Allogeneic human umbilical cord-derived mesenchymal stem cells for severe bronchopulmonary dysplasia in children: Study protocol for a randomized controlled trial(the MSC-BPD Trial)

Background: Bronchopulmonary dysplasia (BPD) is a complex lung pathological lesion secondary to multiple factors and one of the most common chronic lung diseases with a poor prognosis, especially in preterm infants. However, effective therapies for this disease are lacking. Stem cell therapy is a promising way to improve lung injury and abnormal alveolarization, and the human umbilical cord (hUC) is a good source of mesenchymal stem cells (MSCs), which have demonstrated efficacy in other diseases. We hypothesized that intravenous allogeneic hUC-MSCs are safe and effective for severe BPD. Methods: The MSC-BPD trial is a randomized single-center open-label dose-escalation phase II trial designed to investigate the safety and efficacy of hUC-MSCs in children with severe BPD. In this study, 72 patients will be enrolled and randomly divided into two intervention groups and one control group. Patients in the intervention groups will receive a low dose of hUC-MSCs (n = 24; 2.5 million cells/kg) or a high dose of hUC-MSCs (n = 24; 5 million cells/kg) in combination with traditional supportive treatments for BPD. The patients in the control group (n = 24) will be treated with traditional supportive treatments alone without hUC-MSCs. The primary outcome measures will be cumulative duration of oxygen therapy. Follow-up assessments will be performed at 1, 3, 6, 12, and 24 months post-intervention, and the key outcome during follow-up will be changes on chest radiography. Statistical analyses will evaluate the efficacy of the hUC-MSC treatment. Discussion: This will be the first randomized controlled trial to evaluate the safety and efficacy of intravenous hUC-MSCs in children with severe BPD. Its results will provide a new evidence-based therapy for severe BPD. umbilical cord mesenchymal stem cells; LIA-AEs, late infusion–associated adverse events; MSCs, mesenchymal stem cells; NICHD, and Human SAEs, severe adverse events; UC, umbilical cord.

Background Bronchopulmonary dysplasia (BPD) is a common chronic lung disease whose incidence is increasing annually, especially in this time of the two-child policy in China [1,2]. Patients with BPD usually have ventilator or oxygen dependence during the early stage of the disease [3]. Most patients can gradually withdraw from the ventilator or stop oxygen treatment at different times depending on disease severity, but abnormalities in pulmonary structure and lung function may last until late childhood even adulthood, especially in those who are diagnosed as severe BPD according to the diagnostic criteria of the National Institute of Child Health and Human Development (NICHD) [4,5]. The mortality of overall BPD patients is about 15% [6]; nevertheless, the mortality rate of severe BPD reaches 41%, creating an enormous threat to the health of these children [4,7]. More than 50% of survivors with BPD experience hospital readmission for repeated lower respiratory infections in the first year or two, which causes serious economic and labor burdens on families [4,8].Although surfactant treatment [9], prenatal steroid usage [1,10], ventilator strategies [11], and improved nutrition [12] are used in BPD patients, effective therapies are lacking [14]. Therefore, identifying novel effective therapies for severe BPD in children is urgent and significant.
In recent years, the rapid development of stem cell technology and regenerative medicine has identified stem cells as potential treatments for various refractory diseases, which are difficult to treat by traditional medical methods, such as degenerative diseases, cancer, and tissue damage [15][16][17].Mesenchymal stem cells (MSCs) are a class of adult stem cells derived from the mesoderm with   characteristics of non-tumorigenic, low immunogenicity, and powerful paracrine   function, and can be isolated from several sources, including bone marrow, human umbilical cord (UC), adipose tissue, amniotic fluid, and other tissues [18,19].
Among them, hUC-derived mesenchymal stem cells (hUC-MSCs) are useful in clinical application because they are easy to obtain, more proliferative, and have more powerful paracrine function than any other sources; they are effective at relieving lung inflammation, fibrosis, angiogenesis, and apoptosis [20][21][22][23][24]. Interestingly, the therapeutic potential of hUC-MSCs in several animal pulmonary diseases models, including BPD, acute lung injury, and idiopathic pulmonary fibrosis, were confirmed [22,25,26]. Approximately 75% of hUC-MSCs reportedly accumulate in the microvessels of the lungs [27]. Hence, the therapeutic potential of hUC-MSCs for severe BPD in children requires investigation.
A multicenter dose-escalation phase I clinical trial (NCT01775774) investigated the safety and efficacy of bone marrow-derived MSCs for moderate to severe acute respiratory distress syndrome (ARDS) in adults without any adverse events (AEs) related to infusion reported, which had potential efficacy [28]. The study reported that hUC-MSC treatment was safe in patients with moderate to severe chronic obstructive pulmonary disease (COPD) (NCT00683722) [29]. Another phase I trial reported treating 9 BPD patients in Korea with hUC blood-derived mesenchymal stem cells (hUCB-MSCs) (NCT01297205) [30]. The follow-up data from this study showed the safety of hUCB-MSC administration and the ability to reduce the level of profibrotic factors in tracheal aspirates [31].

Study objectives
The goal of this clinical trial is to test the safety and efficacy of hUC-MSCs in children with severe BPD. There are three specific objectives: Evaluate the long-term safety and efficacy of intravenous hUC-MSCs in children with severe BPD; Test the hypothesis that the administration of hUC-MSCs can reduce the duration of mechanical ventilation and oxygen and improve impairment of the pulmonary structure in children with severe BPD; and Explore the potential therapeutic mechanism of hUC-MSCs for severe BPD.

Study design and setting
The MSC-BPD trial (registered at www.clinicaltrials.gov [no. NCT03601416]) is a randomized single-center dose-escalation phase II trial aiming to evaluate the safety and efficacy of hUC-MSCs in a total of 72 children with severe BPD. A study flow chart of the trial is shown in Fig. 1. The study protocol will be reported based on SPIRIT guidelines (Additional file 1).
This trial will be conducted at the Children's Hospital of Chongqing Medical University (CHCMU) in Chongqing, China.

Sample size and calculation
The sample size of this phase II randomized controlled trial was calculated by power analysis using the Power and Sample Size online calculator (http://powerandsamplesize.com/). The trial is designed to investigate the hypotheses of two interventions compared to control, but it is not powered to test differences between the two intervention groups. The larger of the two numbers is the sample size of this trial. The primary outcome measure is the change in the cumulative duration of oxygen therapy. The cumulative mean duration of severe BPD is reportedly 90 ± 15 d [4,13]. This trial is powered to identify differences of 15% (from 90 d in the control group to 77 d in the low dose groups) and 20% (from 90 d in the control group to 72 d in the high dose groups) in participants who accept the hUC-MSC intervention. Meanwhile, the power is set at 0.8, type I error ɑ is 0.05, and type II error β is 0.20. The calculated sample size is 21 for each group. To account for the possibility of 10% of patients being lost to follow-up, the final sample size will be 24 and the total size will be 72.

Patient and public involvement
The participants will not be involved in the development of the trial including the study design, recruitment, and conduct, selection of research question, and outcome measures. The participation will be voluntary and the participants will have freedom to participate or withdraw from this trial at any time throughout the study.
The participants' privacy will be protected.
Participants will be enrolled in this study according to the inclusion and exclusion criteria ( Table 1). The parents or guardians of all participants will provide the written informed consent form approved by the Ethics Committee of Stem Cell Clinical Research of CHCMU.

Inclusion and exclusion criteria
The inclusion and exclusion criteria are listed in Table 1. Participants will be 0-1 year of age. The diagnostic criteria and BPD severity gradation refers to the criteria established by the NICHD workshop [5]. The Silverman and Andersen score is used to assess the severity of abnormal respiratory manifestations [32].

Recruitment
Patients can only be enrolled in this study after passing the citywide consultation resolution and signing the informed consent form.
Participants will be recruited from three sources. First, the parents of potentially eligible hospitalized patients diagnosed with severe BPD will be approached and asked to join this study. Second, physicians will generate lists of patients with a diagnosis of BPD who were discharged within 1 year from the electronic medical records of CHCMU. Investigators or physicians will contact the patients' parents by phone or mail them a research leaflet and recruitment letter. Third, physicians will post study flyers at the outpatient department, the official website, and the WeChat public platform of CHCMU for those diagnosed with severe BPD at other hospitals. If the parents of these patients with severe BPD are interested in this research, we will initiate the screening process.
A multidisciplinary consultation will be held to confirm whether these potential participants meet the general diagnostic criteria of BPD as well as the inclusion and exclusion criteria. The consultation will consist of a neonatologist, a respiratory physician, a radiologist, a laboratory physician, a Department of Critical Care Medicine expert, stem cell treatment center researchers, and medical department staff. If more than 80% experts agree on the hUC-MSCs treatment, these patients will be viewed as potential participants. The researcher will then arrange a meeting to communicate with the legal representative or parents about the clinical trial research detail and sign the written informed consent form.
The following details of the clinical trial will be fully explained to the patients' guardians as follows: (1) study purpose; (2) research background; (3) number of participants and duration of their participation; (4) study procedures; (5) potential discomfort and risks of treatment; (6) expected benefits; (7) protection of confidentiality and privacy; and (8) their participation is voluntary. Each patient's legal representative or parents will sign the informed consent form after all the items above are fully understood. After that, the patients' baseline characteristics will be recorded by the clinicians ( Table 2).

Randomization and blinding
Participants will be randomized into three groups in a 1:1:1 ratio after collection of the baseline data. The allocation sequence will be generated and sent to the investigators by a statistician. Participants will not be blinded during the phase II trial and the patients in the control group will not be given hUC-MSC treatment.

Intervention
The hUC-MSCs produced by Ever Union Biotechnology Co. Ltd. (EUBIO) are transported to the ward on the infusion day. The hUC-MSCs are suspended in 0.9% normal saline. In addition to inspecting the quality of the hUC-MSC product by EUBIO, the staff of the stem cell center in CHCMU will confirm the viability and quality of the hUC-MSC product before the infusion.
There is currently no effective therapy for BPD patients, who are often given traditional supportive treatments such as nutritional support, fluid restriction, and respiratory support (including ventilator support and oxygen supply) so all participants will be given traditional supportive treatments to ensure safety. Thus, the intervention groups will be given the traditional supportive treatments and extra low-or high-dose hUC-MSC infusion and the control group will be given only the traditional supportive treatment. Participants will be unable to use glucocorticoids 3 days before or after the hUC-MSC treatment.
A total of 72 patients in the intervention groups will be randomized in a 1 An insurance policy will be prepared for all participants, who will be provided with ancillary and post-trial care in the case of injury or death as a result of their participation in the trial.

Outcome evaluation
The outcome measures and their time frames of this trial are listed in Table 3. The primary endpoint is the cumulative duration of oxygen therapy, i.e. the duration from starting to stopping oxygen treatment.
The secondary endpoints include the safety and efficacy outcomes. The safety of the study will be assessed by the number of AEs including SAEs, acute infusion-

Follow-up procedures
Follow-up assessments will be performed at 1, 3, 6, 12, and 24 m after the hUC-MSC injection ( Table 2). The five follow-up points will be conducted through telephone and outpatient contact. The first two follow-ups will be performed at 1 and 3 m after the hUC-MSC treatment by phone to ask the parents of the condition of their child(ren). The details of the phone interview are shown in Table 4. The next three follow-ups will be performed at 6, 12, and 24 m at outpatient visits as shown in Table 5. The key outcome during follow-up will be changes on chest radiographs.

Safety monitoring
The independent Data and Safety Monitoring Board (DSMB) will supervise safety during the trial. The members of DSMB are independent of the sponsor and trial investigators and have no competing interests. The DSMB will review and evaluate clinical safety and efficacy data collected according to the specified time intervals in the protocol. If the threshold of the safety data exceeds a predefined threshold, the DSMB will be notified. Furthermore, the DSMB will conduct the interim analysis of all AE occurrences every 6 months during the study. Only the data managers and study designers have access to the data in the trial. The data will be locked by the data management team when the trial is completed. All of the data will be provided to the DSMB. If the trial is terminated earlier than the expected end date, the DSMB will contribute to that decision.

Data collection
The data generated during the trial will be recorded in the original medical record and the CRF. The quality control personnel will check the consistency of the CRF data with the original record to ensure that the data are accurately entered into the CRF. There are nine data collection points: baseline, 1 d, 3 d, 7 d, 1 m, 3 m, 6 m, 12 m, and 24 m ( Table 2). Within 3 days after completion of the data collection, the research records will be submitted to the research leader for review and all data will be submitted to the project leader within 10 days. Next, the auditor will review each original research record to confirm that the clinical trial data records are timely, precise, and standardized. Data checks and entries will then be disposed by the statistical data manager and analyzed by the statisticians.
The data of this trial will be disseminated through national and international conferences and peer-reviewed publications. Our data set will be available after the trial's completion.
Statistical analysis SPSS version 17 (SPSS Inc., Chicago, IL, USA) statistical analysis software will be used to analyze the data in the study. Significant differences will be considered at an α level of 0.05.
The data will be examined at group assignment to the intervention and control groups using the χ2 test, t-test or ANOVA. For the intervention and control groups, between the intervention and control groups will be conducted using the t-test.
Mortality and hospital readmission rates will be tested by the χ2 test, while 24month mortality will be analyzed by Kaplan-Meier curves.  Declarations the trial and collecting the data. XHL will be responsible for the data integrity and analysis.

Discussion
Ethics approval and consent to participate