Cell culture and gene transfection
Orbital adipose tissue specimens were obtained from patients with GO (n = 3) during fat decompression and control individuals without GO history (n = 3) under the consent conditions. Orbital fibroblast preparation was approved by Institutional Review Board of CHA Bundang Medical Center, Seongnam, Republic of Korea. Orbital tissue explants were chopped and treated with 0.25 mg/mL collagenase (Sigma-Aldrich, St. Louis, MO, USA) for 1 h at 37℃ shaking incubator. After collagenase digestion, the orbital tissues were placed in the culture plates with DMEM/F12 supplemented 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, KOREA (IRB 07–18). PD-MSCs were isolated as described previously [18] 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). PRL-1 (protein tyrosine phosphatase type IVA, member 1; PTP4A1) plasmid vector was purchased (#RG200435; Origene, Rockville, MD, USA). To induced overexpression of PRL-1 gene, naïve PD-MSCs (passage = 7) transfected using AMAXA Nucleofector system (Lonza, Basel, Switzerland) according to the manufacturer’s instructions. After transfection for 24 h, cells were selected by 1.5 mg/mL neomycin. All kinds of cells were maintained at 37℃ in humidified atmosphere containing 5% CO2.
Differentiation for PD-MSCsPRL−1 and OF with GO
To analyze the potential of PD-MSCsPRL−1 to differentiate into mesodermal lineages, PD-MSCsPRL−1 (passage = 5) were plated at density of 5ⅹ103 cells/cm2 in each differentiation induction medium using 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) for staining lipids to visualize lipid vesicles and von Kossa staining with 5% silver nitrate (Sigma-Aldrich) under light to evaluate their accumulation of calcium deposits.
To induce adipogenic differentiation, normal and GO-derived orbital fibroblasts (5ⅹ103 cells/cm2) were seeded and changed to 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 dexametasone, and 0.1 mM isobutylmethylxanthine (IBMX; all from Sigma-Aldrich) for the first 4 days only. To induce maturation of adipogenesis, the medium was supplemented except 1 µM dexamethasone and 0.1 mM IBMX (all from Sigma-Aldrich) for 6 days. The media was replaced every other day. Lipid accumulation and adipocyte morphology were visualized by Oil red O staining.
Coculture experiments
For the detection of inhibition of adipogenesis, normal and GO-derived orbital fibroblasts after adipogenesis differentiation were cocultured with naïve and PD-MSCsPRL−1 (5 × 103 cells/cm2
onto 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℃ in humidified atmosphere containing 5% CO
2.
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) according to the manufacturer’s protocol. The concentration and purity of total RNA were determined spectrophotometrically at OD 260 nm and 280 nm. cDNA was synthesized by reverse transcriptase (RT) 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. 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 differences in gene expression. qRT-PCR was performed with primers (Table 2) and SYBR Green PCR Master Mix (Roche, Basel, Switzerland) in CFX Connect™ Real-Time System (Bio-Rad, Hercules, CA, USA). All reactions were performed in triplicate.
Table 1
Primer sequences using reverse transcription polymerase chain reaction
Genes | | Primer sequences | Tm |
Oct4 | Forward | 5'-AGTGAGAGGCAACCTGGAGA-3' | 52 |
Reverse | 5'-GTGAAGTGAGGGCTCCCATA-3' |
Nanog | Forward | 5'-TTCTTGACTGGGACCTTGTC-3' | 52 |
Reverse | 5'-GCTTGCCTTGCTTTGAAGCA-3' |
Sox2 | Forward | 5'-GGGCAGCGTGTACTTATCCT-3' | 52 |
Reverse | 5'-AGAACCCCAAGATGCACAAC-3' |
HLA-G | Forward | 5'-GCGGCTACTACAACCAGAGC-3' | 58 |
Reverse | 5'-GCACATGGCACGTGTATCTC-3' |
TERT | Forward | 5'-GAGCTGACGTGGAAGATGAG-3' | 55 |
Reverse | 5'-CTTCAAGTGCTGTCTGATTCCAATG-3' |
AFP | Forward | 5'-ATGCTGCAAACTGACCACGC-3' | 55 |
Reverse | 5'-GCTTCGCTTTGCCAATGCTT-3' |
Albumin | Forward | 5'-TGAGTTTGCAGAAGTTTCCA-3' | 60 |
Reverse | 5'-CCTTTGCCTCAGCATAGTTT-3' |
β-actin | Forward | 5'-TCCTTCTGCATCCTGTCAGCA-3' | 58 |
Reverse | 5'-CAGGAGATGGCCACTGCCGCA-3' |
Table 2
Primer sequences using quantitative real time polymerase chain reaction
Genes | | Primer sequences | Tm |
OC | Forward | 5'-AGTGAGAGGCAACCTGGAGA-3' | 52 |
Reverse | 5'-GTGAAGTGAGGGCTCCCATA-3' |
COL1A1 | Forward | 5'-TTCTTGACTGGGACCTTGTC-3' | 52 |
Reverse | 5'-GCTTGCCTTGCTTTGAAGCA-3' |
Adipsin | Forward | 5'-GGGCAGCGTGTACTTATCCT-3' | 52 |
Reverse | 5'-AGAACCCCAAGATGCACAAC-3' |
PPAR-γ | Forward | 5'-GCGGCTACTACAACCAGAGC-3' | 58 |
Reverse | 5'-GCACATGGCACGTGTATCTC-3' |
Adiponectin | Forward | 5'-GAGCTGACGTGGAAGATGAG-3' | 55 |
Reverse | 5'-CTTCAAGTGCTGTCTGATTCCAATG-3' |
Leptin | Forward | 5'-ATGCTGCAAACTGACCACGC-3' | 55 |
Reverse | 5'-GCTTCGCTTTGCCAATGCTT-3' |
LPL | Forward | 5'-TGAGTTTGCAGAAGTTTCCA-3' | 60 |
Reverse | 5'-CCTTTGCCTCAGCATAGTTT-3' |
FABP4 | Forward | 5'-GCATGGCCAAACCTAACATGA-3' | 55 |
Reverse | 5'-CCTGGCCCAGTATGAAGGAAA-3' |
IGFBP1 | Forward | 5'-GAGCCCTGCCGAATAGAAC-3' | 60 |
Reverse | 5'-GGATCCTCTTCCCATTCCAAG-3' |
IGFBP2 | Forward | 5'-ACATCCCCAACTGTGACAAG-3' | 60 |
Reverse | 5'-ATCAGCTTCCCGGTGTTG-3' |
IGFBP3 | Forward | 5'-CAGAGCACAGATACCCAGAAC-3' | 60 |
Reverse | 5'-AGCACATTGAGGAACTTCAGG-3' |
IGFBP4 | Forward | 5'-CTGACAGCTTTCGAGAGTGAG-3' | 60 |
Reverse | 5'-GCGCATTTGAGGGAAACTTC-3' |
IGFBP5 | Forward | 5'-ACCCAGTCCAAGTTTGTCG-3' | 60 |
Reverse | 5'-TGTAGAATCCTTTGCGGTCAC-3' |
IGFBP6 | Forward | 5'-GTCTACACCCCTAACTGCG-3' | 60 |
Reverse | 5'-CTCTGTTGGTCTCTGCGG-3' |
IGFBP7 | Forward | 5'-GCCCAGAAAAGCATGAAGTAAC-3' | 60 |
Reverse | 5'-TTTATAGCTCGGCACCTTCAC-3' |
ITGA4 | Forward | 5'-AGAGAGACAATCAGTGGTTGG-3' | 55 |
Reverse | 5'-TCAGTTCTGTTCGTAAATCAGG-3' |
ITGB7 | Forward | 5'-AGCAGCAACAACTCAACTGG-3' | 55 |
Reverse | 5'-TTACAGACCCACCCTTCCTCT-3' |
FAK | Forward | 5'-GAAGCATTGGGTCGGGAACTA-3' | 55 |
Reverse | 5'-CTCAATGCAGTTTGGAGGTGC-3' |
GAPDH | Forward | 5'-TCCTTCTGCATCCTGTCAGCA-3' | 58 |
Reverse | 5'-CAGGAGATGGCCACTGCCGCA-3' |
Flow cytometry analysis
For immunophenotyping of cell surface antigens, third-passage PD-MSCsPRL−1 were detached and stained with fluorescein isothiocyanate- (FITC-) and phycoerythrin- (PE-) conjugated antibodies and were analyzed with FACSablibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). Monoclonal antibodies against the human antigens 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) were used. 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 was not injected (Con; n = 2). After maintaining for 14 weeks, testes were collected and all mice were sacrificed. Testis were homogenized and fixed in 10% Neutral buffered formalin and embedded in paraffin. Sections were stained with haematoxylin and eosin (H&E).
Western blotting
Total protein was acquired by 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, blocked in 5% Bovine serum albumin, and then incubated overnight 4℃ with primary antibodies : anti-PI3K p110 alpha (1:1000, Cell Signaling Technology, Denvers, MA), anti-pAKT (1:1000, Cell Signalling Technology), anti-pmTOR (1,1000, Abcam), anti-pFAK (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). Membranes incubated with horseradish peroxidase- (HRP) conjugated secondary antibodies (Bio-Rad, Hercules, CA, USA). The bands were detected using an enhanced-chemiluminescence reagent (Bio-Rad).
Statistical analysis
The experimental statistics were expressed as the means ± SD. Statistical analysis was performed using Student’s t-test, and the difference was considered statistically significant when the p value was less than 0.05. Each experiment has been conducted in duplicate or triplicate.