Intracoronary infusion of Wharton’s jelly-derived mesenchymal stem cells in acute myocardial infarction: double-blind, randomized controlled trial

Patients with ST-elevation AMI were admitted to cardiology centers in 11 hospitals in China between February 2011 and January 2012. A total of 160 subjects were enrolled in the trial. For inclusion in the study, patients had to satisfy the following eligibility criteria: 18- to 80-years old; a first ST-segment elevation MI; successful reperfusion with stent implantation of the infarct-related artery within 12 hours after the onset of symptoms; a substantial residual LV regional wall-motion abnormality (three or more hypokinetic LV segments observed on an echocardiograph after percutaneous coronary intervention, PCI); and creatine kinase (CK)-MB levels over three-fold the upper limit of the reference values. Exclusion criteria included previous Q-wave MI and severe coexisting conditions, such as advanced renal or hepatic dysfunction, and documented terminal illness or cancer. All subjects were administered medications according to the current updated ACC/AHA/SCAI guidelines along with standard rehabilitation programs for MI [27].

The study protocol conformed to the Declaration of Helsinki and was approved by the ethics committee of Navy General Hospital. All subjects signed written informed consent for enrollment in the study and treatment. The trial was monitored by an independent data and safety monitoring board (DSMB) who met every two months and as needed to assess adverse events.

The eligible patients were assigned randomly to each of two groups (WJMSCs or placebo control) in a 1:1 fashion using a computer-generated randomization of sequence numbers (Fig. 1). Physicians and other clinical personnel remained blind to the treatment assignment throughout the study. Day 0 was defined as the day of PCI. During days 5–7, all subjects were assigned randomly into either the WJMSC group, receiving 6 × 10⁶ WJMSCs through intracoronary infusion as described previously [28], or the placebo group, with a placebo injected via the same delivery method as that of the WJMSC group. Safety was evaluated on days 0 and 3, as well as 1, 4, 12 and 18 months post-treatment. Cardiac nuclear studies were performed pre-treatment and at four months post-treatment. Two-dimensional echocardiograms were measured before cell transfer and 4, 12 and 18 months after cell transplantation or placebo infusion.

The primary end point was safety: the incidence of adverse events (AEs) within 18 months, including death, nonfatal MI, stroke, hospitalization for worsening heart function, severe arrhythmias, repeated coronary intervention, stent thrombosis, coronary artery microvascular obstruction, immune system disorders, or ectopic tissue formation, was monitored and quantified. Laboratory assays, including biochemical assays, hematologic, tumor and immune indexes and Holter monitoring, were performed at the different follow-up times specified above.

The secondary end point was efficacy, which was assessed in terms of the absolute change in myocardial viability and perfusion in the infarcted region from the baseline to 4 months post-treatment, as well as the global LV ejection fraction (LVEF) from baseline to 18 months post-treatment, as measured by F-18-fluorodeoxyglucose positron emission computed tomography (F-18-FDG-PET), 99mTc-sestamibi single-photon emission computed tomography (99mTc-SPECT) and two-dimensional echocardiogram (ECG), respectively.

The protocol of WJMSCs preparation was approved by the General Logistics Department of the PLA and the Navy General Hospital Ethical Review Board. Twenty-one human umbilical cords were obtained, with the consent of the parents, from healthy donors, who had no complications throughout the pregnancy, no history of disease, and a full-term birth by caesarian section, and were aseptically stored in sterile saline and processed within six hours from partum to obtain the umbilical cord. After removal of blood vessels, the abundant extracellular matrix of Wharton’s jelly, which is a mucous tissue continuum from the subamnion to the perivascular region, was scraped off with a scalpel, finely cut and rinsed in sterile phosphate-buffered saline. The WJMSCs were isolated by a non-enzymatic method and cultured as described previously [18]. The WJMSCs were purified in a central cell-processing laboratory following the regulatory guidelines of the International Conference on Harmonization and the US Food and Drug Administration [29]. All procedures were performed and all solutions were prepared under Good Manufacturing Practice (GMP).

The infused WJMSCs were harvested at passage 3, during which ≥95 % of cells expressed CD29, CD73, CD90 and CD105, while the expression of CD45, CD34, CD14, CD79 and HLA-DR was 2 % or less. Released cells were negative for the pathogenic microorganisms HBV, HCV, HIV, cytomegalovirus, syphilis and exhibited ALT and endotoxin levels within 40 IU/L and 0.5 EU/mL, respectively. Final processing incorporated a total cell count and cell viability (≥85 %) determination by trypan blue testing. Based on the results from our animal experiments, we decided on a dose of 6 × 10⁶ WJMSCs by intracoronary transplantation in this trial. In brief, a dose-escalation study for intracoronary delivery of WJMSCs involved 12 pig models of AMI, weighing 28–35 kg, of mixed gender. The dose was escalated at 1, 2, 3, 6 x 10⁶ with a 30-minute interval. A coronary angiogram as well as left ventriculogram was obtained at 15 minutes following each infusion. Blood flow to the distal left anterior descending artery (LAD), measured under fluoroscopy by counting the number of heart beats required to fill this region of the vessel with contrast, was not changed at bolus doses up to 6 × 10⁶ WJMSCs. Significant changes in LV wall motion were revealed until administration of the 3 × 10⁶ dose [30]. The placebo consisted of a vehicle (saline with 10,000 U/L heparin) injected without cells.

The cells were shipped at 4 °C and delivered to each catheterization laboratory at the 11 participating cardiology centers, using a standard operating procedure. After extensive discussion with the enrolled subjects, written informed consent was obtained before initiating treatment. After arterial puncture and administration of 8 000 U heparin, 6 × 10⁶ WJMSCs dispersed in 10 mL heparinized saline (saline with 10,000 U/L heparin), or the placebo, were infused using a stop-flow technique through an over-the-wire balloon catheter positioned within the stent segment as described previously [28]. Contrast medium was injected into the infarct-related artery to ascertain vessel patency after cell infusion.

F-18-FDG- PET (GE Millennum VG Hawkeye, Israel) and 99mTc-sestamibi SPECT (Varicam, GE-Elscint, Haifa, Israel) was performed in patients at one day before and four months after the procedure. After an overnight fast for at least 12 hours, patients were given an oral glucose load according to their serum glucose level. Sequential measurements of serum glucose were made until the serum glucose level reached 7.8 – 8.9 mmol/L and then 18F-FDG (222–296 MBq) and 99mTc-sestamibi (555–740 MBq) were injected intravenously, respectively. Myocardial images were reconstructed using a standard filtered back projection and displayed as a series of short-axis, horizontal and vertical long-axis slices. Mean signal intensities were measured in the respective areas supplied by the three major coronary arteries in three-axis views. Results were calculated using F-18–FDG-PET and 99mTc-sestamibi SPECT bull’s-eye views. All parameters were analyzed independently by two experienced observers who were unaware of the treatment assignment.

Patients who received cell grafts or standard treatment underwent echocardiography consecutively 1–2 days before cell/placebo infusion and at 4, 12 and 18 months follow-up. A 17-segment echocardiogram was performed to measure regional left ventricular wall motion score, end-systolic volume, end-diastolic volume and LVEF by using standard methods of the American Society of Echocardiography [31], and were analyzed independently by two experienced observers who were unaware of patients’ treatment assignments.

The population size of patient enrollment was influenced by the difficulty in selecting eligible patients because the inclusion criteria used in our study selected only those patients with AMI who had undergone primary PCI within 12 hours and who had also agreed to accept a WJMSC intracoronary transplant five days after their first PCI operation. Very few patients were deemed eligible. To address this issue, we expanded the study to include more hospitals. We also performed a statistical analysis specifically to determine whether the study population was normally distributed. To ascertain the efficacy, including PET and SPECT at month 4, and two dimensional-ECG at months 4, 12 and 18, with variables adjusted by baseline values, analysis of covariance (ANCOVA) was performed to assess differences between the placebo and WJMSC treatment groups. To estimate the treatment effect, differences in least-squares means and the corresponding 95 % confidence intervals (CI) were calculated based on the ANCOVA model. The Wilcoxon or Student’s t-test was used to compare changes between the baseline and follow-up values according to the distribution of variables. Categorical variables were analyzed by chi-square or Fisher’s exact test, as appropriate. Continuous variables were expressed as means ± standard errors (SE) unless otherwise stated. Categorical data were presented as frequencies and percentages. All statistical tests were two-sided, and P <0.05 was considered statistically significant. All analyses were performed using SAS software, version 9.3 (SAS Institute, Cary, NC, USA).