The criteria for inclusion and exclusion are summarized in Tables 1 and 2 , respectively. The study aims to enroll 60 men and women, aged 21 to 85 years, diagnosed with ICM, as confirmed by documented coronary artery disease; a left ventricular ejection fraction (LVEF) of ≤40% by magnetic resonance imaging (MRI); and a myocardial scar involving ≥5% the LV mass (infarct volume) with any subendocardial involvement by MRI. Participants must be in New York Heart Association (NYHA) class I-III and be receiving guideline-directed medical therapy (GDMT) at stable doses for ≥1 month before consent. At least 3 months must have elapsed from a percutaneous coronary intervention or implantation of a cardiac resynchronization therapy (CTR) device. Key exclusion criteria include indications for cardiac surgery and/or percutaneous coronary intervention (PCI), severe valvular heart disease, recent stroke, MRI contraindications, HbA1c >10%, and noncardiac conditions projecting a life expectancy of <1 year. The 3 Clinical Centers recruit patients from their clinical practices and the community, with an anticipated randomization and treatment of ∼20 participants per Center. Based on the experience of these Center with previous HF trials CONCERT-HF and SENECA, a 10% dropout is anticipated,

Similar to previous trials of cell therapy in HF, such as RIMECARD, CONCERT-HF and POSEIDON-DCM, CATO includes patients in NYHA class I, II, and III. Intravenous infusion of UC-MSCs has been demonstrated to be safe in many noncardiac conditions ^,, and, despite lack of symptoms, patients with EF <40% still have increased morbidity and mortality, as the underlying cardiomyopathy follows a progressive course. For these reasons, the potential benefits imparted by the anti-inflammatory, antiapoptotic, angiogenic, and antifibrotic actions of UC-MSCs were deemed to outweigh the theoretical and extremely low risk associated with intravenous UC-MSC administration.

CATO is primarily an efficacy study, although safety and feasibility are also evaluated. The primary objective is to assess (1) the efficacy of 4 repeated intravenous infusions of UC-MSCs relative to placebo, and (2) the efficacy of 4 repeated infusions relative to 1 infusion in patients with ICM. Secondary objectives are (1) to assess the safety and feasibility of intravenous infusions of UC-MSCs, and (2) to examine the effects of intravenous infusion of UC-MSCs on the immune system. The 3 primary questions addressed in CATO are: (1) Do 4 intravenous doses of UC-MSCs impart beneficial effects relative to placebo? (2) If so, does 1 dose also impart beneficial effects relative to placebo? (3) If so, are 4 doses superior to 1 dose?

The trial is a Phase IIA randomized, double-blind, placebo-controlled, multicenter study ( Figure 1 ). Briefly, 60 subjects are assigned in a random fashion to 3 groups on a 1:1:1 basis: control (placebo), single dose, and repeated doses. After randomization and baseline testing, subjects receive 4 study product injections (SPIs) via the intravenous route at 2-month intervals. SPIs consist of either 100 × 10 ⁶ UC-MSCs or placebo (Plasma-Lyte A with 1% human serum albumin [HSA]). Subjects in the control group receive 4 doses of placebo; subjects in the single-dose group receive 1 dose of UC-MSCs followed by 3 doses of placebo; subjects in the repeated-dose group receive 4 doses of UC-MSCs ( Figure 1 ).

Schematic flowchart of the CATO trial.

Placebo is infused intravenously at a rate of 1 mL/min for a total of 60 mL over 60 minutes. UC-MSCs (100 × 10 ⁶ cells) are infused intravenously at the same rate (1 mL/min for a total of 60 mL over 60 minutes; 1.67 million cells/mL/min). Subsequent SPIs take place at 2 months (60 ± 7 days) after the previous SPI ( Table 3 ). Patients receive diphenhydramine (25-50 mg) and hydrocortisone (25-50 mg) 15-30 minute before each SPI. After each SPI, subjects are monitored for 2 hours. A 12-lead ECG is obtained before SPI. Subjects are required to maintain a daily temperature log for 7 days after each SPI to assist in early infection detection. Visits occur at 1 week and 2 months after each SPI, with a phone call at 1 month ( Table 3 ). After the second, third, and fourth SPI, the 1-week visit is conducted remotely via telemedicine. The 1-week visit, 1-month phone call, and 2-month follow-up visit are scheduled 7 ± 3, 30 ± 7, and 60 ± 7 days after SPI, respectively. All subjects are then examined at 6 months after the last (fourth) SPI to complete safety and efficacy assessments. All data collections for visits are completed prior to SPI; if necessary, outpatient visits and SPI procedures may span more than 1 day.

Table 3

Schedule of procedures.

1 Chemistry tests: Na ⁺, K ⁺, Cl ^–, HCO ₃ ^– (CO2), glucose, blood urea nitrogen (BUN), creatinine, and eGFR.

2 CBC with differential: WBC, RBC, hemoglobin, hematocrit, MCV, platelets, and differential.

3 Liver function tests (LFTs): albumin, alkaline phosphatase, alanine transaminase (ALT), aspartate transaminase (AST), total bilirubin, direct bilirubin, and total protein.

4 HbA1c will be measured at 2 mo after the first, second, third, and fourth SPI only if the baseline value is >6.5.

5 Infectious disease tests: HBc, HBsAg, HBV, HCV, HIV-1 groups M&O RNA, and Anti-HIV.

6 Cardiac troponin I will be measured at 4-6 h after SPI.

Patients meeting criteria for chronic LV dysfunction secondary to MI, EF ≤40%, and NYHA class I-III without study exclusions undergo baseline testing. The initial baseline testing and the first SPI are scheduled to occur within 60 days of obtaining informed consent, and SPI within 45 days of MRI ( Table 3 ). The randomization is stratified by site. The study maintains a double-blind design, with bias control achieved by securing master randomization lists and utilizing a centralized Core laboratory for MRI analyses to maintain blinding. Although patients with cardiac devices have been traditionally excluded from MRI studies in most HF trials, the experience of CONCERT-HF and SENECA has demonstrated the safety and feasibility of MRI in subjects with MRI-conditional or nonconditional cardiac devices.

Allogenic UC-MSCs are manufactured under GMP conditions at the Clinical Research Cell Manufacturing Program, Interdisciplinary Stem Cell Institute, University of Miami. Donors are screened in compliance with the Food and Drug Administration (FDA) donor eligibility requirements (21 The Code of Federal Regulations [CFR] 1,271 Subpart C), including a comprehensive medical and social history assessment, physical examination, and laboratory testing for communicable diseases such as human immunodeficiency virus (HIV)-1/2, hepatitis B virus (HBV), hepatitis C virus (HCV), syphilis, human T-lymphotropic virus (HTLV)-I/II, and, where applicable, West Nile virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Donors with active infections, high-risk behaviors, or malignancies (excluding nonmelanoma skin cancer) are excluded. The UC-MSCs are derived from UC tissue (either gender) obtained from healthy pregnant women during caesarean delivery, with each cord providing approximately 3 to 4 billion cells. For this study, cords from 4 to 5 donors are processed individually to establish separate single-donor UC-MSC banks; cells from different donors are not pooled within a batch. For each subject, all administered doses are derived from the same donor bank to ensure intrapatient product consistency. Using 4 to 5 donor banks allows us to characterize and manage donor-to-donor variability, which may be important to optimize future commercial use. UC-MSCs are isolated from whole UC tissue, which includes Wharton’s Jelly, perivascular tissue, and sub-amniotic regions. The cord is cleaned, dissected, and enzymatically digested using collagenase. Following digestion, the tissue is mechanically agitated and filtered to remove debris, and the resulting cell suspension is plated into culture flasks to facilitate the attachment and outgrowth of MSCs under standard culture conditions. After approximately 8 to 10 days, the initial adherent population (passage zero, P0) is harvested using enzymatic detachment and expanded into a larger number of flasks. The cells are further cultured for 1 week to reach passage one (P1). At this stage, cells are harvested, washed, counted, and further cultured for P2. They are then recultured a week later for P3.

All UC-MSC batches undergo rigorous quality control testing to ensure they meet predefined release criteria ( Table 4 ), including >80% positivity for cluster of differentiation (CD)73, CD90, and CD105, and <2% for lympho-hematopoietic lineage marker CD45+ subsets, including CD34, CD14, CD19, and human leukocyte antigen- DR isotype (HLA-DR) or class II markers (as per International Society of Cellular Therapy guidelines ). Donor-derived banks are subjected to functional assessment, including colony-forming units-fibroblast (CFU-F) as a supportive measure of clonogenic potential and proliferative capacity and a developing mechanism-linked immunomodulation assay (see below). The cells are cryopreserved at passage 3 using a validated freezing protocol and stored in a vapor-phase liquid nitrogen tank. Cell counts and viability are assessed at both the manufacturing facility and the clinical sites. At the GMP facility, cell number and viability are measured for each lot immediately prior to cryopreservation and again on a post-thaw sample. Final product release is based on these post-thaw data ( Table 4 ). At the clinical site, local cell processing laboratories repeat cell counting and viability assessment after thaw and formulation, immediately before administration. Table 5 shows the FDA-approved post-thaw release criteria.

The frozen UC-MSCs are shipped to the clinical sites in validated liquid nitrogen dry shippers. Upon receipt, local cell processing laboratories thaw and prepare the product for administration according to a standardized protocol.

The placebo control consists of Plasma-Lyte A without cells and is prepared and handled identically to preserve study blinding. Before administration, both the UC-MSCs and placebo undergo final QC checks at the clinical site, including cell counts and viability ( Table 5 ), as well as a visual inspection. All study products (UC-MSCs and placebo) are provided to the treatment teams in identical packaging to ensure proper blinding. The final formulation for the UC-MSC preparation before administration involves thawing the appropriate number of frozen bags in a 37°C ± 1°C water bath. In a biological safety cabinet, the cell suspension is carefully diluted with Plasma-Lyte A supplemented with 1% HSA. The diluted suspension is centrifuged, and the resulting cell pellet is suspended in the dilution buffer. Total viability is determined through cell counting. The cells undergo another round of centrifugation, and then the cell pellet is resuspended in buffer to achieve a cell concentration of 100 million in 60 mL.

In addition to phenotypic characterization (CD73/CD90/CD105 positivity >80% and <2% expression of CD45, CD34, CD14, CD19, and HLA-DR) ( Table 4 ), each donor-derived UC-MSC batch undergoes functional assessment prior to qualification for clinical use. We are implementing a developing mechanism-linked immunomodulation assay as the lead candidate potency readout, consistent with the UC-MSC expected mode of action. This assay is based on the coculture of UC-MSCs with stimulated peripheral blood mononuclear cells (PBMCs), with quantification of pro- and anti-inflammatory mediators (eg, tumor necrosis factor [TNF]-α and vascular endothelial growth factor [VEGF]) by enzyme-linked immunosorbent assay (ELISA). We also perform a CFU-F assay on all donor batches to evaluate clonogenic potential and proliferative fitness. For CFU-F, cells are plated in culture medium, cultured for 14 days, and stained with Wright–Giemsa, and colonies are then counted. Based on historical data, we have established an internal reference range for the readouts. Donor batches must achieve a predefined minimum CFU-F frequency of at least 1 CFU-F per 2,000 MSCs in the harvest sample obtained prior to cryopreservation and fall within these control limits to qualify for clinical manufacturing; donor candidates that do not meet these CFU-F criteria are not advanced. These CFU-F thresholds are internal qualification criteria and are not part of the FDA IND-defined release specifications for the final clinical product.

The endpoints of the study encompass the evaluation of feasibility, safety, and efficacy. Adverse events of grade 2 and above, as per the Common Terminology Criteria for Adverse Events, are systematically documented. These events include major adverse cardiac events (MACE) related to heart failure (HF-MACE), ie, death, hospitalization for worsening HF, and/or exacerbation of HF requiring Emergency Department visit and/or intravenous therapy), along with other clinically significant events. The feasibility of harvesting, preparing, and delivering the designated number of cells, as well as the collection of cardiac MRI variables in patients with cardiac devices, is assessed.

The primary endpoint is the change in LVEF (Δ LVEF) between baseline (M0) and 12 months after the first SPI (M12) (ie, 6 months after the fourth SPI). Three comparisons will be made: (1) 4 UC-MSC doses vs placebo, (2) 1 UC-MSC dose vs placebo, and (3) 4 UC-MSC doses vs 1 dose.

Secondary endpoints include assessments of additional efficacy variables ( Table 6 ), as well as safety and feasibility ( Table 7 ) and immunologic variables ( Table 8 ). Since the study is not powered for these endpoints, they are considered exploratory. To comprehensively assess the efficacy of cell therapy, multiple endpoints have been selected from different categories of effects (domains) encompassing LV function, LV structure, functional capacity, quality of life, clinical outcome, and biomarkers of HF ( Table 6 ). Assessments of LV function and structure, performed by MRI, involve changes in LV end-systolic volume index (LVESVI), LV end-diastolic volume index (LVEDVI), scar mass (in grams and as a percentage of LV), LV sphericity index, global and regional strain (tagged MRI), LV wall thickening as well as assessment of LV function concordance measures (ie, number of individuals who experience an increase in LVEF and a simultaneous decrease in both LVESVI and LVEDVI) ( Table 6 ). Functional capacity and quality of life assessments include changes in VO ₂ max (treadmill test), exercise tolerance (6-minute walk test), NYHA class, and Kansas City Cardiomyopathy Questionnaire (KCCQ) score ( Table 6 ). Clinical outcomes are measured by the incidence of HF-MACE, defined as above ( Table 6 ). Biomarkers include changes in N-terminal pro-B-type natriuretic peptide (NT-proBNP) and high-sensitivity C-reactive protein (hsCRP) ( Table 6 ).

Table 9 .

Safety endpoints encompass HF-MACE, treatment emergent serious adverse events (TE-SAEs), all treatment emergent adverse events (TE-AEs) of at least grade 2 in severity, immune reactions to allogeneic cells measured by panel reactive antibodies (cPRA) (although donor-specific antibodies, rather than cPRA, are the most reliable method for assessing immune responses), abnormal findings in automatic implantable cardioverter-defibrillator (AICD) interrogation before and after MRI, and abnormalities of hematological and clinical chemistry values as well as urinalysis results ( Table 7 ).

Feasibility is assessed by determining whether UC-MSCs can be delivered intravenously 4 times, with planned measurements of safety and efficacy endpoints ( Table 7 ).

Immunologic endpoints include measurements of selected cytokines and immune cells; specifically, plasma levels of TNF-α, TNF-β, IFN-γ, IL-1, IL-2, IL-4, IL-6, IL-10, IL-12, IL-13, IL-17, IL-18, CRP, TGF-β, and GM-CSF, and blood levels of natural killer (NK) cells, T cells (Tc, Th1, Th2, Th17, Treg), B cells, monocytes, exhausted B cells, and terminally differentiated effector memory CD45RA T cells ( Table 8 ). Immune cells are enumerated by flow cytometry, and cytokines are measured by ELISA. The choice of the timing of these measurements was dictated by the need to avoid additional visits in this complex trial; thus, blood samples are obtained at time-points when patients are seen for SPI or follow-up visits. The purpose of these studies is to gain initial insights into the mechanism of action of UC-MSCs, which is the first indispensable step toward formulating mechanistic hypotheses.

The primary endpoint analyses will be tested under an alpha level of 0.05, where alpha will be maintained through a fixed-sequence hierarchical testing. Assuming a standard deviation of 3.3 (based on the previous CONCERT-HF study ), 18 subjects per arm (ie, 54 subjects total) provide 90% power to detect a between-group difference of 3.7 in Δ LVEF and an 80% power to detect a between-group difference of 3.2 in Δ LVEF. To account for a 10% expected dropout, 20 subjects in each arm (60 subjects total) will be enrolled.

Statistical analyses will be performed using SAS version 9.4. All statistical tests will be 2-sided and a 0.05 significance level will be used throughout the analyses, where applicable. The Intent-to-Treat (ITT) population is defined as all subjects who are randomized and receive at least 1 SPI. The ITT population will be the primary population for the analysis of primary and secondary endpoints. All secondary analyses are considered exploratory for the purpose of informing future trial design and no alpha adjustment will be applied to these analyses. A Per Protocol analysis of primary and secondary endpoints will be considered supportive. The Safety Population includes any subject receiving any treatment after randomization. This population will be used for the analysis of safety parameters.

The primary endpoint is the change in LVEF (Δ LVEF) from baseline (M0) to 12 months after the first SPI (M12). There will be 3 comparisons for the primary endpoint:

• Test 1 : The repeated-dose regimen of UC-MSCs improves LVEF compared with the control group. This is addressed by Hypothesis 1 : H ₀ : µ _control = µ _repeated vs µ _control ≠ µ _repeated , where µ _control is the mean Δ LVEF in the control arm and µ _repeated is the mean Δ LVEF in the repeated-dose arm.

• Test 2 : The single-dose regimen of UC-MSCs improves LVEF compared with the control group. This is addressed by Hypothesis 2 : H ₀ : µ _control = µ _{single dose} vs µ _control ≠ µ _{single dose} , where µ _control is the mean Δ LVEF in the control arm and µ _{single dose} is the mean Δ LVEF in single dose arm.

• Test 3 : The repeated-dose regimen of UC-MSCs improves LVEF compared with the single-dose group. This is addressed by Hypothesis 3 : H ₀ : µ _{single dose} = µ _repeated vs µ _{single dose} ≠ µ _repeated , where µ _{single dose} is the mean Δ LVEF in the single-dose arm and µ _repeated is the mean Δ LVEF in the repeated-dose arm.

To maintain alpha for the primary analysis multiple comparisons, a fixed-sequence hierarchical testing approach will be used. A 2-sided t-test (or nonparametric Wilcoxon Rank Sum, as appropriate) will be used for each comparison at a significance level = 0.05. This hierarchal testing will guide the choice of optimal dosing for future studies by addressing the 3 prespecified primary hypotheses: If 4 doses are not effective (Test 1), no further comparison is needed because 1 dose will likewise be ineffective. However, if 4 doses are effective, then it will be important to determine whether 1 dose is sufficient to produce a beneficial effect (Test 2). If 1 dose is not effective, no further comparison is needed. However, if 1 dose is effective, then it is important to test whether it is necessary to use 4 doses to produce a therapeutic effect (Test 3).