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

ABSTRACT Previously we have shown that human abdominal adipose derived-stem cells (ADSCs) could aggregate during the high-density culture in the presence of human serum (HS). In the present study, we observed that human cord blood serum (CBS) and follicular fluid (HFF) also induced aggregation. Similarly, porcine serum could induce aggregation whereas bovine and sheep sera induced little aggregation. qRT-PCR analyses demonstrated that, compared to FBS-cultured ADSCs, HScultured cells exhibited higher level of mRNA expression of CLDN3, -6, -7, -15, and -16 genes among the tight junction proteins. ADSCs examined at the time of aggregation by culture with HS, BSA, HFF, CBS, or porcine serum showed significantly higher level of mRNA expression of JAM2 among JAM family members. In contrast, cells cultured in FBS, bovine serum or sheep serum, showed lower level of JAM2 expression. Immunocytochemical analyses demonstrated that the aggregates of HS-cultured cells (HS-Agg) showed intense staining against the anti-JAM2 antibody whereas neither non-aggregated cells (HS-Ex) nor FBS-cultured cells exhibited weak staining. Western blot results showed that HS-Agg expressed JAM2 protein more prominently than HS-Ex and FBS-cultured cells, both of latter reveled weaker intensity. These results suggest that the aggregation property of ADSCs during high-density culture would be dependent on the specific components of serum, and that JAM2 molecule could play a role in the animal sera-induced aggregation in vitro.


INTRODUCTION
Human mesenchymal stem cells (MSCs) can be isolated from a variety of tissues, such as bone marrow, adipose tissue or other tissues. They are promising sources for cell based therapy because of the potential to differentiate into various cell lineages including adipocytes, osteoblasts and chondrocytes (Galderisi & Giordano, 2014). It has been expected that MSCs will replace damaged tissue and/or induce endogenous repair after homing to the tissue of injury following transplantation in vivo. Compared to other tissues, human adipose tissue is an abundant and easily accessible for the isolation of stem cells. Hence adipose-derived stem cells (ADSCs) have been recognized as a suitable cell source for tissue engineering and regenerative medicine applications (Chen et al., 2013). ADSCs have been examined for their clinical potentials to treat disease such as graft versus host disease (Le Blanc et al., 2008), cartilage damage (Bhumiratana et al., 2014), myocardial infarction (Jiang et al., 2014), liver malfunction (Li et al., 2013) and osteogenesis imperfect (Pereira et al., 2014).
Previously, we observed that human eyelid adiposederived stem cells could form aggregation during highdensity culture in the presence of human serum (HS) in vitro (Song et al., 2012). Since it is well known that FBS alone does not induce an aggregation of plastic-adherent MSCs in vitro, it was questioned whether the aggregation phenomenon might be due to the presence of cell-aggregating factor(s) in HS or due to the absence of the factor in FBS. The present study aimed to give answers to these questions. Furthermore, to unveil the nature of the aggre- shown to affect stem cell behavior (Brizzi et al., 2012). It has been indicated that adherent junctions and gap junctions are essential for the promotion of MSC stemness and proliferation (Wong et al., 2008). For example, MSCs are known to form functional gap junctions with adult cardiac myocytes both in vitro and in vivo (Valiunas et al., 2004;Hahn et al., 2008). Tight junctions (TJs) are specialized plasma membrane microdomains which are highly enriched in cholesterol that encircle cells at the apical end of the lateral membrane are aligned at the apical side of adherent junctions (Yamada et al., 2013). Claudin-6, one of essential component of TJs, has been shown as a cell surface marker of hESCs and hiPSCs due by their specific expression (Ben-David et al., 2013). Present researches focus on the possible involvement and elucidation of the roles of TJs in the aggregation phenomenon of the human adult stem cells during high-density culture.

MATERIALS AND METHODS
1. Human body fluids, animal sera and reagents HS (Millipore, Temecula, CA) was heat-activated at 56°C for 30 min and kept frozen at -20°C until use. Cord

Isolation and culture of ADSCs
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-cm 2 culture flask (Nunc, Roskilde, Denmark) containing 5 mL of the culture medium. Cells were cultivated at 37°C with 5% CO 2 . 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/cm 2 . The culture medium was changed twice a week. ADSCs between passage (p) 2 and p6 were used throughout this study.

Immunocytochemistry
Cell cultures on 8-well slides were fixed in 4% para-
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).

Statistical analysis
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.

Aggregation of ADSCs induced by HS
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,

Effects of other human body fluids
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)  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.

Effects of animal sera
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. Culture period elapsed until the aggregation of ADSCs pre-treated with FBS for 1 day followed by replacing with each animal serum treatment.
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.

Protein expression of JAM2 molecule
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. Scale bar =200 µm. therapy (Aldahmash et al., 2011;Wang et al., 2012). Previously we have observed that human eyelid adiposederived stem cells could aggregate during high-density culture in the presence of HS (Song et al., 2012). In the present study using ADSCs, we observed that, in addition to HS, CBS and HFF could also induce the aggregation.
These results lead to suggest that cell aggregation might be a generalized phenomenon which can take place in other adult stem cells. We also found that the aggregation is not unique to HS treatment, rather is general as seen in cells   Moreover, western blot results also demonstrate that JAM2 protein expression was most prominent in HS-Agg than cells with FBS-culture or HS-Ex.
TJs family include transmembrane proteins such as claudin, occludin, and junctional adhesion molecules (JAM), the latter which are associated with claudin through their cytoplasmic protein, ZO-1 (Bazzoni et al., 2003). JAM family is composed of JAM1, JAM2, JAM3, and JAM4/ JAML, with each have different functions. Of these, JAM2 plays a role in many events such as interaction between embryo and uterine tissue at the time of implantation (Su et al., 2012), melanoma cell metastasis (Ghislin et al., 2011) and the blood-testis barrier between Sertoli cells (Wang Y et al., 2009). These reports indicate a role of JAM2 in cellcell adhesion events. In addition, it plays a crucial role in the adjustment of transendothelial migration and mediated rolling of T lymphocytes dependent on integrin alpha4 and beta1 (Ludwig et al., 2009). Previously, GDF-5 has been shown to enhance the chondrogenic differentiation of human MSCs in vitro as revealed by increased expression of glycosaminoglycan and type II collagen (Zhang et al., 2011). Later study demonstrates that the enhancing effect of GDF-5 might be mediated by the expression of connexin43, which appear to play an important role in in vitro chondrogenesis (Sun et al., 2012). Taken together, it is suggested that tight junction molecules such as JAM and connexin43 plays a role in cell migration as well as cellcell interaction. Present study showed that JAM2 molecule is involved in the aggregation of adult stem cells induced by specific serum or body fluid. Since ADSCs must migrate directing to an area where they aggregate, it is possible that JAM2 molecule in the present study might play dual roles, both migration and adhesion of ADSCs.
One important thing is that while our study shows ADSCs