In recent decades, pediatric liver transplantation has evolved into a state-of-the-art procedure improving prognosis and quality of life for children and adolescents with terminal liver disease. Immunosuppressive pharmacotherapy including calcineurin inhibitors (CNIs) allows the transplantation of solid organ grafts with reasonable patient and graft survival rates [
Mesenchymal stem cells or, according to the terminology of the International Society of Cellular Therapy (ISCT), multipotent mesenchymal stromal cells [
Numerous experimental studies imply that MSCs play a role in modulation of immune responses: in mixed lymphocyte cultures, human MSCs have a significant suppressive effect on T-cell proliferation by means of cell-cell interaction and secretion of soluble factors [
Preclinical models have confirmed these beneficial immunomodulatory effects of MSCs [
Mesenchymal stem (stromal) cells (MSCs) may represent an attractive therapeutic option in solid organ transplantation because they modulate immune response and promote regeneration [
Due to the aforementioned toxicity of CNI and insufficient long-term outcome results after liver transplantation, children may benefit from alternative approaches to immunomodulation that will prolong graft survival while reducing CNI toxicity, improving quality of life and promoting long-term allograft tolerance. However, in the context of pediatric living-donor liver transplantation, the safety and feasibility of intraportal and intravenous application of MSCs remain to be proven.
The primary objective of this pilot trial is to assess the safety of donor-derived MSC infusions in children undergoing LDLT. Safety will be determined by the following:
Incidence, timing, and severity of acute complications related to MSC infusion, using a specific toxicity scoring system (MYSTEP score, Figure Incidence of severe adverse events (SAEs) and their relation to investigational treatment Graft integrity and function after liver transplantation, as measured by aminotransferase and gamma glutamyl transferase activity, bilirubin, albumin, and INR.
Further, this study aims to evaluate the following:
Efficacy
Feasibility and safety of tapering immunosuppressive medication according to standard guidelines [ Time to first biopsy-proven acute rejection (BPAR). Hematologic and immunologic function, as measured by characterization and quantification of mononuclear cell populations, detection of donor-specific antibodies (DSA) and liver autoantibodies, and analysis of a protocol transplant biopsy. Patient and graft survival at 1 and 2 years posttransplantation.
MYSTEP score monitoring infusional toxicity. Cut-off levels are defined upon current publications in adult [
The MYSTEP1 (Mesenchymal Stem Cells in Pediatric Liver Transplantation) trial is a 24-month, nonrandomized, open-label, prospective, single-center pilot trial. In total, a minimum of 7 de novo liver recipients, 0–17 years of age, will be recruited at the University Hospital of Tübingen. We will enroll them in the study upon the consent of their legal representatives and upon meeting the eligibility criteria. The study group may be expanded to as many as 10 patients after consulting the data safety monitoring board (DSMB), which will monitor study progress. The board comprises a pediatrician, a transplant surgeon, and a biometrician not otherwise involved in the trial. We have obtained regulatory approval from the Ethical Committee of the University of Tübingen and from the German Federal Institute for Vaccines and Biomedicines (Paul-Ehrlich-Institut; EudraCT number 2014-003561-15). This trial is registered with Clinicaltrials.gov
Patients eligible for inclusion in this study must fulfill all of the following criteria:
Patient and both parents and/or legal guardian must have given written informed consent. Patients will undergo living-donor liver transplantation for chronic terminal liver failure. Age ≥ 8 weeks and ≤18 years. Body weight > 5 kg.
Patients fulfilling any of the following criteria are not eligible in this study:
Living donor not suitable according to donor and recipient criteria Pregnant or breastfeeding Refusal of adequate contraception (if appropriate) Acute liver failure or highly urgent transplantation Receiving any form of solid organ retransplantation Multi-organ-transplantation Active autoimmune disease Preexisting renal failure with eGFR < 50 ml/min/1.73 m2 or require hemodialysis Reduced pulmonary function (lung function test in children older than 6 years: FEV1 and FVC < 70% of age-appropriate norm) or clinical suspicion of pulmonary disease affecting the patient’s physical performance, requiring invasive or noninvasive mechanical ventilation History of pulmonary embolism Pulmonary hypertension and/or right ventricular load in echocardiography Reduced cardiac function: left ventricular shortening fraction < 25% Clinically significant systemic infections Undergoing critical care treatment like mechanical ventilation, dialysis, or vasopressor agents Seropositivity for HIV, HTLV, or hepatitis B/C Hepatobiliary malignancies or history of any extrahepatic malignancy Thrombophilia Budd-Chiari syndrome Preexisting thrombosis of portal vein Doppler-sonographic evidence for relevant portosystemic shunts, for example, persistent ductus venosus Cold ischemia time > 90 min Known abuse of drugs or alcohol Known allergy to DMSO.
Donor-derived mesenchymal stem cells will be obtained 4 weeks before the planned LDLT via bone marrow puncture of the living donor. All donors will undergo routine examination and screening tests including an extensive infectious disease work-up, according to our transplant center’s donor screening protocol [
The cell product can only be released if the following criteria have been fulfilled: regular surface marker expression (CD105+CD73+CD90+CD45-HLA-DR-cells > 90%; CD3+, CD19+, and CD14+ cells < 0.5%); spindle-shaped morphology; a colorless cell suspension; viability of cells of >80%; absence of microbial contamination using culture, mycoplasma PCR, and endotoxin testing; and absence of cell aggregates. The MSC product is cryopreserved with 10% DMSO until designated application (storage at <−150°C in gas phase of liquid nitrogen). On the day of cell transfusion, the assigned dose of mesenchymal stem cells will be thawed at the GMP stem cell laboratory, washed, and suspended in an appropriate volume of isotonic saline with 0.5% albumin at a cell concentration ranging between about 1 and 1.5 × 106 cells/ml.
The investigational treatment will consist of two transfusions of donor-derived mesenchymal stem cells, each dose ~1 × 106 MSC/kg body weight, with the first infusion intraoperatively (day 0) and the second infusion postoperatively, on days 1–3 after living-donor liver transplantation (Figure
Postoperative venous thrombosis prophylaxis is mandatory using low-dose heparin. After about 10 days, prophylaxis should be switched to aspirin and maintained for 3 months after LDLT. Prophylaxis against bacterial, fungal, and viral infections will adhere to our center’s pLT protocol.
Children enrolled in this study will undergo a standard pretransplant work-up, which consists of baseline clinical data (demographics, medical history, current medication, physical examination, laboratory examinations, thrombophilia screening test, urinalysis, electrocardiogram, abdominal ultrasound, and chest X-ray). Pregnancy tests will be performed using a test for
The first administration of 1 × 106 cells/kg body weight will be performed intraoperatively via portal infusion after complete liver allograft reperfusion (day 0). MSC suspension will be administered via a small venous catheter into the portal vein for 20 minutes while gently waving the syringe to keep cells in suspension. In addition to using Doppler ultrasonography, we will measure portal flow by transit time flow measurement (Medistim®) during cell infusion [
We will see patients frequently for follow-up visits during the first 28 days after transplantation. Study visits will consist of regular clinical examinations, Doppler sonography, and blood tests aimed at early detection of treatment-emergent events. We will assess the patient’s MYSTEP toxicity score on days 0, 2, 4, 7, 10, and 28 after LDLT. Additional study visits will be performed up to 720 days after LDLT to assess allograft survival, incidence of rejection, incidence of (opportunistic) infections, kidney function, and individual need for immunosuppressive medication (Table
Assessment schedule MYSTEP1 study.
Visit | BL | MSC1 | MSC2 | Follow-up visits | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Days after LT | −28 | 0 | 2 ± 1 | 4 | 7 | 10 | 28 | 90 | 180 | 270 | 360 | 540 | 720 |
Informed consent |
x | ||||||||||||
Concomitant medication | x | x | x | x | x | x | x | x | x | x | x | ||
TAC dosage | x | x | x | x | x | x | x | x | x | x | x | ||
Anthropometric parameters | x | x | x | x | x | x | x | x | x | ||||
Vital signs | x | x | x | x | x | x | x | x | x | x | x | x | x |
Physical examination | x | x | x | x | x | x | x | x | x | x | x | x | x |
Tacrolimus blood trough level | x | x | x | x | x | x | x | x | x | x | x | ||
Routine laboratory including liver parameters | x | x | x | x | x | x | x | x | x | x | x | x | x |
eGFR | x | x | x | x | x | x | x | x | x | ||||
Virus PCR: EBV, HCMV, and ADV | x | x | x | x | x | x | x | x | x | ||||
HHV-6 | x | x | x | ||||||||||
Doppler ultrasonography | x | x | x | x | x | x | x | x | x | x | x | x | x |
Infusional toxicity score | x | x | x | x | x | x | |||||||
TNF- |
x | x | x | x | |||||||||
Immune monitoring | x | x | x | x | x | x | |||||||
Antibodies: DSA; ANA, SMA, and LKMA | x | x | x | x | |||||||||
Percutaneous liver biopsy | x | ||||||||||||
MSC administration | x | x |
BL: baseline; MSC1: first intraoperative MSC infusion; MSC2: second MSC infusion on postoperative day 2.
In order to evaluate and quantifiy treatment-emergent adverse events of MSC infusion, we defined a pediatric infusional toxicity score that adopts the MiSOT-I score for adults [
Scheme of investigational treatment and IS tapering strategy. BX = basiliximab; TAC = tacrolimus.
In this study, we will perform a protocol liver biopsy 6 months after LDLT and MSC infusion. A percutaneous liver biopsy will be performed under sonographic control and in analgosedation, according to recommendations of the ESPGHAN Hepatology Committee [
Additional blood samples will be collected to investigate surrogate markers of the participant’s immune response status (Table
MSCs may support induction of allograft tolerance and help to achieve long-term tolerance [
Introduction of current standard immunosuppressive therapies including CNIs has had a major impact on reduction of acute mortality after pediatric liver transplantation [
To our knowledge, this is the first clinical trial of immunomodulating therapy with mesenchymal stem cells in pediatric solid organ transplantation. In this pilot study, we aim primarily to determine safety and feasibility of intraportal and intravenous infusion of donor-derived MSC in children undergoing LDLT. We will assess safety based on incidence of acute infusion-related complications measured by the MYSTEP score, on occurrence of severe adverse events and on allograft function after pLT. We have designed our study protocol to optimize prevention of adverse events and to identify any that arise early. For example, using a “staggered approach” to patient recruitment and ensuring intraoperative quantitative monitoring of graft perfusion by transit time flow measurement will help ensure a very high level of safety for the children who participate. In adults, no toxicity has been observed during intravenous and intraportal infusion to date [
Previous studies of MSCs in kidney transplantation have shown contradictory data on the risk of overimmunosuppression, which may lead to opportunistic infections [
Many details of the mechanisms of immunoregulation by MSCs in transplant recipients remain unknown. It is evident, however, that an increase in the percentage of Foxp3 positive regulatory T-cells is one important mode of MSC action in transplant patients [
The optimal donor source of MSCs is still unclear. In this trial, we will use bone marrow-derived MSCs obtained from the solid-organ donor. Donor-derived MSCs may contribute to donor-derived allograft tolerance [
In addition to the source of MSCs, the time and route of cell application may influence the effectiveness of MSCs. Direct infusion of the cells into the graft can make use of the tissue-repair capacity of MSCs to treat ischemia reperfusion injury. Furthermore, preclinical models showed that in addition to systemic effects, local mechanisms were responsible for transplant tolerance by MSCs [
In keeping with our focus on safety and feasibility, all participants will be treated in combination with the center’s standard immunosuppressive regime, which consists of basiliximab, tacrolimus, and steroids. Preclinical studies suggested that CNIs and glucocorticoids may affect MSC morphology, migration, and immunomodulatory behavior, possibly affecting the success of the cell therapy [
In conclusion, MSCs have the potential to become part of an array of novel treatment options for pediatric LDLT recipients aimed at promoting allograft tolerance and improving long-term allograft survival while reducing toxicity of chronic IS treatment. A positive outcome of the MYSTEP1 trial in terms of safety and allograft survival would constitute a major advancement in pediatric solid organ transplantation. Subsequently, we intend to conduct a second, larger multicenter trial to study the immunomodulatory efficacy of MSC treatment protocols for improving long-term allograft tolerance in pediatric liver transplant recipients.
Adenovirus
Antinuclear antibody
Biopsy-proven acute rejection
Donor-specific HLA-antibody
Human cytomegalovirus
Dimethylsulfoxide
Data safety monitoring board
Epstein-Barr virus
International normalized ratio
Immunosuppression
Intensive care unit
International Society of Cellular Therapy
Living-donor liver transplantation
Anti-liver-kidney microsomal antibody
Liver transplantation
Myeloid-derived immunosuppressive cell
Mesenchymal stem (stromal) cell
Pediatric liver transplantation
Resistance index
Reverse transcription polymerase chain reaction
Time-averaged maximum velocity
Severe adverse event
Anti-smooth muscle antibody.
The MYSTEP1 study was approved by the Medical Ethics Committee of the University of Tübingen (Ref. 003/2016AMG1).
The funding bodies have no influence on the study design and collection, analysis, and interpretation of data.
The authors declare that they have no competing interests.
Steffen Hartleif and Ekkehard Sturm designed the study and will conduct the study as principal investigators. Michaela Döring, Alfred Königsrainer, Silvio Nadalin, Marc H. Dahlke, and Rupert Handgretinger contributed to the idea and design of the study. Michael Schumm, Peter Lang, and Rupert Handgretinger set up and coordinate the MSC culture and quality controls in the GMP facility. Michaela Döring, Markus Mezger, and Steffen Hartleif contributed to the design and implementation of immune monitoring. All authors have read and approved the final manuscript.
The authors thank Cathy Shufro for the text editing. This study is supported by the Center for Rare Diseases, Tübingen; the University of Tübingen; and the Astellas European Foundation.