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Contribution of human adipose tissue–derived stem cells and the secretome to the skin allograft survival in mice

Published:January 31, 2014DOI:https://doi.org/10.1016/j.jss.2013.10.063

      Abstract

      Background

      Despite considerable evidence showing the immunosuppressive properties of mesenchymal stem cells (MSCs) in vitro, such properties have not been fully demonstrated in vivo. The aim of this study was to evaluate the effect of MSCs and/or MSC secretome in inducing tolerance in a mouse skin transplantation model.

      Methods

      After receiving full-thickness skin allotransplantation on the back of the mouse, the recipient mice were infused with phosphate-buffered saline, adipose tissue–derived stem cells (ASCs), conditioned media (CM), and control media. Specifically, ASCs (1.0 × 106/0.1 mL) were transplanted to ASC-infused mice and 25-fold concentrated CM, which had been obtained from ASC culture were infused to CM-infused mice. Graft survival rates and the parameters reflecting immunologic consequences were assessed.

      Results

      The serum level of proinflammatory cytokine interleukin 6 decreased in mice treated with ASCs or CM compared with the control groups after infusion (P < 0.05). Interferon gamma, interleukin 10, and tumor necrosis factor alpha messenger RNA levels in the skin graft seemed to be decreased in the ASC-infused mice and CM-infused mice. Hyporesponsiveness was identified in mixed lymphocyte reaction assay at 30-d posttransplantation in ASC- or CM-infused mice. And, administering ASCs and CM markedly increased skin allograft survival compared with control animals (P < 0.001).

      Conclusions

      These findings suggest that ASCs and their secretome have the potential to induce immunologic tolerance. Moreover, our results demonstrate that the immunosuppressive properties of ASCs are mediated by the ASC secretome. Our approach could provide insights into a promising strategy to avoid toxicities of chemical immunosuppressive regimen in solid organ transplantation.

      Keywords

      1. Introduction

      Immunologic tolerance in the clinical setting can be defined as long-term allograft survival in the absence of immunosuppressive treatment [
      • Fehr T.
      • Sykes M.
      Tolerance induction in clinical transplantation.
      ]. Immunologic tolerance has been considered an ultimate goal in human solid organ transplantation. Currently, the immunologic barriers after organ transplantation have been somewhat successfully overcome by immunosuppressive regimens, which can be toxic and have many side effects, possibly resulting in allograft loss. In such circumstances, recent sporadic reports have indicated overcoming immunologic barriers by using the immunosuppressive properties of stem cells [
      • Lu F.
      • Mizuno H.
      • Uysal C.A.
      • et al.
      Improved viability of random pattern skin flaps through the use of adipose-derived stem cells.
      ,
      • Sbano P.
      • Cuccia A.
      • Mazzanti B.
      • et al.
      Use of donor bone marrow mesenchymal stem cells for treatment of skin allograft rejection in a preclinical rat model.
      ,
      • Wang Y.
      • Liu J.
      • Xu C.
      • et al.
      Bone marrow transplantation combined with mesenchymal stem cells induces immune tolerance without cytotoxic conditioning.
      ,
      • Zografou A.
      • Tsigris C.
      • Papadopoulos O.
      • et al.
      Improvement of skin-graft survival after autologous transplantation of adipose-derived stem cells in rats.
      ].
      Mesenchymal stem cells (MSCs) are derived from fetal and adult organs and have the capacity to self-renew and differentiate into various tissues including muscle, fat, stroma, tendon, cartilage, and bone [
      • Abdallah B.M.
      • Kassem M.
      Human mesenchymal stem cells: from basic biology to clinical applications.
      ]. Numerous in vitro experiments have revealed that MSCs have potent immunosuppressive properties [
      • Atoui R.
      • Chiu R.C.
      Concise review: immunomodulatory properties of mesenchymal stem cells in cellular transplantation: update, controversies, and unknowns.
      ,
      • Trento C.
      • Dazzi F.
      Mesenchymal stem cells and innate tolerance: biology and clinical applications.
      ]. MSCs suppress the T cell response to mitogenic stimuli and directly inhibit T cell proliferation [
      • Di Nicola M.
      • Carlo-Stella C.
      • Magni M.
      • et al.
      Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli.
      ,
      • Tse W.T.
      • Pendleton J.D.
      • Beyer W.M.
      • et al.
      Suppression of allogeneic T-cell proliferation by human marrow stromal cells: implications in transplantation.
      ]. MSCs also inhibit B cell proliferation and differentiation into antibody-secreting cells [
      • Corcione A.
      • Benvenuto F.
      • Ferretti E.
      • et al.
      Human mesenchymal stem cells modulate B-cell functions.
      ]. Furthermore, MSCs affect immunologic functions of antigen-presenting cells and inhibit monocyte differentiation into mature dendritic cells [
      • Nauta A.J.
      • Kruisselbrink A.B.
      • Lurvink E.
      • et al.
      Mesenchymal stem cells inhibit generation and function of both CD34+-derived and monocyte-derived dendritic cells.
      ].
      Although in vitro results support the immunosuppressive properties of MSCs, their effects in vivo have been poorly investigated. Recently, infusing donor bone marrow with epidermal cells was shown to improve skin allograft survival [
      • Petit F.
      • Minns A.B.
      • Nazzal J.A.
      • et al.
      Prolongation of skin allograft survival after neonatal injection of donor bone marrow and epidermal cells.
      ] and bone marrow–derived MSCs were reported to improve skin graft survival [
      • Bartholomew A.
      • Sturgeon C.
      • Siatskas M.
      • et al.
      Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo.
      ]. However, there is also a report to show that MSC infusion did not improve heart allograft survival [
      • Inoue S.
      • Popp F.C.
      • Koehl G.E.
      • et al.
      Immunomodulatory effects of mesenchymal stem cells in a rat organ transplant model.
      ].
      Based on the above-mentioned findings, the purpose of this study was to evaluate the role of MSCs in inducing skin allograft tolerance in a mouse skin transplantation model. In particular, we compared MSCs' immunosuppressive properties with the properties of the conditioned media (CM), most of which include MSC secretome. We think that our approach can provide not only an understanding of how MSCs work but also provide new insight into a novel therapy based on the MSC's mechanism.

      2. Materials and methods

      2.1 Animals and study design

      This study was carried out in compliance with the guidelines of the Institute for Laboratory Animal Research, Korea. Eight-week-old male C57BL/6 mice (Damool Science, Daejeon, Korea) were used as skin graft donors, and 8-week-old male BALB/c mice (Damool Science) were used as skin graft recipients. Obtaining specimens, such as serum samples or tissue, usually kills mice or influences the graft survival, limiting validation of survival analysis. Therefore, we designed two experimental sets: a set for graft survival analysis (n = 48) and a set for specimen attainment (n = 64). The set for graft survival was also used for mixed lymphocyte reaction (MLR) at posttransplantation 30 d. According to the materials injected via tail vein after skin allograft transplantation, each set was divided into four groups: phosphate-buffered saline (PBS) group, adipose tissue–derived stem cells (ASC) group, CM group, and M group. PBS group was infused with PBS; ASC group, ASCs; CM group, CM; and M group, control media. Specifically, ASCs at a concentration of 1.0 × 106/0.1 mL were given to ASC-infused mice, 25-fold concentrated CM which had been obtained from ASC culture to CM-infused mice, and control media (MesenPro RS medium; Gibco. Grand Island, NY) to control media-infused mice, respectively.

      2.2 Preparation of ASCs and CM

      2.2.1 Preparation of ASCs

      Passage-three human MSCs were kindly donated by Hurim Biocell Co. (Seoul, Korea). The MSCs were obtained from human subcutaneous adipose tissue and thus are also called human ASCs. After thawing, the cells were cultured in MesenPro RS medium (Gibco) with growth supplement and antibiotics (amphotericin B, streptomycin, and penicillin) at 37°C in 5% CO2. On reaching 80% confluence, the cells were digested with 0.25% trypsin/ethylenediaminetetraacetic acid at 37°C, centrifuged, and resuspended in the medium. The cell suspensions were plated in new flasks and cultured. Cells were used after three passages in our laboratory.

      2.2.2 Identification of ASCs

      Expression of cell-surface antigens in the cultured cells was analyzed by flow cytometry. The passage 3 cells were incubated with fluorescein isothiocyanate- or phycoerythrin-conjugated anti-CD44, anti-CD45, anti-CD29, or mouse IgG isotype control antibodies (BD Biosciences, Heidelberg, Germany) for 30 min at 4°C in PBS. Cells were washed and analyzed with a FACSCalibur kit (BD Biosciences, Pharmingen, CA).

      2.2.3 4, 6-Diamidino-2-phenylindole labeling

      To allow identification of ASCs in transplant specimens, ASC nuclei were stained with 4, 6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, St. Louis, MO) before infusion to mice. DAPI was added to the medium at a final concentration of 50 mg/mL. The cells were incubated for 30 min at 37°C with 5% CO2 and washed six times in PBS. Cells were detached by digestion with 0.25% trypsin/ethylenediaminetetraacetic acid at 37°C, centrifuged, and resuspended in PBS at 2 × 108 cells/mL.

      2.2.4 Preparation of CM

      ASCs that reached 70%–80% confluence were refed with serum-free MesenPro RS basal medium without growth supplement. CM was prepared by collecting the serum-free medium after culturing for 48 h. The medium was then concentrated approximately 25-fold using ultrafiltration units (Amicon Ultra-PL 3; Millipore, Bedford, MA) with a 3-kDa molecular weight cutoff. The CM was stored at 4°C or −80°C until use.

      2.3 Full-thickness skin graft model and transplantation

      2.3.1 Skin grafting

      After mice were sedated with tiletamine-zolazepam (Zoletil 20) (30 mg/kg intraperitoneally), 3.0 × 3.0 cm full-thickness skin grafts were harvested from the midline of the back including the panniculus carnosus. After removing subcutaneous tissue of the recipient, the skin grafts were placed on the midline of the back of the recipient and fixed with simple separate stitches (4-0 Silk; Ethicon, Somerville, NJ). The graft was monitored daily for 30 d or until it was rejected. Graft rejection was defined when >50% the graft tissue became necrotic. And, if the graft survived 30 d without evidence of necrosis, it was considered accepted.

      2.3.2 Injection of ASCs or CM

      Within 1 h after skin grafting, mice underwent transplantation (or injection) of PBS, ASCs, CM, or controlled media via tail vein. The ASC group received ASCs (0.1 mL) at a concentration of 1.0 × 106 cells per mouse. The CM group received 0.3 mL equivalent of CM concentrated 25-fold. The M group received 0.3 mL equivalent of basal media (MesenPro RS basal medium), which had been also concentrated 25-fold.

      2.4 Detection of cytokine production

      At days 3, 8, and 20 posttransplantation, serum was collected from peripheral blood samples and mouse interleukin (IL)-6 concentrations were measured by using a sandwich enzyme-linked immunosorbent assay (mouse ELISA kits, ab100712; Abcam, Cambridge, MA) according to the manufacturer's instruction.

      2.5 Mixed lymphocyte reaction

      An MLR assay was used to find the difference in alloreactivity according to the treatments at day 30 posttransplantation. For the MLR assay, responder cells were freshly isolated from the spleens of recipient BALB/c mice. Stimulator cells (1.0 × 108) were isolated from the spleens of C57BL/6 mice. CD4+ cells from the donor spleens were isolated by Purple Easysep Magnet (CATALOG #18000; StemCell Technologies, Vancouver, Canada) were used as stimulator cells. Responder cells were cultured in RPMI 1640 medium with 10% fetal bovine serum, 10 mM HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid), 1% nonessential amino acids, 2 mM l-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin (all from Lonza, St. Louis, MO) and 5 μg/mL 2-mercaptoethanol (Sigma-Aldrich). A total of 2 × 105 responder cells were placed in triplicate wells of a 96-well culture plates and incubated for 5 d with stimulator cells which had been inactivated by mitomycin C (Sigma-Aldrich). After 5 d, the proliferation of responder cells was measured by CCK assay (EZ-Cytox Cell Viability Asssay kit; iTSBiO, Seoul, Korea) according to the manufacturer's instruction. The absorbance (450 nm) for each sample was analyzed by a microplate reader (model 680; Bio-Rad Laboratories, Hercules, CA) and was interpolated with a standard curve.

      2.6 RNA extraction and quantitative real-time polymerase chain reaction

      Total RNA was prepared from mouse skin using a NucleoSpin RNAII kit (Macherey-Nagel, Düren, Germany). Complementary DNA (cDNA) was synthesized from 1 μg RNA by using Reverse Transcriptase Premix (Elpis Biotech, Daejeon, Korea). After reverse transcription, the cDNA was used as a template in polymerase chain reactions (PCRs) using gene-specific primer pairs. Glyceraldehyde-3-phosphate dehydrogenase was used as a reference gene. Specific PCR products (Bioneer, Daejeon, Korea) were generated using 5 pmol each of the following sense and antisense primers based on human genome database: IL-10, sense 5′-TGCTATGCTGCCTGCTCTTA-3′ and antisense 5′-TCATTTCCGATAAGGCTTGG-3′; tumor necrosis factor alpha (TNF-α), sense 5′-ACGGCATGGATCTCAAAGAC-3′ and antisense 5′-GTGGGTGAGGAGCACGTAGT-3′; interferon gamma (IFN-λ), sense 5′-GCRACAACATGCATCTTGGCTTT-3′ and antisense 5′-ATTGATGCTCTCTTTGTCTG-3′; and GAPDH, sense 5′-TGCAGTGGCAAAGTGATT-3′ and antisense 5′-CGTGAGTGGAGTCATACTGGAACA-3′. cDNA was amplified in a Power SYBR Green PCR Master Mix (Applied Biosystems, Warrington, UK). Quantitative real-time PCR was performed using an ABI 7500 FAST (Applied Biosystems, Foster City, CA).

      2.7 Histologic examination

      Paraffin-embedded tissue from skin grafts was sectioned 4 mm thick. All sections were stained with hematoxylin and eosin. Immunohistochemistry was used to stain vasoactive endothelial growth factor (VEGF) and mouse macrophages with a VEGF polyclonal antibody (ab1316, 1:500 dilution; Abcam) and F4/80 monoclonal antibody (ab6640, 1:500 dilution; Abcam), respectively. Standard procedures for this staining were performed as recommended by the manufacturers. Stained sections were interpreted by a single pathologist blinded to the treatment regimen.

      2.8 Statistical analysis

      Numeric data were presented as mean and standard deviation. Continuous variables were analyzed using the independent t-test or Wilcoxon rank-sum tests depending on whether they follow a normal distribution or not. All P values were two tailed. Statistical analysis was performed using SPSS ver. 15.0 (SPSS Inc, Chicago, IL). Statistical significance was accepted for P values <0.05.

      3. Results

      3.1 ASC characteristics

      ASCs do express MSC markers such as CD29, CD44, CD73, CD90, and CD105 and do not express hematopoietic markers such as CD34, CD45, and CD14 [
      • Dominici M.
      • Le Blanc K.
      • Mueller I.
      • et al.
      Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement.
      ,
      • Foster L.J.
      • Zeemann P.A.
      • Li C.
      • et al.
      Differential expression profiling of membrane proteins by quantitative proteomics in a human mesenchymal stem cell line undergoing osteoblast differentiation.
      ]. Our results from flow cytometry affirmed that our cultured cells were negative for hematopoietic markers (CD31 and CD34) and positive for a MSC marker (CD 90) (Fig. 1).
      Figure thumbnail gr1
      Fig. 1Flow cytometry of ASCs with mesenchymal and hematopoietic stem cell markers. Whereas hematopoietic stem cell marker (CD31, CD34) was not expressed (A), MSC marker (CD90) was expressed (B).

      3.2 Proinflammatory IL-6 levels determined by enzyme-linked immunosorbent assay

      IL-6, which is secreted by immune cells such as T cells and macrophages, functions as a proinflammatory cytokine stimulating immune and inflammatory responses. Throughout all posttransplantation days (days 1, 2, 7, and 20), the serum IL-6 levels in the treatment groups (ASC or CM) was lower than in the control group (PBS) (P < 0.05) (Fig. 2). The differences between the ASC and CM groups were inconsistent; the CM group had lower IL-6 levels at day 1 posttransplantation, but the ASC group had lower IL-6 levels at day 7 posttransplantation (P < 0.05).
      Figure thumbnail gr2
      Fig. 2Comparison of the serum levels of proinflammatory cytokine IL-6 after transplantation according to the treatments. The serum levels of IL-6 were measured at days 1, 2, 7, and 20 posttransplantation. Four mice from PBS, ASC, and CM groups were randomly selected on posttransplantation days 1, 2, 7, and 20, respectively, for the evaluation. Overall, the serum levels of IL-6 seemed to be decreased in ASC-infused mice and CM-infused mice compared with PBS-infused mice (control). (Color version of figure is available online.)

      3.3 DAPI-labeled ADSC distribution inside flaps

      The presence of DAPI-positive cells in 4-mm tissue sections from the ASC group was assessed by fluorescence microscopy (DM IRE2; Leica Inc, Wetzlar, Germany). Several DAPI-labeled cells were detected in and beneath the graft tissue at day 7 posttransplantation (Fig. 3).
      Figure thumbnail gr3
      Fig. 3Mouse skin allograft specimen containing DAPI-labeled human ASC. The specimen was observed under a fluorescent microscope at 7-d posttransplantation. (Color version of figure is available online.)

      3.4 Cytokine expression analysis

      Using quantitative real-time PCR, messenger RNA (mRNA) expressions of different genes in the allograft at day 7 posttransplantation were investigated. The expression patterns of IFN-λ, IL-2, and TNF-α genes were similar (Fig. 4); the gene expressions were reduced in ASC group and the reduction was most prominent in CM group compared with control groups (PBS group and M group). Furthermore, IFN-λ, IL-2, and TNF-α mRNA levels were significantly lower in CM group than in the PBS group (P = 0.021, P = 0.020, and P = 0.021, respectively).
      Figure thumbnail gr4
      Fig. 4mRNA expressions of proinflammatory IFN-λ, IL-10, and TNF-α genes at day 7 posttransplantation manifested by real-time PCR. Data are expressed as fold changes in relation to the control group (PBS). CM-infused mice (CM) showed the lowest expression of these genes, and ASC-infused mice (ASC) showed the next lowest expression of these genes. (Color version of figure is available online.)

      3.5 Mixed lymphocyte reaction

      An MLR assay was performed to estimate whether there is a change in alloreactivity after ASC or CM infusion compared with the control mice at day 30 posttransplantation. Data are expressed as fold changes related to the control group (PBS) (Fig. 5). The alloreactivity between responder cells (from BALB/c mice of corresponding groups) and stimulating cells (from C57BL/6 mice) reduced after ASC treatment (P < 0.05). CM treatment also showed a similar hyporesponsiveness in MLR as ASC treatment.
      Figure thumbnail gr5
      Fig. 5Result of MLR at day 30 posttransplantation. Data are expressed as fold changes related to the control group (PBS). The alloreactivity between responder cells (from BALB/c mice of corresponding groups) and stimulating cells (from C57BL/6 mice) was measured using CCK assay. ASC group (n = 12) and CM group (n = 12) showed significantly reduced alloreactivity compared with PBS group (n = 12) (P < 0.05). (Color version of figure is available online.)

      3.6 Histologic evaluation

      Histopathologic changes were evaluated on days 7 and 20 after transplantation, respectively. The histopathology was assessed by the degree of lymphocytic infiltration, neutrophil infiltration, and epidermis thickness on a hematoxylin-eosin stain. The inflammatory reactions, which were prominent in the PBS group on day 7 after transplantation, were attenuated by ASC or CM treatment (Fig. 6A–C). Mouse macrophages were stained brown in F4/80 immunostain. Fewer macrophages seemed to infiltrate after ASC treatment than after PBS or CM infusion (Fig. 6D–F). In VEGF immunohistochemistry, VEGF was not expressed in PBS group; however, it was evident in the ASC group and most prominent in the CM group (Fig. 6G–I). On day 20 after transplantation, most full-thickness skin grafts treated with PBS were detached from the recipient subcutaneous tissue, possibly as a result of acute rejection (Fig. 7A–C). However, a considerable number of grafts treated with ACS or CM remained undetached with limited inflammatory cell infiltration. Few macrophages infiltrated after ASC or CM treatment assessed by F4/80 immunohistochemistry (Fig. 7D–F). Less VEGF was expressed on day 20 after transplantation than on day 7 in the ASC and CM groups (Fig. 7G–I).
      Figure thumbnail gr6
      Fig. 6Comparison of histologic changes according to the intravenous infusion materials at day 7 posttransplantation. The inflammatory reactions, which had been prominent in PBS group on day 7 posttransplantation, attenuated after ASC or CM treatment on hematoxylin-esosin stain (A–C). F4/80 stain demonstrated lesser infiltrated macrophage after ASC treatment when it was compared with PBS or CM group (D–F). Finally, in contrast to PBS group in which VEGF was not expressed, VEGF was evidenced in ASC group, and most prominent in CM group (G–I). (Color version of figure is available online.)
      Figure thumbnail gr7
      Fig. 7Comparison of histologic changes according to the intravenous infusion materials at day 20 posttransplantation. Inflammatory reactions were still more prominent in PBS group than in ASC or CM group (A–C). Macrophages showed lesser infiltrated after ASC or CM treatment on F4/80 immunohistochemistry (D–F). And, VEGF was lesser expressed on day 20 posttransplantation than it did on day 7 posttransplantation in ASC group and CM groups (G–I). (Color version of figure is available online.)

      3.7 Skin graft survival

      Grafts were monitored daily for 30 d. Figure 8 includes representative photographs of skin allografts showing graft rejection and graft acceptance. The graft survival curves of each group are illustrated in Figure 9. Skin allograft survival in untreated animals was limited (mean survival duration: 9.3 ± 1.4 d), reflecting strong immunogenicity. However, ASC infusion markedly increased skin allograft survival compared with its control (PBS) group (mean survival duration: 23.9 ± 2.0 d, P < 0.001). In addition, CM infusion also resulted in considerable survival benefits compared with its control (M) group (mean survival durations: 19.6 ± 2.4 d versus 10.3 ± 1.2 d, P = 0.002). There was no significant difference in survival between ASC- and CM-treated animals (P = 0.250).
      Figure thumbnail gr8
      Fig. 8Representative photographs of skin allografts showing graft rejection and graft acceptance. Two CM-infused mice showed graft rejection at day 20 posttransplantation and two ASC-infused mice showed necrosis-free graft acceptance at day 20 posttransplantation. (Color version of figure is available online.)
      Figure thumbnail gr9
      Fig. 9Kaplan-Meier allograft survival curves for mice that had been intravenously administrated PBS, control media (M; MesenPro RS basal medium), ASC (1 × 106), and CM obtained from ASC. ASCs infusion and CM infusion markedly increased skin allograft survivals in comparison with their respective control groups, respectively (P < 0.001, P = 0.002). In the comparison of ASC and CM treated animal, there was no significant survival difference (P = 0.250). (Color version of figure is available online.)

      4. Discussion

      Despite considerable evidence showing the immunosuppressive properties of MSCs in vitro [
      • Di Nicola M.
      • Carlo-Stella C.
      • Magni M.
      • et al.
      Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli.
      ,
      • Tse W.T.
      • Pendleton J.D.
      • Beyer W.M.
      • et al.
      Suppression of allogeneic T-cell proliferation by human marrow stromal cells: implications in transplantation.
      ,
      • Corcione A.
      • Benvenuto F.
      • Ferretti E.
      • et al.
      Human mesenchymal stem cells modulate B-cell functions.
      ,
      • Nauta A.J.
      • Kruisselbrink A.B.
      • Lurvink E.
      • et al.
      Mesenchymal stem cells inhibit generation and function of both CD34+-derived and monocyte-derived dendritic cells.
      ], the immunomodulatory characteristics of MSCs have not been fully determined in vivo. In this study, we examined the effects of intravenous ASC administration on skin allograft survival in a preclinical mouse model. Furthermore, we investigated the effects of the ASC secretome on skin allograft survival. The present study showed that the intravenous ASC infusion improved the mean skin graft survival compared with control animals, suggesting that ASCs have an immunosuppressive role. Furthermore, we showed that infusion of CM without ASCs also effectively induced immunosuppression and prolonged graft survival.
      Recent research has revealed that MSCs are immune-privileged and, therefore, survive longer when transplanted into allogeneic or even xenogeneic recipients [
      • Nauta A.J.
      • Fibbe W.E.
      Immunomodulatory properties of mesenchymal stromal cells.
      ]. This survival benefit is attributed to their expression of major histocompatibility complex (MHC) class I molecules and no expression of MHC class II molecules [
      • Bartholomew A.
      • Sturgeon C.
      • Siatskas M.
      • et al.
      Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo.
      ,
      • Pittenger M.F.
      • Mackay A.M.
      • Beck S.C.
      • et al.
      Multilineage potential of adult human mesenchymal stem cells.
      ]. The MHC class I molecules expression protects MSCs from certain natural killer cells. Not expressing MHC class II molecules, which are strong alloantigens, allow MSCs to evade host immune surveillance. We observed transplanted ASCs in the skin at 7-d posttransplantation (Fig. 3). In the literature, the survival and fate of transplanted stem cells had been reported to be 5 wk [
      • McDonald J.W.
      • Liu X.Z.
      • Qu Y.
      • et al.
      Transplanted embryonic stem cells survive, differentiate and promote recovery in injured rat spinal cord.
      ] to 6 mo [
      • Haddad-Mashadrizeh A.
      • Bahrami A.R.
      • Matin M.M.
      • et al.
      Evidence for crossing the blood barrier of adult rat brain by human adipose-derived mesenchymal stromal cells during a 6-month period of post-transplantation.
      ] in xenografts and 4 wk to [
      • Xia C.
      • Cao J.
      Imaging the survival and utility of pre-differentiated allogeneic MSC in ischemic heart.
      ] 6 mo in allograft [
      • Dai W.
      • Hale S.L.
      • Martin B.J.
      • et al.
      Allogeneic mesenchymal stem cell transplantation in postinfarcted rat myocardium: short- and long-term effects.
      ], suggestive of their immune-privileged property. Furthermore, the undifferentiated ASCs which had been used in our experiment can sidestep host's immunosurveillance easier than differentiated ones [
      • Xia C.
      • Cao J.
      Imaging the survival and utility of pre-differentiated allogeneic MSC in ischemic heart.
      ]. Therefore, it is possible that ASCs could persist and exert their immunosuppressive function throughout the study period of 30 d.
      In addition to the immune-privileged properties of MSCs, mounting evidence indicates that MSCs have the ability to induce immunologic tolerance [
      • Le Blanc K.
      • Ringden O.
      Immunomodulation by mesenchymal stem cells and clinical experience.
      ]. MSCs suppress T cell proliferation and migration, inhibit B-cell activation, suppress migration of various antigen-presenting cells, induce suppressor T-cell formation, and alter the expression of several receptors necessary for antigen capture and processing by releasing secretome [
      • Aggarwal S.
      • Pittenger M.F.
      Human mesenchymal stem cells modulate allogeneic immune cell responses.
      ,
      • Atoui R.
      • Shum-Tim D.
      • Chiu R.C.
      Myocardial regenerative therapy: immunologic basis for the potential “universal donor cells”.
      ]. In addition, MSCs contribute to a shift from T1 cells toward T2 cells, which have anti-inflammatory properties [
      • Aggarwal S.
      • Pittenger M.F.
      Human mesenchymal stem cells modulate allogeneic immune cell responses.
      ].
      Skin graft is a good model for the evaluation of inflammatory process because it undergoes well-known wound healing process—comprised inflammation, proliferation, and maturation—after transplantation. Although transplanted stem cells impacts all phases of wound repair [
      • Maxson S.
      • Lopez E.A.
      • Yoo D.
      • et al.
      Concise review: role of mesenchymal stem cells in wound repair.
      ], we took note of their impacts on the inflammatory phase, which decisively determine the acceptance of the graft. The inflammatory phase peaks between 1 and 3 d after the injury and can last 4–6 d in normally healing wounds. Our results comparing serum IL-6 levels in the inflammatory phase suggested ASC or CM treatment has profound anti-inflammatory effect. We also evaluated mRNA expressions of proinflammatory mediators, such as IFN-λ, IL-10, and TNF-α at 7-d posttransplantation, judging that such a change in the specimens would be prominent at the end of inflammatory phase. We were intended to evaluate MLR, the consequence of all the immunologic events, at the end point of our experiment. Throughout the MLR, we showed that anti-inflammatory effects of MSC or CM could be related with immunologic hyporesponsiveness favoring graft acceptance, which occurred in the later period.
      Although MSCs have the ability of inducing tolerance, directly applying MSCs to clinical use raises several concerns. It is well known that only a small proportion of MSCs, which are administered locally or systemically, is eventually incorporated into injured tissues [
      • Li T.S.
      • Takahashi M.
      • Ohshima M.
      • et al.
      Myocardial repair achieved by the intramyocardial implantation of adult cardiomyocytes in combination with bone marrow cells.
      ,
      • Muraca M.
      • Ferraresso C.
      • Vilei M.T.
      • et al.
      Liver repopulation with bone marrow derived cells improves the metabolic disorder in the Gunn rat.
      ,
      • Newsome P.N.
      • Johannessen I.
      • Boyle S.
      • et al.
      Human cord blood-derived cells can differentiate into hepatocytes in the mouse liver with no evidence of cellular fusion.
      ,
      • Rosario C.M.
      • Yandava B.D.
      • Kosaras B.
      • et al.
      Differentiation of engrafted multipotent neural progenitors towards replacement of missing granule neurons in meander tail cerebellum may help determine the locus of mutant gene action.
      ]. And the actual effect of MSCs on the target microenvironment has not been determined and, therefore, should be further investigated. Furthermore, occurrence of cell-related aftermath, such as immune-mediated rejection, senescence-induced genetic instability, limited cell survival, and possible malignant transformation should be considered [
      • Baglio S.R.
      • Pegtel D.M.
      • Baldini N.
      Mesenchymal stem cell secreted vesicles provide novel opportunities in (stem) cell-free therapy.
      ,
      • Rubio D.
      • Garcia S.
      • Paz M.F.
      • et al.
      Molecular characterization of spontaneous mesenchymal stem cell transformation.
      ]. Therefore, we paid attention to the therapeutic potential of MSC secretome instead of MSC itself. The secretome is the entire set of products secreted by stem cells and, therefore, includes many proteins, such as chemokines and growth factors. Recently, more and more evidences suggest that the stem cells' mechanism of tissue repair and regeneration is based not on their engulfment and transdifferentiation but also on their paracrine function [
      • Hoch A.I.
      • Binder B.Y.
      • Genetos D.C.
      • et al.
      Differentiation-dependent secretion of proangiogenic factors by mesenchymal stem cells.
      ,
      • Ranganath S.H.
      • Levy O.
      • Inamdar M.S.
      • et al.
      Harnessing the mesenchymal stem cell secretome for the treatment of cardiovascular disease.
      ,
      • Woo D.H.
      • Kim S.K.
      • Lim H.J.
      • et al.
      Direct and indirect contribution of human embryonic stem cell-derived hepatocyte-like cells to liver repair in mice.
      ].
      In the present study, the immunosuppressive potential of ASCs and CM infusion were compared with assess whether CM infusion have equivalent tolerogenic potential as ASCs infusion. Most experimental and histologic results revealed that their effects were comparable; there was no significant difference between these two groups in inflammatory cell infiltration in histology, serum levels of proinflammatory cytokine IL-6, and even allograft survival. However, CM treatment decreased gene expression of the proinflammatory cytokines IFN-λ, IL-10, and TNF-α in the skin graft more significantly than ASC did, and therefore, further research is required to confirm these findings. Overall, we could conclude that secretome itself can induce equivocal immunosuppression and anti-inflammation as ASCs.
      Enhanced neovascularization, which is involved in angiogenesis or vasculogenesis, has a significant effect on skin allograft viability [
      • Asahara T.
      • Murohara T.
      • Sullivan A.
      • et al.
      Isolation of putative progenitor endothelial cells for angiogenesis.
      ,
      • Tepper O.M.
      • Callaghan M.J.
      • Chang E.I.
      • et al.
      Electromagnetic fields increase in vitro and in vivo angiogenesis through endothelial release of FGF-2.
      ]. Therefore, we also investigated that whether ASCs or CM infusion enhanced the production of angiogenic growth factors such as VEGF. The control group (PBS) expressed no or minimal VEGF, whereas VEGF expression was evident in the ASC group and pronounced in the CM group, suggesting a vasculogenic role of ASCs.
      The skin, the most antigenic tissue in the body, usually leads transplanted skin graft to acute rejection within 10–20 d unless immunosuppressive therapy is provided [
      • Sbano P.
      • Cuccia A.
      • Mazzanti B.
      • et al.
      Use of donor bone marrow mesenchymal stem cells for treatment of skin allograft rejection in a preclinical rat model.
      ]. In our experiment, ASC or CM infusion markedly increased skin allograft survival compared with control groups (P < 0.001). However, considerable skin grafts in ASC or CM group also experienced acute rejection between days 10 and 25 posttransplantation (Fig. 9). We think that prolonged, however not consistent, survival outcomes in ASC and CM groups are attributed to the difference in the microenvironment where they exert; the mice that overcome the immunologic barrier showed graft acceptance, and the mice that could not overcome the barrier showed rejection. Further studies focusing on adjusting the injection time, frequency, and amount are required to find a way to overcome immunologic barrier completely.
      It was noticeable that CM-infused mice showed equivocal skin graft survival as ASC-infused mice did. This result suggests that the chemokines and many growth factors, which comprise ASC-CM, are sufficient to induce potential and persistent immunosuppression. Osugi et al. [
      • Osugi M.
      • Katagiri W.
      • Yoshimi R.
      • et al.
      Conditioned media from mesenchymal stem cells enhanced bone regeneration in rat calvarial bone defects.
      ] also reported that the infusion of stem-cell-cultured CM promoted bone regeneration, which had been persisted 8 wk after the infusion, showing persistent effect of the secretome.

      5. Conclusion

      We showed the immunosuppressive, anti-inflammatory, and vasculogenic potentials of human ASCs, which had been xenotransplanted into a preclinical skin allograft mouse model. And, we also demonstrated that ASC secretome itself can induce similar tolerogenic effects as ASC. Therefore, the use of ASC or ASC secretome can be a good option to avoid drug toxicity and side effects of immunosuppressive regimens. Further in-depth research is required to fully investigate this novel approach and to reproduce their effects in clinical situations.

      Disclosure

      The authors reported no proprietary or commercial interest in any product mentioned or concept discussed in this article.

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