To our knowledge, we firstly conducted a randomized, open-labelled clinical trial to evaluate the safety of intravenous transfusion of allogeneic MSCs and compared the preliminary outcomes of MSCs and rituximab in recipients with severe hepatic failure receiving emergent ABO-i LT. Overall, this phase I/II study shown that multi-doses of MSCs administration were safe and well-tolerance in this population. Furthermore, we also provided that as an adjunct treatment, transfusion of MSCs not only has comparable ability of rituximab in preventing AMR, but also has lower incidences of post-operative severe infection and biliary complications after ABO-i LT.
With the incredible capabilities of immunomodulation and organ protection, MSCs have been gradually praised as a promising therapy, and increasing clinical trials were allowed to administrate for various diseases, which have shown encouraging outcomes. However, several issues still exist to be discussed, and the source of MSCs are firstly worth considered. Olivier et al. preferred to the transfusion of BM-MSCs for patients after LT. Besides, Wang et al. exhibited the role of MSCs in attenuating acute graft rejection after LT via affecting the percentage of Tregs and Treg/Th17 ratio. In this study, we also used MSCs deriving from umbilical cord for the patients after ABO-i LT, as umbilical cord obtain are more available without any ethical problems. The process of MSCs harvest in our center was performed at the Stem Cell Laboratory Facility of Biotherapy Center of the Third Affiliated Hospital of Sun Yat-sen University and abided by the standardized, aseptic requirement. Secondly, the doses and times of MSCs administration are also controversy. It was believed that the minimum effective cell dosage was 1 × 107 cells/kg, and high rate of mortality was occurred when single-dose was more than 21 × 107/kg[21, 22]. However, due to combined utilization of multiple immunosuppressants, we adopted 1 × 106 cells/kg of single-dose infusion of MSCs in this study, which was similar to the suggestion in the previous experience of MSCs administration in organ transplantation fields[10, 23]. In addition, a single-dose and multiple-dose administration were both reported respectively in the past researches. Our previous clinical study shown the hepatoprotective effects of repeated doses of MSCs transfusion for improving ITBL after LT without any severe side-effects. Thus, we continued to adopt this strategy in the current study and used eight doses of MSCs in the postoperative period. Furthermore, as hyperacute rejection reaction, theoretically, occurs immediately after graft reperfusion, we added a dose in this period during operation, including 10% MSCs administration through portal vein after graft reperfusion and 90% transfused through peripheral vein.
As this is the first report about the administration of stem cell-based therapy for preventing postoperative complications after ABO-i LT, safety issue remains the primary concern for our observation. Due to their characterization, MSCs liable to embolize pulmonary circulation in animal experiments when they were transfused through peripheral or central vein so that increased the burden of pulmonary exchange. In addition, with the abilities of immunosuppression and multi-lineage differentiation, MSCs theoretically exist a potential risk of carcinogenesis. Fortunately, the results from this trial shown that our eleven recipients receiving MSCs therapy did not arise severe infusional toxicity, including allergic reaction and did not develop any signs of pulmonary dysfunction and malignant transformation after 2-year follow-up. Only a few patients were noted limiting fever and completely recover 3 h after the infusions without any special treatment. In generally, our data indicated that allogeneic MSCs administration for these patients was safe, which is adherence to the investigations of our previous study of treating patients with ITBL after LT.
As another primary outcome, we additionally compared the incidence of allograft rejection between the Rituximab group and the MSC group. Traditionally, AR is divided into AMR and ACR. During ABO-i LT, high titer of ABO antibodies may lead to a high risk of AMR. Rituximab is an immune-chimeric monoclonal antibody that specifically points to the transmembrane protein CD20 molecule, which expressed on the majority of B cells, but not on antibody-producing plasma cells, to deplete B cells. Rituximab is also approved for the application in transplantation field, especially in the ABO-i organ transplantation. In 2003, Monteiro et al. firstly reported the administration rituximab for the recipients receiving ABO-i LT, and since then several studies have demonstrated that rituximab obviously reduced graft loss rates and is crucial to prevent the risk of AMR after ABO-i LT[27, 28]. In this setting, we found that MSCs treatment resulted in the lower risk of AMR was comparable to that of rituximab following ABO-i LT. This encourage result also indicated that MSCs might replace rituximab to modulate the functions of B cells.
As the secondary outcome, we prospectively assessed the therapeutic effects of MSCs on ABO-i LT recipients by comparison with the Rituximab group. No difference could be investigated between rituximab and MSCs treatment on the changes of the levels of ALB, ALP, AST, γ-GGT, BUN, CREAT and TBIL during the follow-up periods. Interesting, of these results, we found that a more distinct rise of post-transplant ALB level was observed in the MSC group compared with the Rituximab group, which might speculate that MSCs play an important role in repairing liver function. In addition, we also compared the rate of opportunistic infections between these two groups, due to both are immunosuppressive. Previous study shown that along with the effect on preventing AMR, treatment with rituximab increased the incidence of severe infections. Again, a large-sample size trial of multiple sclerosis shown that the rate of serious infections after used rituximab was higher than that in the control group. And other study showed that MSCs treatment for living-related kidney transplant recipients oppositely exhibited lower rate of infectious complications. In the present study, higher rate of serious infection was observed in the Rituximab group than that in the MSC group. Of all, three cases died from infection after treated with rituximab, and it did not happen in the MSC group. Through liver biopsy, we also detected that compared with MSCs, CD20-postive B cells were obviously depleted in the liver tissue by rituximab, which may explain why rituximab treatment susceptible to opportunistic infection. Other indicators, including survival rate, graft survival rate, acute rejection and arteriostenosis, were also no significant difference from these two groups except biliary complications. As large-size retrospective study by Song et al demonstrated that biliary complication in the only concern in ABO-i LT after treated with rituximab. Likely, high rate of ITBL in the Rituximab group was observed in this study. Consistent with our previous studies, MSCs also exhibited ability in protecting biliary structure following ABO-i LT. To explain this issue, we referred the past researches, and found that after ABO-i LT kidney transplantation, the graft vascular endothelium markedly expressed ABO blood group antigens that is the target of attack. Lacob et al. revealed that the presence of anti-human leucocyte antigen (HLA) class II antibodies was closely associated with biliary injury after LT. And we speculated that MSCs might exhibit critical roles in regulating the level of DSA and affecting the membranous expression of MHC-II of bile duct epithelium following ABO-i LT to improve biliary injury.
There are several important shortcomings in this study which should be acknowledged for improving further investigation. First, this phase I/II trial is the first study only enrolled 11 ABO-i LT recipients in both the MSC group and the Rituximab group. Therefore, a well-designed study with a large sample size, multicenter and long-term investigation is required to further ascertain these outcomes. Second, due to the characteristics of complications of ABO-i LT, we administrated repetitive infusions of MSCs both during and post operation via portal vein and peripheral vein. However, the timing, the dose and the route of MSCs administration remain deserve to deliberation and should be further evaluated. Furthermore, this is an open-labeled study, and neither recipients nor observers were blinded to the therapeutic strategy. Thus, bias was inevitable in the interpretation of adverse events. Finally, although we have detected the changes of several serum cytokines in this two groups during the follow-up period, the deep mechanism of the effect of MSCs in ABO-i LT should be further observed with evaluating the change of immune cell subpopulation in peripheral circulation.