Adipose tissue represents an abundant and accessible source of multipotent adult stem cells and is used by many investigators for tissue engineering applications; however, not all laboratories use cells at equivalent stages of isolation and passage. We have compared the immunophenotype of freshly isolated human adipose tissue-derived stromal vascular fraction (SVF) cells relative to serial-passaged adipose-derived stem cells (ASCs). The initial SVF cells contained colony-forming unit fibroblasts at a frequency of 1:32. Colony-forming unit adipocytes and osteoblasts were present in the SVF cells at comparable frequencies (1:28 and 1:16, respectively). The immunophenotype of the adipose-derived cells based on flow cytometry changed progressively with adherence and passage. Stromal cell-associated markers (CD13, CD29, CD44, CD63, CD73, CD90, CD166) were initially low on SVF cells and increased significantly with successive passages. The stem cell-associated marker CD34 was at peak levels in the SVF cells and/or early-passage ASCs and remained present, although at reduced levels, throughout the culture period. Aldehyde dehydrogenase and the multidrug-resistance transport protein (ABCG2), both of which have been used to identify and characterize hematopoietic stem cells, are expressed by SVF cells and ASCs at detectable levels. Endothelial cell-associated markers (CD31, CD144 or VE-cadherin, vascular endothelial growth factor receptor 2, von Willebrand factor) were expressed on SVF cells and did not change significantly with serial passage. Thus, the adherence to plastic and subsequent expansion of human adipose-derived cells in fetal bovine serum-supplemented medium selects for a relatively homogeneous cell population, enriching for cells expressing a stromal immunophenotype, compared with the heterogeneity of the crude SVF. STEM CELLS 2006;24:376 -385

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The relationship between bone and fat formation within the bone marrow microenvironment is complex and remains an area of active investigation. Classical in vitro and in vivo studies strongly support an inverse relationship between the commitment of bone marrow-derived mesenchymal stem cells or stromal cells to the adipocyte and osteoblast lineage pathways. In this review, we focus on the recent literature exploring the mechanisms underlying these differentiation events and discuss their implications relevant to osteoporosis and regenerative medicine.

Regenerative medical techniques will require an abundant source of human adult stem cells that can be readily available at the point of care. The ability to use unmatched allogeneic stem cells will help achieve this goal.

Studies of adipogenic protein induction have led to a new appreciation of the role of adipose tissue as an endocrine organ. Adipocyte-derived "adipokines" such as adiponectin, leptin, and visceral adipose tissue-derived serine protease inhibitor (vaspin) exert hormone-like activities at the systemic level. In this study, we examined the secretome of primary cultures of human subcutaneous adipose-derived stem cells as an in vitro model of adipogenesis. Conditioned media obtained from four individual female donors after culture in uninduced or adipogenic induced conditions were compared by two-dimensional gel electrophoresis and tandem mass spectrometry. Over 80 individual protein features showing >2-fold relative differences were examined. Approximately 50% of the identified proteins have been described previously in the secretome of murine 3T3-L1 preadipocytes or in the interstitial fluid derived from human mammary gland adipose tissue. As reported by others, we found that the secretome included proteins such as actin and lactate dehydrogenase that do not display a leader sequence or transmembrane domain and are classified as "cytoplasmic" in origin. Moreover we detected a number of established adipokines such as adiponectin and plasminogen activator inhibitor 1. Of particular interest was the presence of multiple serine protease inhibitors (serpins). In addition to plasminogen activator inhibitor 1, these included pigment epithelium-derived factor (confirmed by Western immunoblot), placental thrombin inhibitor, pregnancy zone protein, and protease C1 inhibitor. These findings, together with the recent identification of vaspin, suggest that the serpin protein family warrants further proteomics investigation with respect to

Interferon-␥ (IFN␥) treatment of adipocytes results in a down-regulation of the peroxisome proliferator-activated receptor ␥ (PPAR␥). The decrease in PPAR␥ expression is mediated by inhibition of PPAR␥ synthesis and increased degradation of PPAR␥. In this study, we demonstrate that both PPAR␥1 and PPAR␥2 are targeted to the proteasome under basal conditions and that PPAR␥1 is more labile than PPAR␥2. The IFN␥-induced increase in PPAR␥ turnover is blocked by proteasome inhibition and is accompanied by an increase in PPAR␥-polyubiquitin conjugates. In addition, IFN␥ treatment results in the transcriptional activation of PPAR␥. Similar to ligand-dependent activation of PPAR␥, IFN␥-induced activation was greater in the phosphorylationdeficient S112A form of PPAR␥ when compared with wild-type PPAR␥. Moreover, the inhibition of ERKs 1 and 2 with a MEK inhibitor, U1026, lead to an inhibition in the decay of PPAR␥ proteins, indicating that serine phosphorylation influences the degradation of PPAR␥ in fat cells. Our results also demonstrate that the proteasome-dependent degradation of PPAR␥ does not require nuclear export. Taken together, these results indicate that PPAR␥ is targeted to the ubiquitinproteasome pathway for degradation under basal conditions and that IFN␥ leads to an increased targeting of PPAR␥ to the ubiquitin-proteasome system in a process that is affected by ERK-regulated serine phosphorylation of PPAR␥ proteins. PPAR␥1 is a member of the nuclear hormone receptor family, a group of transcription factors that are activated by small lipophilic ligands (1). PPAR␥ exists as two isoforms, PPAR␥1 and PPAR␥2, which are produced by a combination of different promoters and alternative splicing (2). There is also a PPAR␥3 gene that codes for a protein that is identical to PPAR␥1 (3). PPAR␥1 is predominantly expressed in fat cells but occurs in low levels in multiple tissues. PPAR␥2 has an N-terminal extension of 30 amino acids and is very highly expressed in adipocytes (4, 5). Deletion of the PPAR␥ gene in mice results in placental dysfunction and embryonic lethality (6, 7).PPAR␥ has been implicated in the regulation of systemic insulin sensitivity. This was first demonstrated when PPAR␥ was shown to be a functional receptor for the synthetic antidiabetic thiazolidinediones (TZDs) (8). Thiazolidinediones are specific high affinity ligands for PPAR␥ and the order of their receptor binding affinities in vitro mirrors their antihyperglycemic activity in vivo (9). Direct evidence for the association between PPAR␥ and insulin sensitivity comes from genetic studies showing that mutations in the ligand-binding domain of PPAR␥ are associated with severe insulin resistance. Although not obese, these patients developed type 2 diabetes as well as early onset hypertension (10). Also, insulin has been shown to acutely regulate the expression of PPAR␥ in human adipocytes (11), and mice that only express one copy of the PPAR␥ gene have been shown to be more sensitive to insulin (12). We have recently demonstrated that IFN␥ res...

Adipogenesis plays a critical role in energy metabolism and is a contributing factor to the obesity epidemic. This study examined the proteome of primary cultures of human adipose-derived adult stem (ADAS) cells as an in vitro model of adipogenesis. Protein lysates obtained from four individual donors were compared before and after adipocyte differentiation by two-dimensional gel electrophoresis and tandem mass spectroscopy. Over 170 individual protein features in the undifferentiated adipose-derived adult stem cells were identified. Following adipogenesis, over 40 proteins were up-regulated by >2-fold, whereas 13 showed a >3-fold reduction. The majority of the modulated proteins belonged to the following functional categories: cytoskeleton, metabolic, redox, protein degradation, and heat shock protein/chaperones. Additional immunoblot analysis documented the induction of four individual heat shock proteins and confirmed the presence of the heat shock protein 27 phosphoserine 82 isoform, as predicted by the proteomic analysis, as well as the crystallin ␣ phosphorylated isoforms. These findings suggest that the heat shock protein family proteome warrants further investigation with respect to the etiology of obesity and type 2 diabetes. Molecular & Cellular Proteomics 4:731-740, 2005.Obesity is a health problem of epidemic proportions. It is estimated that in 2000 over 60% of adults are overweight (BMI 1 Ͼ 25) and that 30% are obese (BMI Ͼ 30); this compares to levels of 46 and 14%, respectively, in 1980. Obesity and increased adiposity are associated clinically with the onset of insulin resistance, dysfunctional glucose sensing and utilization, hypertension, and hypertriglyceridemia, all contributing to the pathologic sequelae of type 2 diabetes. Paradoxically type 2 diabetes also occurs in patients with inherited or acquired forms of lipodystrophy or loss of adipose tissue depots (1, 2). Lipodystrophy occurs through defects in genes associated with triglyceride metabolism or as a consequence of antiretroviral therapy in human immunodeficiency viruspositive patients (1, 2). Animal models confirm these clinical observations; multiple strains of transgenic mice with a lipodystrophic phenotype exhibit type 2 diabetes (3-5). Diabetes in these animals responds not to insulin therapy but to transplantation of subcutaneous adipose tissue or to leptin treatment (3-5). These clinical and experimental observations have led to the hypothesis that a failure in adipocyte differentiation is a critical etiologic factor leading to type 2 diabetes (6 -8). Danforth (6) and others postulate that in obese individuals adipose tissue depots have already committed all of their stem cell reserves to the adipocyte lineage and have lost their capacity to create new adipocytic cells (6 -8). In the face of excess energy balance, both obese and lipodystrophic individuals deposit triglycerides in ectopic sites, such as muscle and liver, thereby contributing to the metabolic dysfunction associated with type 2 diabetes (increased hepatic glucon...

The nuclear hormone receptor, REV-ERB, plays an essential role in adipogenesis. Rev-erbalpha expression is induced in 3T3-L1 cells during adipogenesis, and overexpression of this receptor leads to expression of adipogenic genes. We recently demonstrated that the porphyrin heme functions as a ligand for REV-ERB, and binding of heme is required for the receptor's activity. We therefore hypothesized that REV-ERB ligands may play a role in regulation of adipogenesis. We detected an increase intracellular heme levels during 3T3-L1 adipogenesis that correlated with induction of aminolevulinic acid synthase 1 (Alas1) expression, the rate-limiting enzyme in heme biosynthesis. If the increase in Alas1 expression was blocked, adipogenesis was severely attenuated, indicating that induction of expression of Alas1 and the increase in heme synthesis is critical for differentiation. Inhibition of heme synthesis during adipogenesis leads to decreased recruitment of nuclear receptor corepressor to the promoter of a REV-ERB target gene, suggesting alteration of REV-ERB activity. Treatment of 3T3-L1 cells with a synthetic REV-ERB ligand, SR6452, resulted in induction of adipocyte differentiation to a similar extent as treatment with the peroxisomal proliferator-activated receptor-gamma agonist, rosiglitazone. Combination of SR6452 and rosiglitazone had an additive effect on stimulation of adipocyte differentiation. These results suggest that heme, functioning as a REV-ERB ligand, is an important signaling molecule for induction of adipogenesis. Moreover, synthetic small molecule ligands for REV-ERB are effective modulators of adipogenesis and may be useful for treatment of metabolic diseases.