Syngeneic adipose-derived stem cells with short-term immunosuppression induce vascularized composite allotransplantation tolerance in rats HUI-YUN CHENG 1,2 , NICOLAE GHETU 3 , WEI-CHAO HUANG 4 , YEN-LING WANG 1 , CHRISTOPHER GLENN WALLACE 5 , CHIH-JEN WEN 6 , HUNG-CHANG CHEN 7 , LING-YI SHIH 5 , CHIH-FAN LIN 6 , SHIAW-MIN HWANG 8 , SHUEN-KUEI LIAO 9,10 & FU-CHAN WEI 1,5,6 1 Center for Vascularized Composite Allotransplantation, Chang Gung Memorial Hospital, Gueishan, Taiwan, 2 Department of Medical Research and Development Linkou Branch, Chang Gung Medical Foundation, Taoyuan, Gueishan, Taiwan, 3 Former Microsurgery Fellow, Chang Gung Memorial Hospital; Regional Oncological Institute, University of Medicine and Pharmacy. “Grigore T. Popa,” Iasi, România, 4 Division of Plastic and Reconstructive Surgery, Tzu Chi General Hospital at Taipei, New Taipei, Taiwan, 5 Department of Plastic and Reconstructive Surgery, Chang Gung Memorial Hospital, Gueishan, Taiwan, 6 School of Medicine, Chang Gung University, Gueishan, Taiwan, 7 Graduate Institute of Biomedical Sciences, Chang Gung University, Gueishan, Taiwan, 8 Bioresource Collection and Research Center, Food Industry Research and Development Institute, Hsinchu, Taiwan, 9 Graduate Institute of Cancer Biology and Drug Discovery and Center of Excellence for Cancer Research, Taipei Medical University, Taipei, Taiwan, and 10 R&D Division, Vectorite Biomedica Inc, Taipei, Taiwan Abstract Background aims. A clinically applicable tolerance induction regimen that removes the requirement for lifelong immuno- suppression would benefit recipients of vascularized composite allotransplantation (VCA). We characterized the immuno- modulatory properties of syngeneic (derived from the recipient strain) adipocyte-derived stem cells (ADSCs) and investigated their potential to induce VCA tolerance in rats. Methods. ADSCs were isolated from Lewis (LEW, RT1A l ) rats; their immunomodulatory properties were evaluated by means of mixed lymphocyte reactions in vitro and VCAs in vivo across a full major histocompatibility complex mismatch with the use of Brown-Norway (BN, RT1A n ) donor rats. Two control and four experimental groups were designed to evaluate treatment effects of ADSCs and transient immunosuppressants (anti-lymphocyte globulin, cyclosporine) with or without low-dose (200 cGy) total body irradiation. Flow cytometry was performed to quantify levels of circulating CD4 þ CD25 þ FoxP3 þ regulatory T cells (Tregs). Results. Cultured syngeneic ADSCs exhibited CD90.1 þ CD29 þ CD73 þ CD45  CD79a  CD11b/c  phenotype and the plasticity to differentiate to adi- pocytes and osteocytes. ADSCs dramatically suppressed proliferation of LEW splenocytes against BN antigen and mitogen, respectively, in a dose-dependent fashion, culminating in abrogation of allo- and mitogen-stimulated proliferation at the highest concentration tested. Accordingly, one infusion of syngeneic ADSCs markedly prolonged VCA survival in LEW recipients treated with transient immunosuppression; of these, 66% developed tolerance. Total body irradiation provided no additional VCA survival benefit. An important role for Tregs in tolerance induction/maintenance was suggested in vivo and in vitro. Conclusions. Treatment comprising syngeneic ADSCs and transient immunosuppression (i) increased levels of circulating Tregs and (ii) induced tolerance in 66% of recipients of major histocompatibility complexemismatched VCAs. Key Words: adipose-derived stem cell, allotransplantation, regulatory T cell, tolerance, vascularized composite Introduction Vascularized composite allotransplantation (VCA) describes the en bloc reconstruction of a recipient’s anatomical unit, such as hand/forearm, abdominal wall or face, by replacing it with a transplantation of the corresponding part from a deceased donor (1e3). The term “allotransplantation” is applied because the donor and recipient are genetically Correspondence: Shuen-Kuei Liao, PhD, Graduate Institute of Cancer Biology and Drug Discovery, Taipei Medical University, Taipei 110, Taiwan. E-mail: liaosk@h.tmu.edu.tw; Fu-Chan Wei, MD, Center for Vascularized Composite Allotransplantation, Chang Gung Memorial Hospital, Gueishan 333, Taiwan. E-mail: fuchanwei@gmail.com Cytotherapy, 2013; 0: 1e12 (Received 26 February 2013; accepted 27 June 2013) ISSN 1465-3249 Copyright Ó 2013, International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jcyt.2013.06.020