Subcutaneous Adipose-derived Stem Cells Research Articles

Opposite Effects of Mechanical Action of Fluid Flow on Proangiogenic Factor Secretion From Human Adipose-Derived Stem Cells With and Without Oxidative Stress.

Mechanical forces, hypoxia, and oxidative stress contribute to skin renewal, perfusion, and wound healing, but how are they regulating subcutaneous adipose-derived stem cells (ASCs) in the inflammatory microenvironment associated to skin repair and disorders is unknown. In this study, ASCs were isolated from lipoaspirate samples from plastic surgery patients, primary cultured and their differentiation and secretion of a panel of cytokines with pronounced effects on skin repair and angiogenesis were studied under mechanical stimulation by intermittent fluid flow, 1% hypoxia and oxidative stress by glutathione (GSH) depletion with buthionine sulfoximine (BSO) treatment. Mechanical action of fluid flow did not alter mesenchymal phenotype of CD90+ /CD29+ /CD44+ /CD34- /CD106- /CD45- ASCs; however, it remarkably induced ASC secretion of human umbilical vein endothelial cell (HUVEC) migration-stimulating factors. Multiplex Luminex assay further confirmed an increased secretion of VEGF, G-CSF, HGF, Leptin, IL-8, PDGF-BB, Angiopoietin-2, and Follistatin from mechanically-stimulated ASCs via cyclooxygenase-2. Consistent with this mechanism, GSH depletion and hypoxia also increased ASC secretion of VEGF, IL-8, leptin, Angiopoitein-2, and PDGF-BB. However, mechanical action of fluid flow abrogated VEGF and HUVEC migration-stimulating activity from GSH-depleted and hypoxic ASCs. Conversely, GSH depletion and hypoxia abrogated VEGF and HUVEC migration-stimulating activity from mechano-stimulated ASCs. Although mechanical action of fluid flow, hypoxia, and GSH-depletion had independent proangiogenic-stimulating activity on ASCs, mechanical stimulation had opposite effects on proangiogenic factor secretion from ASCs with and without oxidative stress. These data uncover the role of hypoxia and endogenous redox balance during the proangiogenic response of ASCs and other mesenchymal-derived cell types to mechanical action of interstitial fluid flow. J. Cell. Physiol. 232: 2158-2167, 2017. © 2016 Wiley Periodicals, Inc.

Infrapatellar Fat Pad Stem Cells: From Developmental Biology to Cell Therapy.

The ideal cell type to be used for cartilage therapy should possess a proven chondrogenic capacity, not cause donor-site morbidity, and should be readily expandable in culture without losing their phenotype. There are several cell sources being investigated to promote cartilage regeneration: mature articular chondrocytes, chondrocyte progenitors, and various stem cells. Most recently, stem cells isolated from joint tissue, such as chondrogenic stem/progenitors from cartilage itself, synovial fluid, synovial membrane, and infrapatellar fat pad (IFP) have gained great attention due to their increased chondrogenic capacity over the bone marrow and subcutaneous adipose-derived stem cells. In this review, we first describe the IFP anatomy and compare and contrast it with other adipose tissues, with a particular focus on the embryological and developmental aspects of the tissue. We then discuss the recent advances in IFP stem cells for regenerative medicine. We compare their properties with other stem cell types and discuss an ontogeny relationship with other joint cells and their role on in vivo cartilage repair. We conclude with a perspective for future clinical trials using IFP stem cells.

Effects of Streptozotocin-Induced Diabetes on Proliferation and Differentiation Abilities of Mesenchymal Stem Cells Derived from Subcutaneous and Visceral Adipose Tissues.

Introduction: Accumulated evidence indicates that there are intrinsic differences between adipose tissue-derived stem cells (ASCs) obtained from different body fat depots. Here, we compared the proliferation and multipotency of subcutaneous ASCs (SC-ASCs) and epididymal ASCs (ED-ASCs) before and after induction of diabetes by streptozotocin. Methods: The adipogenic and osteogenic abilities of rat SC-ASCs and ED-ASCs were evaluated using Oil Red O and Alizarin Red staining, respectively. The expression of adipocyte (PPAR-γ, LPL) and osteoblast (ALP, SPP1) specific mRNAs was evaluated by quantitative real-time PCR. MTT test was used for determination of cell proliferation capacity. Results: The proliferation of SC-ASCs was higher than ED-ASCs, both before and after diabetes induction (P<0.05). Diabetes increased the proliferative capability of SC-ASCs (P<0.05) but not ED-ASCs. Before diabetes, both adipogenic and osteogenic differentiation of SC-ASCs were higher than ED-ASCs (P<0.05). After diabetes, both SC-ASCs and ED-ASCs were able to differentiate into adipocyte and osteoblast, but the levels of differentiation were higher in SC-ASCs than in ED-ASCs (P<0.05). Diabetes decreased the expression of PPAR-γ and LPL, but increased the SPP1 and ALP expression in both SC-ASCs and ED-ASCs. Conclusion: Our data suggested that diabetes increases the proliferation of ASCs but decreases their adipogenic differentiation. Also, SC-ASCs have higher proliferation and differentiation abilities than ED-ASCs in normal and diabetic conditions so can be more preferable for cell therapy.

Adipose-derived stem cells from both visceral and subcutaneous fat deposits significantly improve contractile function of infarcted rat hearts.

Adipose-derived stem cells (ASCs) from subcutaneous and visceral adipose tissues have been studied individually. No studies have compared their abilities in treatment of heart failure. This study was designed to evaluate whether ASCs from the two sources could provide a long-term improvement of cardiac function in infarcted hearts. Rat subcutaneous and visceral adipose tissues were excised for isolation of ASCs. Morphology, yield, proliferation, surface markers, differentiation, and cytokine secretion of the subcutaneous ASCs (S-ASCs) and visceral ASCs (V-ASCs) were analyzed. Then a rat model of myocardial infarction (MI) was established by a coronary occlusion. Seven days after occlusion, S-ASCs (n = 22), V-ASCs (n = 22), and Dulbecco's modified Eagle medium (DMEM, n = 20) were injected into the infarct rim, respectively. Cardiac function was then monitored with MRI for up to 6 months. The hearts were then removed for histological assessments. The yield of V-ASCs per gram of the visceral adipose depot was significantly greater than that of S-ASCs in 1 g of the subcutaneous adipose depot. On the other hand, the S-ASCs showed a greater proliferation rate and colony-forming unit relative to the V-ASCs. In addition, the infarcted hearts treated with either S-ASCs or V-ASCs showed a significantly greater left ventricular ejection fraction (LVEF) than those treated with DMEM at 4 weeks and 6 months following the cell/DMEM transplantation. Moreover, the infarct sizes of both S-ASC- and V-ASC-treated hearts were significantly smaller than that in the DMEM-treated hearts. MRI showed the implanted ASCs at the end of 6 months of recovery. Despite the differences in cell yield, proliferation, and colony formation capacity, both S-ASCs and V-ASCs provide a long-lasting improvement of cardiac contractile function in infarcted hearts. We conclude that the subcutaneous and visceral adipose tissues are equally effective cell sources for cell therapy of heart failure.

Characterization of adipose-derived stem cells from subcutaneous and visceral adipose tissues and their function in breast cancer cells.

Adipose-derived stem cells are capable of differentiating into multiple cell types and thus considered useful for regenerative medicine. However, this differentiation feature seems to be associated with tumor initiation and metastasis raising safety concerns, which requires further investigation. In this study, we isolated adipose-derived stem cells from subcutaneous as well as from visceral adipose tissues of the same donor and systematically compared their features. Although being characteristic of mesenchymal stem cells, subcutaneous adipose-derived stem cells tend to be spindle form-like and are more able to home to cancer cells, whereas visceral adipose-derived stem cells incline to be "epithelial"-like and more competent to differentiate. Moreover, compared to subcutaneous adipose-derived stem cells, visceral adipose-derived stem cells are more capable of promoting proliferation, inducing the epithelial-to-mesenchymal transition, enhancing migration and invasion of breast cancer cells by cell-cell contact and by secreting interleukins such as IL-6 and IL-8. Importantly, ASCs affect the low malignant breast cancer cells MCF-7 more than the highly metastatic MDA-MB-231 cells. Induction of the epithelial-to-mesenchymal transition is mediated by the activation of multiple pathways especially the PI3K/AKT signaling in breast cancer cells. BCL6, an important player in B-cell lymphoma and breast cancer progression, is crucial for this transition. Finally, this transition fuels malignant properties of breast cancer cells and render them resistant to ATP competitive Polo-like kinase 1 inhibitors BI 2535 and BI 6727.

Obesity reduces the pro-angiogenic potential of adipose tissue stem cell-derived extracellular vesicles (EVs) by impairing miR-126 content: impact on clinical applications.

Background/Objectives:Soluble factors and cell-derived extracellular vesicles (EVs) are crucial tissue repair mediators in cell-based therapy. In the present study, we investigate the therapeutic impact of EVs released by adipose tissue-derived stem cells (ASCs) recovered from obese subjects' visceral and subcutaneous tissues.Methods:ASCs were recovered from 10 obese (oASCs) and 6 non-obese (nASCs) participants and characterized. In selected experiments, nASCs and oASCs were cultured with palmitic acid (PA) or high glucose (HG), respectively. EVs from obese (oEVs) and non-obese (nEVs) subjects' visceral and subcutaneous ASCs were collected after ultracentrifugation and analyzed for their cargo: microRNA-126 (miR-126), vascular endothelial growth factor (VEGF), and matrix metalloproteinase 2 (MMP-2), and for their biological effects on endothelial cells (ECs). Western blotting analysis and loss- and gain-of function experiments were performed.Results:oEVs show impaired angiogenic potential compared with nEVs. This effect depends on EV cargo: reduced content of VEGF, MMP-2 and, more importantly, miR-126. We demonstrate, using gain- and loss-of-function experiments, that this reduced miR-126 content leads to Spred1 upregulation and the inhibition of the extracellular signal–regulated kinase 1/2 mitogen-activated protein kinase pathway in ECs. We also show that PA treatment of nASCs translates into the release of EVs that recapitulate oEV cargo. Moreover, HG treatment of oASCs further reduces miR-126 EV content and EV-mediated in vitro angiogenesis. Finally, impaired pro-angiogenic potential is also detected in EVs released from obese subcutaneous adipose tissue-derived ASCs.Conclusions:These results indicate that obesity impacts on EV pro-angiogenic potential and may raise concerns about the use of adipose tissue-derived EVs in cell-based therapy in the obese setting.

Fat depot-specific gene signature and ECM remodeling of Sca1high adipose-derived stem cells

Stem cell antigen-1 (Sca1 or Ly6A/E) is a cell surface marker that is widely expressed in mesenchymal stem cells, including adipose-derived stem cells (ASCs). We hypothesized that the fat depot-specific gene signature of Sca1high ASCs may play the major role in defining adipose tissue function and extracellular matrix (ECM) remodeling in a depot-specific manner. Herein we aimed to characterize the unique gene signature and ECM remodeling of Sca1high ASCs isolated from subcutaneous (inguinal) and visceral (epididymal) adipose tissues. Sca1high ASCs are found in the adventitia and perivascular areas of adipose tissues. Sca1high ASCs purified with magnetic-activated cell sorting (MACS) demonstrate dendrite or round shape with the higher expression of cytokines and chemokines (e.g., Il6, Cxcl1) and the lower expression of a glucose transporter (Glut1). Subcutaneous and visceral fat-derived Sca1high ASCs particularly differ in the gene expressions of adhesion and ECM molecules. While the expression of the major membrane-type collagenase (MMP14) is comparable between the groups, the expressions of secreted collagenases (MMP8 and MMP13) are higher in visceral Sca1high ASCs than in subcutaneous ASCs. Consistently, slow but focal MMP-dependent collagenolysis was observed with subcutaneous adipose tissue-derived vascular stromal cells, whereas rapid and bulk collagenolysis was observed with visceral adipose tissue-derived cells in MMP-dependent and -independent manners. These results suggest that the fat depot-specific gene signatures of ASCs may contribute to the distinct patterns of ECM remodeling and adipose function in different fat depots.

Diphlorethohydroxycarmalol of Ishige okamurae and Caffeine Modified the Expression of Extracellular Fibrillars during Adipogenesis of Mouse Subcutaneous Adipose Derived Stem Cell.

Although, one of the etiologies of localized lipodystrophy of the subcutaneous connective tissue (cellulite) is the histological alternation of adipose tissue, the characteristics of expression of the components of extracellular matrix (ECM) components during adipogenesis are not uncovered. In this study, the effects of caffeine and Ishige okamurae originated diphlorethohydroxycarmalol (DPHC) on the expression of extracellualr fibers was analyzed with quantitative RT-PCR during differentiation induction of mouse subcutaneous adipose derived stem cells (msADSC) into adipocyte. The expression levels of Col1a, Col3a1, and Col61a were decreased by the adipogenci induction in a time-dependent manners. However, Col2a mRNA and Col4a1 mRNA expressions were oposit to them. Caffeine and DPHC stimulated the changes of the expression of these collagens. Eln mRNA expression was increased by induction. DPHC stimulated the expression of it. Mfap5 mRNA expression was deceased in both adipogenic cell and matured adipocytes. Caffeine suppressed the expression of Mfap5 but the effect of DPHC was different by the concentration. The expression of bioglycan, decorin, and lumican were also modified by caffeine and DPHC in a concentration-dependent manner. Based on this study, we revealed firstly the effects of caffeine and DPHC on the expression of collagens, elastin, and glycoproteins during adipogenesis of msADSCs. Those results suggest that DPHC may have antiadipogenic effect and has more positive effets on normal adipose tissue generation and work as suppressor the abnormality of ECM structure. Such results indicate that DPHC can be applied in keeping the stability of the ECM of adipogenic tissues.

Body mass index affects proliferation and osteogenic differentiation of human subcutaneous adipose tissue-derived stem cells

BackgroundObesity is associated with a higher risk of developing cancer and co-morbidities that are part of the metabolic syndrome. Adipose tissue is recognized as an endocrine organ, as it affects a number of physiological functions, and contains adipose tissue-derived stem cells (ASCs). ASCs can differentiate into cells of multiple lineages, and as such are applicable to tissue engineering and regenerative medicine. Yet the question of whether ASC functionality is affected by the donor’s body mass index (BMI) still exists.ResultsASCs were isolated from patients having different BMIs (BMI-ASCs), within the ranges of 18.5-32.8. It was hypothesized that overweight BMI-ASCs would be more compromised in early adipogenic and osteogenic potential, and ability to form colonies in vitro. BMI was inversely correlated with ASC proliferation and colony forming potential as assessed by CyQUANT proliferation assay (fluorescence- based measurement of cellular DNA content), and colony forming assays. BMI was positively correlated with early time point (day 7) but not later time point (day 15) intracytoplasmic lipid accumulation as assessed by Oil-Red-O staining. Alizarin red staining and RT-PCR for alkaline phosphatase demonstrated that elevated BMI resulted in compromised ASC mineralization of extracellular matrix and decreased alkaline phosphatase mRNA expression.ConclusionsThese data demonstrate that elevated BMI resulted in reduced ASC proliferation, and potentially compromised osteogenic capacity in vitro; thus BMI is an important criterion to consider in selecting ASC donors for clinical applications.

Hepatogenic differentiation of human mesenchymal stem cells from peritoneal adipose tissue

Backgrounds/AimsIt has been reported that functional hepatogenic differentiation has the possibility to occur in subcutaneous adipose tissue-derived stem cells. However, no studies have investigated whether the adipose tissue-driven stem cells present in various body parts differ according to hepatogenic differentiations. In this study, stem cells were separated from body visceral fat and abdominal subcutaneous adipose tissue, and cultured, and then hepatogenic differentiation was induced. We aim to investigate the possibilities and aspects of hepatogenic differentiations within the two types of fat cells.MethodsOmental fat tissues were obtained as visceral fat and abdominal subcutaneous adipose tissues were obtained from patients who had suction-assisted lipectomy. Stem cells were separated from the obtained fat tissues, and then, hepatogenic differentiation was carried out by utilizing 2-step differentiation protocols.ResultsAfter the differentiation, two types of cultured cells that showed the similar neuron-like shapes were changed to cuboidal shapes and included several binucleated cells which could be characteristics of mature hepatocytes. We confirmed that hepatocyte specific genes and proteins such as albumin and CYP3A4 were being expressed. By utilizing the ELISA test, we were able to observe that the albumin was secreted into the culture fluids in both cells. After completing the differentiation, we observed the presence of the hepatocyte specific properties by confirming glycogen storage within the cells and the ICG reagent uptake.ConclusionsWe confirmed that hepatogenic differentiation was possible to occur in the omental fat as well as subcutaneous adipose tissue.

Decreased contact inhibition in mouse adipose mesenchymal stem cells.

The proliferation of embryonic cells or adult stem cells in tissue is critically regulated during development and repair. How limited the proliferation of cells, so far, is not much explored. Cell-cell contact proliferation inhibition is known as a crucial mechanism regulating cell proliferation in vitro and in vivo. In this study we examined the characters of mouse subcutaneous adipose derived stem cells (msADSC) whether they lost or get contact inhibition during in vitro culture. The characters of msADSC growth after confluence were analyzed using confocal microscope and the expression profiles of contact inhibition related genes were analyzed according to the morphological changes using real-time PCR method. msADSC showed overlapping growth between them but not after passage 14. The cell shapes were also changed after passage 14. The expression profiles of genes which are involved in contact inhibition were modified in the msADSC after passage 14. The differentiation ability of msADSCs to adipocyte, chondrocyte and osteocyte was not changed by such changes of gene expression profiles. Based on these results, it is revealed that smADSC were characterized by getting of strong cell-cell contact inhibition after passage 14 but the proliferation and developmental ability were not blocked by the change of cell-cell contact proliferation inhibition. These finding will help to understand the growth of adipose tissue, although further studies are needed to evaluate the physiological meaning of the cell-cell contact proliferation inhibition during in vitro culture of msADSC.

Diet-induced obesity alters the differentiation potential of stem cells isolated from bone marrow, adipose tissue and infrapatellar fat pad: the effects of free fatty acids

IntroductionObesity is a major risk factor for several musculoskeletal conditions that are characterized by an imbalance of tissue remodeling. Adult stem cells are closely associated with the remodeling and potential repair of several mesodermally derived tissues such as fat, bone, and cartilage. We hypothesized that obesity would alter the frequency, proliferation, multipotency, and immunophenotype of adult stem cells from a variety of tissues.Materials and MethodsBone marrow-derived mesenchymal stem cells (MSCs), subcutaneous adipose-derived stem cells (sqASCs), and infrapatellar fat pad-derived stem cells (IFP cells) were isolated from lean and high-fat diet induced obese mice, and their cellular properties were examined. To test the hypothesis that changes in stem cell properties were due to the increased systemic levels of free fatty acids (FFAs), we further investigated the effects of FFAs on lean stem cells in vitro.ResultsObese mice showed a trend toward increased prevalence of MSCs and sqASCs in the stromal tissues. While no significant differences in cell proliferation were observed in vitro, the differentiation potential of all types of stem cells was altered by obesity. MSCs from obese mice demonstrated decreased adipogenic, osteogenic, and chondrogenic potential. Obese sqASCs and IFP cells showed increased adipogenic and osteogenic differentiation, but decreased chondrogenic ability. Obese MSCs also showed decreased CD105 and increased PDGFRα expression, consistent with decreased chondrogenic potential. FFA treatment of lean stem cells significantly altered their multipotency but did not completely recapitulate the properties of obese stem cells.ConclusionsThese findings support the hypothesis that obesity alters the properties of adult stem cells in a manner that depends on the cell source. These effects may be regulated in part by increased levels of FFAs, but may involve other obesity-associated cytokines. These findings contribute to our understanding of mesenchymal tissue remodeling with obesity, as well as the development of autologous stem cell therapies for obese patients.

S1756 Subcutaneous Adipose Tissue-Derived Stem Cells Facilitate Colonic Mucosal Recovery from 2, 4, 6-Trinitrobenzene Sulfonic Acid (TNBS)-Induced Colitis in Rats

S1756 Subcutaneous Adipose Tissue-Derived Stem Cells Facilitate Colonic Mucosal Recovery from 2, 4, 6-Trinitrobenzene Sulfonic Acid (TNBS)-Induced Colitis in Rats

Subcutaneous adipose tissue–derived stem cells facilitate colonic mucosal recovery from 2,4,6-trinitrobenzene sulfonic acid (TNBS)–induced colitis in rats

Adipose tissue-derived stem cells (ADSCs) can be easily obtained from subcutaneous adipose tissue, and ADSCs can be demonstrated to display multilineage developmental plasticity. In this study, using TNBS-induced colitis rats, we show the feasibility of repairing injured intestinal mucosa with adipose tissue-derived stem cells. The subcutaneous adipose tissue of F344 rats was obtained and digested by collagenase. The digested tissue was cultured in DMEM containing 10% FBS for 1 month. ADSCs were confirmed to differentiate under appropriate conditions into various lineages of cells, including bone, neural cells, adipocytes, and epithelial cells. HGF, VEGF, TGF-beta, and adiponectin in the culture supernatants of ADSCs were determined by ELISA. ADSCs (10(7) cells) were injected into the submucosa of the colon to examine their capacity to repair intestinal mucosa injured by TNBS. In the experimental colitis model, the injection of ADSCs facilitated colonic mucosal repair and reduced the infiltration of inflammatory cells. High levels of HGF, VEGF, and adiponectin were detected in the culture supernatants of ADSCs. Moreover, injected ADSCs distributed to several layers of the colon, and some of them differentiated into mesodermal lineage cells. ADSCs can accelerate the regeneration of injured regions in experimental colitis. HGF, VEGF, and adiponectin might be responsible for the regeneration of injured regions in the colon.

Induction of Circadian Gene Expression in Human Subcutaneous Adipose‐derived Stem Cells

Genes encoding the circadian transcriptional apparatus exhibit robust oscillatory expression in murine adipose tissues. This study tests the hypothesis that human subcutaneous adipose-derived stem cells (ASCs) provide an in vitro model in which to monitor the activity of the core circadian transcriptional apparatus. Primary cultures of undifferentiated or adipocyte-differentiated ASCs were treated with dexamethasone, rosiglitazone, or 30% fetal bovine serum. The response of undifferentiated ASCs to dexamethasone was further evaluated in the presence of lithium chloride. Lithium inhibits glycogen synthase kinase 3, a key component of the circadian apparatus. Total RNA was harvested at 4-hour intervals over 48 hours and examined by real-time reverse transcription polymerase chain reaction (RT-PCR). Adipocyte-differentiated cells responded more rapidly to treatments than their donor-matched undifferentiated controls; however, the period of the circadian gene oscillation was longer in the adipocyte-differentiated cells. Dexamethasone generated circadian gene expression patterns with mean periods of 25.4 and 26.7 hours in undifferentiated and adipocyte-differentiated ASCs, respectively. Both rosiglitazone and serum shock generated a significantly longer period in adipocyte-differentiated ASCs relative to undifferentiated ASCs. The Bmal1 profile was phase-shifted by approximately 8 to 12 hours relative to Per1, Per3, and Cry2, consistent with their expression in vivo. Lithium chloride inhibited adipogenesis and significantly lengthened the period of Per3 and Rev-erbalpha gene expression profiles by >5 hours in dexamethasone-activated undifferentiated ASCs. These results support the initial hypothesis and validate ASCs as an in vitro model for the analysis of circadian biology in human adipose tissue.

VEGF is critical for spontaneous differentiation of stem cells into cardiomyocytes

Cardiomyocyte regeneration is limited in adult life and is not sufficient to compensate for cell loss with myocardial infarction. Hence, the identification of a useful source of cardiomyocyte progenitors is of great interest for possible use in regenerative therapy. In this study, we isolated stem cells derived from human subcutaneous adipose tissue. The expression of Nkx2.5 and GATA-4 can be observed by PCR directly after extraction and during cultivation in some of these cells. Cardiac Troponin T and myosin light chain-2v become positive after 12 days of cultivation. To define respective factors responsible for spontaneous differentiation, we measured VEGF level in ADSC conditioned medium. Our data showed that ADSC secrete significant amount of VEGF (283.5pg per microgram DNA) and that anti-VEGF receptor antibodies blocked the cardiac differentiation. In conclusion, we demonstrated the spontaneous differentiation of human subcutaneous adipose-derived stem cells into a cardiomyocyte phenotype under standard culturing conditions.

Secretome of Primary Cultures of Human Adipose-derived Stem Cells: Modulation of Serpins by Adipogenesis

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 > or =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 the etiology of obesity and type 2 diabetes.