Adipose-derived cellular therapies prolong graft survival in an allogenic hind limb transplantation model

Abstract

Background: The long-term survival after vascularized composite allotransplantation (VCA) is often limited by systemic rejection as well as the adverse effects of immunosuppressants. The stromal vascular fraction (SVF) can be expanded to produce adipose-derived stem cells (ADSC) which represents a combination of endothelial cells, preadipocytes, immune cells, and ADSC. It has been demonstrated that ADSC possess consistently reliable clinical results. However, literature is scarce regarding SVF in VCA. This study seeks to determine the impact of ex vivo allograft pretreatment in combination with SVF cells in the ability to promote composite tissue allotransplantation immunotolerance.

Methods: A rat hind limb allotransplant model was used to investigate the influence of ex vivo pretreatment of SVF and ADSC on VCA survival. Intravascular cell-free saline, ADSC, or SVF was infused into the models with immunosuppressants. The histopathological examination and duration that the allografts went without displaying symptoms of rejection was documented. Peripheral T lymphocytes and Tregs were quantified with flow cytometry while allotissue expressions of CD31 were quantified with immunohistochemical staining (IHC). ELISA was used to detect vascular endothelial growth factor (VEGF)-A as well as anti- and pro-inflammatory cytokines.

Results: We demonstrated that ex vivo treatment of allografts with SVF or ADSC prolonged allograft survival in contrast to medium control cohorts. There were also enhanced levels of immunomodulatory cytokines and increased VEGF-A and CD31 expression as well as reduced infiltration and proliferation of T lymphocytes along with raised Treg expressions.

Conclusion: These studies demonstrated that adipose-derived cellular therapies prolong graft survival in an allogenic hind limb transplantation model and have the potential to establish immunotolerance.

Keywords: Immune modulation; Stromal vascular fraction (SVF); Vascularized composite allograft (VCA).

Fig. 1

a Allograft images in control, ADSC, and SVF groups at post-operative days 10, 17, 26, and 35. b Survival rate of control, ADSC, and SVF groups. c Survival data summarized in a table. Significant differences are indicated by *P < 0.05

Fig. 2

Skin and muscle samples of different groups are stained with hematoxylin and eosin (scale bar = 100 μm)

Fig. 3

Concentrations of anti-inflammatory and pro-inflammatory cytokines in serum detected by ELISA. a IL-10, b TGFβ1, and c TNF-α (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001)

Fig. 4

Gating strategy and percentages of T cells. a Gating strategy for CD4+, CD8+, and Treg cells. b Percentage of CD4+ T cells of three groups at post-operative days 10 and 14. c Percentage of CD8+ T cells of three groups at post-operative days 10 and 14. d Percentage of Treg cells of three groups at post-operative days 10 and 14 (*P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 5

CD31 expression in allografts and VEGF-A expression in serum. a Images of allograft skin tissue subjected to immunohistochemical of three groups at post-operative day 7 (scale bar = 50 μm). b Quantitative analysis of integrated optical density (IOD) of CD31-stained area. c Quantification of blood vessel number in CD31-stained tissue sections. d Concentrations of VEGF-A in serum (*P < 0.05, **P < 0.01, ***P < 0.001)