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Stem Cell & Translational Investigation 2015; 2: e504. doi: 10.14800/scti.504; © 2015 by Ken-ichiro Seino, et al. http://www.smartscitech.com/index.php/scti REVIEW New immunosuppressive strategies for transplantation based on pluripotent stem cell (PSC)-derived immunoregulatory cells Ken-ichiro Seino, Haruka Wada, Muhammad Baghdadi Division of Immunobiology, Institute for Genetic Medicine, Hokkaido University, Kita-15 Nishi-7, Sapporo 060-0815, Japan Correspondence: Ken-ichiro Seino E-mail: [email protected] Received: January 03, 2015 Published online: February 11, 2015 Pluripotent stem cells (PSCs) such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) have the potential to give rise to cells from all three germ layers that can be utilized as new reliable sources in transplantation medicine. However, when allogeneic PSCs are used for transplant generation, an effective immunosuppressive strategy is required to protect transplants from rejection induced by immune responses of recipient to donor alloantigen after transplantation. Cell-based immunosuppressive strategies offer effective, safe and specific methods to inhibit recipient’s immune response against donor transplant and help to avoid side effects accompanied with conventional immunosuppressant drugs. In this context, we hypothesized that allogeneic PSCs-derived transplants can be protected from immune rejection if accompanied with adoptive transfer of immunosuppressive cells generated from the same PSCs. Indeed, we have developed several differentiation protocols to generate immunosuppressive cells such as regulatory T cells (Tregs) or macrophages from PSCs, which showed promising results to prolong same PSCs-derived graft survival. In this review, we introduce new findings related to PSCs-based cellular immunosuppressive therapy and its applications in several transplantation models. Keywords: Pluripotent stem cells; embryonic stem cells; induced pluripotent stem cells; immunosuppressive cells; allograft rejection; immuneregulation To cite this article: Ken-ichiro Seino, et al. New immunosuppressive strategies for transplantation based on pluripotent stem cell (PSC)-derived immunoregulatory cells. Stem Cell Transl Invest 2015; 2: e504. doi: 10.14800/scti.504. Introduction Recent progress in stem cell research has opened up the potential to create organs and tissues for transplantation from pluripotent stem cells (PSCs) such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) [1,2]. This promising technology can help to generate transplants from autologous cells and thus overcome the problem of immune rejection. However, it is difficult to obtain autologous ESCs from all patients. The development of iPS technology has facilitated the generation of stem cells by introducing a specific set of reprogramming factors into adult somatic cells which can be obtained anytime from the patient. Theoretically, this excellent resource of autologous cells can help to generate transplants with limited immunogenicity and thus avoid the risk of immune rejection. Practically, the generation of autologous iPSCs faces many difficulties represented by the cost and time needed to evaluate safety, stability and efficacy for each individual case. To overcome such problems, a pioneer project of generating a bank of ready-to-use PSCs collected from healthy donors has been triggered for future therapeutic use [3]. In such cases, transplants are expected to be derived from allogeneic PSCs, and thus must be accompanied with an effective immunosuppressive therapy to protect transplant from undesired immune responses induced by mismatching in histocompatibility between donor and recipient. Page 1 of 6 Stem Cell & Translational Investigation 2015; 2: e504. doi: 10.14800/scti.504; © 2015 by Ken-ichiro Seino, et al. http://www.smartscitech.com/index.php/scti Recent advances in cell-based therapies such as donor-specific blood transfusion and bone marrow transplantation have shown promising effects on conditioning the immune response of recipients to donor alloantigen after transplantation [4-6]. Additionally, newer methods are being developed to facilitate the ex vivo induction, expansion and purification of tolerance-promoting immune cells [7-9]. These novel strategies offer more effective, safe and specific immunosuppressive methodologies to help protecting transplanted organ or tissue from acute and chronic immune rejection, and minimizing the side effects accompanied with conventional immunosuppressant drugs [10]. From these backgrounds, we hypothesized that immune protection of ESCs- or iPSCs-derived transplants can be achieved by adoptive transfer of immune-regulatory cells differentiated from the same cellular origin of the transplant. Efforts in our laboratory focus on developing new protocols to generate immune-regulatory cells from ESCs and iPSCs for promoting transplant tolerance in clinical settings. In this brief review, we introduce protocols used to differentiate immunosuppressive cells from ESCs and iPSCs, and its applications in allogeneic transplantation models. Induction of immunosuppressive cells from PSCs Regulatory T cells Immune rejection of transplanted grafts is an adaptive immune response mediated by killer T cells that induce apoptosis in target cells. To prevent organ rejection, immunosuppressive drugs such as cyclosporins have been used to inhibit T cell activation, thus preventing T cells from attacking the transplanted organ. However, the use of immunosuppressant drugs is associated with a wide range of side effects, including increased risk of infections, cancers, diabetes, in addition to neurotoxicity and nephrotoxicity [9]. Thus, it is critically important to develop new methods to limit or reduce T cells function in an alloantigen-specific manner, while other effector functions are kept intact. FoxP3-positive regulatory T cells (Tregs), also known as suppressor T cells, are a subset of T cells that play important roles in the regulation of the immune response, and maintain tolerance to self-antigens [11]. The utilization of alloantigen-primed Tregs has been considered as a promising non-toxic immunosuppressive strategy to inhibit transplant rejection reactions. Indeed, the adoptive transfer of exvivo expanded Tregs effectively prevents graft versus host disease after allogeneic bone marrow transplantation in mice and human. Tregs have also the potential to establish transplant tolerance to MHC molecules of cellular origin used for Tregs generation. In this regard, we investigated Tregs differentiation from iPSCs in vitro as a possible cellular therapy for the induction of tolerance to iPSCs-derived transplants. Tregs are classified into two distinct subsets: naturally occurring Tregs and induced Tregs. Naturally occurring Tregs develop in the thymus, while induced Tregs develop in the periphery under the influence of transforming growth factor β (TGFβ) [9]. TGFβ can be used to induce generation of Tregs from peripheral T cells in in vitro culture [11]. To generate Tregs from mouse iPSCs, we developed a culture protocol based on the induction of hematopoietic cells from iPSCs using Flt3L, followed by induction of T cells from these hematopoietic cells using IL-7, and finally induction of Tregs from T cells using IL-2 and TGFβ (figure. 1A) [12]. Using this protocol, we could generate FoxP3 positive T cells at an efficiency reached up to 10%. These Tregs induced from iPSCs have immunosuppressive features as shown by effective inhibition of T cell proliferation [12]. Thus, adoptive transfer of iPSCs-derived Tregs may contribute to decrease the risk of allogeneic immune rejection of same iPSCs-derived transplants, which should be examined in details in future studies. As indicated above, Tregs play essential roles in transplantation tolerance, and the therapeutic effects of Tregs have been reported in several animal models [13]. Human Tregs can be isolated from peripheral blood or cord blood, and expanded in ex vivo cultures as a therapeutic product [14]. For PSCs-derived transplants, Tregs can be generated from the same PSCs and thus are expected to suppress allogeneic immune responses. The efficiency of Treg induction from iPSCs in our protocol is still unsatisfying; however, accumulating knowledge of Tregs cell biology will contribute to the improvement of this culture protocol using several cytokines and their cocktails. Immunosuppressive macrophages Allograft rejection results from immune attack against the transplanted graft, and is characterized by multiple inflammatory features. Macrophage accumulation has been considered as a prominent feature of allograft rejection [15]. Recent progress in macrophage biology has improved our understanding of the roles of macrophages in allogeneic immune response [16]. Macrophages may contribute to allograft injury by inducing both innate and adaptive immune responses during the process of acute and chronic allograft rejection [15]. On the other hand, macrophages may also promote tissue repair mechanisms and suppression of immune responses [17]. Thus, macrophages serve as an attractive and effective target to improve outcomes in organ Page 2 of 6 Stem Cell & Translational Investigation 2015; 2: e504. doi: 10.14800/scti.504; © 2015 by Ken-ichiro Seino, et al. http://www.smartscitech.com/index.php/scti Figure. 1 Generation of immunosuppressive cells from PSCs. A scheme describes culture protocol used to generate regulatory T cells from iPSCs (A) or immunosuppressive macrophage-like cells from ESCs (B). transplantation. Indeed, several approaches based on macrophages are being examined in preclinical and clinical models [18-22]. For example, in clinical renal transplantation, adoptive transfer of donor-derived regulatory macrophages was effective to maintain excellent graft functions in patients who were minimized to low-dose tacrolimus monotherapy [21,22] . In this context, we examined the ability to generate macrophages with immunosuppressive features from PSCs, in order to suppress allogeneic immune responses against transplants derived from the same PSCs [23]. Arginase-1, Nos-2 and Tgfβ1 (table.1) [23]. Indeed, this fraction of cells showed significant abilities to inhibit T cell proliferation, and thus refer to as ES-derived immunosuppressive cells (ES-SCs). ES-SCs efficiently suppressed T cell proliferation in allogeneic lymphocyte reaction (MLR), which was related to iNOS expression23. More importantly, systemic administration of ES-SCs was effective to delay allogeneic immune rejection of ES-derived embryoid bodies or cardiomyocytes [23], indicating that ES-SCs have the potential to prolong survival of allogeneic transplanted grafts. To evaluate this concept, we developed a culture protocol based on the induction of hematopoietic cells from ESCs, followed by stimulation with cytokines related to myeloid cell differentiation including M-CSF, GM-CSF and IL-4, and finally LPS (figure. 1B) [23]. Using this protocol, we obtained floating cells with dendritic cells-like features and thus refer to as ES-derived dendritic cells (ES-DCs), as previously described [20]. On the other hand, adherent cells were characterized by increased expression of macrophage markers such as F4/80 and CD115, and showed potent immunosuppressive capacities as indicated by the high expression levels of immunosuppressive genes including In summary, macrophages play important roles in the determination of fate of transplanted grafts, and thus serve as a promising therapeutic target for improving the outcome of organ transplantation. Using our culture protocol, macrophage-like cells with immunosuppressive features can be generated from PSCs and contribute to the survival of same PSCs-derived transplants. Further analysis of characterization, differentiation and function of various macrophages subsets may provide novel therapeutic targets to improve transplant survival. Page 3 of 6 Stem Cell & Translational Investigation 2015; 2: e504. doi: 10.14800/scti.504; © 2015 by Ken-ichiro Seino, et al. http://www.smartscitech.com/index.php/scti Table 1. Comparison between ES-DCs and ES-SCs obtained from ESCs Induction of hematopoietic stem cells from PSCs Combination of hematopoietic stem cell (HSC) transplantation and T cell inhibition has been reported to induce indefinite allograft survival [24]. Therefore, it seems of great interest to generate HSCs from PSCs. A recent study has reported the ability to induce hematopoietic progenitor cells from HOXB4-transduced mouse ESCs25. HOXB4 is a hematopoietic transcription factor, which contributes to the expansion of HSCs [26]. CD45+ hematopoietic progenitor cells isolated from HOXB4-transduced ESCs show low expression of MHC molecules [25]. This feature allows the long-term engraftment of these CD45+ MHClow hematopoietic progenitor cells in sublethally irradiated recipients across MHC barriers without the need of immunosuppressive drugs [25]. Low levels of chimerism were observed in the bone marrow of the recipients over 100 days, and the recipients were protected from rejection of donor-derived cardiac allografts [25]. serially transplantable in vivo [27]. It is anticipated that iHSCs described above could be also derived from PSCs with similar protocol, and might help to facilitate the engraftment of PSCs-derived grafts. In another recent study, HSCs were successfully induced directly from human iPSCs by the manipulation of Wntβ-catenin signal pathway during hematopoietic programs transition [28]. The generation of KDR+ CD235a+ primitive progenitors depends on stage-specific activin-nodal signaling and inhibition of the Wnt–ß-catenin pathway, whereas specification of KDR+ CD235a− definitive progenitors requires Wntβ-catenin signaling during this same timeframe. The manipulation of Wntβ-catenin signal pathway offers a simple and effective differentiation strategy for the development of PSC-derived hematopoietic progenitors [28]. Together, these differentiation protocols are expected to enable the generation of HSCs from PSCs as a promising future strategy for immune regulation. Concluding remarks More recent study has reported the ability to impart HSC potential onto committed mouse blood cells by transient expression of six transcription factors including Run1t1, Hlf, Lmo2, Prdm5, Pbx1 and Zfp37 in committed lymphoid and myeloid progenitors [27]. The efficacy of this direct reprogramming protocol was further improved by addition of Mycn and Meis1 and use of polycistronic viruses. These induced-HSCs (iHSCs) showed a gene expression profile similar to endogenous HSCs. Additionally, iHSCs reconstituted stem/progenitor compartments, and were The use of several immunosuppressive drugs over the past decade has helped to overcome the problems of acute rejection and improved short-term allograft survival. However, in addition to their precautions and side effects, the contribution of these immunosuppressive drugs on long-term allograft survival remains unsatisfying. Current immunosuppressive strategies are focusing on conditioning immune responses of organ transplanted-recipients to donor-derived alloantigen using various cell-based therapies. Page 4 of 6 Stem Cell & Translational Investigation 2015; 2: e504. doi: 10.14800/scti.504; © 2015 by Ken-ichiro Seino, et al. http://www.smartscitech.com/index.php/scti Figure. 2 PSCs-derived immunosuppressive cells as a promising strategy for future transplantation medicine. Recent progress in the field of regenerative medicine has enabled the generation of tissues and organs for transplantation from PSCs such as ESCs and iPSCs. The induction of recipient’s tolerance to donor alloantigen after transplantation can be mediated by the adoptive transfer of immunosuppressive cells generated from the same allogeneic PSCs used for transplants generation. These novel strategies can help to specifically suppress immune responses against alloantigens, and thus keep other effector immune functions intact avoiding the nonspecific immunosuppression mediated by conventional immunosuppressive drugs. Current works in our laboratory focus on developing new culture protocols that help to facilitate the ex vivo induction, expansion, and purification of tolerance-promoting cells from PSCs such as ESCs or iPSCs (figure.2). Compared to other established techniques such as donor-specific blood transfusion and bone marrow transplantation, theses protocols offer the advantages of obtaining desired doses and qualities of target cells in a short-term cell culture. In addition to Tregs and immunosuppressive macrophages described in this review, it is of great interest to examine if other immunoregulatory cells such as tolerogenic dendritic cells, mesenchymal stem cells can be generated from PSCs. In conclusion, we hope that PSCs-derived immunosuppressive cells would be useful for the development of safe and effective immunotherapy for transplantation medicine, and that this experimental technology will graduate to clinic in the near future. References 1. 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