Adipose-derived stromal cells promote allograft tolerance induction

Amputations and unsalvageable injuries with devastating tissue loss are common in the combat wounded. Reconstructive transplantation in the civilian setting using vascular composite allotrans-plants (VCAs) with multiple tissues (skin, muscle, nerve, bone) combined with long-term multidrug im-munosuppression has been encouraging. However, skin rejection remains a critical complication. Adipose-derivedstromal/stemcells(ASCs)are easily obtained from normal individuals in high numbers, precluding ex vivo expansion. The reparative function and paracrine immunomodulatory capacity of ASCs has gained considerable attention. The present study investigated whether ASCs facilitate long-term skin allograft survival. ASCs were isolated from fresh human subcutaneous adipose lipoas-pirate. Full-thickness skin grafts from BALB/c mice were transplanted onto the dorsal flanks of C57BL/6 mice treated with five doses of anti-CD4/CD8 monoclonal antibodies (10 mg/kg) on days 0, +2, +5, +7, and +14 relative to skin grafting. A single nonmyeloablative low dose of busulfan(5mg/kg) was given on day +5. Seven days after skin transplantation, ASCs (3 × 10⁶) were infused i.v. with or without donor bone marrow cells (BMCs; 5 × 10⁵). ASC+BMC coinfusion with minimal conditioning led to stable lym-phoid and myeloid macrochimerism, deletionof alloreactive T cells, expansion of regulatory Tcells, and long-term allograft survival (>200 days). ASCs constitutively produced high levels ofanti-inflammatory/ immunoregulatory factors such as prostaglandin E₂, indoleamine 2,3-dioxygenase, APO-1/Fas (CD95), and programmed cell death-1 ligand-2. These findings serve as a foundation for developing a translational advanced VCA protocol, embodying both ASCs and low-dose donor BMCs, in nonhuman primates, with the goal of enhancing functional outcomes and eliminating the complications associated with long-term immunosuppression.