Liver transplantation remains the only definitive treatment for a number of diseases, including end-stage chronic liver disease, acute liver failure, or limited hepatic neoplasms, with patient and graft survival rates exceeding 75% after five years [1, 2]. However, liver transplantation is burdened by the need for life-long immunosuppression in order to prevent graft rejection. All drugs currently used for immunosuppression cause significant clinical side effects. Besides their well-known intrinsic toxicities (e.g., neurotoxicity of tacrolimus and renal toxicity of ciclosporin [[3–5]]), they also increase the risk for cancer and opportunistic infections [[6–11]]. The long-term overall success of liver transplantation is frequently determined by complications related to immunosuppressive drug therapy. Yet, immunosuppressants are indispensable to maintain graft function and to cover aberrations in immune reactions that may result in rejection of the transplanted organ.
Growing numbers of patients in need of a liver graft are faced with a continuous shortage of donor organs. In the Eurotransplant area, for instance, only 1631 transplant livers were available for 2641 patients on the waiting list in 2009 . To overcome this shortage, criteria for the acceptance of donors have been liberalized, e.g., in terms of prolonged ischemia time, increased donor age, or the presence of clinically significant donor liver steatosis. While increasing the donor pool, these "marginal" organs are also associated with higher incidences of primary graft dysfunction and major complications [[13–15]]. Here, we propose a novel protocol involving treatment of liver transplant recipients with multipotent adult progenitor cells (MAPCs) with the goal of reducing the dose of immunosuppressive drugs and of supporting liver regeneration in marginal grafts.
Multipotent adult progenitor cells
MAPCs belong to the family of mesenchymal stem cells (MSCs) and are cultured from bone marrow aspirates [[16–18]]. The clinical-grade MAPC product (MultiStem®, Athersys Inc., Cleveland, Ohio, USA) to be used in this study is isolated from a single bone marrow aspirate and cultured with heat inactivated fetal bovine serum (FBS) and growth factors EGF and PDGF. Cells display a linear expansion rate to 65 population doublings or greater before senescence. Doubling times average 20 hours during expansion. Cells are used after 30 population doublings and tested by flow cytometry, in vitro immunomodulatory assays and cytogenetics. Moreover, extensive safety testing in immunodeficient animal models is performed [[19–21]].
MAPCs share immunosuppressive functions with MSCs , they have been shown to suppress T-cell proliferation in vitro and ameliorate graft-versus-host disease (GvHD) in small animal models . First clinical trials with MAPCs have already been initiated to treat GvHD and Crohn's disease . Moreover, MAPCs have regenerative properties, contributing to vascular regeneration in models of limb ischemia , improving cardiac function after myocardial infarction , and contributing to the regeneration of injured livers through their ability to differentiate into hepatocyte-like cells .
MSCs and MAPCs have been successfully applied in preclinical heart transplantation models in combination with various immunosuppressants [[26–29]]. Our group has demonstrated that MSCs and MAPCs induce long-term graft acceptance when applied together with mycophenolic acid [26, 27]. In contrast, calcineurin inhibitors (CNIs) have been shown to abrogate the immunosuppressive effect of MSC therapy in this and other animal models . The current study protocol therefore calls for a CNI-free, "bottom-up" immunosuppressive regimen combined with the MAPC infusions.
Current standard clinical protocols for post-transplant immunosuppression vary between institutions, continents and indications. However, most induction therapies include corticosteroids that are subsequently tapered over the first months. CNIs, such as ciclosporin A or tacrolimus, are the mainstay of immunosuppression, sometimes in combination with mycophenolic acid (MPA). Further treatment options are also available, like e.g. thymoglobulin. In addition, anti-CD25 monoclonal antibodies can be used to block activated T cells in the first week after the operation . Because standard immunosuppressive treatment is often reliant on CNI-based regimens, which can cause among other things renal impairment, hypertension, and hyperglycemia [[32–35]], efforts have been made to reduce CNI exposure for liver transplant recipients . Indeed, a proportion of patients can achieve graft acceptance without CNIs, while acute rejection episodes in the remaining patients can be treated with high-dose steroids and intensification of the baseline immunosuppressive regimen, without graft loss.
"Bottom-up" immunosuppression, then, refers to a CNI-free induction protocol consisting of steroids, mycophenolic acid and basiliximab. CNIs are introduced only when needed, e.g. in case of biopsy-proven acute rejection. This approach is feasible in liver transplantation, because of its lower immunogenicity in comparison to other types of organ transplants and because of the low risk of graft loss or permanent graft damage by acute rejection episodes. The "bottom-up" regimen has already been applied successfully in clinical studies [37, 38] and is particularly valuable for high-MELD (Model for End-stage Liver Disease) patients with increased risk of infections or renal dysfunction. In view of the synergistic interplay of MSCs with mycophenolic acid, and because CNIs have been shown to abolish the beneficial effect of MSCs in animal models, this study will use "bottom-up" immunosuppression in combination with MAPCs. We hypothesize that MAPC infusions will help to significantly delay the introduction of CNIs or allow to avoid them altogether.