脂肪分化相关

Differences in Gene Expression and Cytokine Release Profiles Highlight the Heterogeneity of Distinct Subsets of Adipose Tissue-Derived Stem Cells in the Subcutaneous and Visceral Adipose Tissue in Humans Sebastio Perrini1, Romina Ficarella1, Ernesto Picardi3, Angelo Cignarelli1, Maria Barbaro1, Pasquale Nigro1, Alessandro Peschechera1, Orazio Palumbo5, Massimo Carella5, Michele De Fazio2, Annalisa Natalicchio1, Luigi Laviola1, Graziano Pesole3,4, Francesco Giorgino1* 1Department of Emergency and Organ Transplantation, Section on Internal Medicine, Endocrinology, Andrology and Metabolic Diseases, University of Bari Aldo Moro, Bari, Italy, 2 Section on General Surgery and Liver Transplantation, University of Bari Aldo Moro, Bari, Italy, 3Department of Biosciences, Biotechnology and Pharmacological Sciences, University of Bari Aldo Moro, Bari, Italy, 4 Institute of Biomembranes and Bioenergetics, National Research Council, Bari, Italy, 5 Istituto di Ricovero e Cura a Carattere Scientifico Casa Sollievo della Sofferenza, San Giovanni Rotondo (FG), Italy Abstract Differences in the inherent properties of adipose tissue-derived stem cells (ASC) may contribute to the biological specificity of the subcutaneous (Sc) and visceral (V) adipose tissue depots. In this study, three distinct subpopulations of ASC, i.e. ASCSVF, ASCBottom, and ASCCeiling, were isolated from Sc and V fat biopsies of non-obese subjects, and their gene expression and functional characteristics were investigated. Genome-wide mRNA expression profiles of ASCSVF, ASCBottom and ASCCeiling from Sc fat were significantly different as compared to their homologous subsets of V-ASCs. Furthermore, ASCSVF, ASCCeiling and ASCBottom from the same fat depot were also distinct from each other. In this respect, both principal component analysis and hierarchical clusters analysis showed that ASCCeiling and ASCSVF shared a similar pattern of closely related genes, which was highly different when compared to that of ASCBottom. However, larger variations in gene expression were found in inter-depot than in intra-depot comparisons. The analysis of connectivity of genes differently expressed in each ASC subset demonstrated that, although there was some overlap, there was also a clear distinction between each Sc-ASC and their corresponding V-ASC subsets, and among ASCSVF, ASCBottom, and ASCCeiling of Sc or V fat depots in regard to networks associated with regulation of cell cycle, cell organization and development, inflammation and metabolic responses. Finally, the release of several cytokines and growth factors in the ASC cultured medium also showed both inter- and intra-depot differences. Thus, ASCCeiling and ASCBottom can be identified as two genetically and functionally heterogeneous ASC populations in addition to the ASCSVF, with ASCBottom showing the highest degree of unmatched gene expression. On the other hand, inter-depot seem to prevail over intra-depot differences in the ASC gene expression assets and network functions, contributing to the high degree of specificity of Sc and V adipose tissue in humans. Citation: Perrini S, Ficarella R, Picardi E, Cignarelli A, Barbaro M, et al. (2013) Differences in Gene Expression and Cytokine Release Profiles Highlight the Heterogeneity of Distinct Subsets of Adipose Tissue-Derived Stem Cells in the Subcutaneous and Visceral Adipose Tissue in Humans. PLoS ONE 8(3): e57892. doi:10.1371/journal.pone.0057892 Editor: Andrea Vergani, Children’s Hospital Boston, United States of America Received October 14, 2012; Accepted January 28, 2013; Published March 5, 2013 Copyright: � 2013 Perrini et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by grants from the Ministero dell’Universita` e Ricerca (Italy), the COST Action BM0602, the Fondazione della Societa` Italiana di Diabetologia per la Ricerca in Diabetologia e Malattie Metaboliche (Fo.Ri.SID), and NovoNordisk to F. Giorgino. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: This study was partly funded by NovoNordisk. Francesco Giorgino is a PLOS ONE Editorial Board member. However, there are no patents, products in development or marketed products to declare. This does not alter the adherence of the authors to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors. * E-mail: francesco.giorgino@uniba.it Introduction The biological diversity of adipose tissue depots has become a fundamental issue in recent years, in light of its potential impact on human health [1]; [2]. It is known that visceral adipose tissue is morphologically and functionally different from subcutaneous adipose tissue [3]; [4]. Depot-related variations have long been described for a variety of biological endpoints, such as signaling reactions [5], glucose metabolism [6]; [7] and cytokine secretion [8]. It has been also proposed that extrinsic factors, including depot-specific blood flow, cell density, cell heterogeneity and/or innervation [9], could contribute to distinct gene expression patterns and metabolic profiles of adipocytes from different anatomical regions. Alternatively, variations in the inherent properties of undifferentiated fat cell progenitors may dictate the biological specificity between the two fat depots bringing about the innate characteristics of Sc and V adipose cells. This concept has been supported by the demonstration that pre-adipocytes from distinct fat depots retain specific dynamic characteristics and gene expression patterns even after 40 cell doublings [10]. More recently, we have shown that depot-related differences in gene expression, adiponectin secretion, and insulin signaling and action PLOS ONE | www.plosone.org 1 March 2013 | Volume 8 | Issue 3 | e57892 were still evident when precursor cells isolated from bioptic adipose tissue fragments were differentiated in vitro [11]. Altogether, these findings support the concept that there could be an early commitment of fat precursor stromal cells able to influence the biological responses of the resulting adipocytes, independently of extrinsic influences deriving from tissue micro- environment. However, little is known about the identity, localization, or specific characteristics of endogenous adipocyte progenitors. Adipocyte progenitors recognizably reside in the adipose stromal-vascular fraction (SVF), a heterogeneous mixture of cells operationally defined by enzymatic dissociation of fat tissue followed by density separation from adipocytes [12]. Initially, the stromal-derived cells were designated as «pre-adipocytes», but since 2004 the International Fat Applied Technology Society adopted the term «adipose-derived stem cells» (ASC) to define the plastic-adherent cells isolated from SVF of adipose tissue with self- renewal properties and able to regenerate adipocytes (ASCSVF) [12]; [13]. Further complexity to this scenario, however, is provided by the observation that, during the procedure of fat tissue enzymatic digestion and centrifugation, two additional ASC populations can be isolated from the «fat cake» at the top of the supernatant [13]. One ASC population is obtained from a pre- adipocyte fraction in the fat cake not previously collected together with the ASCSVF; these cells can be grown at the bottom surface of the culture flask (ASCBottom). Another ASC population develops from mature adipocytes of the fat cake adherent to the ceiling surface of the culture flask filled with growth medium (ASCCeiling). It has been suggested that ASCCeiling may derive from mature adipocytes through an asymmetric mitosis [14]; [15]. In culture, ASCCeiling and ASCBottom display both cell-surface markers that are similar to those expressed by ASCSVF, including CD105, SH3, Stro-1, CD49d and CD44, and show similar potential for unlimited self-renewing proliferation and differentiation along the mesenchymal lineage to produce adipocytes, osteoblasts, and chondrocytes [15]. In addition to their ability to differentiate into multiple mesenchymal cell lineages, ASC actively produce paracrine factors, hormones and metabolic signals, in a distinct manner from that of differentiated fat cells. Indeed, non-fat cells and ASC are considered one of the main sources of pro-inflammatory adipokine released by the adipose tissue [16], with great potential for repercussions on distant target tissues and metabolic and cardiovascular regulation [17]. Several studies have reported that ASC and adipocytes contribute roughly similarly to the overall secretion/expression of adipokines, except for adiponectin and leptin [16]; [18]; [19]. Thus, the different production of adipokines, such as IL-6 or MCP-1, between subcutaneous and visceral adipose tissues may reflect either intrinsic properties of the resident ASC or a different ASC proportion within each fat depot [20]; [21], underlying the contribution of ASC to the biological diversity of specific adipose tissue depots. In this study, ASCSVF, ASCBottom and ASCCeiling were isolated from abdominal subcutaneous (Sc) and visceral omental (V) adipose tissue biopsies obtained from non-obese subjects and studied through a genome-wide differential gene expression analysis followed by an in-depth bioinformatics examination. In addition, 27 cytokines were measured in the culture medium collected from each of the 6 ASC populations. By performing comparative intra-depot (i.e., ASCSVF vs. ASCCeiling vs. ASCBot- tom) and inter-depot (i.e., Sc-ASC vs. V-ASC) analyses, differences in transcripts data and cytokines output emerged, which allowed us to identify six ASC subsets in the Sc and V fat depots that differ among them for gene expression profiles e cell functions. Materials and Methods Materials All tissue culture reagents were purchased from Life Technol- ogies (Carlsbad, CA, US). Unless otherwise stated, all chemicals used were obtained from Sigma-Aldrich (St. Louis, MO, US). Recombinant human insulin was purchased from Roche Diag- nostics (Mannheim, Germany). The thiazolinedione (TZD) compound rosiglitazone was kindly provided by GlaxoSmithKlein (Middlesex, UK). Recombinant human fibroblast growth factor 2 (FGF2) and EGF were obtained from Miltenyi Biotec (Bergisch Gladbach, Germany). Human Donors and Adipose Tissue Biopsies Paired Sc and V adipose tissue biopsies were obtained from 15 non-obese subjects undergoing elective open-abdominal surgery (9 men, 6 women; age 6867 yrs; BMI 27.061.5 kg/m2; fasting plasma glucose 85611 mg/dl). None of the patients had diabetes or severe systemic illness, and none were taking medications known to affect adipose tissue mass or metabolism. The protocol was approved by the Independent Ethical Committee at the Azienda Ospedaliero-Universitaria Policlinico Consorziale, Bari, Italy. All patients gave their written informed consent. ASC Isolation Human ASC were isolated as previously described [11]; [14], with minor modifications. Briefly, fat tissue fragments were minced and digested in medium containing 1 mg/ml collagenase, type I, with gentle shaking at 37uC for 1 h. Resulting material was filtered through 250 mm mesh, and adipocytes and free oil were separated from stromovascular components by centrifugation at 1,200 rpm for 5 min at room temperature. The floating fraction consisting of pure isolated adipocytes was placed in 25-cm2 culture flasks completely filled with DMEM/Ham’s F12 1:1 supplemented with 20% fetal bovine serum, and cells were incubated at 37uC in 5% CO2. The primary ASC grown at top and bottom of flask, respectively, were cultured for 7 days until they reached confluence (defined as passage 0), and were then split into 60- mm plates. The stromovascular pellet was resuspended in erythrocyte lysis buffer, consisting of 154 mM NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA, for 5 min. The cell suspension was centrifuged at 1,200 rpm for 5 min and then resuspended in a culture medium consisting of DMEM/Ham’s F12, 10% FCS and antibiotics. This cell suspension was filtered through a 25-mm sterile nylon mesh before being plated. After a 16-h incubation for cell attachment, cells were cultured in ASC growth medium (DMEM/Ham’s F12 1:1 supplemented with 100 units/ml penicillin, 0.1 mg/ml streptomycin, 2.5% FCS, 1 ng/ml FGF2, and 10 ng/ml EGF). Except when indicated, all cells were used in the experimental procedures at passage 4. Mesenchymal Differentiation For adipogenic differentiation, confluent ASC at passage 4 were differentiated in a chemically defined serum-free medium contain- ing antibiotics, 2 nM triiodotyronine (T3), 100 nM human insulin, 100 nM dexamethasone, and 1 mM rosiglitazone, as previously reported [11]; for the first 4 days of the differentiation period, 0.5 mM of methyl-isobutylxanthine was also added. Osteogenic differentiation was induced as previously described [22]. Differ- entiation into chondrocytes was induced by StemPro Chondro- genesis Differentiation Medium, according to the manufacturer’s instructions. Heterogeneity of Human Adipose Tissue Precursors PLOS ONE | www.plosone.org 2 March 2013 | Volume 8 | Issue 3 | e57892 Histochemical Staining of Differentiated Cells Oil-Red-O, Alizarin Red, and Alcian Blue staining, respective- ly, were performed as described previously [11]; [22]; [23], and pictures were taken on wide-field microscopes (Nikon) with a color CCD camera. RNA Isolation and Quantitative RT-PCR (qRT-PCR) RNA from the ASC populations was isolated by using an RNeasy kit (Qiagen, Hamburg, Germany). 250 ng of RNA were reverse-transcribed with standard reagents (Applied Biosystems). One microliter of each reverse-transcription reaction was ampli- fied by using SYBR Green PCR master mix from Applied Biosystems, using the ABI 7500 real-time PCR system. For each gene, mRNA expression was calculated relative to 18S rRNA. Amplification of specific transcripts was confirmed by melting curve profiles at the end of each PCR. Primer sequences for each gene are given in Table S1. Microarray Analysis Total RNA was isolated from the ASC of 5 lean subjects. The quality and integrity of total RNA (RNA Integrity Number [RIN] $8.0) was evaluated on an Agilent Bioanalyzer (Agilent Technol- ogies, Waldbronn, Germany). RNA was then processed for hybridization on Human Gene 1.0 ST Array chip (Affymetrix, High Wycombe, UK), covering 28,869 well-annotated genes with 764,885 distinct probes, using standard Affymetrix protocols. Raw signal intensities of gene expression data were processed, analyzed, and linearly scaled using GeneChip Operating Software 1.1 (GCOS, Affymetrix) to a mean hybridization intensity of 500 units. For each array, GCOS output was imported as CEL files into Partek Genomic Suite (Partek GS, Partek Inc., 2008, Revision 6.3) software, and data were normalized using the RMA (Robust Multichip Averaging) algorithm. RNA hybridization intensity data obtained from the array analysis were concordant for all ASCBottom, and for 4 of the 5 ASCCeiling and ASCSVF from both Sc and V fat depot. The remaining one ASC samples were excluded from the array analysis, since their data were ambiguous or incorrect, resulting in cell-mismatched design when compared with the homologous Sc and V-ASC subsets. Similarities and differences among gene expression profiles were assessed by hierarchical clustering using Partek GS software. Statistical significance was defined as being differentially expressed with an adjusted p-value,0.01. The array data have been deposited at the Gene Expression Omnibus archive, accession number GSE37324. A further analysis to identify the biological mechanisms, pathways and functions and the most relevant overexpressed genes was performed using the Ingenuity Pathways Analysis (IPA) software and database (Ingenuity Systems, http://www.ingenuity.com). Briefly, the dataset containing gene identifiers and corresponding fold changes was uploaded into the web-delivered application, and each gene identifier was mapped to its corresponding gene object. The threshold for a significant association was determined by the p-value ,0.01, becoming significant any score above 3 [24]; [25]. For all analyses, Fisher’s exact test was used to calculate a p-value and to determine a score for all networks that were ranked on the probability that a collection of genes equal to or greater than the number in a network could be achieved by chance alone. Multiplex Analysis of Cytokine Production ASC-derived conditioned medium (CM) was obtained by incubating confluent cells in serum-free DMEM/Ham’s F12 for 16 h, after which time all CM was collected, pooled, cleared by centrifugation, frozen in dry ice, and stored at 280uC. Concen- trations of various cytokines in CM were analyzed using a multiplex biometrix immunoassay from Bio-Rad, containing fluorescent dyed microspheres conjugated with a monoclonal antibody specific for the target proteins according to the manufacturer’s instructions (Bio-Plex Human Cytokine Assay; Bio-Rad, Munich, Germany). The following cytokines were assayed: IL-1b, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 (p70), IL-13, IL-15, IL-17, FGF basic, eotaxin, G- CSF, GM-CSF, IFN-c, IP-10, MCP-1 (MCAF), MIP-1a, MIP-1b, PDGF-BB, RANTES, TNF-a, and VEGF. The amount of protein in the medium was normalized for 106 cells [26]. The inter- and intra-assay coefficients of variation for all cytokines under investigation were less than 10%. Statistical Analysis Data are presented as mean 6 SE. Comparisons between the values were performed using a two-tailed Student’s t-test. For the comparison of multiple groups, a one-way ANOVA test followed by Fisher’s post-hoc test was applied. For all statistical analyses, the level of significance was set at a probability of p,0.05. All experiments were repeated at least 3 times. These analyses were performed using SPSS 16 (SPSS, Chicago, IL). Results Phenotypic Analysis and Stem Cell Properties of ASC Populations ASCSVF, ASCCeiling and ASCBottom isolated from the Sc and V fat depots grew as a characteristic cell monolayer in culture dishes (Fig. 1A). To identify these cells as adipocyte progenitors and to rule out potential contamination by other adipose tissue cell types, analysis by qRT-PCR for a selection of known hematopoietic, endothelial and stem cell-associated markers was carried out. The expression of the key ASC markers CD105, CD49d, and CD44 was noticeably pronounced and expressed at similar magnitude in ASCSVF, ASCCeiling and ASCBottom from both Sc and V fat depots (Fig. 1B; Table S2). The initial Sc-ASCSVF and V-ASCSVF cultures contained a subset of cells that were also positive for the endothelial and hematopoietic lineage markers CD11b, CD45, and CD31 (Fig. 2A). However, with successive passages, the expression of CD11b, CD45, and CD31 declined significantly, becoming negligible by passage 4 (p,0.001 vs. passage 0; Fig. 2A), whereas expression levels of ASC markers remained constant (p=0.860 vs. passage 0; Fig. 2B). To confirm the multipotent mesenchymal stem cell properties of the distinct ASC populations, a comparative in vitro tri-lineage differentiation assay was carried out by exposing culture-expanded ASC derived from Sc and V adipose tissue to osteogenic, chondrogenic, or adipogenic differentiation media, respectively. The qualitative histochemical evaluation of functional outcomes indicated that ASCSVF, ASCCeiling and ASCBottom from both adipose tissue depots underwent dif