Study design and participants
The current study was a prospective, monocentric, open-label, randomized, rituximab-controlled, phase I/II clinical trial that featured a total 2-year follow-up period. This trial was approved by the local ethics committee on clinical trials of the Third Affiliated Hospital of Sun Yat-sen University (Guangzhou, China), which piloted conforming to the ethics guideline of the 1975 Declaration of Helsinki and registered the trial at chictr.org.cn (ChiCTR2000037732). The target population involved adults with severe hepatic failure who were hospitalized for an emergency ABO-i LT. The detailed inclusion and exclusion criteria are listed in Fig. 1.
All participants were recruited at the Department of Liver Transplantation of the Third Affiliated Hospital of Sun Yat-sen University. Between August 2016 and August 2018, 22 patients were enrolled after screening a total of 47 patients receiving ABO-i LT at our center, and the enrolled patients were randomly assigned equally to the MSC or rituximab group. All patients in this trial provided written informed consent.
All enrolled patients in these two groups received standard immunosuppressive regimen treatment according to previously described methods, including steroids, baliximab, tacrolimus, mycophenolate mofetil, and intravenous immunoglobulin (IVIG) [13]. Specifically, 1 g of steroid was administered during the operation, followed by 500 mg on day 1 and 240 mg on day 2. The dose was tapered by 40 mg/d until the daily dose reached 40 mg/d. Subsequently, the steroid was administered at 48 mg/d p.o., with the dose tapered by 8 mg every 3 days until reaching 8 mg/day, and this does was then maintained at 8 mg/d for more than 1 year. IVIG (10 g/d) was used in the first 7 days after LT. Tacrolimus (2.0 mg/d) and mycophenolate mofetil were initially administered on the third day after ABO-i LT to maintain the blood concentrations of the drugs. Finally, baliximab was given twice, once during surgery and again on the fourth day after transplantation (Fig. 2).
Liver transplant procedures
The following details regarding the enrolled deceased liver graft donors were prospectively recorded: sex, age, donor after circulatory or brain death (DCD or DBD), cause of death, body mass index (BMI), ABO blood type, terminal serum sodium level, terminal hepatic and renal function tests, sojourn time in the intensive care unit (ICU), ventilator settings, and need for vasopressors.
The ABO-i LT procedures, Piggy-back LT, were standardly performed at the authors’ single centers, which were specified in detail previously [14]. The following recipient characteristics were collected: sex, age, liver, and renal function tests; coagulation function tests; model for end-stage liver disease (MELD) score; and titers of specific antibodies upon hospitalization for LT. During transplantation, the operation time, cold graft ischemic time, intraoperative blood loss, and blood transfusion volume were recorded.
Preparation, culture, and identification of allogeneic MSCs
Umbilical cord donors
Human umbilical cords were obtained from healthy donors who understood the study, met the inclusion and exclusion criteria and provided written informed consent. Before preparing the MSCs, the umbilical cords were initially confirmed to be negative for cytomegalovirus (CMV) antigen, anti-human T lymphotrophic virus (HTLV) I/II antibody, anti-hepatitis A virus (HAV) IgM antibody, hepatitis B virus (HBV) antigen, anti-hepatitis C virus (HCV) antibody, hepatitis D virus (HDV) antigen, anti-hepatitis E virus (HEV) IgM/IgG antibodies, syphilis, anti-HIV-1/2 antibodies, fungi, and bacteria. In addition, we limited the age of the donors from 18 to 35.
Preparation, culture, and identification of MSCs
Third-party MSC preparation was approved by the Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University. Following good manufacturing practice (GMP), the cell preparation and culture processes were performed under standardized and aseptic conditions at the Stem Cell Laboratory Facility of the Biotherapy Center at our hospital [12]. Fresh UCs were obtained from healthy pregnant women and immersed in 4 °C phosphate-buffered saline (PBS). The umbilical cords were washed twice with PBS to remove the remnant blood until they became white, cut into 10 mm3/piece in 0.1% type I collagenase with CaCl2 (3 mM) containing 0.1% hyaluronidase (Invitrogen, USA), and then incubated on a shaker (220 rpm) at 37 °C for 4 h digestion. Subsequently, the isolated cells were cultured in low-sugar Dulbecco’s modified Eagle’s medium (1 g/L DMEM, Gibco, Life, Austria) with 10% fetal bovine serum (FBS, Gibco, Life, Austria) in a humidified atmosphere of 5% CO2 and 37 °C. The medium was refreshed every 3 days to remove nonadherent cells. To assess the phenotype on the cell surface, flow cytometric analysis of CD105, CD73, CD44, CD90, CD45, CD34, CD166, and CD29 was performed. The differential potential was detected according to criteria established by the 2006 International Society of Cellular Therapy, which investigated their ability to differentiate into osteocytes and adipocytes [15]. At 70–80% confluence, MSCs were passaged by trypsin treatment. MSCs were collected and clinically used at passages 3–5. Before injection, the cells were tested again and confirmed to be negative for HBV, HCV, HIV, syphilis, mycoplasma, fungi, and endotoxins.
Allogeneic MSC transfusion
Allogeneic MSCs were collected and suspended in 100 ml of 0.9% NaCl at a density of 1.0 × 106 cells/kg body weight. The enrolled patients received nine total doses of MSC infusion, including the first time, which involved the infusion of 10% of the MSCs through the portal vein after graft reperfusion and 90% of the MSCs being transfused through the peripheral vein during LT; the subsequent 8 infusions (1 week, 2 weeks, 4 weeks, 8 weeks, 12 weeks, 16 weeks, 20 weeks, and 24 weeks after operation) were administered intravenously via the forearm. During the infusion, the MSC suspension was transfused within 30 min and swung gently every 3 min to avoid cell deposition. All patients were observed for 2 h after the transfusion to screen for any adverse events.
Primary and secondary endpoints
The primary endpoints of the investigation were the safety and tolerability of multidose MSC administration in study subjects with assessments of MSC-related adverse events (fever, headache, rash, vomiting, diarrhea, and carcinogenesis) and the incidence of allograft rejection, including antibody-mediated rejection (AMR) and acute cellular rejection (ACR), at the 2-year follow-up period. The secondary endpoints were the preliminary observations of MSC efficacy in patients who underwent ABO-i LT compared with rituximab, including (1) the evaluation of graft and recipient survival and (2) the incidence of postoperative complications, including biliary complications and specific infections.
Graft biopsy and immunohistochemistry
At week 2 and month 6, surveillance biopsies were performed, and 4-μm formalin-fixed, paraffin-embedded sections were prepared. First, hematoxylin and eosin (H&E) staining was performed to characterize graft rejection according to the Banff criteria by double-blind scoring [16]. In addition, the sections were stained with cytokeratin 19 (CK19) to observe biliary formation, stained with C4d to indirectly assess the severity of AMR, and immunostained with antibodies against human CD4, CD8, and CD20 to assess immune cell infiltration. At least three fields (×200 magnification) from each patient were randomly selected to calculate the mean number of positive cells. Archival tonsil sections were used as positive controls. Sections treated with only the secondary antibody and diaminobenzidine (no primary antibody) served as the negative controls. All of the primary antibodies were purchased from Abcam (USA), and the secondary antibody and diaminobenzidine were purchased from DAKO (USA).
Definitions
Acute cellular rejection (ACR) was scored using the Banff criteria. AMR was serologically diagnosed by acute tissue injuries, such as vascular inflammation and bile duct inflammation damage, and significantly increased titers of specific antibodies and histologically diagnosed by C4d staining [17]. Hepatic arterial stenosis was defined by visualizing Doppler ultrasonography and computed tomographic (CT) angiography. Biliary leakage was suspected with the persistent drainage of bile from the abdominal cavity and diagnosed by postoperative cholangiography. Bile duct anastomotic stenosis was diagnosed by magnetic resonance cholangiopancreatography (MRCP). ITBL was suspected based on laboratory examinations and elevated levels of serum ALP and γ-GGT and diagnosed by contrast-enhanced ultrasonography (CEUS) and MRCP [18, 19]. Post-transplant septic shock was diagnosed in patients who suffered from severe sepsis, which was determined by a positive culture of pathogenic forms of bacteria or fungi, with hyperlactatemia and obvious hemodynamic changes requiring vasopressor therapy.
Statistical analysis
As appropriate, summary data with continuous variables are presented as descriptive statistics, including n, mean ± standard deviation (SD), median/interquartile, and maximum/minimum, whereas the categorical variables are summarized using frequency and percentages. A mixed model (repeated measures) was used to analyze the outcomes from multiple follow-up times to compare the efficacies between the two groups, whereas comparisons within the same group were performed using the model-estimated contrasts. If the outcomes were highly skewed, a Wilcoxon signed rank sum test was used to make comparisons of each intragroup follow-up effect by covariance adjustment of the baseline. In addition, categorical variables were analyzed by Fisher’s exact tests. All data from this study were analyzed by SPSS 23.0 software (SPSS Inc., Chicago, IL, USA) or GraphPad software version 7.0 (CA, USA), as appropriate. Two-sided p < 0.05 was considered statistically significant.