Expression of Tight Junction Molecule In The Human Serum-Induced Aggregation of Human Abdominal Adipose-Derived Stem Cells In Vitro

HS (Millipore, Temecula, CA) was heat-activated at 56°C for 30 min and kept frozen at –20°C until use. Cord blood serum (CBS) and human follicular fluid (HFF) were kindly donated from CHA Gangnam Medical Center in Seoul. Porcine serum (PS, Sigma), sheep serum (SS, Millipore), bovine serum (BS, Gibco, Grand Island, NY) were used after treatment like HS. Unless indicated elsewhere, all reagents were purchased from Sigma (Seoul, Korea).

Human abdominal adipose tissues were obtained from the patients undergoing liposuction with informed consent. All experiments were approved by Institutional Review Board of Seoul Women’s University. After removal of red blood cells by several washing with Dulbecco’s phosphatebuffer saline (DPBS, Gibco), adipose tissue was mixed with 1x volume of 0.15% type I collagenase (Gibco) and incubated at 37°C for 1 h with gentle shaking. Then 1x volume of culture medium consisting of DMEM-low glucose type (DMEM-LG; Gibco), 10% FBS (Gibco), 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 3.7 mg/mL sodium bicarbonate was added and centrifuged at 3,000 rpm for 10 min. Cell pellets were washed twice, and then plated in 25-cm² culture flask (Nunc, Roskilde, Denmark) containing 5 mL of the culture medium. Cells were cultivated at 37°C with 5% CO₂. When adherent cells were 70~80% confluent, they were detached using Hank’s balanced salt solution containing 0.125% trypsin (Gibco) and 1 mM EDTA (trypsin/EDTA) at 37°C for 2 min and re-suspended in the culture medium at a density of 4,000 cells/cm². The culture medium was changed twice a week. ADSCs between passage (p) 2 and p6 were used throughout this study.

ADSCs were seeded in 6-well or 12-well plate (Nunc, Jiangsu, China) at a density of 2.8×10⁴ cells/cm². Cells were cultivated with DMEM-LG containing 5% or 10% of FBS, HS, CBS, HFF, PS, SS or 1% of bovine serum albumin (BSA). Sometimes cells were seeded in 24-well dish (Nunc) at same density. After culture in HS, aggregates of ADSCs (HS-Agg) were isolated using a pipette tip. HStreated but non-aggregated cells (HS-Ex) were harvested after detachment using trypsin/EDTA solution. Collected cells were kept at –20°C before use.

Total RNA was isolated using Tri-reagent (Ambion, Carlsbad, CA) according to the manufacturer’s instructions. RT-PCR was carried out using a Technic PCR system TC- 100 (Bibby Scientific, Staffordshire, UK). Total 7.5 μg (1 μg/μL) of RNA was reverse-transcribed using the following RT mixture: 20 mM MgCl₂ (Bio Basic, Ontario, Canada), 10 × PCR buffer (Bio Basic), 10 mM dNTPs mixture (Bio Basic), 0.5 mg/mL oligo (d)T¹⁵ (Bionics, Korea) for RTPCR or 0.5 mg/mL random hexamer (Roche, Branchburg, NJ) for qRT-PCR , 40 U/μL RNase inhibitor (Bio Basic) and 200 U/μL AMV-RT (Invitrogen, Carlsbad, CA). RT reaction was allowed to occur for 60 min at 37°C. PCR was performed using the following mixture: 25 mM MgCl₂, 10 × PCR buffer, 5 U/μL Taq polymerase, 2.5 mM dNTP, 10 μM sense and antisense primer. Amplification was performed for 35 cycles at a denaturing temperature of 94°C for 30 sec and an extension temperature of 72°C for 30 sec. Annealing temperature was set depending on the species of primer. The PCR products electrophoretically separated on agarose gels were analyzed using an image analyzer (ULTima, Hoefer, Leicestershire, UK). qRT-PCR was performed using a buffer containing 1 μL of template cDNA, 10 μL SYBR Green I Master (Roche, Mannheim, Germany), and 10 pM of each primer using Light Cycler 480 Real-Time System (Roche). Relative expression levels of cDNA were normalized to the expression of 18s rRNA using the comparative CT (2^-ΔΔCT) method. Primers used in this study are shown in Table 1. PCR mixture components were purchased from Takara (Shiga, Japan).

Cell cultures on 8-well slides were fixed in 4% paraformaldehyde for 1 h at 4°C. They were washed with washing buffer (0.1% BSA in DPBS) and incubated in a blocking solution consisting of 2% BSA in PBS for 1 h at room temperature with gentle shaking. Then cells were incubated with rabbit polyclonal anti-JAM2 antibody (1:400) (Abcam, Cambridge, UK) for overnight at 4°C with gentle shaking. They were then incubated with the goat antirabbit IgG-FITC (1:100) (Santa Cruz, CA) for 1 h. After washing, cell nuclei were stained with 1 μg/mL DAPI in 0.1 M PBS for 2 min. Fluorescent images were visualized by confocal microscopy (C1+, Nikon, Melville, NY) or fluorescence microscope (Axioskop2+, Carl Zeiss, Thornwood, NY).

Cells were homogenized in lysis buffer consisting of 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% sodium deoxycholate, 1% Triton X-100, 1% sodium dodecyl sulfate (SDS), and 25x protease inhibitor (Roche, Mannheim, Germany) for 30 min on ice. Protein sample was mixed with 2× sample buffer (12.5 mM Tris-HCl (pH 6.8), 4% SDS, 10% 2-mercaptoethanol, 20% glycerol, 0.004% bromophenol blue) and heated at 90°C for 5 min. Following separation by 10% SDS polyacrylamide gel electrophoresis (SDS-PAGE), proteins on the gels were electrically transferred to nitrocellulose membranes (Millipore, Bedford, MA). Then membranes were incubated with the rabbit polyclonal anti-JAM2 antibody or mouse monoclonal antibody against β-actin (1:800, Santa Cruz) in blocking buffer (4.9% BSA in washing buffer) for overnight at 4°C. Then the membrane was incubated in HRP-conjugated goat anti-mouse IgG for 1 h at 37°C. Membranes were the visualized by ECL (BioFX, Aden Prairie, MN). Data were analyzed using ImageJ program (NIH, Bethesda, MD).

Data were shown as a mean±SEM. Statistical significance was analyzed by student’s t-test. P-value<0.05 was considered to be statistically significant.

During culture of ADSCs in the presence of 10% FBS, none of the cells from three different lines showed any aggregation or morphological change in every experiment. However, same cells but cultivated in HS exhibited aggregation, some of which were located in the center of wells whereas others were formed in the vicinity of the wells (Fig. 1A). Cell aggregation was observed within 5 days from the beginning of culture (Fig. 1B). However, when cells were pre-treated with FBS for 1 day before replacing with HS-medium or BSA-medium, the aggregation formation was delayed to 8-10 days. To examine whether FBS could inhibit the HS-induced aggregation, cells were cultivated in the mixture of FBS and HS. In the presence of 10% mixture, most wells exhibited aggregation-positive within 12 days. In the presence of 5% mixture, only 60% of wells showed aggregation-positive after 15 days without further aggregation (Fig. 1C). In contrast, all wells containing cells with either 5% or 10% HS exhibited aggregationpositive within 8 days. These results demonstrate that FBS could not inhibit the aggregation of ADSCs induced by HS, implying that cell aggregation might be induced by certain component(s) present in the HS.

Fig. 1. Aggregation of ADSCs during culture in HS-medium. (A) Aggregation of ADSCs during culture in HS-medium. FBS-culture did not induce any aggregation (a), whereas HS-culture induced cell aggregation in the vicinity (b) or in the center (c) of the culture well. Scale bar =200 μm. (B) Culture period elapsed until the aggregation of three different lines of ADSCs occurred in the presence of HS. (C) Culture period elapsed until the aggregation of ADSCs occurred in the presence of both FBS and HS. The data were obtained from three independent experiments.

Download Original Figure

It was further examined whether other human body fluids could induce similar aggregation of ADSCs. All culture wells containing either CBS or HFF showed the appearance of cell aggregation (Fig. 2A). However, while HS showed 100% of aggregation-positive wells within 4 days of culture, CBS or HFF induced 100% or 80% aggregation 6 or 8 days after culture, respectively. Pre-treatment of ADSCs with FBS delayed the aggregation by 6 days during the subsequent culture in HFF whereas pre-treatment with FBS resulted in little difference between HS-culture and CBS-culture (Fig. 2B). These results demonstrate that human body fluids such as CBS and HFF might contain same or similar cell-aggregating factors as HS, while the aggregating activity of HFF seemed weaker than other two.

Fig. 2. Effect of human body fluids on the aggregation of ADSCs. (A) Culture period at the time of aggregation of ADSCs in various human body fluids. (B) Culture period at the time of aggregation of ADSCs pre-treated with FBS for 1 day followed by human body fluids treatment.

Download Original Figure

To see if animal serum other than HS could induce similar aggregation of ADSCs, effects of sheep serum (SS), porcine serum (PS) and bovine serum (BS) were examined. Similar to HS, PS-medium induced distinctive aggregation (Fig. 3A). However, the aggregation seemed to take place 2 days later compared to the HS-medium. In contrast, neither SS nor BS showed any appearance of aggregation at all under the same culture condition. When cells were pre-treated with FBS, again it delayed the aggregation. However, there was little difference in the culture periods required for the 100% aggregation-positive wells between HS- and PS-treated groups (Fig. 3B). These results showed that cell aggregating activity by body fluids is not humanspecific, rather is depending on the animal species.

Fig. 3. Effect of selected animal sera on the aggregation of ADSCs. (A) Culture period required for the aggregation of ADSCs in a medium containing 10% of human, sheep, porcine or adult bovine serum. (B) Culture period elapsed until the aggregation of ADSCs pre-treated with FBS for 1 day followed by replacing with each animal serum treatment.

Download Original Figure

aggregated cells maintained tightly adhering structures during culture for many days, it was questioned what kinds of cell adhesion molecules might be involved in the rigid aggregation. As shown in Fig. 4A, conven-tional RT-PCR analyses showed that ADSCs consistently expressed mRNAs of CLDN7, CLDN11, CLDN12, CLDN14, TJP1, and JAM3 genes. In contrast, cells inconsistently expressed genes of CLDN3, CLDN4, CLDN6, CLDN8, CLDN9, CLDN10, CLDN16, OCLN, JAM1, TJP2, and TJP3, depending on cell lines. They never expressed CLDN2 and CLDN5 genes. Quantitative RT-PCR analyses using three different cell lines showed greater expression of CLDN3, -6, -7, -15, -16 genes and JAM2 gene in HS-Agg than cells cultivated with FBS or HS-Ex (Fig. 4B). Of these mRNAs, mRNA of JAM2 showed the greatest expression. Compared to the JAM2 mRNA expression in cells cultivated with FBS, more than five times greater expression was observed in HS-Agg. Cells cultivated with HFF, CBS or PS also showed greater expression than cells with FBS. In contrast, cells with SS-culture or BS-culture showed similar expression level of JAM2 mRNA as cells cultivated with FBS (Fig. 4C).

Fig. 4. Comparative mRNA expression level of TJ molecules in cells cultivated under various conditions. (A) conventional RT-PCR analyses of TJ genes in ADSCs after culture for 4 days in FBS- or HScontaining medium. (B) qRT-PCR analyses of expression of selected TJ genes in the same samples as in (A). Note that, regardless of the cell lines, JAM2 gene expression level in HS-Agg is consistently greater than that in HS-Ex and FBS-culture. (C) qRT-PCR analyses of JAM2 expression in ADSCs after culture in FBS, human body fluids or various animal sera. After culture for up to 8 days, all cells were harvested and their relative expression level of JAM2 gene was assessed by comparing to the FBS-cultured cells. * P < 0.05.

Download Original Figure

Immunocytochemical staining showed that human JAM2 protein was distinctly expressed in ADSCs that has begun the aggregation during HS-culture, whereas none of the cells cultivated in FBS exhibited staining (Fig. 5). Western blot analysis demonstrated that JAM2 protein was expressed in cells cultivated with FBS or HS (Fig. 6A). However, the staining intensity of the protein was most prominent in HS-Agg compared to FBS group or HS-Ex group (Fig. 6B). Taken together, these results suggest that HS treatment could induce an aggregation in ADSCs, and only cells undergoing aggregation appear to enhance the JAM2 protein expression unlike remaining cells which do not undergo aggregation during the HS treatment.

Fig. 5. Localization of JAM2 protein in ADSCs after culture with FBS or HS. ADSCS were cultivated in the presence of FBS (A) or HS (B, C) for 7 days, and then stained with antibody against human JAM2 protein. Green color denotes the positive staining and nuclei were counterstained with DAPI (blue). Arrows indicate the area of cells undergoing aggregation. BF, bright field; NC, negative control. Scale bar =200 μm.

Download Original Figure

Fig. 6. Western blot analysis of JAM2 protein in ADSCs after culture with FBS or HS. (A) Protein expression was analyzed after culture for 4 days. Note that staining intensity of JAM2 protein (48kDa) was the most prominent in HS-Agg compared to cells of FBS and HS-Ex. (B) Relative intensity of JAM2 protein from the above is shown as bar. The results were obtained from three independent experiments. * P < 0.05.

Download Original Figure