cells is unknown.