Cell culture and gene transfection
Orbital adipose tissue specimens were obtained from patients with GO (n = 3) during fat decompression and from control individuals without a history of GO (n = 3) under consent conditions. OF preparation was approved by the Institutional Review Board of CHA Bundang Medical Center, Seongnam, Republic of Korea (IRB-2018-01-007). Orbital tissue explants were minced and treated with 0.25 mg/mL collagenase (Sigma-Aldrich, St. Louis, MO, USA) for 1 h at 37 °C in a shaking incubator. After collagenase digestion, the orbital tissues were placed in culture plates with DMEM/F12 supplemented with 20% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) and 1% penicillin/streptomycin (P/S; Gibco).
Placentas were collected for research purposes by the Institutional Review Board of CHA Gangnam Medical Center, Seoul, Republic of Korea (IRB 07-18). PD-MSCs were isolated as previously described  and cultured in α-modified minimal essential medium (α-MEM; HyClone Logan, UT, USA) supplemented with 10% FBS (Gibco), 1% P/S (Gibco), 1 μg/mL heparin (Sigma-Aldrich), and 25 ng/mL human fibroblast growth factor-4 (hFGF-4; Peprotech, Rocky Hill, NJ, USA). The PRL-1 plasmid vector was purchased from Origene (#RG200435; Rockville, MD, USA). To induce overexpression of the PRL-1 gene, naïve PD-MSCs (passage = 7) were transfected using the AMAXA nucleofector system (Lonza, Basel, Switzerland) according to the manufacturer’s instructions. After transfection for 24 h, the cells were selected by 1.5 mg/mL neomycin. All cells were maintained at 37°C in a humidified atmosphere containing 5% CO2.
Differentiation of PD-MSCsPRL-1 and OFs from GO patients
To analyze the potential of PD-MSCsPRL-1 to differentiate into mesodermal lineages, PD-MSCsPRL-1 (passage = 5) were plated at a density of 5×103 cells/cm2 in various differentiation induction media using the StemPro adipogenesis and osteogenesis differentiation kit (Gibco) according to the manufacturer’s instructions. After approximately 21 days, PD-MSCsPRL-1 were fixed in 4% paraformaldehyde and incubated for 1 h with Oil Red O (Sigma-Aldrich) to stain lipids to visualize lipid vesicles and von Kossa with 5% silver nitrate (Sigma-Aldrich) under the light to evaluate the accumulation of calcium deposits.
To induce adipogenic differentiation, normal and GO-derived OFs (5×103 cells/cm2) were seeded and incubated in serum-free DMEM/F12 supplemented with 33 μM biotin, 17 μM pantothenic acid, 10 μg/mL transferrin, 0.2 nM triiodothyronine (T3), 1 μM insulin (all from Sigma-Aldrich), 0.2 μM carbaprostacyclin (cPGI2; Cayman Chemical, Ann Arbor, MI, USA), 1 μM dexamethasone, and 0.1 mM isobutylmethylxanthine (IBMX; all from Sigma-Aldrich) for the first 4 days. To induce the maturation of adipocytes, the medium was supplemented except 1 μM dexamethasone and 0.1 mM IBMX (all from Sigma-Aldrich) for 6 days and was replaced every other day. Lipid accumulation and adipocyte morphology were visualized by Oil Red O staining.
To detect the inhibition of adipogenesis, normal and GO-derived OFs underwent adipogenic differentiation and were cocultured with naïve and PD-MSCsPRL-1 (5×103 cells/cm2) in Transwell inserts (8 μm pore size; Corning, NY, USA) in α-MEM (HyClone) supplemented with 10% FBS and 1% P/S (all from Gibco) for 24 h at 37 °C in a humidified atmosphere containing 5% CO2.
Reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qRT-PCR)
Total RNA was extracted using TRIzol LS reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The concentration and purity of the total RNA were determined spectrophotometrically by measuring the ODs at 260 nm and 280 nm. cDNA was reverse transcribed from total RNA (500 ng) by using SuperScript III reverse transcriptase (Invitrogen). To analyze stemness markers in PD-MSCsPRL-1, PCR amplification was performed with specific primers (Table 1). β-actin was used as an internal control. The amplified PCR products were electrophoresed on 2% agarose gels containing 1.5 μg/mL ethidium bromide and visualized under UV light. qRT-PCR analysis was used to determine differences in gene expression. qRT-PCR was performed with primers (Table 2) and SYBR Green PCR master mix (Roche, Basel, Switzerland) in a CFX Connect™ Real-Time System (Bio-Rad, Hercules, CA, USA). All reactions were performed in triplicate.
Flow cytometry analysis
For immunophenotyping of cell surface antigens, third-passage PD-MSCsPRL-1 were detached, stained with antibodies conjugated with fluorescein isothiocyanate (FITC) and phycoerythrin (PE) and analyzed with a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). The following monoclonal antibodies were used: CD34-PE, CD90-PE, HLA-ABC-FITC, HLA-DR-FITC (BD Bioscience, San Jose, CA, USA), CD13-PE (BioLegend, San Diego, CA, USA), CD105-FITC (R&D Systems, Minneapolis, MN, USA), and HLA-G (Abcam, Cambridge, UK). For each sample, at least 10,000 events were acquired.
Teratoma formation and histological analysis
Nine-week-old male NOD/SCID mice (Laboratory Animal Research Center, Bungdang CHA Medical Center, CHA University, Seongnam, Republic of Korea) were maintained in an air-conditioned animal house under specific pathogen-free conditions. To investigate teratoma formation, PD-MSCsPRL-1 (5×105 cells) were directly injected into each testis (TP; n = 2). Control mice were not injected with cells (Con; n = 2). After 14 weeks, the testes were collected, and all mice were sacrificed. The testes were fixed in 10% neutral buffered formalin and embedded in paraffin. Sections were stained with hematoxylin and eosin (H&E).
Total protein was isolated lysis buffer (Sigma-Aldrich). The protein lysates were separated by 8 to 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes, which were then blocked in 5% bovine serum albumin and incubated overnight at 4 °C with the following primary antibodies: anti-phospho-PI3K p110α (1:1000, Cell Signaling Technology, Danvers, MA), anti-phospho-AKT (1:1000, Cell Signaling Technology), anti-phospho-mTOR (1,1000, Abcam), anti-phospho-FAK (1:1000, Cell Signaling Technology), anti-PPAR-γ (1:500, Santa Cruz Biotechnology, Dallas, TX), anti-leptin (1:500, R&D systems), anti-TNF-α (1:500, Santa Cruz Biotechnology), and anti-GAPDH (1:3000, AbFrontier, Seoul, Republic of Korea). The membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Bio-Rad, Hercules, CA, USA), and the bands were detected using an enhanced-chemiluminescence reagent (Bio-Rad).
Naïve and PD-MSCsPRL-1 analyzed karyotypes, respectively using G-banding techiniques. The karyotype analysis was interpreted according to the International System for Human Cytogenomic Nomenclature (ISCN 2016) and visualized under light microscope (Axioskop2 plus, Zeiss, Germany).
Human cytokine array
Cell culture supernatants in naïve and PD-MSCsPRL-1 were collected and analyzed according to the manufacturer’s protocol using Human Proteome Profiler™ Cytokine Array Kit (R&D Systems).
Data analyses were performed using GraphPrism version 5.0 (GraphPad Software, CA, USA) and statistically significant differences were assessed using two-tailed unpaired Student’s t-test or nonparametric statistical test by Mann-Whitney U and Kruskal-Wallis test at a significance level of less than 0.05. All experiments were analyzed in duplicate or triplicate.