International Journal of Molecular Sciences Article Crohn’s Disease Increases the Mesothelial Properties of Adipocyte Progenitors in the Creeping Fat Ana Madeira 1,2,† , Carolina Serena 1,2,† , Miriam Ejarque 1,2 , Elsa Maymó-Masip 1,2 , Monica Millan 1,2,3 , M. Carmen Navarro-Ruiz 4,5,6 , Rocío Guzmán-Ruiz 4,5,6 , María M. Malagón 4,5,6 , Eloy Espin 7 , Marc Martí 7 , Margarita Menacho 8 , Ana Megía 1,2 , Joan Vendrell 1,2,9, * and Sonia Fernández-Veledo 1,2, * 1 Department of Research, Institut d’Investigació Sanitària Pere Virgili, Hospital Universitari de Tarragona Joan XXIII, 43005 Tarragona, Spain; madeira.aps@gmail.com (A.M.); carolserena@gmail.com (C.S.); miriam.ejarque@gmail.com (M.E.); elsamaymomasip@gmail.com (E.M.-M.); monica.millan@ymail.com (M.M.); amegia.hj23.ics@gencat.cat (A.M.) 2 Instituto de Salud Carlos III, CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), 08029 Madrid, Spain 3 Department of Surgery, Colorectal Surgery Unit, Hospital Universitari i Politècnic La Fe, 46026 Valencia, Spain 4 Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC), University of Cordoba, Reina Sofia University Hospital, Edificio IMIBIC, Avda. Menéndez Pidal s/n, 14004 Cordoba, Spain; b02narum@uco.es (M.C.N.-R.); bc2gurur@uco.es (R.G.-R.); bc1mapom@uco.es (M.M.M.) 5 Department of Cell Biology, Physiology and Immunology, University of Cordoba, 14004 Cordoba, Spain 6 CIBER Fisiopatología de la Obesidad y Nutrición (CIBEROBN), Instituto de Salud Carlos III, 28029 Madrid, Spain  7 Colorectal Surgery Unit, General Surgery Service, Hospital Vall d’Hebron, Universitat Autònoma de  Barcelona, 08035 Barcelona, Spain; eespin@vhebron.net (E.E.); marcmartig@gmail.com (M.M.) 8 Digestive Unit, Hospital Universitari Joan XXIII, 43007 Tarragona, Spain; mmenacho.hj23.ics@gencat.cat Citation: Madeira, A.; Serena, C.; 9 Department Medicine and Surgery, Medicine School, Universitat Rovira i Virgili, 43003 Tarragona, Spain Ejarque, M.; Maymó-Masip, E.; * Correspondence: jvo@comt.es (J.V.); sonia.fernandezveledo@gmail.com (S.F.-V.); Millan, M.; Navarro-Ruiz, M.C.; Tel.: +34-977-295-800 (ext. 3401) (J.V. & S.F.-V.); Fax: +34-977-295-823 (J.V. & S.F.-V.) Guzmán-Ruiz, R.; Malagón, M.M.; † These authors contributed equally to this work. Espin, E.; Martí, M.; et al. Crohn’s Disease Increases the Mesothelial Abstract: Our understanding of the interplay between human adipose tissue and the immune system Properties of Adipocyte Progenitors in the Creeping Fat. Int. J. Mol. Sci. is limited. The mesothelium, an immunologically active structure, emerged as a source of visceral 2021, 22, 4292. https://doi.org/ adipose tissue. After investigating the mesothelial properties of human visceral and subcutaneous 10.3390/ijms22084292 adipose tissue and their progenitors, we explored whether the dysfunctional obese and Crohn’s dis- ease environments influence the mesothelial/mesenchymal properties of their adipocyte precursors, Academic Editor: Giovanni Latella as well as their ability to mount an immune response. Using a tandem transcriptomic/proteomic approach, we evaluated the mesothelial and mesenchymal expression profiles in adipose tissue, Received: 10 March 2021 both in subjects covering a wide range of body-mass indexes and in Crohn’s disease patients. We Accepted: 16 April 2021 also isolated adipose tissue precursors (adipose-derived stem cells, ASCs) to assess their mesothe- Published: 20 April 2021 lial/mesenchymal properties, as well as their antigen-presenting features. Human visceral tissue presented a mesothelial phenotype not detected in the subcutaneous fat. Only ASCs from mesenteric Publisher’s Note: MDPI stays neutral adipose tissue, named creeping fat, had a significantly higher expression of the hallmark mesothelial with regard to jurisdictional claims in genes mesothelin (MSLN) and Wilms’ tumor suppressor gene 1 (WT1), supporting a mesothelial published maps and institutional affil- nature of these cells. Both lean and Crohn’s disease visceral ASCs expressed equivalent surface iations. percentages of the antigen-presenting molecules human leucocyte antigen—DR isotype (HLA-DR) and CD86. However, lean-derived ASCs were predominantly HLA-DR dim , whereas in Crohn’s dis- ease, the HLA-DR bright subpopulation was increased 3.2-fold. Importantly, the mesothelial-enriched Crohn’s disease precursors activated CD4+ T-lymphocytes. Our study evidences a mesothelial signa- Copyright: © 2021 by the authors. ture in the creeping fat of Crohn’s disease patients and its progenitor cells, the latter being able to Licensee MDPI, Basel, Switzerland. This article is an open access article present antigens and orchestrate an immune response. distributed under the terms and conditions of the Creative Commons Keywords: Crohn’s disease; adipose tissue; mesothelium; adipose-derived stem cells Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Int. J. Mol. Sci. 2021, 22, 4292. https://doi.org/10.3390/ijms22084292 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 4292 2 of 19 1. Introduction Excessive and dysfunctional adipose tissue is responsible for many of today’s health epidemics, especially obesity. Additionally, obesity-associated chronic low-grade inflam- mation is a key contributing factor to systemic metabolic dysfunction and comorbidities such as type 2 diabetes (T2D), fatty liver disease, cardiovascular disease, and cancer [1]. Fat accumulation and inflammation can also be locally restricted. Such is the case in Crohn’s disease, a chronic inflammatory bowel disease characterized by the expansion of mesenteric adipose tissue, known as “creeping fat”, which correlates with the disease severity [2–4]. Recent findings indicate similarities between the complex pathological features of Crohn’s disease and obesity/T2D with respect to adipose tissue behavior [5], especially in terms of inflammation [5–7], and it is clear that adipose tissue holds yet unrealized functions in the setting of these diseases. In fact, a recent publication used a novel mesenteric creeping fat index based on computed tomography to characterize accurately the extent of mesenteric fat wrapping in surgical specimens, which correlated with intestinal fibrostenosis [8]. White adipose tissue (WAT) is a highly plastic and dynamic tissue composed of various cell types, including mature adipocytes, adipose-derived stem cells (ASCs, the precursor cells that give rise to adipocytes), vascular cells, and a diverse array of innate and adaptive immune cells. In addition to being a highly active endocrine organ that can orchestrate whole-body energy homeostasis [9,10], WAT participates in immune surveillance and host defense. Indeed, recent research placed mammalian WAT at the center of immunological memory: WAT is a reservoir of memory T-cells, which are able to sustain long-term immune responses to infection [11]. Increasing evidence suggests that an appreciation of the complex heterogeneity among adipose tissue progenitors is key to fully understand the functional and biological dif- ferences between fat depots [12–17]. Depending on its anatomical location, WAT can be broadly classified as visceral adipose tissue (VAT) or subcutaneous adipose tissue (SAT). Global differences in the gene expression and functional properties (including metabolic activity and adipokine secretion profile) of adipose tissue are well-documented for visceral and subcutaneous depots [18]. Nevertheless, significant gaps remain in our understand- ing of the developmental origins of the different WAT depots, including creeping fat in Crohn’s disease patients. Recent evidence indicates that creeping fat expands to prevent the dissemination of translocated gut bacteria across the leaky intestinal barrier [19]. Adipocytes are generally believed to derive from the mesoderm, and distinct develop- mental timings are described for SAT and VAT [9,20]. Additionally, adipocytes have long been thought to derive exclusively from tissue-resident ASCs/mesenchymal stem cells (MSCs); however, there is growing evidence that individual adipose tissue depots may arise through distinct lineages [9,13–16,21–25]. The mesothelium—a layer of specialized epithe- lial (mesothelial) cells that forms the lining of all serous cavities—was recently proposed as a source of adipocyte progenitors for VAT, but not SAT, hinting at the diversity among visceral adipocytes and precursor cells [17,21]. A recent single-cell sequencing study iden- tified a mesothelial subpopulation within the human visceral stromal vascular fraction [17]. However, little is known about the functionality of these mesothelial-enriched precursors, and whether their proportion is changed by a pathological environment. Mesothelial cells are capable of recognizing pathogens and tissue damage and initiating inflamma- tory responses through antigen presentation and cytokine production [26–31]. These cells are thought to represent the first line of defense against bacterial insults to the adipose tissue [32], which is a particularly troublesome event in Crohn’s Disease and is possibly the trigger of creeping fat expansion [19]. The mesothelium is one of the fundamental histological structures that composes the mesentery [33]. Mesentery´s fat is abnormally expanded in Crohn’s Disease, leading to the formation of creeping fat around the intestinal damaged area. Given the recent link established between the mesothelium and the development of VAT, we hypothesized that human visceral ASCs, especially those from the creeping fat, derive from the mesothelial lineage. Upon microbial dysregulation and the breakdown of the intestinal barrier (both Int. J. Mol. Sci. 2021, 22, 4292 3 of 19 hallmarks of Crohn’s Disease), ASCs migrate towards the intestinal lesions to participate in tissue repair [19]. This event, which would be beneficial in the first instance, also induces an accumulation of adipose tissue surrounding the intestine (“creeping fat”), which is a consequence of the natural fate of ASCs to differentiate into adipocytes. In this work we hypothesized that the proportion of mesothelial-derived precursors is increased in the creeping fat and that the newly recruited precursors are able to process and present antigens and mount an immune response. Accordingly, we set out to investigate the mesothelial/mesenchymal properties of human adipose tissue and their precursors in Crohn’s disease and obesity (an established inflammatory condition). Finally, our goal was to demonstrate that ASCs’ populations with different abundances of mesothelial precursors would also differ in their immune phenotypic and antigen-presentation features. 2. Results 2.1. A Mesothelial Gene Signature Distinguishes Visceral Adipose Tissue and Its Precursors from Subcutaneous Adipose Tissue To investigate the mesothelial properties of human adipose tissue, we first sur- veyed the expression of a panel of mesothelial marker genes in the SAT and VAT de- pots of lean individuals using an inventoried TaqMan array. We found that the ex- pression of keratin 8 (KRT8), cadherin 1 (CDH1), mesothelin (MSLN), leucine rich repeat neuronal 4 (LRRN4), and Wilms’ tumor suppressor gene 1 (WT1) was significantly higher in VAT than in SAT (Figure 1A), which is consistent with the finding of a mesothelial gene expression pattern in omental fat in a previous microarray-based analysis (GEO accession number GSE20950; Supplementary Table S1) [34]. By contrast, no differences were found in the expression of typical mesenchymal genes between VAT and SAT samples (Figure 1A). We then performed qualitative liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) proteomic analysis in lean adipose tissue to gain further insight into the differences in mesothelial marker expression between SAT and VAT. Results showed that the mesothelial proteins MSLN, tight junction protein 1 (TJP1), keratin 7 (KRT7), KRT8, and calponin 1 (CNN1) were detected in VAT but not in SAT samples (Supplementary Figure S1), in accordance with the transcription profile. Having established a mesothelial signature for VAT from lean subjects, we extended this analysis to examine the gene expression profile of precursor cells isolated from the two distinct adipose tissue depots. As shown in Figure 1B, expression of the mesothelial genes CNN1, CDH1, MSLN, and WT1 was significantly higher in ASCs obtained from lean intra-abdominal adipose tissue than from SAT, supporting a mesothelial nature for these cells. Moreover, a reduction in the expression of mesenchymal markers was also noted for lean visceral-derived ASCs when compared with their SAT counterparts [17,35–37]. Overall, these results support a mesothelial origin for human VAT and its precursors. 2.2. Creeping Fat Precursor Cells Have Mesothelial-Enriched Properties Obesity and Crohn’s disease are distinguished by intra-abdominal fat accumulation, which is a well-recognized forerunner of disease pathology [5]. Given our identification of a mesothelial profile for VAT in lean subjects, we next tested whether this held true in the context of obesity and Crohn’s Disease. We again analyzed a panel of marker genes in adipose tissue and ASCs obtained from obese and morbid obese subjects, and from patients with Crohn’s Disease. We found a consistent upregulation of multiple mesothelial markers in VAT samples when compared with equivalent SAT samples, and these differences were maintained irrespective of the BMI or whether samples were from patients with Crohn’s disease (Figures 2 and 3). We also observed a non-significant downregulation in the expres- sion of several mesenchymal genes, when comparing VAT and SAT samples from obese, morbid obese, and Crohn’s disease patients. No significant differences were observed in the expression of mesothelial markers in obese, morbid obese, and Crohn’s disease VAT samples with respect to lean-derived VAT (Supplementary Figure S2). Interestingly, the expression of several mesothelial genes in mesenteric fat-derived ASCs from patients with Int. J. Mol. Sci. 2021, 22, 4292 4 of 19 Crohn’s disease showed a clear upregulation with respect to equivalent lean VAT-derived ASCs, namely LRRN4, carbonic anhydrase 2 (CA2), MSLN, and KRT8 (Figure 4), with a trend for greater expression for others including CDH1, KTR7, WT1, and THBD. No differ- ences were observed for the expression of mesenchymal markers in obese, morbid obese, andREVIEW Int. J. Mol. Sci. 2021, 22, x FOR PEER Crohn’s disease VAT and VAT-derived ASCs with respect to equivalent lean-derived 4 of 21 samples (Supplementary Figure S3). Figure 1. Mesothelial markers are Figure 1. Mesothelial elevated in leanmarkers VAT and are elevated in lean VAT in VAT-derived and inqPCR ASCs. VAT-derived analysis ASCs. qPCR analysis of mesothelial and of mesothelial and mesenchymal marker expression in (A) Lean VAT versus lean SAT samples mesenchymal marker expression(VAT in (A) Lean VAT versus lean SAT samples (VAT n = 10; SAT n = 12) and n = 10; SAT n = 12) and (B) VAT-derived ASCs versus SAT-derived ASCs (VAT-ASCs n = 8, (B) VAT-derived ASCs versus SAT-derived ASCsexcept (VAT-ASCs n= n = 14 for 8, except LRRN4, MSLN,n =KRT8, LRRN4, 14 forCDH1, WT1,MSLN, CDH2, KRT8, and CA2;CDH1, WT1,n CDH2, SAT-ASCs = 8). Meanand CA2; lean SAT-ASCs n = 8). Mean lean SAT SATvalues values were were normalized normalized to 1, toand are represented 1, and as a redas are represented dotted a redline. n represents dotted line. nbiologi- represents biologically independent samples. cally independent samples. Abbreviations: ANPEP,Abbreviations: ANPEP, alanyl alanyl aminopeptidase; aminopeptidase; ASCs, adipose-derivedASCs,stem adipose-de- cells; CNN1, rived stem cells; CNN1, calponin 1 ; CDH1, cadherin 1; ENG, endoglin; ITGA4, integrin subunit calponin 1; CDH1, cadherin 1; ENG, endoglin; ITGA4, integrin subunit alpha 4; KRT8, keratin 8; LRRN4, alpha 4; KRT8, keratin 8; LRRN4, leucine rich repeat neuronal 4; MME, membrane metalloendo- leucine rich repeat neuronal 4; MME, membrane metalloendopeptidase; peptidase; MSLN, mesothelin; MSLN, mesothelin; NT5E, NT5E, 5’-nucleotidase 5’-nucleotidase ecto; SAT, subcutaneousecto;adipose SAT, subcutaneous tissue; TJP1, adipose tissue; TJP1, tight junction tight junction protein 1; protein UPK3, 1; UPK3, uroplakin uroplakin 3A; VAT, 3A;visceral VAT, visceral adipose adipose tissue; tissue; WT1, WT1, Wilms Wilms tumor tumor protein. protein. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 (Mann-Whitney U test). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 (Mann-Whitney U test). Int. J. Mol. Sci. 2021, 22, 4292 5 of 19 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 6 of 21 Figure Figure2.2.Mesothelial Mesothelialmarkers markersare upregulated are upregulated in in obese, obese,morbid morbid obese, andand obese, Crohn’s disease Crohn’s VAT disease VAT with with respect to SAT. qPCR analysis of mesothelial and mesenchymal marker gene expression ininVAT respect to SAT. qPCR analysis of mesothelial and mesenchymal marker gene expression VAT versus SAT samples in (A) obese (ob) (ob VAT n = 10, except n = 14 for LRRN4, MSLN, KRT8, versus SAT samples in (A) obese (ob) (ob VAT n = 10, except n = 14 for LRRN4, MSLN, KRT8, CDH1, CDH1, WT1, CDH2, and CA2; ob SAT n = 10), (B) morbid obese (mob) (mob VAT n = 13 or 8, and WT1, CDH2, and CA2; ob SAT n = 10), (B) morbid obese (mob) (mob VAT n = 13 or 8, and mob SAT mob SAT n = 8) and (C) Crohn’s disease (CD VAT n = 10, except n = 20 for LRRN4, MSLN, KRT8, n = 8) and CDH1, WT1,(C) Crohn’s CDH2, anddisease CA2; CD (CD VAT SAT n =n 8). = 10, except Mean SATn levels = 20 for LRRN4, were MSLN, to normalized KRT8, CDH1, 1, and WT1, are rep- CDH2, CA2; CD SAT n resented as a red dotted line. Abbreviations: CA2, carbonic anhydrase 2; CALB2, calretinin; CD, red and = 8). Mean SAT levels were normalized to 1, and are represented as a dotted line. Crohn’s Abbreviations: disease; CA2,morbid ob, obese; mob, carbonic anhydrase obese; 2; CALB2, calretinin; SAT, subcutaneous adiposeCD, Crohn’s tissue; VAT, disease; visceral ob, obese; mob, adipose morbid tissue. obese; Data are SAT, subcutaneous expressed as mean ± SEM;adipose * p 96%), CD73 (ecto-50 -nucleotidase; >98%), CD105 (Endoglin; >82%), and CD36 (platelet glycoprotein 4; >43%), and negative for CD34 (hematopoietic progenitor cell antigen CD34; <0.1%), CD45 (leukocyte common antigen; <0.1%), CD14 (monocyte differentiation antigen; <2%), and CD31 (platelet endothelial cell adhesion Int. J. Mol. Sci. 2021, 22, 4292 14 of 19 molecule; <1%). In addition, we analyzed those related to antigen presentation such as HLA-DR (Human Leukocyte Antigen—DR isotype), CD86 (T-lymphocyte activation antigen CD86), and CD40 (Tumor necrosis factor receptor superfamily member 5). 4.4. Gene Expression Analysis Fat tissue samples were homogenized using a conventional battery-operated, hand- held homogenizer and solubilized in Tris Reagent (Sigma, St. Louis, MO, USA). Total RNA was isolated from adipose tissue and cells using the miRNeasy Mini kit (Qiagen Science, Hilden, Germany). RNA quantity was measured at 260 nm, and purity was assessed by the OD260/OD280 ratio. One microgram of RNA was transcribed to cDNA with random primers using the Reverse Transcription System (Applied Biosystems, Foster City, CA, USA). Quan- titative gene expression was evaluated by real-time polymerase chain reaction (qPCR) on a 7900HT Fast Real-Time PCR System, using 384-well microfluidic TaqMan low- density array cards with an inventoried panel of gene expression assays from Applied Biosystems (Carlsbad, CA, USA). We performed an extensive literature search to assem- ble the mesothelial and mesenchymal expression profiles with well-established markers (Supplementary Table S2). Relative expression levels were calculated using the compara- tive Ct method and normalized to the expression of the housekeeping genes cyclophilin 1A (PPIA; reference gene for the tissue samples) and 18S (reference gene for ASCs samples). 4.5. Proteomics Paired SAT and VAT samples (100 mg) from three lean male subjects were pooled and processed for proteomic analysis, as described [71]. Samples were pooled to reduce biologi- cal variance among individuals. LC-MS/MS analysis was carried out at the Proteomics Unit of the University of Córdoba (Central Services Research Support, Córdoba, Spain). Adipose tissue samples were analyzed using a Dionex Ultimate 3000 nano-liquid chromatography system (nano UHPLC; Thermo Fisher Scientific, Sunnyvale, CA, USA) in tandem with a linear quadrupole ion trap Orbitrap (LTQ Orbitrap XL) mass spectrometer equipped with a nanoelectrospray ion source (Thermo Fisher Scientific, Waltham, MA, USA). Acquired data were analyzed with Proteome Discoverer 2.1 software (Sequest HT algorithm; Thermo Fisher Scientific, Waltham, MA, USA), using a specific human database extracted from the non-redundant UNIPROT website (https://www.uniprot.org (accessed on 20 April 2021).). Peptide searches were performed with a maximum of one missed cleavage site. Car- bamidomethylation of cysteines was set as a fixed modification, and methionine oxidation and lysine acetylation were set as variable modifications. Finally, the identified peptides were filtered using a 1% false-discovery rate calculated using a decoy database strategy. 4.6. Antigen Presentation Assay between ASCs and Autologous CD4+ T-lymphocytes This assay required a coordinated extraction of visceral adipose tissue and isolation of ASCs with the collection of blood samples from the same patient. ASCs were kept for approximately three weeks in culture, and when they reached passage number 3, an additional appointment with the patient took place to collect the blood samples and isolate peripheral blood mononuclear cells (PBMCs) and CD4+ T-lymphocytes. The detailed antigen presentation protocol was as follows: Day 1: ASCs activation. Confluent ASCs cultures were treated or not with Inter- feron gamma (IFNγ) (10 ng/mL) for 1 h before the addition or not of ovalbumin (OVA) (1 mg/mL) for 18 h [72,73]. Day 2: CD4+ T-cells isolation and co-culture. CD4+ T-cells were isolated from PBMCs, which were obtained from heparinized blood by gradient centrifugation on a Ficoll solution (density 1.077 g/mL, Biochrom, Germany) at 800× g for 15 min at room temperature. PBMCs’ count and viability was assessed by trypan blue dye exclusion, and cells were immediately used for CD4+ T-cells’ isolation through the autoMACS Pro Cell Separator using the human CD4+ T-cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacture’s protocol. Autologous CD4+ T-cells were labeled with 2 µM Int. J. Mol. Sci. 2021, 22, 4292 15 of 19 CellTrace violet dye (CTV) (CellTrace kits; Invitrogen, Waltham, MA, USA) according to manufacturer’s protocol, after which were added to ASCs at a 1:4 ratio in RPMI medium. Day 4: assay. T-cells’ proliferation was followed by flow cytometry by examining CTV intensity in viable CD4+ T-lymphocytes. ASCs were harvested and immunophenotyped, as previously described. Data were acquired on FACSAria™ III (BD Biosciences, Franklin Lakes, NJ, USA) and were analyzed using FACSDiva™ v8 (BD Biosciences, Franklin Lakes, NJ, USA) and FlowJo™ v10 softwares (Ashland, OR, USA). 4.7. Statistical Analysis Statistical analysis was performed with the statistical computation system R, Version 2.6.2 [74]. The results were expressed as mean ± SEM of n biologically independent samples. Significance was tested by the unpaired Mann-Whitney U test with Benjamin- Hochberg adjustment. Linear models with empirical Bayes statistic (Limma) were used for data obtained from the National Center for Biotechnology Information Gene Expression Omnibus (GEO) database. 5. Conclusions We found a mesothelial gene signature that distinguishes visceral adipose tissue and its precursors from subcutaneous adipose tissue. Furthermore, we demonstrated that the creeping fat precursor cells have mesothelial-enriched properties including an altered immunophenotypic profile and may act as antigen-presenting cells. Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/ijms22084292/s1, Figure S1: Heatmap of mesothelial marker proteins in paired samples of lean SAT and VAT, Figure S2: Mesothelial expression profile in VAT from obese, morbid obese and Crohn’s disease patients does not significantly differ from that of lean individuals, Figure S3: Mesenchymal marker expression in VAT and VAT-derived ASCs from obese, morbid obese and Crohn’s disease patients does not significantly differ from that of lean individuals, Figure S4: Flow cytometry analysis of lean subcutaneous (SAT)-derived ASCs, lean visceral (VAT)-derived ASCs, and Crohn’s disease visceral-derived ASCs, Table S1: Human adipose tissue and ASCs Microarray Data, Table S2: Assays contained in TaqMan microfluidic card. Author Contributions: J.V. and S.F.-V.: conception and design, revision, and final approval of manuscript. A.M. (Ana Madeira): collection of data, data analysis and interpretation, and manuscript writing. C.S.: patients’ recruitment, sample processing, collection of data, data analysis and in- terpretation, and manuscript revision. M.E.: collection of data, data analysis and interpretation, and manuscript revision. E.M.-M.: patients’ recruitment and sample processing. M.M. (Margarita Menacho), M.M. (Monica Millan), E.E., and M.M. (Marc Martí): human samples collection and manuscript revision. M.C.N.-R. and R.G.-R.: collection of data and data analysis and interpretation. M.M.M. and A.M. (Ana Megía): scientific discussion and manuscript revision. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by grants from the Spanish Ministry of Science, Innovation and Universities (PI14/00228 and PI17/01503 to JV, SAF2015-65019-R to SF-V, BFU2015-70454- REDT and BFU2017-90578-REDT to SF-V and MMM, BFU2016-76711-R to MMM and PI15/00143 and PI18/00037 to CS) co-financed by the European Regional Development Fund (ERDF) and by Fundació La Marató de TV3 (PV170125) to SF-V and the European Crohn’s and Colitis Organization (ECCO) grant to CS. The Spanish Biomedical Research Center in Diabetes and Associated Metabolic Disorders (CIBERDEM) (CB07708/0012) is an initiative of the Instituto de Salud Carlos III. AM is a recipient of a postdoctoral fellowship from FJCI-2014-23060 and IJCI-2016-30572. SF-V acknowledges support from the Miguel Servet tenure-track program (CP10/00438 and CPII16/00008) of the Fondo de Investigación Sanitaria, co-financed by the ERDF. C.S. acknowledges support from the ‘Ramón y Cajal’ program from MINECO (RYC2013-13186), co-financed by the ERDF. Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of University Hospital Joan XXIII (Tarragona, Spain) and University Hospital Vall d’Hebrón (Barcelona, Spain) (references CEIM Int. J. Mol. Sci. 2021, 22, 4292 16 of 19 177/2018 and CEIM 41p/2015). All samples are stored in a tissue biobank registered at the National Register of Biobanks (C.0003609). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: Original imaging data have been deposited to Mendeley Data and are available http://dx.doi.org/10.17632/w6cnxgyrph.1 (accessed on 20 April 2021). Acknowledgments: The authors acknowledge the patients and the BioBank IISPV (PT17/0015/0029) integrated in the Spanish National Biobanks Network for its collaboration. The authors also thank Alfonso Saera for his help in the preparation of the figures, Margarida Terrón and Joan Sabadell for the flow cytometry technical support and Kenneth McCreath for his helpful comments on the manuscript. Conflicts of Interest: The authors declare no conflict of interest. References 1. Tchernof, A.; Despres, J.P. Pathophysiology of human visceral obesity: An update. Physiol. Rev. 2013, 93, 359–404. [CrossRef] [PubMed] 2. Peyrin-Biroulet, L.; Chamaillard, M.; Gonzalez, F.; Beclin, E.; Decourcelle, C.; Antunes, L.; Gay, J.; Neut, C.; Colombel, J.F.; Desreumaux, P. Mesenteric fat in Crohn’s disease: A pathogenetic hallmark or an innocent bystander? Gut 2007, 56, 577–583. [CrossRef] 3. Buning, C.; von Kraft, C.; Hermsdorf, M.; Gentz, E.; Wirth, E.K.; Valentini, L.; Haas, V. Visceral Adipose Tissue in Patients with Crohn’s Disease Correlates with Disease Activity, Inflammatory Markers, and Outcome. Inflamm. Bowel Dis. 2015, 21, 2590–2597. [CrossRef] 4. Feng, Q.; Xu, X.T.; Zhou, Y.; Yan, Y.Q.; Ran, Z.H.; Zhu, J. Creeping fat in patients with ileo-colonic Crohn’s disease correlates with disease activity and severity of inflammation: A preliminary study using energy spectral computed tomography. J. Dig. Dis. 2018, 19, 475–484. [CrossRef] 5. Jurjus, A.; Eid, A.; Al Kattar, S.; Zeenny, M.N.; Gerges-Geagea, A.; Haydar, H.; Hilal, A.; Oueidat, D.; Matar, M.; Tawilah, J.; et al. Inflammatory bowel disease, colorectal cancer and type 2 diabetes mellitus: The links. BBA Clin. 2016, 5, 16–24. [CrossRef] [PubMed] 6. Serena, C.; Keiran, N.; Ceperuelo-Mallafre, V.; Ejarque, M.; Fradera, R.; Roche, K.; Nunez-Roa, C.; Vendrell, J.; Fernandez-Veledo, S. Obesity and Type 2 Diabetes Alters the Immune Properties of Human Adipose Derived Stem Cells. Stem Cells 2016, 34, 2559–2573. [CrossRef] 7. Serena, C.; Keiran, N.; Madeira, A.; Maymo-Masip, E.; Ejarque, M.; Terron-Puig, M.; Espin, E.; Marti, M.; Borruel, N.; Guarner, F.; et al. Crohn’s Disease Disturbs the Immune Properties of Human Adipose-Derived Stem Cells Related to Inflamma- some Activation. Stem Cell Rep. 2017, 9, 1109–1123. [CrossRef] 8. Li, X.H.; Feng, S.T.; Cao, Q.H.; Coffey, J.C.; Baker, M.E.; Huang, L.; Fang, Z.N.; Qiu, Y.; Lu, B.L.; Chen, Z.H.; et al. Degree of Creeping Fat Assessed by CT Enterography is Associated with Intestinal Fibrotic Stricture in Patients with Crohn’s Disease: A Potentially Novel Mesenteric Creeping Fat Index. J. Crohn’s Colitis 2021. [CrossRef] 9. Schoettl, T.; Fischer, I.P.; Ussar, S. Heterogeneity of adipose tissue in development and metabolic function. J. Exp. Biol. 2018, 221. [CrossRef] 10. Ghaben, A.L.; Scherer, P.E. Adipogenesis and metabolic health. Nat. Rev. Mol. Cell Biol. 2019, 20, 242–258. [CrossRef] 11. Han, S.J.; Glatman Zaretsky, A.; Andrade-Oliveira, V.; Collins, N.; Dzutsev, A.; Shaik, J.; Morais da Fonseca, D.; Harrison, O.J.; Tamoutounour, S.; Byrd, A.L.; et al. White Adipose Tissue Is a Reservoir for Memory T Cells and Promotes Protective Memory Responses to Infection. Immunity 2017, 47, 1154–1168.e6. [CrossRef] 12. Tchkonia, T.; Tchoukalova, Y.D.; Giorgadze, N.; Pirtskhalava, T.; Karagiannides, I.; Forse, R.A.; Koo, A.; Stevenson, M.; Chin- nappan, D.; Cartwright, A.; et al. Abundance of two human preadipocyte subtypes with distinct capacities for replication, adipogenesis, and apoptosis varies among fat depots. Am. J. Physiol. Endocrinol. Metab. 2005, 288, E267–E277. [CrossRef] 13. Burl, R.B.; Ramseyer, V.D.; Rondini, E.A.; Pique-Regi, R.; Lee, Y.H.; Granneman, J.G. Deconstructing Adipogenesis Induced by beta3-Adrenergic Receptor Activation with Single-Cell Expression Profiling. Cell Metab. 2018, 28, 300–309.e4. [CrossRef] 14. Gao, Z.; Daquinag, A.C.; Su, F.; Snyder, B.; Kolonin, M.G. PDGFRalpha/PDGFRbeta signaling balance modulates progenitor cell differentiation into white and beige adipocytes. Development 2018, 145. [CrossRef] 15. Hepler, C.; Shan, B.; Zhang, Q.; Henry, G.H.; Shao, M.; Vishvanath, L.; Ghaben, A.L.; Mobley, A.B.; Strand, D.; Hon, G.C.; et al. Identification of functionally distinct fibro-inflammatory and adipogenic stromal subpopulations in visceral adipose tissue of adult mice. eLife 2018, 7. [CrossRef] 16. Merrick, D.; Sakers, A.; Irgebay, Z.; Okada, C.; Calvert, C.; Morley, M.P.; Percec, I.; Seale, P. Identification of a mesenchymal progenitor cell hierarchy in adipose tissue. Science 2019, 364. [CrossRef] 17. Vijay, J.; Gauthier, M.F.; Biswell, R.L.; Louiselle, D.A.; Johnston, J.J.; Cheung, W.A.; Belden, B.; Pramatarova, A.; Biertho, L.; Gibson, M.; et al. Single-cell analysis of human adipose tissue identifies depot and disease specific cell types. Nat. Metab. 2020, 2, 97–109. [CrossRef] Int. J. Mol. Sci. 2021, 22, 4292 17 of 19 18. Lafontan, M. Differences Between Subcutaneous and Visceral Adipose Tissues. In Physiology and Physiopathology of Adipose Tissue; Bastard, J.-P., Fève, B., Eds.; Springer: Paris, France, 2013; pp. 329–349. [CrossRef] 19. Ha, C.W.Y.; Martin, A.; Sepich-Poore, G.D.; Shi, B.; Wang, Y.; Gouin, K.; Humphrey, G.; Sanders, K.; Ratnayake, Y.; Chan, K.S.L.; et al. Translocation of Viable Gut Microbiota to Mesenteric Adipose Drives Formation of Creeping Fat in Humans. Cell 2020. [CrossRef] 20. Berry, R.; Jeffery, E.; Rodeheffer, M.S. Weighing in on adipocyte precursors. Cell Metab. 2014, 19, 8–20. [CrossRef] 21. Chau, Y.Y.; Bandiera, R.; Serrels, A.; Martinez-Estrada, O.M.; Qing, W.; Lee, M.; Slight, J.; Thornburn, A.; Berry, R.; McHaffie, S.; et al. Visceral and subcutaneous fat have different origins and evidence supports a mesothelial source. Nat. Cell Biol. 2014, 16, 367–375. [CrossRef] 22. Marcelin, G.; Ferreira, A.; Liu, Y.; Atlan, M.; Aron-Wisnewsky, J.; Pelloux, V.; Botbol, Y.; Ambrosini, M.; Fradet, M.; Rouault, C.; et al. A PDGFRalpha-Mediated Switch toward CD9(high) Adipocyte Progenitors Controls Obesity-Induced Adipose Tissue Fibrosis. Cell Metab. 2017, 25, 673–685. [CrossRef] 23. Sebo, Z.L.; Jeffery, E.; Holtrup, B.; Rodeheffer, M.S. A mesodermal fate map for adipose tissue. Development 2018, 145. [CrossRef] 24. Schwalie, P.C.; Dong, H.; Zachara, M.; Russeil, J.; Alpern, D.; Akchiche, N.; Caprara, C.; Sun, W.; Schlaudraff, K.U.; Soldati, G.; et al. A stromal cell population that inhibits adipogenesis in mammalian fat depots. Nature 2018, 559, 103–108. [CrossRef] [PubMed] 25. Raajendiran, A.; Ooi, G.; Bayliss, J.; O’Brien, P.E.; Schittenhelm, R.B.; Clark, A.K.; Taylor, R.A.; Rodeheffer, M.S.; Burton, P.R.; Watt, M.J. Identification of Metabolically Distinct Adipocyte Progenitor Cells in Human Adipose Tissues. Cell Rep. 2019, 27, 1528–1540.e7. [CrossRef] [PubMed] 26. Valle, M.T.; Degl’Innocenti, M.L.; Bertelli, R.; Facchetti, P.; Perfumo, F.; Fenoglio, D.; Kunkl, A.; Gusmano, R.; Manca, F. Antigen-presenting function of human peritoneum mesothelial cells. Clin. Exp. Immunol. 1995, 101, 172–176. [CrossRef] [PubMed] 27. Hausmann, M.J.; Rogachev, B.; Weiler, M.; Chaimovitz, C.; Douvdevani, A. Accessory role of human peritoneal mesothelial cells in antigen presentation and T-cell growth. Kidney Int. 2000, 57, 476–486. [CrossRef] 28. Shaw, T.J.; Zhang, X.Y.; Huo, Z.; Robertson, D.; Lovell, P.A.; Dalgleish, A.G.; Barton, D.P. Human Peritoneal Mesothelial Cells Display Phagocytic and Antigen-Presenting Functions to Contribute to Intraperitoneal Immunity. Int. J. Gynecol. Cancer Off. J. Int. Gynecol. Cancer Soc. 2016, 26, 833–838. [CrossRef] 29. Katz, S.; Balogh, P.; Kiss, A.L. Mesothelial cells can detach from the mesentery and differentiate into macrophage-like cells. Apmis Acta Pathol. Microbiol. Immunol. Scand. 2011, 119, 782–793. [CrossRef] 30. Isaza-Restrepo, A.; Martin-Saavedra, J.S.; Velez-Leal, J.L.; Vargas-Barato, F.; Riveros-Duenas, R. The Peritoneum: Beyond the Tissue—A Review. Front. Physiol. 2018, 9, 738. [CrossRef] 31. van Baal, J.O.; Van de Vijver, K.K.; Nieuwland, R.; van Noorden, C.J.; van Driel, W.J.; Sturk, A.; Kenter, G.G.; Rikkert, L.G.; Lok, C.A. The histophysiology and pathophysiology of the peritoneum. Tissue Cell 2017, 49, 95–105. [CrossRef] 32. Gupta, O.T.; Gupta, R.K. Visceral Adipose Tissue Mesothelial Cells: Living on the Edge or Just Taking Up Space? Trends Endocrinol. Metab. 2015, 26, 515–523. [CrossRef] [PubMed] 33. Coffey, J.C.; O’Leary, D.P. The mesentery: Structure, function, and role in disease. Lancet Gastroenterol. Hepatol. 2016, 1, 238–247. [CrossRef] 34. Hardy, O.T.; Perugini, R.A.; Nicoloro, S.M.; Gallagher-Dorval, K.; Puri, V.; Straubhaar, J.; Czech, M.P. Body mass index- independent inflammation in omental adipose tissue associated with insulin resistance in morbid obesity. Surg. Obes. Relat. Dis. Off. J. Am. Soc. Bariatr. Surg. 2011, 7, 60–67. [CrossRef] [PubMed] 35. Perrini, S.; Ficarella, R.; Picardi, E.; Cignarelli, A.; Barbaro, M.; Nigro, P.; Peschechera, A.; Palumbo, O.; Carella, M.; De Fazio, M.; et al. 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 2013, 8, e57892. [CrossRef] [PubMed] 36. Ong, W.K.; Tan, C.S.; Chan, K.L.; Goesantoso, G.G.; Chan, X.H.; Chan, E.; Yin, J.; Yeo, C.R.; Khoo, C.M.; So, J.B.; et al. Identification of specific cell-surface markers of adipose-derived stem cells from subcutaneous and visceral fat depots. Stem Cell Rep. 2014, 2, 171–179. [CrossRef] 37. Russo, V.; Yu, C.; Belliveau, P.; Hamilton, A.; Flynn, L.E. Comparison of human adipose-derived stem cells isolated from subcutaneous, omental, and intrathoracic adipose tissue depots for regenerative applications. Stem Cells Transl. Med. 2014, 3, 206–217. [CrossRef] 38. Ford, M.L.; Larsen, C.P. Translating costimulation blockade to the clinic: Lessons learned from three pathways. Immunol. Rev. 2009, 229, 294–306. [CrossRef] 39. Chan, J.L.; Tang, K.C.; Patel, A.P.; Bonilla, L.M.; Pierobon, N.; Ponzio, N.M.; Rameshwar, P. Antigen-presenting property of mesenchymal stem cells occurs during a narrow window at low levels of interferon-gamma. Blood 2006, 107, 4817–4824. [CrossRef] 40. Stagg, J.; Pommey, S.; Eliopoulos, N.; Galipeau, J. Interferon-gamma-stimulated marrow stromal cells: A new type of non- hematopoietic antigen-presenting cell. Blood 2006, 107, 2570–2577. [CrossRef] 41. Benvenuto, F.; Ferrari, S.; Gerdoni, E.; Gualandi, F.; Frassoni, F.; Pistoia, V.; Mancardi, G.; Uccelli, A. Human mesenchymal stem cells promote survival of T cells in a quiescent state. Stem Cells 2007, 25, 1753–1760. [CrossRef] 42. Lenardo, M.; Chan, K.M.; Hornung, F.; McFarland, H.; Siegel, R.; Wang, J.; Zheng, L. Mature T lymphocyte apoptosis–immune regulation in a dynamic and unpredictable antigenic environment. Annu. Rev. Immunol. 1999, 17, 221–253. [CrossRef] Int. J. Mol. Sci. 2021, 22, 4292 18 of 19 43. Hepler, C.; Vishvanath, L.; Gupta, R.K. Sorting out adipocyte precursors and their role in physiology and disease. Genes Dev. 2017, 31, 127–140. [CrossRef] 44. Macartney-Coxson, D.; Benton, M.C.; Blick, R.; Stubbs, R.S.; Hagan, R.D.; Langston, M.A. Genome-wide DNA methylation analysis reveals loci that distinguish different types of adipose tissue in obese individuals. Clin. Epigenetics 2017, 9, 48. [CrossRef] 45. Jiang, Y.; Berry, D.C.; Tang, W.; Graff, J.M. Independent stem cell lineages regulate adipose organogenesis and adipose homeostasis. Cell Rep. 2014, 9, 1007–1022. [CrossRef] 46. Chau, Y.Y.; Hastie, N. Wt1, the mesothelium and the origins and heterogeneity of visceral fat progenitors. Adipocyte 2015, 4, 217–221. [CrossRef] 47. van Tuyn, J.; Atsma, D.E.; Winter, E.M.; van der Velde-van Dijke, I.; Pijnappels, D.A.; Bax, N.A.; Knaan-Shanzer, S.; Gittenberger-de Groot, A.C.; Poelmann, R.E.; van der Laarse, A.; et al. Epicardial cells of human adults can undergo an epithelial-to-mesenchymal transition and obtain characteristics of smooth muscle cells in vitro. Stem Cells 2007, 25, 271–278. [CrossRef] 48. Lansley, S.M.; Searles, R.G.; Hoi, A.; Thomas, C.; Moneta, H.; Herrick, S.E.; Thompson, P.J.; Newman, M.; Sterrett, G.F.; Prele, C.M.; et al. Mesothelial cell differentiation into osteoblast- and adipocyte-like cells. J. Cell. Mol. Med. 2011, 15, 2095–2105. [CrossRef] 49. Lachaud, C.C.; Lopez-Beas, J.; Soria, B.; Hmadcha, A. EGF-induced adipose tissue mesothelial cells undergo functional vascular smooth muscle differentiation. Cell Death Dis. 2014, 5, e1304. [CrossRef] 50. Lachaud, C.C.; Rodriguez-Campins, B.; Hmadcha, A.; Soria, B. Use of Mesothelial Cells and Biological Matrices for Tissue Engineering of Simple Epithelium Surrogates. Front. Bioeng. Biotechnol. 2015, 3, 117. [CrossRef] 51. Lachaud, C.C.; Pezzolla, D.; Dominguez-Rodriguez, A.; Smani, T.; Soria, B.; Hmadcha, A. Functional vascular smooth muscle-like cells derived from adult mouse uterine mesothelial cells. PLoS ONE 2013, 8, e55181. [CrossRef] 52. Ariza, L.; Carmona, R.; Canete, A.; Cano, E.; Munoz-Chapuli, R. Coelomic epithelium-derived cells in visceral morphogenesis. Dev. Dyn. Off. Publ. Am. Assoc. Anat. 2016, 245, 307–322. [CrossRef] 53. Deng, T.; Lyon, C.J.; Minze, L.J.; Lin, J.; Zou, J.; Liu, J.Z.; Ren, Y.; Yin, Z.; Hamilton, D.J.; Reardon, P.R.; et al. Class II major histocompatibility complex plays an essential role in obesity-induced adipose inflammation. Cell Metab. 2013, 17, 411–422. [CrossRef] 54. Morris, D.L.; Cho, K.W.; Delproposto, J.L.; Oatmen, K.E.; Geletka, L.M.; Martinez-Santibanez, G.; Singer, K.; Lumeng, C.N. Adipose tissue macrophages function as antigen-presenting cells and regulate adipose tissue CD4+ T cells in mice. Diabetes 2013, 62, 2762–2772. [CrossRef] [PubMed] 55. Xiao, L.; Yang, X.; Lin, Y.; Li, S.; Jiang, J.; Qian, S.; Tang, Q.; He, R.; Li, X. Large adipocytes function as antigen-presenting cells to activate CD4(+) T cells via upregulating MHCII in obesity. Int. J. Obes. 2016, 40, 112–120. [CrossRef] [PubMed] 56. Charriere, G.; Cousin, B.; Arnaud, E.; Andre, M.; Bacou, F.; Penicaud, L.; Casteilla, L. Preadipocyte conversion to macrophage. Evidence of plasticity. J. Biol. Chem. 2003, 278, 9850–9855. [CrossRef] 57. Zuk, P. Adipose-Derived Stem Cells in Tissue Regeneration: A Review. ISRN Stem Cells 2013, 2013, 35. [CrossRef] 58. McIntosh, K.; Zvonic, S.; Garrett, S.; Mitchell, J.B.; Floyd, Z.E.; Hammill, L.; Kloster, A.; Di Halvorsen, Y.; Ting, J.P.; Storms, R.W.; et al. The immunogenicity of human adipose-derived cells: Temporal changes in vitro. Stem Cells 2006, 24, 1246–1253. [CrossRef] 59. Gimble, J.M.; Bunnell, B.A.; Frazier, T.; Rowan, B.; Shah, F.; Thomas-Porch, C.; Wu, X. Adipose-derived stromal/stem cells: A primer. Organogenesis 2013, 9, 3–10. [CrossRef] 60. Vogel, I.T.; van Gool, S.W.; Ceuppens, J.L. CD28/CTLA-4/B7 and CD40/CD40L costimulation and activation of regulatory T cells. World J. Immunol. 2014, 4, 63–77. [CrossRef] 61. Galipeau, J.; Krampera, M.; Barrett, J.; Dazzi, F.; Deans, R.J.; DeBruijn, J.; Dominici, M.; Fibbe, W.E.; Gee, A.P.; Gimble, J.M.; et al. International Society for Cellular Therapy perspective on immune functional assays for mesenchymal stromal cells as potency release criterion for advanced phase clinical trials. Cytotherapy 2016, 18, 151–159. [CrossRef] 62. Gimble, J.; Guilak, F. Adipose-derived adult stem cells: Isolation, characterization, and differentiation potential. Cytotherapy 2003, 5, 362–369. [CrossRef] [PubMed] 63. Dominici, M.; Le Blanc, K.; Mueller, I.; Slaper-Cortenbach, I.; Marini, F.; Krause, D.; Deans, R.; Keating, A.; Prockop, D.; Horwitz, E. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 2006, 8, 315–317. [CrossRef] [PubMed] 64. Zimmerlin, L.; Donnenberg, V.S.; Pfeifer, M.E.; Meyer, E.M.; Peault, B.; Rubin, J.P.; Donnenberg, A.D. Stromal vascular progenitors in adult human adipose tissue. Cytom. Part A J. Int. Soc. Anal. Cytol. 2010, 77, 22–30. [CrossRef] 65. Bourin, P.; Bunnell, B.A.; Casteilla, L.; Dominici, M.; Katz, A.J.; March, K.L.; Redl, H.; Rubin, J.P.; Yoshimura, K.; Gimble, J.M. Stromal cells from the adipose tissue-derived stromal vascular fraction and culture expanded adipose tissue-derived stromal/stem cells: A joint statement of the International Federation for Adipose Therapeutics and Science (IFATS) and the International Society for Cellular Therapy (ISCT). Cytotherapy 2013, 15, 641–648. [CrossRef] [PubMed] 66. Pachon-Pena, G.; Serena, C.; Ejarque, M.; Petriz, J.; Duran, X.; Oliva-Olivera, W.; Simo, R.; Tinahones, F.J.; Fernandez-Veledo, S.; Vendrell, J. Obesity Determines the Immunophenotypic Profile and Functional Characteristics of Human Mesenchymal Stem Cells From Adipose Tissue. Stem Cells Transl. Med. 2016, 5, 464–475. [CrossRef] [PubMed] 67. Kester, L.; van Oudenaarden, A. Single-Cell Transcriptomics Meets Lineage Tracing. Cell Stem Cell 2018, 23, 166–179. [CrossRef] 68. Kredel, L.I.; Jodicke, L.J.; Scheffold, A.; Grone, J.; Glauben, R.; Erben, U.; Kuhl, A.A.; Siegmund, B. T-cell Composition in Ileal and Colonic Creeping Fat—Separating Ileal from Colonic Crohn’s Disease. J. Crohn’s Colitis 2019, 13, 79–91. [CrossRef] Int. J. Mol. Sci. 2021, 22, 4292 19 of 19 69. Silverberg, M.S.; Satsangi, J.; Ahmad, T.; Arnott, I.D.; Bernstein, C.N.; Brant, S.R.; Caprilli, R.; Colombel, J.F.; Gasche, C.; Geboes, K.; et al. Toward an integrated clinical, molecular and serological classification of inflammatory bowel disease: Report of a Working Party of the 2005 Montreal World Congress of Gastroenterology. Can. J. Gastroenterol. J. Can. Gastroenterol. 2005, 19 (Suppl. A), 5A–36A. [CrossRef] 70. Stylianou, E.; Jenner, L.A.; Davies, M.; Coles, G.A.; Williams, J.D. Isolation, culture and characterization of human peritoneal mesothelial cells. Kidney Int. 1990, 37, 1563–1570. [CrossRef] 71. Diaz-Ruiz, A.; Guzman-Ruiz, R.; Moreno, N.R.; Garcia-Rios, A.; Delgado-Casado, N.; Membrives, A.; Tunez, I.; El Bekay, R.; Fernandez-Real, J.M.; Tovar, S.; et al. Proteasome Dysfunction Associated to Oxidative Stress and Proteotoxicity in Adipocytes Compromises Insulin Sensitivity in Human Obesity. Antioxid. Redox Signal. 2015, 23, 597–612. [CrossRef] 72. Francois, M.; Romieu-Mourez, R.; Stock-Martineau, S.; Boivin, M.N.; Bramson, J.L.; Galipeau, J. Mesenchymal stromal cells cross-present soluble exogenous antigens as part of their antigen-presenting cell properties. Blood 2009, 114, 2632–2638. [CrossRef] [PubMed] 73. Naqvi, A.R.; Fordham, J.B.; Ganesh, B.; Nares, S. miR-24, miR-30b and miR-142-3p interfere with antigen processing and presentation by primary macrophages and dendritic cells. Sci. Rep. 2016, 6, 32925. [CrossRef] [PubMed] 74. Team, R.C. R: A Language and Environment for Statistical Computing; R Foundation for Statistical Computing: Vienna, Austria, 2019.