Comparing the use of differentiated adipose-derived stem cells and mature adipocytes to model adipose tissue in vitro

Differentiation 110 (2019) 19–28

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Comparing the use of differentiated adipose-derived stem cells and mature adipocytes to model adipose tissue in vitro

T

Ann-Cathrin Volza,b, Birgit Omengoc, Sandra Gehrked, Petra Juliane Klugera,e,∗ a

Reutlingen Research Institute, Reutlingen University, Alteburgstrasse 150, 72762, Reutlingen, Germany University of Hohenheim, Schloss Hohenheim 1, 70599, Stuttgart, Germany c Institute of Interfacial Process Engineering and Plasma Technology IGVP, University of Stuttgart, Nobelstrasse 12, 70569, Stuttgart, Germany d Research & Development, Research Special Skincare, Beiersdorf AG, Unnastrasse 48, 20253, Hamburg, Germany e Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB, Cell and Tissue Engineering, Nobelstrasse 12, 70569, Stuttgart, Germany b

A R T I C LE I N FO

A B S T R A C T

Keywords: Mature adipocyte Adipogenic differentiated adipose-derived stem cell 3D model Adipose tissue engineering mRNA transcription

In vitro models of human adipose tissue may serve as beneficial alternatives to animal models to study basic biological processes, identify new drug targets, and as soft tissue implants. With this approach, we aimed to evaluate adipose-derived stem cells (ASC) and mature adipocytes (MA) comparatively for the application in the in vitro setup of adipose tissue constructs to imitate native adipose tissue physiology. We used human primary MAs and human ASCs, differentiated for 14 days, and encapsulated them in collagen type I hydrogels to build up a three-dimensional (3D) adipose tissue model. The maintenance of the models was analyzed after seven days based on a viability staining. Further, the expression of the adipocyte specific protein perilipin A and the release of leptin and glycerol were evaluated. Gene transcription profiles of models based on dASCs and MAs were analyzed with regard to native adipose tissue. Compared to MAs, dASCs showed an immature differentiation state. Further, gene transcription of MAs suggests a behavior closer to native tissue in terms of angiogenesis, which supports MAs as preferred cell type. In contrast to native adipose tissue, genes of de novo lipogenesis and tissue remodeling were upregulated in the in vitro attempts.

1. Introduction Adipose tissue is an endocrine organ and metabolically very active (Coelho et al., 2013). With the incorporated triglycerides it is not only the body's main energy store but actively regulates the body's energy balance. A standardized and validated in vitro model of adipose tissue would facilitate various investigations o within biomedical and pharmaceutical research. These models would help to gain details on the development and causes of adipose tissue related diseases like obesity and diabetes. Furthermore, they would be applicable in therapeutic screening for innovative drugs and for the improvement of diagnostic tools. In a somewhat distant future, an application as in vivo tissue replacement in Regenerative Medicine to treat severe burn wounds or replace removed tissue is as well conceivable. Clinical attempts for the replacement of damaged or lost adipose tissue currently use methods like the Coleman's technique to transfer autologous fat from less affected body sites (Coleman, 2006; SerraRenom et al., 2013; Yoshimura et al., 2008). However, these procedures

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are linked to more intense surgical interventions, associated to a greater risk of complications. Beyond that, the long-term graft survival rate is low (Mahoney et al., 2018). In vitro test systems are often based on the use of mouse-derived 3T3 cells (Brett et al., 2017; Ruiz-Ojeda et al., 2016). Such cell lines offer advantages like well defined characteristics, high homogeneity and the possibility to be extended almost limitless in vitro (Green and Meuth, 1974). Nevertheless, it has to be kept in mind that the modified genome of cell lines, additionally to existing interspecies variances, does not allow a direct transfer of the results to the native human system (Vickers et al., 2011). Conclusively, in vitro models of adipose tissue based on human primary cells are urgently needed. Adipose-derived stem cells (ASCs) are mesenchymal stem cells found in adipose tissue. ASCs are available in high amounts, are easily accessible and the adipose tissue removals show little donor site morbidity and are safer and, less invasive compared to other biopsies (e.g. bone marrow aspiration). The last mentioned points are especially relevant for the autologous transfer for patients (Tan et al., 2016). ASCs are usually differentiated into the adipogenic lineage in vitro, for up to

Corresponding author. Alteburgstrasse 150, 72762, Reutlingen, Germany. E-mail address: [email protected] (P.J. Kluger).

https://doi.org/10.1016/j.diff.2019.09.002 Received 7 May 2019; Received in revised form 19 August 2019; Accepted 3 September 2019 Available online 06 September 2019 0301-4681/ © 2019 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

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Abbreviations 3D ACLY ANGPT ASC AGT CEBP CT DAPI DLK DMEM FA FABP FASN

FATP GLUT IL IRS LDA LPL MA PAI PPAR RIN SREB TNF TGF TMB VEGF

three dimensional Adenosin triphosphate citrate lyase Angiopoietin Adipose-derived stem cell Angiotensinogen CCAAT/enhancer binding protein Critical threshold 4, 6-diamidin-2-phenylindol Delta-like non-canonical Notch ligand Dulbecco's Modified Eagle Medium Fatty acid Fatty acid binding protein Fatty acid synthase

Fatty acid transport protein Glucose transporter Interleukin Insulin receptor substrate Low density array Lipoprotein lipase Mature adipocyte Plasminogen activator inhibitor Peroxisome proliferator-activated receptor RNA Integrity number Sterol regulatory element-binding transcription factor Tumor necrosis factor Transformal growth factor 3,3′,5,5′-tetramethylbenzidine Vascular endothelial growth factor

concerning their morphology, viability and their gene transcription profile. The in vitro models based on MAs or dASCs were compared to native adipose tissue in order to be able to make a statement on their suitability to adequately model adipose tissue in vitro. The following hypotheses were posted to examine the in vitro adipose tissue models:

14 days (Bunnell et al., 2008; Diascro et al., 1998; Ruiz-Ojeda et al., 2016). Mature adipocytes (MAs) share many advantageous features with adipogenic differentiated ASCs (dASCs). However, compared to ASCs, MAs are terminally differentiated and hence can not be further expanded after their isolation (Nie et al., 2015). Also handling of MAs is difficult as these cells are vulnerable to mechanical influence and are unable to adhere to culture surfaces as they float on top of the medium (Flynn and Woodhouse, 2008). Additionally, MAs easily dedifferentiate in vitro (Tholpady et al., 2005). Nonetheless, we optimized the maintenance of MAs in vitro by adapting the culture medium (Huber and Kluger, 2015). Moreover, MAs already show characteristics of functional adipocytes like the univacuolar appearance with only one big lipid vacuole, characteristic protein expression, and hormone release (Blanchette-Mackie et al., 1995). Adipogenic differentiation of ASCs is cost and time consuming and eventually only yealdingin low differentiation rates. Both cell types show advantages as cell source in adipose tissue engineering. Models based on MAs promise a better representation of native adipose tissue, while dASCs are easier in handling and may be proliferated almost infinitely. Both cell types are frequently used in scientific investigations, with promising outcomes (Abbott et al., 2015, 2016, 2017; Aubin et al., 2015; Bellas et al., 2013; Huttala et al., 2018; Sorrell et al., 2011; Wiesner et al., 2015). Therein, dASCs as well as directly isolated adipocytes are usually declared as mature adipocytes. However, having in mind their deviant features, it appears questionable how comparable the two cell types actually behave in vitro and which is more appropriate to model native tissue. Current attempts take into account the importance of the three-dimensional (3D) orientation of adipocytes on the development of a physiological cell and tissue behavior (Pope et al., 2016). Adipocytes or ASCs were encapsulated in natural polymers like collagen type I, gelatin or silk protein (reviewed in (O'Halloran et al., 2017)). Recently, Harms et al. were able to show the positive influence of 3D culture on the maintenance of adipocyte identity and function even in the absence of supporting matrix components (Harms et al., 2019). Some of the developed models are already used as a test systems to study adipose tissue behavior, or evaluate the tissues' reaction to endogenous factors or drugs (Abbott et al., 2016, 2017; Proulx et al., 2016; Rogal et al., 2019). To confirm their predictive power and suitability as tissue implants, a direct comparison of the models to native adipose tissue is needed. A direct comparison was only performed in very few recent studies focusing on white adipose tissue explants which were maintained in ex vivo culture (Abbott et al., 2016; Kim et al., 2015). In this work, it was sought to comprehensively evaluate and compare in vitro adipose tissue models of human MAs and human dASCs to reveal existing differences between the two cell sources. Therefore, 3D adipose tissue models including either MAs or dASCs were set up in vitro in 3D matrices consisting of collagen type I. The cells were analyzed

- MAs are in a higher differentiation and maturity status compared to dASCs - dASCs show a higher potential to initiate vascularization in vitro - models with MAs mimic native adipose tissue more accurately compared to dASCs 2. Materials and methods 2.1. Human tissue samples All research was carried out in accordance with the rules for investigation of human subjects as defined in the Declaration of Helsinki. Patients gave a written agreement according to the permission of the Landesärztekammer Baden-Württemberg (F-2012-078; for normal skin from elective surgeries). 2.2. Cell isolation MAs and ASCs were isolated from human fatty tissue of elective surgeries received from Dr. Ziegler (Klinik Charlottenhaus, Stuttgart). The isolation procedures were previously described by us (Huber et al., 2016; Volz et al., 2017). ASCs and MAs were derived from male or female patients age 18–64 and either taken from abdomen, thigh or upper arm. 2.3. Setup and culture of adipose tissue models MAs or ASCs of at least three donors (deviating between MAs and dASCs) were seeded in collagen type I hydrogels (9 mg/mL from rat tail, Fraunhofer IGB) in 24 well plate transwell inserts (Brand, Germany). A neutralization buffer consisting of 10 x Dulbecco's Modified Eagle Medium (DMEM)/Ham's F-12 (Biochrom, Germany) and 50 mM sodium hydroxide in Aqua dest. (1:1) with 0.2 M sodium carbonate and 0.225 M HEPES (Roth, Germany) was prepared. The collagen was mixed with the MAs or ASCs (5 x 105 cells/gel) in medium and the neutralization buffer in a ratio of 4:4:1. 300 μL of the mixture was pipetted into each insert of a 24 well plate and gelled for 20 min at 37 °C. MAs were cultured in 1.5 mL adipocyte maintenance medium (AM-1; ZenBio, USA) at 37 °C for one day. A schematic illustration of the experimental setup is displayed in Fig. 1. ASCs were differentiated into the adipogenic lineage in the 3D setup for 14 days while culturing them 20

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or stained by a hematoxylin and eosin staining. Therefore, the diameter of the area containing lipid vacuoles, was measured manually in images taken with a 20-x lens. Values of three independent donors were analyzed and averaged. Per donor, 30 cells were included in the evaluation. 2.7. Leptin-ELISA An ELISA against human leptin (Pepro Tech, Germany) was performed according to the manufacturer's protocol based on 24 h culture supernatants. Samples were frozen directly after their collection and stored at −20 °C until analyzed. For color development, 100 μL 3,3′,5,5′-tetramethylbenzidine (TMB)-substrate (Sigma-Aldrich, Germany) for each well was used. Incubation occurred at RT for approx. 15 min. Enzymatic reaction was stopped with 50 μL sulphuric acid (1 M) and color development was measured at 450 nm with a wavelength correction set at 570 nm with a plate reader (Tecan, Germany). 2.8. Glycerol release Basal lipolysis was detected with a glycerol kit (Randox, Ireland). Samples of 24 h culture supernatants were frozen directly after the collection and stored at −20 °C until analyzed. Briefly, 5 μL samples were mixed with 100 μL reagent and incubated for 10 min at RT. Color development was analyzed at 520 nm (Tecan, Germany). Fig. 1. Schematic illustration of the experimental setup. MAs were embedded in hydrogels of collagen type I on day 0. ASCs were integrated on day −14 and differentiated for two weeks. Both groups were continuously cultured for seven days and compared to each other and to native adipose tissue.

2.9. RNA extraction from engineered adipose tissue/human adipose tissue and gene transcription measurement Total RNA of engineered adipose tissue (in vitro) and human adipose tissue (ex vivo) was isolated using the RNeasy Lipid Tissue Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Before starting RNA isolation, a stainless steel bead (5 mm, Qiagen) was added to a 2 mL tube (containing 1 mL Qiazol) and gels were disrupted for 2 min by TissueRuptor (Retsch, Germany). TissueRuptor is a handheld rotor-stater homogenizer that provides efficient disruption of each sample. RNA was quantified using the NanoDrop ND-1000 instrument (Peqlab Biotechnologies, Wilmington, USA). To check the RNA quality, we used the Experion RNA Analysis Kit (BioRad). The Experion automated electrophoresis system employs LabChip microfluidic technology to automate electrophoresis for RNA analysis and offers determination of total RNA and mRNA purity and concentration at nanogram levels. Results showed clearly that RNA quality was not affected by the hydrogel material. The RNA Integrity Number (RIN) is a numerical assessment of the integrity of RNA, a RIN over 8 indicates a high quality of RNA. In our test, we observed RIN numbers of 8.0–9.8. Gene transcription levels of genes associated with fat metabolism were determined using a Custom Taqman Low Density Array (LDA, Applied Biosystems) specifically compiled for the evaluation of adipogenic and endothelial markers. This experiment was carried out with engineered adipose tissue samples including MAs or dASCs and ex vivo human adipose tissue. All critical threshold (CT) values were normalized to 18S and the expression was referred to ex vivo human adipose tissues (control). Transcription levels were calculated using the 2ΔΔCT-method. Columns with values higher than 1 indicate a higher transcription in engineered adipose tissue and columns with values below 1 indicate lower transcription in engineered adipose tissue (compared to human adipose tissue). Average values of all samples were formed. Data points that show average values with different transcription levels (high as well as lower transcription of individual sample) were considered to be outliers. Analyzed genes were divided into four groups: adipogenic differentiation, fatty acid (FA) metabolism and lipid intake, adipocyte functionality, and angiogenesis.

in adipogenic differentiation medium consisting of DMEM with 10% fetal bovine serum, 1 μg/mL insulin (Sigma Aldrich, Germany), 500 μM 3-isobutyl-1-methylxanthine (IBMX, Sigma Aldrich, Germany), 100 μM indomethacin (Cayman Chemicals, USA) and 1 μM dexamethasone (Sigma, Germany). Adipocyte gels were then cultured for seven days in AM-1. Medium exchange was performed twice a week for the MAs and the diffASCs models equally. 2.4. Viability staining The cell viability of the encapsulated adipocytes was examined with a live staining. Gels were stained with 10 μg/mL fluorescein diacetate (FDA, Sigma Aldrich, Germany) and Hoechst 33342 with 0.1 μg/mL in DMEM for 10 min at 37 °C. All fluorescing samples were analyzed in PBS in an imaging μ-dish (ibidi, Germany) using an Axio Observer (Carl Zeiss, Germany). 2.5. Immunofluorescence staining 3D adipocyte gels were fixed with 4% paraformaldehyde for 4 h. Gels were embedded into paraffin and cut into slices of 3 μm. Sections were deparaffinized and unmasked with a target retrieval solution (10 mM Tris base, 1 mM EDTA solution, 0.05% Tween 20, pH 9.0) for 20 min in a preheated steamer (Unold, Germany). For the immunofluorescence staining, samples were blocked in 3% bovine serum albumin in PBS for 30 min and incubated with a primary antibody against perilipin A (1:300, Sigma-Aldrich, Germany) overnight at 4 °C. The goat-derived secondary antibody against rabbit (Chromeo 488: 1:300, Abcam) was incubated for 30 min. Nuclei were stained with 1 μg/mL 4, 6-diamidin-2-phenylindol (DAPI) in PBS for 15 min. Tissue sections were mounted in ProLong®Gold (Invitrogen, Germany). 2.6. Analysis of lipid vacuole diameter The diameter of the area occupied by lipid vacuoles per cell was analyzed with Image J based on the sections stained against perilipin A 21

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3.1.3. MAs and dASCs show endocrine and metabolic activity Metabolically active adipocytes release a broad range of hormones. Leptin release correlates with the stored fat mass (Cole et al., 2003). Both, MAs and dASCs showed a significantly increased leptin release between day 1 and 7 (Fig. 5). MAs probably suffered from the harsh enzymatic and mechanic forces during the isolation and encapsulation procedure. On day 1, they had probably not yet fully recovered and their metabolism had not yet returned to a physiological level. The significant increase on day 7 confirmed their rehabilitation. DASCs were encapsulated 14 days prior to the 7 day culture period, but did not exhibit comprehensive leptin release on day 1. This again underlines the immature state of dASCs after only 14 days of adipogenic differentiation. The leptin release of dASCs significantly increases between day 1 and day 7. This supports their ongoing differentiation and lipid incorporation. However, the levels did not reach those of MAs. Equally, the levels for glycerol release (Fig. 6), as a measurement of degraded triglycerides by basal lipolysis, increased significantly between day 1 and day 7, however only for MAs. Since leptin release of MAs increased and the average diameter of the lipid vacuole did not change significantly, it can be concluded, that MAs may have increased lipogenic pathways along with increased lipolytic activities. Therefore, elevated glycerol values may in this case not be attributed to enhanced degradation of triglycerides but may rather be a sign of increased lipid turnover. As no increase of glycerol release was observed in dASCs, it has to be assumed, that dASCs did not reach a level of differentiation in which excess lipid storage resulted in a measurable increase of the basal lipolysis.

2.10. Statistics Gene transcription analysis was performed with four MA and three dASC donors (non-matching). Glycerol and leptin release were analyzed for three donors of each cell type in two replicates with a student's Ttest using Origin Pro 8.5 G. Lipid vacuole size was evaluated with a one way ANOVA and a Tukey post-hoc test. All results are displayed as mean ± standard deviation. Statistical significances were stated as * p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001 3. Results and discussion 3.1. MAs and dASCs stay viable and keep their phenotype in cultured 3D models To evaluate adipocytes’ in vitro behavior, the expression of the cell specific protein perilipin A, the release of leptin and glycerol as indicators of cell functionality and lipolytic state and the cell viability were analyzed. 3.1.1. MAs and dASCs stay viable After one week, adipocyte morphology and viability were evaluated with a viability staining (Fig. 2). Throughout the hydrogel, many viable adipocytes were visible in all attempts. MAs displayed a round and voluminous morphology on day 1. On day 7 some few elongated cells were present, which had lost the typical adipocyte morphology. This is very likely attributed to an emerging tissue homeostasis, in which some adipocytes dedifferentiate to form a regenerative cell reservoir. In the hydrogels with dASCs some undifferentiated cells were present equally on day 1 and 7. DASCs exhibited several lipid vacuoles, whereas MAs showed only one big lipid vacuole. However, accumulated lipids and consequently cell volume increased in dASCs over time.

3.2. Representation of native adipose tissue by cultured in vitro models Gene transcription profiles of in vitro models including either MAs or dASCs were evaluated quantitatively with a LDA including genes associated to adipose tissue. Thereby existing advances in the use of either MAs or dASCs for the setup of a representative adipose tissue model were sought to be revealed. For interpretation, genes were assigned into four relevant groups, including relevant genes for adipogenic differentiation, adipocyte metabolism, adipocyte functionality, and angiogenesis (Figs. 7 and 8). The gene transcription levels were compared to those of native human adipose tissue. Red lines indicate relative quantities of 2 or 0.5 which equals a 2-fold higher or 2-fold lower gene transcription of engineered adipose tissue models compared to human adipose tissue. In Figs. 7 and 8, statistical significance is indicated when p ≤ 0.05, independent of the level of significance.

3.1.2. MAs and dASCs maintain cell specific perilipin A expression Sections of embedded hydrogels, including adipocytes were stained against perilipin A and compared to sections of native tissue to evaluate whether the adipocyte specific phenotype was retained throughout the culture period (Fig. 3). In native tissue, each cell was in contact with several other cells and the surrounding matrix was arranged in thin layers between the cells. The in vitro attempts exhibited a significantly lower cell to matrix ratio. MAs occupied a larger volume within the hydrogel compared to dASCs and were therefore located closer to each other cell. While the lipid vacuoles in native tissue made up almost the complete cell body, lipid vacuoles in dASCs still only seemed to cover part of it. This became apparent in the direct comparison of dASCs’ perilipin A expression in Fig. 3 with the cell morphology in the viability staining in Fig. 2. The cell body seemed to exceed the vacuoles' membrane circumference. Based on the tissue sections prepared from the cultured hydrogels consisting of either MAs or dASCs, the diameter of the area occupied by lipid vacuole(s) was analyzed per cell. Fig. 4 shows the averaged values for three independent donors. MAs show an average lipid vacuole diameter of 60.9 ( ± 18.7) μm on day 1. The size of the lipid vacuoles does not appear to change during the 7 day culture period, as the average diameter on day 7 stays comparable with 56.9 ( ± 17.0) μm. The diameters for MAs match with those found in literature (Jernås et al., 2006; Verboven et al., 2018). DASC show significantly lower average diameters of 15.2 ( ± 6.2) μm right from the beginning of the comparison on day 1. The diameter of the lipid vacuole containing area increased significantly until day 7 to 23.1 ( ± 11.8) μm. Despite this increase, the lipid vacuole size of dASCs still stayed significantly below the values observed in MAs. This supports the hypothesis that dASCs are not terminally maturated after a period of two weeks and the development continues during week 3. Additionally, it underlines morphological differences between MAs and dASCs.

Fig. 2. Imaging of adipocyte viability in the 3D culture of MAs and dASCs in hydrogels of collagen type I. Live staining with fluorescein diacetate of viable MAs and dASCs (in green) on day 1 and day 7 of the culture, cell nuclei were stained with Hoechst33342 (in blue), n = 3, scale bar = 200 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) 22

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Fig. 5. Quantitative analysis of leptin release by MAs or dASCs cultured in hydrogels of collagen type I. 24 h leptin release was analyzed on day 1 and day 7. Statistical significances were analyzed with a student's T-test and significant differences of p < 0.01 are marked with ** respectively p < 0.001 with ***, MA: n = 4, dASCs: n = 3.

Fig. 3. Imaging of perilipin A expression in the 3D culture of MAs and dASCs in hydrogels of collagen type I. Immunofluorescence staining of perlipin A (shown in green) in 3 μm sections of adipose tissue models on day 1, day 7 and native adipose tissue, n = 3, scale bar: 200 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6. Quantitative analysis of glycerol release by MAs or dASCs cultured in hydrogels of collagen type I. 24 h glycerol release was analyzed on day 1 and day 7. Statistical significances were analyzed with a student's T-test and a significant difference of p < 0.001 is marked with ***, n.s. = non significant, MA: n = 4, dASCs: n = 3.

vitro attempts on day 7 and are also within the range of 50–200% compared to native adipose tissue (Fig. 7A). This suggests a comparable differentiation level. The upregulation of IRS-1 in dASCs suggests increased glucose metabolisms compared to MAs and native adipose tissue. Angiotensinogen (AGT) is mainly secreted by adipocytes and also positively associated to adipogenic differentiation (Ailhaud et al., 2000). It was similarly transcribed in MAs and dASCs. In comparison to native adipose tissue it was noticeably upregulated. The general upregulation in all attempts might be attributed to the dependency of AGT transcription on the stimulation with glucocorticoids (Ailhaud et al., 2000), which were possibly present in the culture attempt in higher concentrations compared to native adipose tissue. Transforming growth factor β2 (TGF-β2) is known to be secreted in an autocrine manner by adipocytes to inhibit further adipogenic differentiation (Rahimi et al., 1998). Its transcription in the in vitro culture of MAs is noticeably down regulated. The difference to the only slightly decreased value in dASCs does not become significant. Therefore, no cell-specific behavior was observed here. Delta-like non-canonical Notch ligand 1 (DLK1), also known as Pref-1, is associated to an immature tissue state and to inhibited adipogenesis (Mitterberger et al., 2012). During adipogenic differentiation and maturation, DLK1 is required to be turned down (Sul et al., 2000). DLK-1 is highly upregulated in dASCs compared to MAs. This indicates the immature state of dASCs. Summarized, dASCs show signs of incomplete adipogenic differentiation and incomplete maturation.

Fig. 4. Average lipid vacuole diameter. Tissue sections of collagen type I hydrogels containing MAs or dASCs were immunostained against perilipin A. The diameter of the area occupied by lipid vacuoles per cell was measured. Statistical differences were analyzed with a one way ANOVA and a Tukey posthoc test. Significant differences of p < 0.01 and p < 0.001 are marked with ** and *** respectively, n = 3.

3.2.1. DASCs show an immature state despite comparable transcription of adipogenic genes A complex cascade of transcriptional factors regulates the transformation of uncommitted cells to the adipogenic lineage. Peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhance binding protein α (CEBPα) describe the central elements in the initiation of adipogenic differentiation (Ayala-Sumuano et al., 2011). They are pushed by CEBPβ and –δ (Rosen and Spiegelman, 2000). In combination, these genes are following responsible for the transcription of adipogenic genes like insulin receptor substrate 1 (IRS-1), FA binding protein 4 (FABP4) or glucose transporter 4 (Glut-4), leading to enhanced uptake of glucose and the assembly and storage of triglycerides (Liu et al., 2012; Petersen et al., 2008; Rosen and Spiegelman, 2000). All of the named genes are transcribed comparably by MAs and dASCs in the in 23

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Fig. 7. Relative gene transcription of MAs and dASCs in collagen type I hydrogels on day 7 in comparison to native adipose tissue. Adipose tissue equivalents consisting of MAs or dASCs were cultured for seven days and transcription of genes was compared to human adipose tissue. Transcription of genes was referred to the level of native human adipose tissue (=1) for genes responsible for A) adipocyte differentiation and B) fatty acid metabolism and lipid intake. Red lines at 0.5 and 2 indicate an expression of 50% and 200%, compared to native human adipose tissue respectively. Statistical significance analyzed by a student's T-test is marked with *p ≤ 0.05, n = 3. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

translocated to the plasma membrane to fulfill this function in reaction to insulin signaling. In contrast to MAs, which transcribed Glut-4 comparably to native adipose tissue, dASCs showed a slight upregulation. Adenosin triphosphate citrate lyase (ACLY) has long been known for its essential role in de novo lipogenesis (the FA generation based on glucose). Another enzyme which participates in the formation of (especially long chain) FAs is the FA synthase (FASN). Increased levels of these enzymes indicate enhanced de novo lipogenesis (Berndt et al., 2007). The predominant use of de novo lipogenesis for lipid accumulation in both dASCs and MAs may be linked to the in vitro conditions and may e.g. derive from the media composition. It is also noticeable that genes of de novo lipogenesis were transcribed to a higher extent compared to enzymes of FA uptake. De novo generated lipids may have had to compensate for reduced FA uptake from the culture medium or increased lipolysis in all the attempts. This potentially led to an increased lipid turnover at bottom line. The increased transcription of some genes with important roles in fatty acid metabolism and lipid incorporation in dASCs became significant for ACLY, FATP1 and SCREBF1. This could be attributed to an increased anabolic state of dASCs. As dASCs are expected to have remained in a still immature state 21 days after induction, an increased occurrence of these metabolic processes is very likely.

3.2.2. 3D adipose tissue models show enhanced transcription of de novo lipogenesis markers The sterol regulatory element-binding transcription factor 1 (SREBF1) is organized upstream of the very important and well known adipogenic genes named PPARγ and CEBPα. It is classified as the master regulator of lipid metabolism and was increasingly transcribed in the attempt based on dASCs (Fig. 7B). DASCs are thought to be not finally maturated and still within the differentiation process and therefore within a phase of intense lipid accumulation. FA binding protein 4 (FABP4) is responsible for binding long-chain FAs and other hydrophobic ligands functioning towards the uptake, transport and metabolism of FAs. FABP4 transcription was comparable to native tissue for both cell types. Similar results were obtained for lipoprotein lipase (LPL). LPL is produced by adipocytes and, after its release into the extracellular space, responsible for the split up of triglycerides from lipoproteins for their further uptake by adipocytes. FA transport protein 1 (FATP1) is a member of the FATP/Slc27 protein family. FATP1 and FATP4 also enhance the cellular uptake of long-chain FAs (LCFAs) and are expressed in several insulin-sensitive tissues (Wiczer et al., 2008). High transcription of FATP4 in all attempts suggests a considerable induction of FA uptake in vitro. FATP1 was, however, significantly downregulated in MAs, indicating a transfer of FATP1 function to other enzymes or a slight downregulation of LCFA uptake. Glut-4 facilitates glucose uptake from the extracellular space and is 24

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Fig. 8. Relative gene transcription of MAs and dASCs in collagen type I hydrogels on day 7 in comparison to native adipose tissue. Adipose tissue equivalents consisting of MAs or dASCs were cultured for seven days and transcription of genes was compared to human adipose tissue. Transcription of genes was referred to the level of human adipose tissue (=1) for genes responsible for A) adipose tissue functionality and B) for angiogenesis. Red lines at 0.5 and 2 indicate an expression of 50% and 200%, compared to native human adipose tissue respectively. Statistical significance analyzed by a student's T-test is marked with *p ≤ 0.05, n = 3. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

the same level until day 7. Adipsin was down regulated in both in vitro attempts. In vivo, it mainly suppresses infectious agents in the humoral immune response. Another known effect of adipsin is the stimulation of glucose uptake, triglyceride accumulation and the inhibition of lipolysis (Song et al., 2016; Van Harmelen et al., 1999). The low values found in all in vitro attempts are most likely associated to the almost completely sterile and immundeficient conditions in cell culture experiments, which prevent an immunological activation of the adipocytes. The slightly higher levels found in dASCs, compared to MAs, may be drawn back to the ongoing adipogenic development which in MAs only takes place to a minimal extent. Simultaneously, resistin and tumor necrosis factor – α (TNFα) were transcribed to a very low extent in all experiment. Resistin transcription is known to be directly pushed by CEBPα transcription (Hartman et al., 2002), wherefore a noticeable resistin transcription was expected in all attempts. Adipocytes are however in question as the main origin of resistin. In fact, macrophages were assumed to produce more resistin compared to adipocytes (Curat et al., 2006). As the here evaluated models simply include a mono-culture of adipocytes rather than the complex population found in vivo, the low transcription of resistin might be lead back to the absence of its main cellular source, presumably macrophages. Equally, the absence of TNFα transcription may be associated to the absence of an immunologic component like

3.2.3. Adipocyte functionality is altered in 3D adipose tissue culture in vitro Adiponectin is a known indicator of low lipid deposits (Nedvidkova et al., 2005), and therefore a promoter of glucose uptake, adipogenic differentiation of preadipocytes and lipid accumulation (Coelho et al., 2013). The transcription of adiponectin was therefore expected to be elevated in dASCs compared to MAs. However, both model types showed an in vivo-like transcription of adiponectin (Fig. 8A). The consistently low adiponectin concentration is most likely observed due to the still immature state of the dASCs. Leptin is a potent indicator of filled lipid stores and is hypothesized to function as a negative feedback regulator of the body's energy balance. Thereby the released concentration correlates with the extent of the fat stores in adipose tissue (Acosta et al., 2016). The leptin transcription was slightly upregulated in MAs while only showing minimal transcription in dASCs. This result is in accordance with the analysis of leptin secretion, showing significantly lower values for dASCs compared to MA, which additionally underlines the immature state of dASCs. Hence, dASCs have to be further maturated (min. three weeks total differentiation) in vitro to serve for corresponding investigations. This conclusion is confirmed by the results of Ambele et al. who received a further increase of adipogenic characteristics on day 21 (Ambele et al., 2016) and our own previous results (Volz and Kluger, 2018). MAs already included a high amount of lipids at the beginning of the culture period, which were remained at 25

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initiation of vascular structures compared to dASCs. However, the concrete contribution of MAs and dASCs during the different steps of angiogenesis has to be analyzed in co-culture experiments to obtain results from a more physiological setup. Plasminogen activator inhibitor-1 (PAI-1) as well participates in the regulation of adipose tissue angiogenesis. Its main function, the inhibition of fibrolysis, is tissue-independent (Kaji, 2016). It is also involved in extracellular matrix (ECM) proteolysis and remodeling (Milenkovic et al., 2017). ECM in this in vitro attempt is solely consisted of collagen. Collagen includes various Arg-Gly-Asp (RGD) cell adhesion and matrix metalloproteinase (MMP) cleavage sites. But in contrast to the in vivo environment, important components for cell adhesion, phenotypic maintenance and development are missing (Pierleoni et al., 1998; Song et al., 2018). An increase in both in vitro attempts might be linked to the immature tissue environment. Obviously, both cell types tried to initiate the remodeling of the surrounding matrix towards an adipose tissue specific composition.

macrophages, which in the human body are responsible for the inflammatory state of the adipocytes. In contrast, interleukin 6 (IL-6) transcription is upregulated in MAs compared to dASCs and native adipose tissue. Why IL-6 transcription of MAs is at a high level in vitro despite the absence of stimulating cellular components like macrophages remains unclear (Lauterbach and Wunderlich, 2017). The upregulation in MAs above 200% compared to the in vivo-like transcription in dASCs might again be attributed to the advanced differential state of MAs, for which IL-6 is a known indicator (Vicennati et al., 2002). Further MAs could have reacted to insulin from the culture medium with increased IL-6 secretion as shown before by Vicennati et al. Visfatin accomplishes multiple functions like blood vessel maturation, neutrophil maintenance or the improvement of insulin sensitivity. It stimulates glucose utilization in adipocytes (Adeghate, 2008). Its consistent upregulation in all in vitro attempts could therefore be associated to the enhanced glucose uptake of adipocytes and is in accordance with the further findings, indicating an elevated de novo lipogenesis in the in vitro models. To summarize, the evaluated players in adipose tissue functionality in vitro, slightly deviated compared to the values from native tissue. This condition might be mainly attributed to the simplified nature of the in vitro models, the unknown influence of the used media or the lack of endocrine or paracrine signals of other cellular components.

4. Conclusion and outlook In this study, we successfully set up 3D adipose tissue models in vitro based on dASCs and MAs. The models maintained viable and active cells, which exhibited cell-specific protein expression and endocrine activity. MAs and dASCs comparably transcribed genes associated to adipogenic differentiation. However, dASCs still exhibited an immature differentiation state and a increased transcription of anabolic genes. By the elongation of the differentiation period of ASCs in vitro, the application of both cell types may be recommended equivalently in approaches concerning adipogenesis and adipocyte metabolism. Independent of the cell type, in vitro attempts in general exhibited a slight shift towards de novo lipogenesis rather than FA uptake. Based on this result, a further adaptation of the culture medium to a more physiological composition, best excluding the use of sera should be pursued. Since both cell types showed remarkably enhanced levels of PAI1, indicating the pronounced initiation of ECM remodeling, the addition of tissue specific matrix components, like fibronectin or collagen type IV should be considered. Additionally, transcription of genes associated to adipose tissue functionalities, like e.g. immunological signaling, deviated remarkably in some cases for both cell types. Therefore, the suitability of MAs and dASCs to model adipose tissue should be evaluated further and in greater detail based on advanced models. Such models should include additional cell types like ECs or macrophages.

3.2.4. Angiogenic marker transcription of dASCs deviates from native tissue and MAs in vitro Important counter players in the balance of angiogenesis and vessel maturation are angiopoietin-1 (ANGPT1) and −2 (ANGPT2). While ANGPT1 induces endothelial cell (EC) interaction and thereby the maturation of existing vessels (Gaengel et al., 2009), ANGPT2 was reported to loosen these contacts and thereby facilitate EC migration and proliferation in the presence of VEGF-A (Augustin et al., 2009). MAs express comparable amounts of ANGPT1 and -2 as native adipose tissue (Fig. 8B). In contrast, dASCs simultaneously show an elevated transcription of ANGPT1 and a decreased transcription of ANGPT2. It was reported before, that ANGPT2 transcription in ASCs rises only when held in co-culture with endothelial cells (Merfeld-Clauss et al., 2015). It was additionally shown, that released leptin in turn enhances the transcription of ANGPT2 (Cohen et al., 2001). The transcription of ANGPT2 therefore possibly rather has be attributed to MAs than preadipocytes. In this attempt, dASCs show an immature development. Additionally it is assumed that there are still some completely undifferentiated cells in the dASC attempt. The immature state of dASCs might explain the reduced transcription of ANGPT2. The increased transcription of ANGPT1 is as well plausible, as undifferentiated ASCs are known to take over the role of perivascular cells in vivo, thereby supporting EC cell survival and mural cell attachment (Mendel et al., 2013). VEGFA is the key regulator of angiogenic and vasculogenic processes (Ferrara et al., 2003). VEGFD is equally responsible for angiogenesis and EC growth, by stimulating EC proliferation and migration. It shows high similarity to VEGFC and both factors are responsible for lymphogenesis (Roy et al., 2006). While all tested factors from the VEGF family are transcribed within the range from 50 to 200% in MAs compared to native tissue, dASCs showed an elevated transcription of VEGFD and VEGFC and a decreased transcription of VEGFA. MAs have been identified as the main source of VEGFA in adipose tissue before (Helmrich et al., 2011; Mick et al., 2002). Equally, VEGFD was found to be highly transcribed in ASCs, at least compared to BMSCs (Hsiao et al., 2011). Whether VEGFC transcription is rather associated to ASCs compared to fully differentiated adipocytes was not described in literature before. The results obtained in this approach however point to a prominent transcription of VEGFC by dASCs or ASCs in general and thus a fundamental role of ASCs in lymphangiogenesis. These results suggest a higher expected support by MAs in the

Declarations of interest None. Funding This work was supported by the European Commission [#263416] and the Federal Ministry of Education and Research [#03FH012PX4]. Funding sources had no involvement in the study design, the collection, analysis and interpretation of the data, the writing of the report or the decision to submit it to Differentiation. Acknowledgements The authors thank Dr. Ziegler (Klinik Charlottenhaus, Stuttgart) for the kind provision of human fatty tissue and skin samples from elective surgery. Also a warm thank you to Silvia Kolbus-Hernandez and Ursula Csacsko for their extraordinary helpful support in the laboratories (Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB and Reutlingen Research Institute). We further thank Brigitte Höhl (Fraunhofer IGB) for her help in all the histology issues. 26

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