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

Abstract

Donor mesenchymal stem cells (MSCs) could prolong vascularized composite allotransplantation (VCA) survival in our previous studies. However, recipient adipose tissue is easier to harvest than donor tissue for preconditioning modulation. Hence, this study investigated the efficacy of recipient autologous adipose-derived stem cells (rADSCs) for VCA survival. The heterotopic hind-limb transplantation from female donor to male recipient was performed in outbred miniature swine. Group I ( n = 6) was untreated controls. Group II ( n = 4) obtained rADSCs infusions (given on weeks 0, +1, +2, and +3). Group III ( n = 4) obtained tacrolimus (FK506, weeks 0 to +4). Group IV ( n = 8) received irradiation (IR; day -1), FK506 (weeks 0 to +4), and rADSC infusions (weeks 0, +1, +2, and +3). The results revealed treatment with multiple injections of rADSCs along with IR and FK506 resulted in a statistically significant increase in allograft survival. The percentage of CD4⁺/CD25⁺/Foxp3⁺ regulatory T cells were significantly increased in the rADSC-IR-FK506 group as compared to controls. Analysis of recipient peripheral blood revealed that transforming growth factor β1 (TGFβ1) was significantly increased in the rADSC-IR-FK506 group. The polymerase chain reaction (PCR) analysis and immunohistochemical staining showed recipient sex-determining region of Y (SRY) chromosome gene expression existed in donor allotissues in the rADSC-IR-FK506 group. These results indicate that rADSCs in addition to IR and transient immunosuppressant could prolong allotransplant survival, modulate T-cell regulation, and enhance recipient cell engraftment into the allotransplant tissues.

Keywords: adipose-derived stem cells; graft rejection; regulatory T cell; vascularized composite allotransplantation.

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.

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.

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.

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.

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.

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.