Transplantation of mesenchymal stem cells for prevention of acute myocardial infarction induced heart failure: Study protocol of a phase III randomized clinical trial

Armin Attar (  attar_armin@yahoo.com ) Shiraz University of Medical Sciences https://orcid.org/0000-0002-4133-4870 Ahmad Monabati Shiraz University of Medical Sciences Mohammad Montaseri Shiraz University of Medical Sciences Massoud Vosough Royan Institute for Stem Cell Biology and Technology Seyed Ali Hosseini Shiraz University of Medical Sciences Javad Kojouri Shiraz University of Medical Sciences Alireza Abdi-Ardekani Shiraz University of Medical Sciences Peyman Izadpanah Shiraz University of Medical Sciences Negar Azarpira Shiraz University of Medical Sciences Gholamreza Pouladfar Shiraz University of Medical Sciences Mani Ramzi Shiraz University of Medical Sciences


Introduction
Myocardial infarction (MI) represents a leading cause of mortality worldwide (1). With a reduction in the rate of mortality due to MIs in recent decades, the incidence of heart failure (HF) has been on the rise (2). This incidence ranges between 14 and 36% among those hospitalized due to an acute MI (AMI) (3). HF exerts a considerable effect on healthcare systems in America, accounting for 6 million cases, 300,000 deaths, and roughly 40 billion USD worth of costs every year (4).
Despite the therapeutic efforts (5), post-MI HF still leads to a high rate of morbidities and mortalities (6,7). Although we have been successful in prolonging the life of HF patients and relieving symptoms, we are yet to regenerate the infarcted cardiac tissues. Hence, a gap exists in the literature as restoring the standard histological architecture of the heart should theoretically lead to improved outcomes for patients with MI-induced HF (8). This may be possible using stem cell-based therapies (9).

Cell-based therapy in cardiovascular disease
Toward the close of the 20th century, scientists signaled a new era in cardiovascular disease treatment through preclinical investigations in which skeletal myoblasts (10) and fetal cardiomyocytes (11) were transplanted into ischemic myocardium. Afterward, the intracardiac implantation of bone marrow (BM) cells was assessed in murine MI models (12,13). Human studies commenced following the turn of the century, with skeletal myoblasts being used in HF patients in 2001 (14) and BM cells being used for AMI patients in 2002 (15). From then on, numerous investigations have aimed to amend the cardiovascular damage caused by diseases like MI and cardiomyopathy through the use of different cell-based therapies.

Mesenchymal stem cells (MSCs)
The BM, heart, Wharton's jelly, and adipose tissue are among the prime sources of MSCs (16) (17) (18). MSCs offer ease of isolation, ex vivo growth, in vitro proliferation, and immune-privileged properties, which is why their use is in clinical trials is expanding rapidly (19). According to the POSEIDON clinical trial on MSC transplantation, allogeneic MSCs are safe and as effective as autologous MSCs (20). Notably, the TAC-HFT trial revealed the two-fold effectiveness of MSCs relative to BM-derived mononuclear cells (BM-MNCs) (21). Accordingly, MSCs appear to be an excellent candidate for cardiac regeneration trials.

Cell-based therapy in acute myocardial infarction (AMI)
To date, BM-MNCs have been used in the majority of research on cell-based therapy following AMI. The TIME trials established that the optimal time for cell implantation following AMI is within 3-7 days (22,23). Fisher et a., in a meta-analysis, proved that BM-MNCs augment the left ventricular ejection fraction (LVEF) following AMI by roughly 2.72%, yielding bene ts both in terms of survival and function in AMI patients younger than 55 years of age with LVEF < 37% (24).
Trials involving the use of MSCs in patients following AMI have shown promising yet controversial results. Gao and coworkers conducted the largest clinical trial in this regard with 116 patients, demonstrating that umbilical cord-derived Wharton's jelly MSCs (WJ-MSCs) led to an almost ve percent improvement in the LVEF (25). This gure was held slightly lower at 3.84% in a related meta-analysis (26). These are in agreement with the ndings of the TAC-HFT trial, which indicated the roughly two-fold effectiveness of MSCs relative to BM-MNCs (21).

BAMI trial
For over two decades, autologous cell-based treatments have been assessed in managing cardiovascular diseases through preclinical and clinical studies. However, phase III trials have been infrequent. Furthermore, the phase II trials have involved different methodologies in terms of the type of stem cells and the method and timing of delivery.
The BAMI trial was the rst phase III trial conducted to clarify whether or not post-MI intracoronary transplantation of BM-MNCs would reduce all-cause mortality. Although the trial was designed to involve 3000 patients, it was stopped prematurely due to futility after the enrollment of 375 patients. Among them, 185 received BM-MNCs (intracoronary infusion) 2-8 days following primary percutaneous coronary intervention (PPCI), and the remaining 190 patients received optimal medical therapy as the control group. All-cause mortality after two years was 3.26% [n = 6; 95% con dence interval (CI): 1.48-7.12%] with BM-MNCs compared to 3.82% (n = 7; 95% CI: 1.84-7.84%) with optimal medical therapy. The main reason behind such results was a signi cant reduction in post-AMI mortality. At the start of the project in 2011, the literature held that following an AMI, the mortality rate from all causes after two years would be approximately 12% among those with an LVEF ≤ 45% post-reperfusion therapy (3). However, the researchers noticed a 3.85% mortality rate while conducting the study, re ecting the evolution of primary angioplasty procedures in those years. Importantly, the investigators noticed that only ve patients (2.7%, 95% CI: 1.0-5.9%) who received BM-MNCs were hospitalized due to HF during the two years of follow-up compared with 15 patients (8.1%, CI: 4.7-12.5%) who received optimal medical therapy (HR: 0.33, 95% CI: 0.12-0.88), representing the sole clinical bene t observed. BAMI showed us that taking mortality as an endpoint for stem cell therapy trials is futile, and the best clinical endpoint to assess is HF incidence.

Hypothesis generation
Since the e cacy of MSCs is higher than BM-MNCs after AMI in the improvement of LVEF, it would be probable that these cells may have a better clinical effect as well. However, no study has evaluated the impact of the transplantation of MSCs on a clinical endpoint such as HF.

Study design
A randomized, multicenter, single-blinded phase III trial will be conducted to assess whether the intracoronary infusion of umbilical cord WJ-MSCs demonstrates a superior effect in reducing HF incidence following AMIs compared to standard treatment. The Ethics Committee of Shiraz University of Medical Sciences approved the study protocol (code: IR.SUMS.REC.1400.409). The trial is registered with https://clinicaltrial.gov under the code NCT05043610. This protocol was conceived following the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) guidelines (Online supplement 1). Figure 1 depicts the SPIRIT ow diagram of the study.

Sample size
In line with the related literature, we made the following assumptions in determining the sample size required considering the aims of this phase III randomized clinical trial: 5% error, 80% power, a one-year HF incidence rate of 1.3-4%, and division between two groups in a 1:1 ratio. Using the formula , 272 people in each group were estimated for a follow-up period of two years and 108 people in each group for four years or 328 patients in 2:1 ratio (control/case) for 3 years. This sample size is only to compare the incidence rate based on the hypothesis test. If we intend to use the Cox regression model, ten people should be added for each independent variable.

Study participants
A total of 360 patients with a history of an anterior ST-elevation MI (STEMI) treated successfully with PPCI 3-7 days earlier will be enrolled. Patients must be below 65 years old and must have severely impaired function of the left ventricle (LV), represented by an LVEF of < 40%. The participating hospitals will be the Al-Zahra Heart, Namazee, and Faghihi hospitals of Shiraz, Iran.
The inclusion criteria are as follows: . Written informed consent The exclusion criteria are as follows: 1. A history of any prior cardiac conditions (valvular, ischemic, or congenital disorders) 2. Regional wall motion abnormalities outside the region of the infarction 3. LV dysfunction due to other etiologies like non-ischemic cardiomyopathy, anthracycline use, or ethanol abuse (> 6 oz./day regularly) 4. Poor echocardiography window 5. Active infection, malignancy, or autoimmune disease Randomization and blinding Randomization will be done via permuted block randomization through a web-based service. A block size of 4 will be considered. Two groups of with 2:1 proportion will be formed, where only one will receive an intracoronary infusion of WJ-MSCs besides the conventional therapy provided to both groups. Those who assess the study outcomes will remain unaware of the allocation (single-blind).

Intervention
This study will use cGMP-certi ed clinical-grade human WJ-MSCs (Cell Tech Pharmed Co. Ltd., Tehran, Iran). The cells will be transferred to the hospital on the same day as the infusion and are to be suspended in normal saline (0.9%). Each batch will be analyzed and certi cated by a quali ed individual, ensuring that the cells comply with the product speci cations. Standard operational protocols will be followed during shipment and handling.
In the intervention group, all 120 patients will receive a single intracoronary infusion of 10 7 WJ-MSCs alongside the conventional treatment that will be provided to the same number of patients in the control group. Patients in the intervention group will be taken to the cardiac catheterization lab, where the infusion of 10 7 WJ-MSCs will be done through the intracoronary route. A bolus heparin dose (weightbased) is to be administered to patients with an activated clotting time below 200 seconds.
For catheterization, we will insert a therapeutic 6 Fr guiding catheter into the left coronary artery. Following the infusion of nitroglycerin (200 µg) through the catheter, we will assess the left anterior descending (LAD) artery and document the TIMI ow. A 0.014-inch soft-tipped guidewire wire will be inserted into the LAD at the distal edge of the stent. After passing through an over-the-wire balloon to the stented area, the balloon will be in ated until achieving occlusion. Following the removal of the guiding wire, we will connect an infusion syringe to the infusion catheter. WJ-MSCs will be infused at a rate of 2.5 ml/min. Low-pressure in ation (2-4 bar) will be performed to achieve occlusion with the balloon catheter, with complete coronary artery occlusion being ensured ahead of cell infusion through the use of dye. After the infusion of each third of the cells, we will pause the infusion, and TIMI coronary ow will be assessed with the contrast agent before the resumption of cell infusion. Once the cells are delivered across the three portions, we will place the coronary ow wire via the microinfusion catheter.

Follow-up and endpoints
Patients will receive daily visits from a cardiologist during hospitalization. The results of all physical examinations will be recorded, and patients will be monitored for early manifestations signaling arrhythmia, pulmonary embolism, or coronary artery injury. Blood tests will be done to measure fasting blood sugar, complete blood count, C-reactive protein, urea and electrolytes, liver function test, creatine kinase, and cardiac troponin T. An electrocardiogram (ECG) will also be obtained. Prior to the MSC infusion process, the cardiac evaluation will be completed using cardiac magnetic resonance imaging and echocardiography. The initial EF will be established according to the wall motion score and Simpson's rule. After the MSCs are delivered, once stable, a beta-blocker, angiotensin-converting enzyme (ACE) inhibitor, aldosterone antagonist, aspirin, ticagrelor, statin, and glyceryl trinitrate (spray or tablets) will be prescribed for the patient to use at home. A cardiac rehabilitation program will also be completed. Subsequent visits will be at ten days after discharge and then every three months, where an ECG and blood tests will be requested. Echocardiography will be done during the six-month follow-up and the nal visit, facilitating the evaluation of LV systolic function.
Our primary endpoint to assess the e cacy of the intervention will be the incidence of HF. Secondary endpoints include the improvement in LV function (through calculation of LVEF) after six months and after three years alongside echocardiographic changes in the left ventricular mass, left ventricular enddiastolic volume, left ventricular end-systolic diameter, and global longitudinal strain (measured via automated formulas in standard views) indices.

Adjudication of study measures
Before statistical analysis, adjudication of all measurements will be done by an experienced cardiology department member excluded from the research group. The adjudicator will assess the quality of each measurement and will exclude those with inadequate quality from the analysis, where they will be regarded as missing. An independent, blinded safety committee will evaluate potential major adverse cardiac events (MACEs). Once the adjudication process is complete, the nalized database will be unblinded.

Statistical analysis
Data will be kept anonymous until analysis, which is to be performed by an independent statistician external to the research group. Treatment e cacy will be assessed according to the decrement in HF with the help of Cox regression analysis. We will consider the EF to have improved signi cantly if a minimum increment of 3% is achieved after six months. The analysis will follow the intention-to-treat approach. The baseline characteristics of the two study groups will also be compared. Continuous variables will be summarized using the mean and standard deviation, while frequencies and percentages will be given for categorical data. The EF, as the primary outcome, will be compared between the study groups using the independent t-test and one-way analysis of variance (ANOVA). The therapeutic effect will be estimated with a 95% CI. Two-sided P-values will be used. Safety will be compared between the two groups according to the occurrence of MACEs (death, recurrent AMI, ICD insertion, non-target vessel revascularization, etc.) and serious adverse events (SAEs). These events will be followed over time with Kaplan-Meier curves, which will allow us to understand their patterns. With the help of the Cox proportional hazards model, we will assess the statistical signi cance and 95% CI.

Adverse events
Adverse events will be reported by the study's executive committee to an independent Data and Safety and Monitoring Board (DSMB). The DSMB will have the authority to stop the trial early if patient safety is compromised or if the primary research objective is met. All safety issues (unanticipated SAEs, mortality, intracoronary infusion complications, and severe arrhythmias, etc.) will be monitored by the DSMB, and the DSMB statistician will report the occurrence of safety issues in each study group quarterly. All deaths will be reported.

Ethical considerations
We discussed all ethical issues with the Institutional Review Board of Shiraz University of Medical Sciences, which ultimately approved the study protocol (IR.SUMS.REC.1400.409). Informed consent will be obtained once patients are clinically stable and sedatives or strong analgesics do not alter their consciousness. Importantly, the use of low balloon in ation pressure and divided (three-part) infusions will prevent complications related to intracoronary cell infusion. The principles of the Declaration of Helsinki will be upheld throughout this study.

Dissemination
Data will be collected until Nov 2024. Thereafter, the analysis will be conducted. Results are expected to be ready by Dec 2024. We will prepare and submit the related manuscript in accordance with the SPIRIT guidelines. This study is registered with clinicaltrials.gov under the code NCT05043610.

Trial Status
We began enrolling patients in Jan 2021 and expect to nish the recruitment process by Nov 2022. This protocol (version 1) was approved in Oct 2020.

Study design
A randomized, multicenter, single-blinded phase III trial will be conducted to assess whether the intracoronary infusion of umbilical cord WJ-MSCs demonstrates a superior effect in reducing HF incidence following AMIs compared to standard treatment. The Ethics Committee of Shiraz University of Medical Sciences approved the study protocol (code: IR.SUMS.REC.1400.409). The trial is registered with https://clinicaltrial.gov under the code NCT05043610. This protocol was conceived following the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) guidelines (Online supplement 1). Figure 1 depicts the SPIRIT ow diagram of the study.

Sample size
In line with the related literature, we made the following assumptions in determining the sample size required considering the aims of this phase III randomized clinical trial: 5% error, 80% power, a one-year HF incidence rate of 1.3-4%, and division between two groups in a 1:1 ratio. Using the formula , 272 people in each group were estimated for a follow-up period of two years and 108 people in each group for four years or 328 patients in 2:1 ratio (control/case) for 3 years.
This sample size is only to compare the incidence rate based on the hypothesis test. If we intend to use the Cox regression model, ten people should be added for each independent variable.

Study participants
A total of 360 patients with a history of an anterior ST-elevation MI (STEMI) treated successfully with PPCI 3-7 days earlier will be enrolled. Patients must be below 65 years old and must have severely impaired function of the left ventricle (LV), represented by an LVEF of < 40%. The participating hospitals will be the Al-Zahra Heart, Namazee, and Faghihi hospitals of Shiraz, Iran.
The inclusion criteria are as follows:

Randomization and blinding
Randomization will be done via permuted block randomization through a web-based service. A block size of 4 will be considered. Two groups of with 2:1 proportion will be formed, where only one will receive an intracoronary infusion of WJ-MSCs besides the conventional therapy provided to both groups. Those who assess the study outcomes will remain unaware of the allocation (single-blind).

Intervention
This study will use cGMP-certi ed clinical-grade human WJ-MSCs (Cell Tech Pharmed Co. Ltd., Tehran, Iran). The cells will be transferred to the hospital on the same day as the infusion and are to be suspended in normal saline (0.9%). Each batch will be analyzed and certi cated by a quali ed individual, ensuring that the cells comply with the product speci cations. Standard operational protocols will be followed during shipment and handling.
In the intervention group, all 120 patients will receive a single intracoronary infusion of 10 7 WJ-MSCs alongside the conventional treatment that will be provided to the same number of patients in the control group. Patients in the intervention group will be taken to the cardiac catheterization lab, where the infusion of 10 7 WJ-MSCs will be done through the intracoronary route. A bolus heparin dose (weightbased) is to be administered to patients with an activated clotting time below 200 seconds.
For catheterization, we will insert a therapeutic 6 Fr guiding catheter into the left coronary artery. Following the infusion of nitroglycerin (200 µg) through the catheter, we will assess the left anterior descending (LAD) artery and document the TIMI ow. A 0.014-inch soft-tipped guidewire wire will be inserted into the LAD at the distal edge of the stent. After passing through an over-the-wire balloon to the stented area, the balloon will be in ated until achieving occlusion. Following the removal of the guiding wire, we will connect an infusion syringe to the infusion catheter. WJ-MSCs will be infused at a rate of 2.5 ml/min. Low-pressure in ation (2-4 bar) will be performed to achieve occlusion with the balloon catheter, with complete coronary artery occlusion being ensured ahead of cell infusion through the use of dye. After the infusion of each third of the cells, we will pause the infusion, and TIMI coronary ow will be assessed with the contrast agent before the resumption of cell infusion. Once the cells are delivered across the three portions, we will place the coronary ow wire via the microinfusion catheter.
Patients will receive daily visits from a cardiologist during hospitalization. The results of all physical examinations will be recorded, and patients will be monitored for early manifestations signaling arrhythmia, pulmonary embolism, or coronary artery injury. Blood tests will be done to measure fasting blood sugar, complete blood count, C-reactive protein, urea and electrolytes, liver function test, creatine kinase, and cardiac troponin T. An electrocardiogram (ECG) will also be obtained. Prior to the MSC infusion process, the cardiac evaluation will be completed using cardiac magnetic resonance imaging and echocardiography. The initial EF will be established according to the wall motion score and Simpson's rule. After the MSCs are delivered, once stable, a beta-blocker, angiotensin-converting enzyme (ACE) inhibitor, aldosterone antagonist, aspirin, ticagrelor, statin, and glyceryl trinitrate (spray or tablets) will be prescribed for the patient to use at home. A cardiac rehabilitation program will also be completed. Subsequent visits will be at ten days after discharge and then every three months, where an ECG and blood tests will be requested. Echocardiography will be done during the six-month follow-up and the nal visit, facilitating the evaluation of LV systolic function.
Our primary endpoint to assess the e cacy of the intervention will be the incidence of HF. Secondary endpoints include the improvement in LV function (through calculation of LVEF) after six months and after three years alongside echocardiographic changes in the left ventricular mass, left ventricular enddiastolic volume, left ventricular end-systolic diameter, and global longitudinal strain (measured via automated formulas in standard views) indices.

Adjudication of study measures
Before statistical analysis, adjudication of all measurements will be done by an experienced cardiology department member excluded from the research group. The adjudicator will assess the quality of each measurement and will exclude those with inadequate quality from the analysis, where they will be regarded as missing. An independent, blinded safety committee will evaluate potential major adverse cardiac events (MACEs). Once the adjudication process is complete, the nalized database will be unblinded.

Statistical analysis
Data will be kept anonymous until analysis, which is to be performed by an independent statistician external to the research group. Treatment e cacy will be assessed according to the decrement in HF with the help of Cox regression analysis. We will consider the EF to have improved signi cantly if a minimum increment of 3% is achieved after six months. The analysis will follow the intention-to-treat approach. The baseline characteristics of the two study groups will also be compared. Continuous variables will be summarized using the mean and standard deviation, while frequencies and percentages will be given for categorical data. The EF, as the primary outcome, will be compared between the study groups using the independent t-test and one-way analysis of variance (ANOVA). The therapeutic effect will be estimated with a 95% CI. Two-sided P-values will be used. Safety will be compared between the two groups according to the occurrence of MACEs (death, recurrent AMI, ICD insertion, non-target vessel revascularization, etc.) and serious adverse events (SAEs). These events will be followed over time with Kaplan-Meier curves, which will allow us to understand their patterns. With the help of the Cox proportional hazards model, we will assess the statistical signi cance and 95% CI.

Adverse events
Adverse events will be reported by the study's executive committee to an independent Data and Safety and Monitoring Board (DSMB). The DSMB will have the authority to stop the trial early if patient safety is compromised or if the primary research objective is met. All safety issues (unanticipated SAEs, mortality, intracoronary infusion complications, and severe arrhythmias, etc.) will be monitored by the DSMB, and the DSMB statistician will report the occurrence of safety issues in each study group quarterly. All deaths will be reported.

Ethical considerations
We discussed all ethical issues with the Institutional Review Board of Shiraz University of Medical Sciences, which ultimately approved the study protocol (IR.SUMS.REC.1400.409). Informed consent will be obtained once patients are clinically stable and sedatives or strong analgesics do not alter their consciousness. Importantly, the use of low balloon in ation pressure and divided (three-part) infusions will prevent complications related to intracoronary cell infusion. The principles of the Declaration of Helsinki will be upheld throughout this study.

Dissemination
Data will be collected until Nov 2024. Thereafter, the analysis will be conducted. Results are expected to be ready by Dec 2024. We will prepare and submit the related manuscript in accordance with the SPIRIT guidelines. This study is registered with clinicaltrials.gov under the code NCT05043610.

Trial Status
We began enrolling patients in Jan 2021 and expect to nish the recruitment process by Nov 2022. This protocol (version 1) was approved in Oct 2020.

Discussion
Currently, the primary focus of post-AMI treatment is to prevent remodeling and avert any further loss of myocytes (5). However, a revolution can potentially be achieved by regenerative medicine, aiming to restore cardiac function by inhibiting and even reversing the process of remodeling through the use of stem cells (25). Despite the fact that some investigations were not very promising in this regard (9), other studies have shown that stem cell therapy may be of value in certain populations.
Although a Cochrane meta-analysis revealed that the LVEF of young AMI patients does not increase following BM-MNC therapy, survival and functional bene ts may be present (24). Importantly, research with MSCs has yielded more promising results, with the TAC-HFT trial indicating the roughly two-fold higher e cacy of MSCs relative to BM-MNCs (21). According to meta-analyses of the various clinical trials, MSCs can improve the LVEF by 3.84% (26), while BM-MNCs can achieve an inferior improvement of 2.72% (24).
Currently, scientists are yet to understand the exact mechanisms behind the therapeutic impact of stem cells, especially MSCs. However, the most commonly suggested mechanism is paracrine signaling, where the implanted stem cells alter the activity of the nearby cells in the heart via mediators like cytokines (27).
Also, it is still unclear whether or not the mechanical improvements in LV function after MSC transplantation would be translated into a clinical bene t by reducing major cardiovascular events. Our trial provides essential insights into the eld by including selected patients who develop reduced LVEFs after AMIs.

Conclusions
Our trial by, enrolling 360 patients, would be the largest clinical trial ever conducted in the eld of regenerative medicine in cardiology. We hope it would help clarify whether MSC transplantation is clinically useful or not.

List Of Abbreviations
Acute

Declarations
Ethics approval and consent to participate: We discussed all ethical issues with the Institutional Review Board of Shiraz University of Medical Sciences, which ultimately approved the study protocol (IR.SUMS.REC.1400.409). Informed consent will be obtained once patients are clinically stable and sedatives or strong analgesics do not alter their consciousness.
Consent for publication: Written consent will be obtained from all patients for publication of their data following deidenti cation.
Data availability: Data will be made available upon reasonable request following the completion of the study.
Competing interests: M. V. is the regulatory affairs manager in Cell Tech Pharmed. Cell Tech Pharmed Co. will provide us with cGMP grade WJ-MSCs but is not involved in the design or interpretation of the research. Authors' contributions: All authors are involved in data collection, manuscript preparation, and critical revision. A. A. and A. M. designed the study. M. V. will provide the project with stem cells. All authors accept responsibility and accountability for the whole project. Figure 1 SPIRIT ow diagram of the study.