Recipient Adipose-Derived Stem Cells Enhance Recipient Cell Engraftment and Prolong Allotransplant Survival in a Miniature Swine Hind-Limb Model

Animals

Thirty-four outbred domestic miniature swine purchased form Taitung Animal Propagation Station, Taitung, Taiwan (Lan-Yu strain; age: 3 mo; weight: 18 to 25 kg) were included in the study. The Lan-Yu strain is an indigenous breed from Lau-Yu Island, southeast of Taiwan, with the genotype of GPI (glucose-6-phosphate isomerase)-BB subtype, and the genotype of PGD (glucose-6-phosphate dehydrogenase)-AA subtype. ^13,14 The inherited differences in donors and recipients from the original parental generation are identified and reported by the National Tai-Tung Veterinary Research Institute, Taiwan. This study was conducted in accordance with the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health, Bethesda, Maryland, USA. The experimental protocols were approved by the Institutional Animal Care and Use Committee of the Kaohsiung Chang Gung Memorial Hospital.

Heterotopic Hind-Limb Swine Model

Heterotopic hind-limb transplantation was performed as previously described. ^13,20 In brief, animals were premedicated with an intramuscular injection of ketamine (10 mg/kg; Sigma-Aldrich, St Louis, MO, USA) and xylazine (1.5 mg/kg; Tai Yu Chemical & Pharmaceutical Co., Hsinchu, Taiwan.) Inhalational anesthesia was maintained with isoflurane (0.5% to 3% inhaled, Forane; AbbVie, Taipei, Taiwan) and oxygen throughout the procedure. A composite tissue consisting of tissues from the tibia, fibula, knee joint, distal femur, and surrounding muscle was harvested from the donor swine. A skin paddle, measuring approximately 8 × 8 cm², was preserved on the medial aspect of the knee supplied by the superficial femoral vessels. The tibia and fibula were divided at approximately 5 cm below the knee, and the femur was divided 5 cm above the knee. The thigh muscles were divided at the mid-femur level. Upon division of the vascular pedicle, heparinized saline (BD Pharmingen, San Diego, CA, USA) was flushed through the femoral artery. The recipient swine were prepared in a similar manner as described for the donor swine. A subcutaneous pocket was created in the anterolateral abdominal wall, and the limb graft was placed into the pocket. The vessels were anastomosed in an end-to-end fashion onto the host femoral vessels with a 9-0 nylon interrupted suture using microscopic magnification. A defect was created in the skin of the host’s flank, and the skin paddle was sutured in place on the donor limb.

Culture of the rADSCs

The groin fat pad from recipients was harvested, washed, and digested with 0.2% type I collagenase (Sigma-Aldrich) for 30 min at 37 °C. After centrifugation (260g, 5 min), the pellet was resuspended in phosphate-buffered saline (PBS), following the red blood cell (RBC) lysis process with ammonium chloride lysis buffer (155 mM ammonium chloride, 0.1 mM ethylenediaminetetraacetic acid [EDTA], and 10 mM sodium bicarbonate, pH 7.2; all from Sigma-Aldrich). After being washed and centrifuged, the rADSC-containing pellet was resuspended in low-glucose Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies, Grand Island, NY, USA) and plated in a 75T flask. The isolated primary culture (P0) rADSCs were cultured in low-glucose DMEM containing 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (both from Invitrogen, Carlsbad, CA, USA) at 37 °C in a humidified incubator with 5% CO₂. The rADSCs were expanded up to passage 5 and characterized by flow cytometry (Becton Dickinson, San Jose, CA, USA) following positive surface staining for CD44, CD90, major histocompatibility complex (MHC) class I, and CD106, but not for CD45, MHC class II, and CD80/B7-1. Before experimental use, the multilineage differentiation ability of rADSCs toward adipocytes, osteoblasts, and chondrocytes was tested. Osteoblasts were identified by von Kossa staining, Oil Red O was used for lipid droplet staining, and chondrogenic differentiation was visualized by Alcian blue staining ¹⁶ (all from Abcam, Taipei, Taiwan).

Experimental Design

Miniature swine underwent heterotopic hind-limb transplantation. Twenty-two male swine were recipients and 12 female were donors; 2 hind limbs harvested from a single donor were given to 2 different recipients. Group I (n = 6) was the control cohort and hence did not undergo immunosuppressive therapy. Group II (n = 4) received the rADSC treatment (1 × 10⁶ cells/kg/dose, given on weeks 0, +1, +2, and +3). Group III (n = 4) received oral administration of FK506 (tacrolimus; Sigma-Aldrich) for 4 wk (weeks +1 to +4; 1 mg/kg/d for 2 wk followed by 0.5 mg/kg/d for 2 wk). Group IV (n = 8) received preconditioning IR (day −1; 150 cGy for total body IR and 700 cGy for intrathymus IR), FK506 for 4 wk (same protocol as group III; weeks +1 to +4), and rADSCs (1×10⁶ cells/kg/dose, weeks 0, +1, +2, and +3). The timing of the immunosuppressant was that used in a previous study. ¹⁴ The dosage and timing of ADSC injections were similar to our previous donor ADSC infusion protocol. ¹⁸ The clinical experimental end point was defined as desquamation and necrosis of the entire area of donor skin. The definition of immune tolerance for the allograft induced by recipient cells was allograft survival for more than 15 wk posttransplantation without any signs of rejection.

Histological Evaluation of Graft Rejection

The transplanted limb was observed daily for signs of rejection as defined by the well-characterized sequence of epidermolysis, dyskeratosis, and necrosis. Full-thickness 3-mm biopsies of donor skin and muscle were obtained +2 and +6 postoperative weeks. The harvested tissues for biopsies were provided around 0.5 × 0.5 cm² by punch. The biopsy specimens were fixed and embedded in paraffin blocks for hematoxylin and eosin (H&E; Sigma-Aldrich) and immunohistochemical (IHC) staining. Rejection grades were scored using Banff classification, according to the severity of pathological changes. ²¹

Evaluation of CD25⁺/Forkhead Box p3 (Foxp3)⁺ T-Cell Expression

Frozen sections were obtained from the alloskin of the grafted limbs on +2 and +6 weeks posttransplantation. To assess the effects of regulatory T cells after transplantation, alloskin samples were subjected to IHC staining. A horseradish peroxidase–diaminobenzidine (HRP-DAB) kit (Bio SB, Santa Barbara, CA, USA) was used in all immunostaining experiments to visualize the expression profiles. Tissue sections were stained with mouse antiporcine CD25 (Serotec, Oxford, UK) or with mouse antirat Foxp3 (Serotec) antibodies. The reacted sections were incubated with biotinylated antimouse antibody (BD Pharmingen) as a secondary antibody. After counterstaining with H&E, the tissue sections were mounted, cleared, and coverslipped.

Tissue sections obtained from 6 individual specimens were analyzed. For immunostaining quantification, sections were analyzed using a Zeiss Axioskop 2 plus Microscope (Carl Zeiss, Göttingen, Germany). Areas (3 mm²) containing Foxp3⁺ T cells were calculated. Four areas were randomly captured at 400× magnification by a Cool CCD camera (SNAP-Pro cf Digital Kit; Media Cybernetics, Silver Spring, MD, USA). Images were analyzed using Image-Pro Plus image analysis software (version 7) (Media Cybernetics Rockville, Maryland, USA).

Flow Cytometric Assessment of CD4⁺/CD25⁺/Foxp3⁺ T Cells

Flow cytometric analyses were performed on the peripheral blood samples of recipients collected on weeks +2, +6, and +15 posttransplant. The whole blood was incubated for 20 to 30 min in the dark (room temperature) with 5 μL of mouse antiporcine CD25-fluorescein isothiocyanate (BD Pharmingen) and combinations of mouse antipig CD4-phycoerythrin (BD Pharmingen) and antimouse/rat Foxp3 PerCP-Cyanine5.5 (eBiosciences, San Diego, CA, USA). After incubation, RBCs were lysed (fluorescent-activated cell sorting (FACs) lysing solution; BD Pharmingen), and the remaining cells were centrifuged twice at 500g for 5 min. Cells were then analyzed by flow cytometry (FACScan; Becton Dickinson). Data were analyzed using the BD FACScan™ program (Becton Dickinson).

Engraftment Was Measured by Labeling rADSCs with 5-bromo-2’-deoxyuridine (BrdU)

In vitro BrdU-incorporated rADSCs were used to analyze rADSCs’ engraftment into the donor allograft. rADSCs were labeled with 2 rounds of 7.5 μg/mL BrdU in culture medium (−3 and −1 days before single injection). Multiple injections of BrdU-labeled ADSCs were performed intravenously (+0, +1, +2, and +3 wk). After incorporation, a 1 × 10⁷ cells/mL suspension was intravenously injected into the recipient animals in order to assess the rADSC population in the donor transplanted skin tissue. Biopsy bilateral samples of recipient tissue in the IR/rADSC/FK (FK506 [tacrolimus]) group were harvested at weeks +2, +6, and +15 after injection. Biopsy tissue sections were IHC stained with goat antimouse BrdU antibody (Sigma-Aldrich), and the signals were visualized using an HRP-DAB staining kit (BioGeneX Laboratories, Fremont, CA, USA).

Expression of Transforming Growth Factor β1 (TGF-β1) and Tumor Necrosis Factor α (TNF-α) in Peripheral Blood of Recipients

The expression of the soluble form of TGF-β1 and TNF-α in peripheral blood was determined by an enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, USA). Blood serum samples were collected at predetermined specific days posttransplantation (+2, +6, and +15 wk).

Polymerase Chain Reaction (PCR) Amplification of Sex-Determining Region of Y Chromosome (SRY)

The punched biopsy skin and muscle from the donor graft (female) and recipient tissues in the IR/rASC/FK group (male) were cut into small pieces, and around 1 mg of tissues was transferred to a 1.5 mL Eppendorf tube containing 100 μL of lysis buffer (10 mM Tris–HCl, pH 7.6, 10 mM KCl, 2 mM EDTA, 4 mM MgCl₂; all from Sigma-Aldrich) for genomic DNA extraction. The male-specific SRY gene to the donor allograft was amplified using the following forward primer set: Sus scrofa SRY_F (5′-CTG GGA TGC AAG TGG AAA AT-3′) and Sus scrofa SRY_R (5′-GGC TTT CTG TTC CTG AGC AC-3′). The SRY primers were designed from a sequence available on GenBank assembly ID: GCA_000003025.4. PCR amplification was performed under the following conditions: an initial denaturation at 95 °C for 5 min, followed by 40 cycles of denaturation at 94 °C for 20 s, annealing at 65 °C for 20 s, extension at 72 °C for 30 s, and a final extension at 72 °C for 7 min in a thermal cycler (GeneAmp 9700; PerkinElmer, Foster City, CA, USA). All the amplified DNA samples were electrophoresed in a 1.5% agarose gel (Merck KGaA, Darmstadt, Germany) and visualized by ethidium bromide staining (Sigma-Aldrich) and photographed. The SRY protein profiles in donor allografts were determined by using IHC staining (Santa Cruz Biotechnology, Dallas, TX, USA).

Data Management and Statistical Analysis

Student’s t test or analysis of variance (ANOVA) was utilized to assess the change and to compare the difference between these groups from normal distributions. Log transformation was performed prior to variance analysis and Student’s t tests to reconfirm normality and equal variances for flow cytometry data. Graft survival was compared between the different groups of transplanted animals using Kaplan–Meier analysis and the log-rank test. A P value < 0.05 was considered statistically significant.

rADSCs Combined with Short-Term Immunosuppressants Prolonged Allograft Survival

The results showed the progressive rejection of the swine hind-limb allograft by weeks 1.1 to 2.3 with a mean of 1.6 wk in the control group. Allograft recipients treated with multiple autologous rADSC injections alone in the absence of immunosuppressant treatment revealed a statistical increase in allograft survival as compared to controls (weeks 2.5 to 3.3 with a mean of 2.93 wk, P = 0.003). Allograft transplantation along with short-term FK506 treatment resulted in delayed rejection (weeks 6 to 10 with a mean of 8.3 wk, P = 0.001). However, treatment with multiple rounds of rADSCs in addition to IR and short-term FK506 resulted in significant prolongation of allotransplant survival (>28 wk), as compared to other experimental groups (P < 0.001; Fig. 1).

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Fig. 1.

Recipient adipose-derived stem cells (rADSCs) in combination with irradiation (IR) and transient FK506 (tacrolimus) therapy prolonged allotransplant survival. (A) Flowchart for the treatment protocol used in this study. Recipient pig received preconditioning IR (day −1; 150 cGy for total body IR and 700 cGy for intrathymus IR), FK506 (tacrolimus) for 4 wk, and rADSCs (1 × 10⁶ cells/kg/dose, weeks 0, +1, +2, and +3). (B) Kaplan–Meier vascularized composite allotransplant survival curves for control and experimental groups. The results show early rejection of the swine hind-limb allograft by weeks 1.1 to 2.3 in the control group. Recipients treated with multiple autologous rADSCs alone revealed a statistical increase in allograft survival as compared to controls (P = 0.003). Allotransplant treated with short-term FK506 resulting in delayed rejection (P = 0.0014). Treatment with rADSCs/IR/FK506 showed significant prolongation of allotransplant survival (>28 wk), as compared to other experimental groups (P < 0.001). **P < 0.01; ###P < 0.001; ***P < 0.001.

rADSCs in Combination with Transient Immunosuppressant Therapy Suppressed Allograft Rejection

Histopathological evaluation of the allograft biopsies revealed severe rejection signs (grades III to IV) and mononuclear infiltration in the alloskin and muscle biopsies of the control group at 2 wk posttransplantation. Histological examination of the allograft biopsies of the rADSC groups revealed less inflammatory cell infiltration in the interstitial muscle layers as compared to that in controls at 2 wk posttransplantation (grade II to III). However, lymphocyte infiltration was nonetheless observed in the alloskin and interstitial muscle layers in group IV with rADSC-IR-FK506 treatment at 2 and 6 wk posttransplantation (grades 0 and I; Fig. 2).

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Fig. 2.

Recipient adipose-derived stem cells (rADSCs) in combination with irradiation (IR) and transient FK506 (tacrolimus) therapy suppressed allograft rejection. Histopathological evaluation revealed severe rejection (grades III and IV) and mononuclear infiltration in the alloskin and muscle biopsies of the control group at 2 wk posttransplantation. The groups with allograft biopsies of rADSCs alone revealed less inflammatory cell infiltration in the interstitial muscle layers as compared to controls (grades II and III). Lymphocyte infiltration was nonetheless observed in the alloskin and muscle layers in group IV with rADSC/IR/FK506 treatment. At 15 wk posttransplantation, the allotransplant biopsy of group IV revealed rare mononuclear infiltration in the alloskin and interstitial muscle layers as compared to that in the other groups (grades 0 and I). Ctrl, control.

rADSC Modulated Regulatory T-Cell Expression in Circulating Blood and Allotissues

IHC staining of graft skin tissue biopsies revealed significant numbers of Foxp3+ T cells (P < 0.001) in the subcutaneous and dermis layers in animals treated with rADSCs, IR, and FK506, as compared to other groups at 2 and 6 wk posttransplantation (Fig. 3A). In parallel, flow cytometric analysis of recipient peripheral blood revealed that CD4⁺/CD25⁺/Foxp3 ⁺ regulatory-like T-cell populations had a significant increase in the rADSC-IR-FK506 group at 6 wk posttransplantation as compared to other treatment groups (Fig. 3B). The populations of CD4 ⁺/CD25⁺/Foxp3 ⁺ T cells were equal to normal in animals treated with rADSCs-IR-FK506 at 15 wk posttransplant. These results demonstrated that treatment with rADSCs combined with a short-term immunosuppressant regimen could increase regulatory T-cell populations.

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Fig. 3.

Recipient adipose-derived stem cells (rADSCs) modulated regulatory T-cell expression in allotissues and circulating blood. (A) Immunohistochemical (IHC) staining of graft skin tissue biopsies revealed significant numbers of CD25⁺ T cells and forkhead box p3 (Foxp3)+ T cells (*P < 0.05) in the subcutaneous and dermis layers in animals treated with rADSCs/irradiation/FK506 (tacrolimus), as compared to other groups at 2 and 6 wk posttransplantation. (B) Flow cytometric analysis of recipient peripheral blood revealed that CD4⁺/CD25⁺/Foxp3 ⁺ T-cell populations have significant increase in rADSC/IR/FK506 treatment group at 6 wk posttransplantation as compared to that in other groups, but skewing down at 15 wk posttransplantation (*P < 0.05). Ctrl or con, control.

rADSCs and Transient Immunosuppressive Therapy Regulated the Pro-Inflammatory and Anti-Inflammatory Cytokines

The concentrations of TGF-βl and TNF-α in peripheral blood were determined by ELISA after the various treatments. Analysis of recipient peripheral serum revealed that TGF-βl was apparently increased at 6 and 15 wk posttransplantation in animals treated with rADSCs, IR, and FK506, as compared to controls (Fig. 4A). The TNF-α level revealed a statistically significant decrease in the group treated with rADSCs, FK506, and IR at 2 and 6 wk posttransplantation, as compared to controls (Fig. 4B).

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Fig. 4.

Recipient adipose-derived stem cells (rADSCs) and transient immunosuppressive therapy that regulated the pro-inflammatory and anti-inflammatory cytokines were determined by enzyme-linked immunosorbent assay (ELISA) after the various treatments. (A) Analysis of recipient peripheral serum revealed that transforming growth factor βl was significantly increased at 6 and 15 wk posttransplantation in animals treated with rADSCs/irradiation (IR)/FK506 (tacrolimus), as compared to controls (*P < 0.05). (B) The tumor necrosis factor (TNFα) level revealed a statistically significant decrease in rADSCs/IR/FK506 group at 2 and 6 wk posttransplant, as compared to controls (ctrl; *P < 0.05), but there was no significant difference at 15 wk posttransplant.

BrdU-Labeled Recipient ADSCs Engrafted into the Allotransplant Tissue

BrdU-labeled rADSCs were intravenously injected into the recipient swine to investigate the BrdU-rADSC engraftment to donor tissue. IHC staining of allotransplant tissue showed the population of BrdU-positive cells in the subcutaneous layers of alloskin was apparently expressed early (2 and 6 wk posttransplantation) (2.62% and 3.06%, respectively; Fig. 5). However, BrdU-positive cells could not be detected in alloskin 15 wk posttransplantation.

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Fig. 5.

Bromodeoxyuridine (BrdU)-labeled recipient adipose-derived stem cells (rADSCs) engrafted into the allotransplant tissue. Immunohistochemical (IHC) staining of allotransplant tissue showed the population of BrdU-positive rADSCs in the subcutaneous layers of alloskin was apparently increased at 2 and 6 wk posttransplantation (2.62% and 3.06%, respectively; *P < 0.05). However, BrdU-positive ADSCs could not be detected in alloskin at 15 wk posttransplantation. Ctrl means negative control without BrdU-labeling normal skin tissue.

rADSCs Increased Recipient SRY-Positive Expression in Cells in Allotransplant Tissue

To detect the engraftment of the recipient cells to the donor transplant tissue, donor cells from the hind limbs of female swine were transplanted into male recipients. A recipient male-specific SRY gene situated in the Y chromosome sex-determination region was detected in allotransplanted tissue. The IHC staining for recipient SRY protein expressions in alloskin revealed significant expression in donor allotissue at 6 and 15 wk posttransplantation (Fig. 6A and B). In contrast, PCR analysis revealed that the recipient SRY gene had strong expressions in the donor alloskin and muscle at 6 wk posttransplantation in the group with rADSC-IR-FK506 treatment (Fig. 6C).

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Fig. 6.

Recipient adipose-derived stem cells (rADSCs) increased the recipient sex-determining region of Y chromosome (SRY)-positive cells in allotransplant tissue. Donor hind limbs from female swine were transplanted into male recipient swine (A and B) Immunohistochemical (IHC) staining for the recipient SRY protein expressions in alloskin revealed significant expression in donor alloskin at 6 and 15 wk posttransplantation (*P < 0.05). (C) Polymerase chain reaction (PCR) analysis revealed that the recipient SRY gene exhibited strong expressions in the donor alloskin and muscle at 6 wk posttransplantation in the group with rADSC/irradiation/FK506 (tacrolimus) treatment.