ExAdEx: A simple method to maintain human adipose tissue in long-term cultures reveals differential expansion of adipose progenitor cell subpopulations in fibrotic and pro-inflammatory microenvironments.

doi:10.21203/rs.3.rs-1733405/v2

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ExAdEx: A simple method to maintain human adipose tissue in long-term cultures reveals differential expansion of adipose progenitor cell subpopulations in fibrotic and pro-inflammatory microenvironments.

https://doi.org/10.21203/rs.3.rs-1733405/v2

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A relevant model to explore how to promote the expansion of adipose progenitor cells (APCs) in lean and obese contexts is still lacking, as current adipocyte models have serious limitations. In this work, we describe a novel adipose tissue model, named ExAdEx, that can be obtained from cosmetic surgery wastes. ExAdEx products are adipose tissue units maintaining the characteristics and organization of adipose tissue as it presents in vivo. The model was viable and metabolically active for up to two months and could adopt a pathological-like phenotype. The results revealed that inflammatory and fibrotic microenvironments differentially regulated the expansion of the CD26 APC subpopulation and its CD54 and CD142 APC progenies. The approach used significantly improves the method of generating adipose tissue models, and ExAdEx constitutes a relevant model of human adipose tissue that could be used to identify pathways promoting expansion of APCs in physiological and pathological contexts.

Cell & Tissue Engineering

Adipose tissue

adipose cell progenitors

microphysiological models

obesity

inflammation

fibrosis

Lipids begin to accumulate in hepatocytes and muscles when the adipose tissue expansion limit is reached. This ectopic lipid accumulation in non-adipocyte cells causes lipotoxic damage and contributes to insulin resistance, steatosis and other disorders associated with obesity. Many factors have been reported to be involved in the regulation of adipose tissue expansion, including the maintenance of a pool of adipose progenitor cells (APCs) that is critical to generate new adipocytes in response to physiological and pathological conditions¹. APCs comprise a heterogeneous group of cell subpopulations that have different degrees of commitment to adipocytes. The major role of CD26-expressingAPCs in the generation of adipocytes has recently been highlighted as the developmental hierarchy of APCs described in murine models, revealing that the CD26 APC subtype displays features of multipotent stem cells, giving rise to CD54–, identified as ICAM1, and CD142-expressing preadipocytes. Interestingly, CD26, CD54 and CD142 APCs were localized in distinct niches. CD26 APCs were localized in the fluid-filled network surrounding the adipose tissue, i.e., the reticular interstitium, whereas CD54 and CD142 preadipocytes were localized between mature adipocytes². CD26, identified as dipeptidyl peptidase 4 (DPP4), is a ubiquitous glycoprotein bound to the cell membrane that can be cleaved in a soluble form with peptidase activity. CD26 also displays non-enzymatic functions. CD26 is highly expressed in APCs, in which it promotes proliferation by affecting growth factor signalling through an independent peptidase activity³. CD26 APCs have a high proliferative capacity and are relatively resistant to differentiation into adipocytes, allowing renewal of the pool of APCs. The depletion of CD26 APCs in visceral adipose tissues compared to subcutaneous fat in obese mice strongly suggests a link between the low abundance of CD26 APCs and the pathological remodelling of adipose tissue². Therefore, understanding how to promote the expansion of APCs in an obese microenvironment could help to protect against metabolic diseases. However, a relevant in vitro model for studying the expansion of CD26 APCs in physiological and pathological contexts is still missing. Current three-dimensional (3D) adipose-tissue-like models are generated by engineering strategies, basically by assembling APCs and synthetic or natural scaffolds⁴. The main limitations of these adipose tissue substitutes engineered in vitro are as follows: i) APCs are typically purified from enzymatically dissociated tissues and then expanded in adherent 2D cell culture conditions that rapidly change the natural ratio of APC subpopulations⁵; and ii) due to the enzymatic dissociation of the tissue, current adipose tissue models lose native vascularization, the extracellular matrix and critical cell types, such as mature adipocytes. Biomaterials partially reconstitute the 3D structure and the extracellular matrix, which are known to govern APC behaviour and their response to external stimuli⁶. The gold standard of the in vitro adipose tissue model is explants of adipose tissue, but the use of adipose tissue fragments is seriously limited by their weak cell viability in culture⁷.

We propose a novel adipose tissue model, named ExAdEx (for Ex vivo Adipocyte Expansion), in which APC subpopulations could expand in the cellular complexity and the 3D vascularized extracellular matrix of native adipose tissue. A schema of the method we applied to generate ExAdEx is shown in Fig. 1a. First, we hypothesized that the movement of the cannula by surgeons during liposuction helps to mechanically dislodge CD26 cells from the reticular interstitial niche and therefore release them in the infranatant, the fluid fraction of the lipoaspirate discarded by practitioners. Indeed, we show that CD26 APCs were dramatically enriched in the infranatant fraction compared to the tissue fraction. In contrast, CD54 APCs were primarily localized in the tissue fraction (Fig. 1b). The differential distribution of CD26 and CD54 APCs in the infranatant and tissue fractions, respectively, fits well with their anatomical localization as mentioned above². The innovativeness of the process to generate the model also stems from the use of emulsified tissues as a natural bioactive matrix for CD26 APC expansion. The rationale for non-enzymatic dissociation, such as the emulsification step, is first that it conserves the native 3D structure of the tissue and second that previous publications have reported the ability of mechanical stimuli to activate quiescent progenitor cells to proliferate to restore tissue homeostasis⁸. In agreement with this claim, no proliferative cells were detectable in the lipoaspirate, whereas a high number of Edu-positive cells were detectable in the tissue after emulsification (Fig. 1c). The final step of the process was then to combine cells from the infranatant fraction depleted from red blood cells with the emulsified tissue fraction to generate the ExAdEx product. As shown in Fig. 1d, the total APC population, isolated from the ExAdEx product using collagenase digestion and then identified as APCs as described in the Methods section, could be expanded by more than 18-fold after 2 weeks in in vitro culture, as demonstrated by producing ExAdEx from 27 donors aged from 21 to 75 years old, with a mean age of 40 years and a BMI of 23.4 kg/m². At that stage, the abundance of CD26 APCs in the total APC population was 36.3%, whereas the percentage of CD54 APCs was 3.9%. Thanks to the emulsification method allowing minimal manipulation of the tissue, APC expansion occurred in a microenvironment where the extracellular matrix and the endothelial cell networks of the native adipose tissue were conserved (Fig. 1e and Supplemental Fig. 1). Analysis of LDH released in culture medium, reflecting dead or damaged cells, showed that less than 20% of the maximum LDH release was detectable after 60 days in culture, whereas 90 days appeared to be the limit of the tissue viability, as two ExAdEx products on three tested presented more than 20% of the maximum release (Fig. 1f). These data support the long-term viability of the model over a period of 8 weeks. This characteristic contrasts with the viability of explants of adipose tissue that can be maintained ex vivo for only 14 days⁷. Moreover, ExAdEx remained metabolically functional during the long–term period in culture, as revealed by the levels of secretion of the anti-diabetic adipokine adiponectin, which were similar to the lipoaspirate and the derived ExAdEx products, and the lipolysis capacity measured by glycerol release upon b-adrenergic receptor stimulation (Fig. 1 g, h). Finally, when maintained in appropriate culture conditions, ExAdEx conserved the physiological potential of beiging, i.e., the capacity to generate clusters of brown-like adipocytes dispersed in white adipose tissue⁹ (Fig. 1 i). Interestingly, adipocytes with lipid droplet diameters of approximately 100 mm could be observed in the ExAdEx model (see Fig. 1 c, e and i). This lipid droplet size is similar to the size that can be observed in native tissue and is substantially larger than the 20-40 mm lipid droplets generated in other 3D models of adipocyte spheroids⁴ or by micropatterning preadipocytes ¹⁰. Altogether, the data support the claim that the ExAdEx model significantly improves on the existing models of human adipose tissue.

The ExAdEx model derived from adipose tissue of healthy donors was then modified to generate a pathological-like model. Fibrosis and inflammation are hallmarks of obesity¹¹ and can be induced separately and in a controlled manner from healthy samples. Therefore, the regulation of APC subpopulations could be investigated and compared in healthy and pathological microenvironments derived from the same donor. Inflammation and fibrosis were modeled by treatment of ExAdEx with tumor necrosis factor-alpha (TNFa) and transforming growth factor beta (TGFb1), respectively. The rationale for the choice of these cytokines is that elevated secretion of TNFa by macrophages has been shown to play a critical role in the low-grade chronic inflammation of obese adipose tissue¹². We and others have previously shown that TGFb superfamily members, such as TGFb1, secreted by macrophages are key mediators that promote fibrosis of adipose tissue¹³. As expected, treatment of ExAdEx products with TNFa promoted major changes: adipocyte gene expression, such as PLIN1 and Glut4, was reduced (see Supplemental Fig. 2), as was the secretion of adiponectin (Fig. 2 a). The proinflammatory cytokine IL6 was secreted at low levels in the untreated condition, suggesting a weak inflammatory state in the healthy ExAdEx model. In contrast, IL6 secretion was dramatically increased in the inflammatory model and interestingly could be reversed in the presence of the anti-inflammatory molecule dexamethasone (Fig. 2 b). Remodelling of the extracellular matrix in the inflamed ExAdEx model was also visualized by laminin staining (Fig. 2c). Treatment of ExAdEx with TGFb1 to generate a fibrotic-like model induced inhibition of PLIN1 and Glut 4 gene expression (Supplemental Fig. 2), secretion of adiponectin (Fig. 2d), and stimulation of INHBA gene expression, a marker of APCs having the potential to differentiate into myofibroblasts, a source of fibrosis¹³ (Supplemental Fig. 2). TGFb1, but not TNFa, treatment induced MMP14 gene expression (Fig. 2e), which has been previously reported to be increased in the early stages of obesity¹⁴. Finally, TGFb1 treatment promoted remodelling of the extracellular matrix, as revealed by the appearance of fibrotic collagen fibres observed with second-harmonic generation imaging (Fig. 2 f). All these data support an inflammatory- and fibrotic-like microenvironment in the pathological ExAdEx model.

The impact of the pathological-like microenvironment on the percentages of CD26, CD54 and CD142 APC subpopulations was then addressed by flow cytometry. The gating strategy used to quantify the different APC subpopulations is shown in Supplemental Fig. 3. The data showed that the abundance of APC subpopulations was differentially regulated in the fibrotic and inflamed ExAdEx models. The abundance of the CD26⁺/CD54^-/CD142^- subpopulation, denoted CD26⁺, was inhibited in both inflammatory and fibrotic-like microenvironments. It is interesting to note that TGFb1 has been shown to promote the proliferation of CD26 cells purified from adipose tissue², whereas CD26 cell abundance was dramatically reduced in the TGFb1-treated ExAdEx model. This difference observed for the TGFb1 effects underlines the relevance of analysing APC regulation in the fully adipose tissue microenvironment.

The CD26+/CD142+ population was also inhibited in both pathological models, whereas the CD26+/CD54+ population was increased in the inflamed microenvironment but not in the fibrotic microenvironment (Supplemental Fig. 4). In contrast, the abundance of the CD54⁺ subpopulation increased in both conditions. The CD142⁺ subpopulation was reduced by inflammation but increased by fibrosis (Fig. 2 g), whereas the CD54+/CD142+ population increased in the inflamed microenvironment only (Supplemental Fig. 4). A schema reporting the effects of TNFa and TGFb1 treatments on APC subpopulations is presented in Supplemental Fig. 5. The impact on the proliferative and adipogenic capacities of the CD54 and CD142 subpopulations as well as on metabolic and endocrine capacities of their adipocyte progenies remains to be investigated¹⁵. However, knowing the potential of APCs to undergo differentiation into myofibroblasts when exposed to an inflammatory or fibrotic environment, it is tempting to speculate that the CD54 subpopulation will display a profibrogenic/proinflammatory phenotype in the pathological model. The accumulation of CD54⁺ APCs in subcutaneous adipose tissue of high-fat-diet-induced obese mice without a corresponding increase in mature adipocyte differentiation supports this hypothesis¹⁶. The role of CD142 APCs in adipogenesis is more controversial because this subpopulation is described as fully adipogenic or as an inhibitor of adipogenic differentiation via paracrine effects¹⁷.

ExAdEx thus represents a robust model with which fully integrated responses could be investigated. It could help in identifying mechanisms that promote healthy expansion of APCs and a more in-depth functional characterization of APC subpopulations in healthy and obese-like contexts. More generally, ExAdEx is a relevant human adipose tissue model that could be used as an in vitro preclinical platform in the early stages of pharmaceutical drug testing to accelerate the pharmaceutical development of molecules to counteract pathological remodelling of human adipose tissue.

Acknowledgements

We thank Dr. L. Formicola for insightful discussions and Dr. P. Leterttre for having given the first liposuctions. This project was funded by the SATT-Sud Est, by the French Government (National Research Agency, ANR) and the University Cote d’Azur through the “Investments for the Future” programs LABEX SIGNALIFE ANR-11-LABX-0028-01 and IDEX UCA^JediANR-15-IDEX-01.

Our thanks to the Université Côte d’Azur Office of International Scientific Visibility for English language editing of the manuscript.

Competing interests statement

VD, AD, BCS and CD declare to be founders of the ExAdEx-innov, a start-up commercially developing the ExAdEx models described in the paper.

Author information:

Affiliations

Université Côte d’Azur, INSERM, CNRS, iBV, Nice, France

Vincent Dani, Solène Bruni-Favier, Agnès Loubat, Christian Dani

Université Côte d’Azur, UPR7354 MICORALIS, UFR Odontologie, Nice, France and Centre Hospitalier Universitaire de Nice, Unité de Thérapie Cellulaire et Génique, Nice, France

Alain Doglio

Clinique Saint Georges, Nice, France

Bérengère Chignon-Sicard.

Contributions:

VD and CD conceived and designed the study. VD and SBF performed the experiments. AL performed the FACS analyses, and BCS provided liposuction. BCS and AD provided conceptual advice. CD supervised the work and wrote the manuscript with input from all the authors. All authors read, discussed and approved the final manuscript.

Corresponding author

Christian Dani

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Human samples:

The method was developed from lipoaspirates of subcutaneous adipose tissues obtained from elective cosmetic surgery of 27 healthy donors. The range of donor ages was 40.7±3.34 years, with a body mass index of 23.4± 0.4 kg/m2. All donors gave informed consent. Samples were processed following approval of the French Institute of Medical Research and Health Ethics Evaluation Committee and Institutional Review Board (IRB00003888, IORG0003254, FWA00005831) under the file reference 19-554.

Isolation of the infranatant cell pellet depleted from red blood cells:

Lipoaspirated adipose tissues were centrifuged at 3000 rpm for 3 min, resulting in separation into four layers: a superior layer containing oil, a middle layer consisting of compacted yellow adipose tissue, the infranatant fluid composed of infiltration liquids and a pellet containing APCs and red blood cells (RBCs).

Infranatant cells were treated with 10 volumes of ammonium chloride (NH4Cl)-containing buffer (RBC Lysing buffer, Invitrogen, France) for 5 min at 37°C. Then, the effect of the RBC lysis buffer was stopped by adding 20 volumes of PBS, and the cells were centrifuged at 3000 rpm for 3 min. RBC and APC viabilities were measured with the Count and Viability Assay Kit of the Muse® Cell Analyzer according to the manufacturer's instructions (Luminex Corporation).

Emulsification of the adipose tissue fraction

The adipose tissue fraction was mechanically emulsified by shifting it between two 10 cc syringes connected to each other by a Luer Lock connector. After 30 passages, the emulsified tissue was mixed with the infranatant cells prepared as described above and maintained in EGM-plus media (CC-4133 from Promocell, Germany) for the indicated times.

In vitro maintenance of the ExAdEx model, beiging and treatment with TNFa or TGFb1

ExAdEx was maintained in Ultra Low Attachment flasks or plates (Corning, France) in EGM-plus media at 37°C/5% CO2 on a shaken balance.

APC expansion was visualized in situ by EdU (PK-CA724-594FM from Promocell, Germany). Briefly, ExAdEx tissues were maintained ex vivo for 14 days and then incubated overnight with EdU 10 mM. Samples were fixed, and EdU incorporation into proliferating cells was detected using a Click-iT EdU Alexa Fluo reaction and imaging kit according to the manufacturer’s instructions (Molecular Probes™ # C10339). Images were acquired with an LSM 710 confocal microscope in z-stack mode and reconstructed by ImageJ.

For beiging, 100 mg of the model expanded for 4 weeks was encapsulated in agarose as previously described¹⁸ and maintained for 2 weeks in DMEM/F12 medium supplemented with 1 µM rosiglitazone, 2 µM T3 and 2,5 µg/ml insulin.

To generate inflamed and fibrotic ExAdEx models, the healthy ExadEx model expanded for 4 weeks was maintained for 7 days in EBM (Cat: C-22211; PromoCell, Germany) supplemented with 2% FCS, 22.5 mg/ml ascorbic acid,1mg/ml heparin and 10 ng/ml TNFa (Cat: H8916, Sigma-Aldrich), or 5 ng/ml TGFb1 (Cat: 100-21, Preprotech, France).

Viability and functional assays

Lactate dehydrogenase activity (LDH)

Cell viability of the model was measured using the lactate dehydrogenase release assay. One hundred milligrams of ExAdEx-processed tissue was cultured in a well of a ULA 24-well plate, and culture supernatant was collected 24 h after a culture medium change. Tissues were then treated with 10% Triton X-100 for 4 h to obtain the maximum level of LDH release and to calculate the percentage of cytotoxicity for each assay. LDH release assays were conducted according to the manufacturer's instructions (LDH-Glo Cytotoxicity Assay, Promega, #J2380).

Measurement of Adiponectin and IL-6 secretion

Levels of secreted adiponectin and interleukin 6 (IL-6) were measured using ELISA. Conditioned media were collected 24 h after the medium was changed and stored at -80°C. Adiponectin and IL-6 doses were determined using commercial ELISA kits (R&D Systems, Minneapolis, MN, USA, #DY1065 and #DY206, respectively) according to the manufacturer’s instructions.

Lipolysis

Tissues were washed once with PBS and maintained for 24 h in RPMI supplemented with 2% fatty acid free BSA, 5 µM triascin C, and 1 ng/ml ascorbic acid. Lipolysis was stimulated with 1 µM isoproterenol for 2 h or 24 h. Glycerol release into the culture medium was determined as an index of lipolysis according to the manufacturer’s instructions using the Glycerol Detection Assay (Promega, #J3150).

RNA Extraction and Reverse Transcription Quantitative Polymerase Chain Reaction

One hundred milligrams of tissue was disrupted using TissueLyser LT, and total RNA was extracted using the RNeasy Plus Universal kit (Qiagen) according to the manufacturer's instructions. Reverse transcription‐polymerase chain reaction (RT‐PCR) analysis and real‐time PCR assays were conducted as previously described¹⁹. All primer sequences are detailed in supplemental online Table 1. Quantification was performed using the comparative DCt method. The housekeeping gene TATA box-binding protein (TBP) was used as a reference.

Isolation of APCs and flux cytometry analysis

Collagenase digestion was only used to quantify APC expansion or to investigate the impact of inflammation and fibrotic environment on APC subtypes. In brief, 100 mg of ExAdEx model was dissociated for 45 min at 37°C in 2 ml PBS containing 2 mg/ml collagenase and 20 mg/ml bovine serum-albumin. Then, APCs were separated from the tissue by low-speed centrifugation (200 g, 10 min), resuspended in EGM and seeded as 2D monolayer culture. Adherent cells were identified as APCs thanks to their immunophenotype (positive for CD105, CD90, CD73) and their capacity to differentiate into adipocytes (see Supplemental Figures 6-7). APCs were counted or fixed with 4% formaldehyde for 15 min the day after plating for FACS analysis with CD26 (Clone BA5b/FITC; Ozyme, France), CD54 (Clone 1H4/APC; Molecular probe) and CD142 (Clone HTF-1/PE; Invitrogen, France).

Confocal microscopy and second-harmonic generation (SHG) imaging

ExAdEx samples were fixed with 4% PAF and then incubated with primary anti-collagen 1 (Abcam ab260043), anti–elastin (Abcam ab21610), anti-laminin (ab11575), anti-fibronectin Santa Cruz sc8422), and anti-CD31 (Abcam ab28364) antibodies overnight at 4°C and then with the corresponding secondary antibody for 45 min at room temperature. Lipid droplets were stained with Oil Red O, and nuclei were stained with DAPI. Samples were visualized on an LSM 780 NLO inverted Axio Observer Z1 confocal microscope (Carl Zeiss Microscopy GmbH, Jena, Germany) using a Plan Apo 25X multi immersion (oil, glycerol, water) NA 0.8 objective. The SHG light source was a Mai Tai DeepSee (Newport Corp., Irvine, CA, USA) tuned to 880 nm. A forward SHG signal was detected with an oil condenser (1.4 NA), bandpass filter 440/40 nm and transmission PMT. Backward SHG was collected with a GaAsP (BIG) nondescanned module of 440/10 nm.

Statistical analysis

The results are presented as the mean ± SEM. To determine statistical significance, the results were analyzed using GraphPad Prism version 9. Groups were compared using the Wilcoxon signed-rank test with n <10 per group or using a paired t-test with n >10 per group, unless indicated otherwise. All data are shown, and no outlier removal was performed. For all data, statistical significance was defined as *p ≤0.05; **p ≤0.01; ***p ≤0.001.

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ExAdEx: A simple method to maintain human adipose tissue in long-term cultures reveals differential expansion of adipose progenitor cell subpopulations in fibrotic and pro-inflammatory microenvironments.

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