Todorčević et al. BMC Genomics 2010, 11:39 http://www.biomedcentral.com/1471-2164/11/39 involved in the regulation of a broad range of physiolo- gical processes but at obesity increased production of cytokines and adipokines lead to the chronic low-grade inflammatory state. The extent of conservation of endo- crine WAT functions in the cold-blooded vertebrate, including its capacity to influence systemic inflammatory responses and development of life style related disorders has not been studied until now. In salmonid fish, precursor cells differentiate into adi- pocytes continuously [4], however, the exact origin of preadipocytes is not known. In mammals, development of white adipocytes is thought to begin with progenitor cells from the perivascular compartment (pericytes) in WAT, characterised by expression of smooth muscle actin 22a, also known as transgelin (TAGLN), and other vascular markers. Isolated pericytes differentiate into mesenchymal stem cells (MSCs), which in turn, give rise to various cell types including osteoblasts, chondrocytes, smooth muscle cells, fibroblasts, macrophages and adi- pocytes [5-9]. Mammalian adipose stromal-vascular frac- tion (aSVF) harbours a population of progenitor cells that is also capable to differentiate ex vivo into cells and tissues of mesodermal origin, thus suggesting their peri- vascular origin. Atlantic salmon WAT contains a large number of fibroblast-like precursor cells that can differ- entiate to mature adipocytes in vitro [10,11], however, their stem cell features have not been characterized yet. In mammals, adipogenesis includes three distinct stages. Following active proliferation and the phase of determination, cells reach confluence, followed by hor- monal induction and terminal differentiation. Re-entry into the cell cycle of growth-arrested cells at confluence involves several rounds of proliferation, referred to as mitotic clonal expansion. Secondary growth arrest and induction of the transcription factors CCAAT-enhancer- binding protein (C/EBP) a and peroxisome proliferator- activated receptor (PPAR) g mark the end of the mitotic clonal expansion phase and entry into terminal differen- tiation with transcriptional activation of genes defining the mature adipocyte phenotype. The few performed studies indicate that adipogenesis in fish bears many resemblances to that of terrestrial vertebrates [10-12], though there certainly exist as yet undiscovered species-specific differences. Atlantic sal- mon precursor cells take longer than mammalian to acquire the mature adipocyte phenotype in culture, due to low incubating temperature, typical of salmonid fish habitat. Further, mammalian cells are able to produce lipid droplets (LDs) from glucose alone, while salmon preadipocytes require lipids in order to achieve the mature phenotype [10]. Hence it is to be expected that white adipocytes and their precursors in a cold-blooded vertebrate may have both evolutionarily conserved and specific features. Page 2 of 17 The aSVF primary culture enabled investigation of early events involved in the adipogenic determination as well as processes characteristic of the later terminal dif- ferentiation phase. Particular emphasis was placed on genes governing nutrient metabolism and stress and inflammatory responses. Results Cell culture characterization A short summary of adipocyte development and cultiva- tion conditions is presented in Fig. 1. In the subconflu- ent stage during the first seven days of culture, aSVF cells had the highest proliferative activity (Fig. 2A). Approximately at day 7, cells reached the confluent stage. Two-day adipogenic hormonal induction was applied at that time point. This was followed by the terminal differentiation stage during which cells acquired a more rounded shape. Morphological changes observed by electron microscopy images at days 15 and 30 were characteristic of the terminally differentiating phenotype of adipocytes, including a relatively low mito- chondrial number, large size of LD and the nucleus located between LDs and cell membrane (Fig. 3A, B). Oil red O staining also showed a high degree of lipid accumulation in mature adipocytes at day 30 (Fig. 4A). A steady decrease in the extracellular superoxide dismu- tase (SOD) activity was observed from day 1 to day 30 (Fig. 4B). Subconfluence (days 1 and 4) Microarray analyses of aSVF in the first days of culture, revealed concurrent expression of genes characteristic of MSCs, immune cells and cells of perivascular origin (Fig. 5). Platelet-derived growth factor A (PDGFA) is a major regulator of proliferation and migration of mam- malian adipose-derived MSCs [13]. Glomulin is essential for normal development of the vasculature while the lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1) is a marker of lymph vessels and endothelial cells in mammals. Chemokine receptor 4 (CXCR4) and other chemokine/cytokine receptors are involved in cell mobilisation and retention in several populations of MSCs and in immune cells [14-16]. We have selected eight co-expressed genes encoding proteins involved in nucleotide metabolism, DNA replication and regulation of cell cycle and their averaged profile, due to their involvement in the similar cellular functions, was shown in Fig. 2B. The lowest expression of genes from this category was at day 1 (also see Fig. 5). The subsequent increase was in concordance with the results of prolifer- ating cell nuclear antigen (PCNA) assay that revealed the highest proliferation rate at day 4 (Fig. 2A). In paral- lel, microarray analyses showed a decreased expression of non-adipogenic cell markers. These genes had high levels at day 1 (Fig. 5). Tumour necrosis factor (TNF) a,