Multiple Injections of Adipose-Derived Stem Cells Improve Graft Survival in Human-to-Rat Skin Xenotransplantation through Immune Modulation

Abstract

Background: Adipose-derived stem cells (ADSCs) exert immunomodulatory effects in the treatment of transplant rejection. This study aimed to evaluate the effects of ADSCs on the skin graft survival in a human-to-rat xenograft transplantation model and to compare single and multiple injections of ADSCs.

Methods: Full-thickness human skin xenografts were transplanted into the backs of Sprague-Dawley rats. The rats were injected subcutaneously on postoperative days 0, 3, and 5. The injections were as follows: triple injections of phosphate-buffered saline (PBS group), a single injection of ADSCs and double injections of PBS (ADSC × 1 group), and triple injections of ADSCs (ADSC × 3 group). The immunomodulatory effects of ADSCs on human skin xenografts were assessed.

Results: Triple injections of ADSCs considerably delayed cell-mediated xenograft rejection compared with the PBS and ADSC × 1 groups. The vascularization and collagen type 1-3 ratios in the ADSC × 3 group were significantly higher than those in the other groups. In addition, intragraft infiltration of CD3-, CD4-, CD8-, and CD68-positive cells was reduced in the ADSC × 3 group. Furthermore, in the ADSC × 3 group, the expression levels of proinflammatory cytokine interferon-gamma (IFN-γ) were decreased and immunosuppressive prostaglandin E synthase (PGES) was increased in the xenograft and lymph node samples.

Conclusion: This study presented that triple injections of ADSCs appeared to be superior to a single injection in suppressing cell-mediated xenograft rejection. The immunomodulatory effects of ADSCs are associated with the downregulation of IFN-γ and upregulation of PGES in skin xenografts and lymph nodes.

Keywords: Adipose-derived stem cells; IFN-γ; PGES; Skin xenograft.

Fig. 1

Study design and macroscopic findings of human skin xenografts. A Rats were randomized into three groups: PBS, ADSC × 1, and ADSC × 3 groups (n = 21 per group). Each 1 ml of PBS and/or ADSCs suspended in PBS was locally injected on POD 0, 3, and 5. After the removal of the tie-on dressing on POD 7, rats were sacrificed on POD 7, 10, 14 for subsequent analysis (n = 6 per subgroup); and on POD 56 for long-term observation (n = 3 per subgroup). Blue arrows, PBS; red arrows, ADSCs (1 × 10⁶ cells/ml). B Representative images of the skin xenografts of each group on POD 0, 7, 10, and 14. The ADSC × 3 group showed considerably delayed xenograft rejection compared to the ADSC × 1 group. C Quantitative evaluation of the relative surface area of the skin xenografts (n = 6 per group). Statistical analysis by two-way ANOVA. ***p < 0.001, **p < 0.01, compared to the PBS group. ^###p < 0.001, ^##p < 0.01, compared to the ADSC × 1 group. PBS phosphate-buffered saline, ADSC adipose-derived stem cell, POD postoperative day

Fig. 2

Histopathology of human skin xenografts. A Representative images of hematoxylin and eosin (H&E) and Masson’s trichome (MT) staining on POD 7, 10, 14, and 56. Scale bar, 50 μm. B Assessment of skin xenografts using the Banff classification (0–4). The Banff score was assessed based on the degree of cellular infiltration, edema, fibrosis, apoptosis, necrosis and disorganization of the epidermis and dermis, with higher scores indicating more severe rejection. Statistical analysis by two-way ANOVA. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05. ns not significant, PBS phosphate-buffered saline, ADSC adipose-derived stem cell, POD postoperative day

Fig. 3

ADSCs significantly improved graft survival and vascularization. A Representative images and B quantitation of IHC staining for proliferating cell nuclear antigen (PCNA) in the epithelium of the skin xenografts on POD 7 (n = 6 per group). Scale bar, 50 μm. C Representative images and D, E quantitation of IHC for CD31 on POD 14 (n = 6 per group). Scale bar, 100 μm. F Representative images and G quantitation of IHC for collagen type 1 and 3 on POD 14 (n = 6 per group). Scale bar, 100 μm. Statistical analysis by one-way ANOVA. ***p < 0.001, *p < 0.05, ns not significant, PBS phosphate-buffered saline, ADSC adipose-derived stem cell, POD postoperative day, HPF high-power field

Fig. 4

Immunohistochemical (IHC) analysis of immune cell markers. A Representative images and B quantitation of IHC for CD3 on POD 7, 10, and 14 (n = 6 per group). C Representative images and D quantitation of IHC for CD8 on POD 7, 10, and 14 (n = 6 per group). E Representative images and F quantitation of IHC for CD4 on POD 7, 10, and 14 (n = 6 per group). G Representative images and H quantitation of IHC for CD68 on POD 7, 10, and 14 (n = 6 per group). Scale bar, 50 μm. Statistical analysis by two-way ANOVA. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns not significant, PBS phosphate-buffered saline, ADSC adipose-derived stem cell, POD postoperative day

Fig. 5

Downregulation of INF-γ expression levels in the skin xenografts, regional lymph nodes and serum. A Ifng mRNA expression in the skin xenografts on POD 7, 10, and 14 (n = 6 per group). B Ifng mRNA expression in the regional lymph nodes on POD 7, 10, and 14 (n = 6 per group). C Representative images and D quantitation of immunohistochemistry staining for INF-γ protein expression in the skin xenografts on POD 14 (n = 6 per group). Scale bar, 100 μm. E Western blot analysis and F quantification of PGES protein levels in the skin xenografts (n = 4 per group). G INF-γ protein concentrations in the serum on POD 14 (n = 4 per group). Statistical analysis by two-way ANOVA for (A, B), and one-way ANOVA for (D–G). ***p < 0.001, **p < 0.01, *p < 0.05, #p < 0.05 versus PBS group, ns not significant, PBS phosphate-buffered saline, ADSC adipose-derived stem cell, POD postoperative day, Ifng, IFN-, interferon gamma

Fig. 6

Upregulation of PGES expression levels in the skin xenografts and regional lymph. A Ptges mRNA expression in the skin xenografts (n = 6 per group). B Ptges mRNA expression in the regional lymph nodes (n = 6 per group). C Western blot analysis and D quantification of PGES protein levels in the skin xenografts (n = 4 per group). Statistical analysis by two-way ANOVA for (A, B), and one-way ANOVA for (D). **p < 0.01, *p < 0.05, ns not significant, PBS phosphate-buffered saline, ADSC adipose-derived stem cell, POD postoperative day, PGES prostaglandin E synthase

Fig. 7

Proposed mechanisms of the immunomodulatory effects of ADSCs in human-to-rat skin xenotransplantation. Triple injections of ADSCs prolonged the survival of skin xenografts by modulating T cell proliferation and macrophage activation, which was associated with downregulation of IFN-γ expression and upregulation of PGES expression. ADSC adipose-derived stem cell, IFN-γ interferon gamma, PGES prostaglandin E synthase