Background: Post-operative tachycardia is a common and poorly understood complication following the Fontan procedure. Post-operative factors such as surgical scarring and venous hypertension can contribute to tachycardia risk, but the specific molecular signaling cascades triggering acute tachycardia remain uncharacterized, limiting therapeutic innovation and leaving clinicians with limited strategies. Here, we present a retrospective translational study leveraging serum proteomics and machine learning to identify molecular drivers of post-operative Fontan tachycardia. Methods: We integrated a clinically relevant ovine animal model Fontan circulation with continuous telemetric heart rate monitoring and human patient data. Serum proteomics coupled with machine learning algorithms were employed to identify protein panels predictive of post-operative tachycardia. Cross-species validation was performed by comparing proteomic signatures from sheep and pediatric patients undergoing Glenn or Fontan surgery. Results: Ovine Fontan animals demonstrated significant heart rate elevation beginning on post-operative day (POD) 1, peaking at POD 3 (159.4 ± 11.7 bpm vs. pre-operative 105.3 ± 10.5 bpm, p<0.0001), before trending toward baseline by POD 10. This pattern was similar in human patients, though more modest. Proteomic analysis identified distinct separation between pre- and post-operative serum profiles. Principal component analysis revealed that the principal components most correlated with heart rate were significantly enriched for inflammatory and neural pathways. We leveraged the Boruta algorithm to identify a seven-protein panel (ACE, ANGT, ITIH4, SELENOP, W5PHP7, PTX3, and F5) with superior predictive power (AUC=0.926). A cross-species comparison between human and sheep demonstrated that three, angiotensinogen (ANGT), angiotensin-converting enzyme (ACE), and pentraxin 3 (PTX3), were similarly dysregulated in both species. Conclusions: This study provides the first direct molecular evidence implicating a dysregulated neurohormonal-inflammatory axis as a principal driver of acute post-operative Fontan tachycardia. The identified protein signature offers novel mechanistic insights and establishes a foundation for targeted diagnostics and therapeutics to predict and mitigate this significant clinical complication. ### Competing Interest Statement The authors have declared no competing interest.

Background: Fontan-associated liver disease (FALD) is a universal complication of the Fontan palliation characterized by chronic congestion and progressive hepatic fibrosis. Current diagnostics rely on invasive biopsies or non-specific biomarkers that fail to capture early fibrogenesis, creating a need for non-invasive biomarkers to stratify disease severity. Methods: We utilized an ovine Fontan model (n = 19) to investigate circulating serum extracellular vesicles (sEVs) as reporters of hepatic pathology. Longitudinal samples paired with liver elastography were collected, and sEVs were subjected to multi-omic profiling including small RNA sequencing and proteomics. Regularized regression was used to identify transcriptomic predictors, which were integrated with time post-surgery into an ordinal logistic regression framework to construct the Fontan EV Score (FES). Model performance was evaluated on a held-out test cohort and benchmarked against established fibrosis indices. To validate the biological relevance of the FES , TGF-β-treated human liver organoids were generated and scored miRNA expression was assessed. Results: The sEV proteome exhibited robust separation by surgical physiology, while the small RNA cargo was primarily stratified by fibrotic status. Bioinformatics confirmed a high hepatic origin for these transcripts and identified enrichment of inflammatory pathways including Toll-like receptor and Interleukin-17 cascades in fibrotic subjects. The FES, incorporating time post-surgery and eleven small RNA biomarkers, demonstrated high predictive accuracy in the independent testing cohort with an AUC of 0.876 for moderate and 0.963 for severe fibrosis, substantially outperforming APRI (AUC = 0.618) and FIB-4 (AUC = 0.731). In human liver organoids, several scoring miRNAs, including miR-125a-5p and miR-193b-5p, were directionally responsive to profibrotic stimulation. Conclusions: Circulating sEVs carry a liver-associated cargo that can be leveraged for the non-invasive prediction of FALD severity. The FES provides a biologically validated scoring system that substantially outperforms existing serological indices and offers a new avenue for early detection and risk stratification of FALD.

Background:Heart failure with preserved ejection fraction (HFpEF) remains a major therapeutic challenge due to its complex pathophysiology and pronounced heterogeneity. Regenerative approaches using neonatal mesenchymal stromal cells (nMSCs) and their secretome (SEC) have shown promise in other heart failure contexts. Objectives:However, the effect of these therapies in HFpEF, and the underlying molecular mechanisms and causal pathways remain poorly understood. Methods:HFpEF was established in two distinct murine models, followed by treatment with either nMSCs or SEC. Functional and histological endpoints were assessed. We developed a novel machine learning framework, VIPcell, which integrates data augmentation, Partial Least Squares (PLS) regression, and causal structure inference to identify genes causally linked to cardiac function using single-nucleus RNA sequencing (snRNA-seq) data. VIPcell was applied to heart tissues from treated HFpEF animals to uncover key regulators of cardiac remodeling. Results:Both nMSC and SEC therapies significantly improved diastolic function in two independent rodent HFpEF models. These improvements were associated with reduced inflammation, attenuated myocardial fibrosis, and improved exercise capacity. Intercellular communication analysis revealed widespread, system-level signaling in nMSC-treated hearts, compared to more localized endothelial-cardiomyocyte crosstalk in SEC-treated hearts. Causal inference via VIPcell suggested overlapping upstream regulators in both treatment groups, particularly genes involved in regulatory T cell (T reg ) biology and immunomodulatory signaling pathways, including FOXO signaling, NLRP3 inflammasome inhibition, and Tie2 activation. In vivo validation confirmed selective expansion of T regs following nMSC and SEC therapy. In vitro, nMSCs induced significantly greater T reg expansion compared to multiple adult stem cell types. Critically, chemical depletion of T regs abrogated the therapeutic effects of both treatments, establishing T regs as central mediators of diastolic function recovery in the HFpEF preclinical model. Conclusions:nMSC and SEC therapies improve diastolic function in HFpEF through distinct remodeling mechanisms converging on T reg -mediated immune modulation. VIPcell supported identification of causal regulators, highlighting T reg -related signaling as a key driver of myocardial recovery in HFpEF. These findings offer mechanistic insight into cellular therapies for HFpEF and support the development of targeted, T reg -focused interventions.

IntroductionCardiovascular disease (CVD) is a leading cause of mortality worldwide. The potency of cell-based therapies for CVD is increasingly attributed to the release of small extracellular vesicles (sEVs) which consist of a lipid/protein membrane and encapsulate nucleic cargo. Specifically, sEVs from ckit + progenitor cells (CPCs) and mesenchymal stromal cells (MSCs) are shown to be pro-reparative, with clinical trials conducted. Despite copious research into sEV cargo, the role of parent cell type on sEV membrane composition and its effects on sEV uptake mechanism by recipient cells remain unclear. This is crucial for designing sEV-based therapeutics as uptake mechanism dictates the functionality of the cargo.MethodsIn this study we investigate the role of sEV parent cell and membrane composition on the mechanism of EV uptake by recipient cells.ResultsWe find that sEV membrane lipid and protein composition varies by parent cell type. Further, vesicle uptake mechanism varies by both sEV parent cell type and recipient cell type, with clathrin-mediated uptake being the most variable across parent cell conditions. Using a partial least squares regression model, we observe that proteins important in clathrin-mediated uptake (e.g., TPM1, MRC2, FSTL1, LTBP1) are dissimilar to other vesicle uptake mechanisms.DiscussionThis work underscores the importance of the sEV source and membrane composition on uptake, and in turn the importance of selecting specific sEVs based on the target recipient cells for CVD therapies.

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) possess tremendous advantage for cardiac regeneration. However, cell survival challenging upon cell transplantation. Since microgravity can profoundly affect cellular properties, we investigated the effect of spaceflight on hiPSC-CMs. spheroids derived from hiPSCs were transported to the International Space Station (ISS) via the SpaceX Crew-8 mission and cultured under space microgravity days. Beating cardiac spheroids were observed on the ISS and upon successful experimentation by the astronauts in space, the live cultures were returned These cells had normal displacement (an indicator of contraction) and Ca2+ transient parameters in 3D live cell imaging. Proteomic analysis revealed that spaceflight upregulated many proteins involved in metabolism (n = 90), cellular component of mitochondrion (n = 62) and regulation of proliferation (n = 10). metabolic pathways enriched by spaceflight included glutathione metabolism, biosynthesis of amino acids, and pyruvate metabolism. In addition, the top gulated proteins in spaceflight samples included those involved in cellular stress response, cell survival, and metabolism. Transcriptomic profiles indicated spaceflight upregulated genes associated with cardiomyocyte development, and cellular components of cardiac structure and mitochondrion. Furthermore, flight upregulated genes in metabolic pathways associated with cell survival such as glycerophospholipid metabolism and glycerolipid metabolism. These indicate that short-term exposure of 3D hiPSC-CMs to the space environment led to significant changes in protein levels and gene expression involved in cell and metabolism.

Despite the promising potential of stromal cell therapy in treating myocardial infarction (MI), its effectiveness is limited by poor cell retention and engraftment in ischemic environments. This study introduces a novel strategy that combines the preconditioning of human adipose-derived stromal cells (hADSCs) using OLED-based photobiomodulation (OPBM) and culturing these cells into 3D spheroids. The preconditioned 3D spheroids (APCS group) exhibit significantly enhanced angiogenic, arterialized, and tissue remodeling capabilities compared with those of traditional 2D cultures and non-preconditioned spheroids. In vivo transplantation of these spheroids into the border zone of infarcted area significantly improve cardiac function and reduce adverse remodeling by enhancing anti-fibrosis and angiogenesis including arterialization. The combined strategy with OPBM preconditioning and 3D spheroid culture system can enhance therapeutic potential of hADSCs with multiple paracrine effects for cardiac repair. This novel approach provides next generation of cell therapeutics to overcome the limitation of adult stromal cell therapy in patients with post-MI heart failure.

Novel cardiac patch designs achieved by advanced 3D manufacturing continue to have favorable impacts on the repair and regeneration of the myocardium after injury. Briefly, auxetic units with a negative Poisson's ratio have already shown remarkable promise for serving as a next-generation complex scaffold in left ventricular disease. In this study we biofabricated a 3D printed polycaprolactone (PCL) cardiac auxetic patch loaded with high density contractile induced pluripotent stem cell-derived cardiomyocytes (iCMs) and examined the synergist effect of iCM auxetic patches on a chronic myocardial infarct rodent model compared to a stiffer non-auxetic control patch architecture. A week after the induction of a temporary left anterior descending artery ligation, we administered the treatment groups in the form of patch implantation over the ischemic area after initial acute inflammation was complete and prior to granulation tissue formation following the infarct for clinical relevance. Our findings highlight that auxetic patches can provide additional ventricular support and diminished adverse ventricular remodeling, as seen through ejection fraction outputs and histology, and iCM-laden auxetics show localized regenerative potential through increased vascularization compared to controls with no patch or a non-auxetic patch architecture. Exploration on the impact of a negative Poisson's ratio on both global functional outcomes and local therapeutic benefit highlights that iCM-laden auxetics should be further surveyed for other cardiac pathophysiologic conditions, including more in-depth studies on infarction or right ventricular disease.

Heart valve disease poses a significant clinical challenge, especially in pediatric populations, due to the inability of existing valve replacements to grow or respond biologically to their microenvironment. Tissue-engineered heart valves (TEHVs) provide a solution by facilitating patient-specific models for self-repair and remodeling. In this study, a 3D-bioprinted TEHV is designed to emulate the trilayer leaflet structure of an aortic valve. A cell-laden hydrogel scaffold made from gelatin methacrylate and polyethylene glycol diacrylate (GelMA/PEGDA) incorporates valvular interstitial-like (VIC-like) cells, being reinforced with a layer of polycaprolactone (PCL). The composition of the hydrogel scaffold remains stable over 7 days, having increased mechanical strength compared to pure GelMA. The scaffold maintains VIC-like cell function and promotes extracellular matrix (ECM) protein expression up to 14 days under two dynamic culture conditions: shear stress and stretching; replicating heart valve behavior within a more physiological-like setting and suggesting remodeling potential via ECM synthesis. This TEHV offers a promising avenue for valve replacements, closely replicating the structural and functional attributes of a native aortic valve, leading to mechanical and biological integration through biomaterial-cellular interactions.

This study evaluates the effectiveness of myocardial matrix (MM) hydrogels in mitigating negative right ventricular (RV) remodeling in a rat model of RV heart failure. The goal was to assess whether a hydrogel derived from either the right or left ventricle could promote cardiac repair. Injured rat right ventricles were injected with either RV-or left ventricular–derived MM hydrogels. Both hydrogels improved RV function and morphology and reduced negative remodeling. This study supports the potential of injectable biomaterial therapies for treating RV heart failure.

There is a critical need for biomarkers of acute cellular rejection (ACR) in organ transplantation. We hypothesized that ACR leads to changes in donor-reactive T cell small extracellular vesicle (sEV) profiles in transplant recipient circulation that match the kinetics of alloreactive T cell activation. In rodent heart transplantation, circulating T cell sEV quantities (P < .0001) and their protein and mRNA cargoes showed time-specific expression of alloreactive and regulatory markers heralding early ACR in allogeneic transplant recipients but not in syngeneic transplant recipients. Next generation sequencing of their microRNA cargoes identified novel candidate biomarkers of ACR, which were validated by stem loop quantitative reverse transcription polymerase chain reaction (n = 10). Circulating T cell sEVs enriched from allogeneic transplant recipients mediated targeted cytotoxicity of donor cardiomyocytes by apoptosis assay (P < .0001). Translation of the concept and EV methodologies to clinical heart transplantation demonstrated similar upregulation of circulating T cell sEV profiles at time points of grade 2 ACR (n = 3 patients). Furthermore, T cell receptor sequencing of T cell sEV mRNA cargo demonstrated expression of T cell clones with intact complementarity determining region 3 signals. These data support the diagnostic potential of T cell sEVs as noninvasive biomarker of ACR and suggest their potential functional roles.

Notch signaling, an important signaling pathway in cardiac development, has been shown to mediate the reparative functions of c-kit+ progenitor cells (CPCs). However, it is unclear how each of the four canonical Notch-activating ligands affects intracellular processes in c-kit+ cells when used as an external stimulus. Neonatal c-kit+ CPCs were stimulated using four different chimeric Notch-activating ligands tethered to Dynabeads, and the resulting changes were assessed using TaqMan gene expression arrays, with subsequent analysis by principal component analysis (PCA). Additionally, functional outcomes were measured using an endothelial cell tube formation assay and MSC migration assay to assess the paracrine capacity to stimulate new vessel formation and recruit other reparative cell types to the site of injury. Gene expression data showed that stimulation with Jagged-1 is associated with the greatest pro-angiogenic gene response, including the expression of VEGF and basement membrane proteins, while the other canonical ligands, Jagged-2, Dll-1, and Dll-4, are more associated with regulatory and epigenetic changes. The functional assay showed differential responses to the four ligands in terms of angiogenesis, while none of the ligands produced a robust change in migration. These data demonstrate how the four Notch-activating ligands differentially regulate CPC gene expression and function.

Single-ventricle defects are treated with the Glenn and Fontan procedures, which offer lifesaving relief but result in lifelong complications. To address the lack of outcome predictors, we conducted an untargeted transcriptomic analysis to identify RNA biomarkers in serum and circulating sEVs from 25 Glenn or Fontan patients with three samples exclusively used for experimental assays. Unsupervised analysis revealed a distinction between pre-op and post-op samples in both surgical groups. Differential gene expression and pathway analysis showed enrichment for pro-angiogenic cargo in post-op sEVs compared to pre-op sEVs. Wound healing assays revealed post-op Fontan sEVs induce a stronger pro-angiogenic response than pre-op Fontan sEVs. A PLSR-guided approach revealed MAPK6, GLE1, hsa-miR-340-5p, and hsa-miR-199b-5p as key transcripts in the observed wound healing response. Lastly, EV-Origin revealed decreased secretion of sEV from cardiac tissue and increased secretion from brain tissue for both Fontan and Glenn samples. This work demonstrates the potential of sEV RNAs as biomarkers for patients with Fontan physiology, enabling quicker diagnosis for Fontan-associated complications.

Objectives: Our goal was to conduct a hemodynamic analysis of a novel animal model of Fontan physiology. Poor late-term outcomes in Fontan patients are believed to arise from Fontan-induced hemodynamics, but the mechanisms remain poorly understood. Recent advances in surgical experimentation have resulted in the development of a chronic sheep model of Fontan physiology; however, detailed analysis of this model is lacking. Methods: We created a single-stage Fontan model in juvenile sheep with normal biventricular circulation. The superior vena cava was anastomosed to the main pulmonary artery, and the inferior vena cava was connected to the main pulmonary artery using an expanded polytetrafluoroethylene conduit. Longitudinal hemodynamics, including catheterization and magnetic resonance imaging were evaluated. Results: Four out of 12 animals survived, with the longest surviving animal living 3 years after single-stage Fontan. We showed a significant era effect regarding survival (1 out of 8 and subsequently 3 out of 4 animals surviving beyond 2 months) attributed in large part to the procedural learning curve. Key characteristics of Fontan hemodynamics, namely systemic venous hypertension and low normal cardiac output, were observed. However, recapitulation of passive human Fontan hemodynamics is affected by volume loading of the right ventricle given an anatomic difference in sheep azygous venous anatomy draining to the coronary sinus. Conclusions: A significant learning curve exists to ensure long-term survival and future surgical modifications, including banding of the main pulmonary artery and ligation of the azygous to coronary sinus connection are promising strategies to improve the fidelity of model hemodynamics. (JTCVS Open 2024;21:268-78)

Soft Tissue Engineering In article number 2215220, Scott J. Hollister, Michael E. Davis, and co-workers report, 3D printing of a novel architected auxetic with enhanced mechanical and biological performance. Specifically, the printed auxetics have high flexibility, permeability, and nonlinear deformation behavior as demonstrated through computational, mechanical, and biological analysis. These characteristics provide a great potential to create clinically viable implants for the reconstruction of large volume soft tissues.

Cell therapies involving c-kit+ progenitor cells (CPCs) and mesenchymal stem cells (MSCs) have been actively studied for cardiac repair. The benefits of such therapies have more recently been attributed to the release of small extracellular vesicles (sEVs) from the parent cells. These sEVs are 30-180 nm vesicles containing protein/nucleic acid cargo encapsulated within an amphiphilic bilayer membrane. Despite their pro-reparative effects, sEV composition and cargo loading is highly variable, making it challenging to develop robust therapies with sEVs. Synthetic alternatives have been developed to allow cargo modulation, including prior work from the laboratory, to design sEV-like vehicles (ELVs). ELVs are synthesized from the sEV membrane but allow controlled cargo loading. It is previously shown that loading pro-angiogenic miR-126 into CPC-derived ELVs significantly increases endothelial cell angiogenesis compared to CPC-sEVs alone. Here, they expand on this work to design MSC-derived ELVs  and study the role of the parent cell type on ELV composition and function. It is found that ELV origin does affect the ELV potency and that ELV membrane composition can affect outcomes. This study showcases the versatility of ELVs to be synthesized from different parent cells and highlights the importance of selecting ELV source cells based on the desired functional outcomes.

Hypoplastic left heart syndrome (HLHS) is a univentricular congenital heart defect that has high morbidity and mortality and requires three-stage surgical reconstruction so that the right ventricle (RV) delivers systemic circulation. Poor outcomes result from RV failure in the systemic position. Accordingly, we conducted a phase I clinical trial (called ELPIS) of Lomecel-B™, an allogeneic bone-marrow derived cell-based therapy designed to improve cardiovascular performance, delivered as a one-time treatment during the Stage II (Glenn) surgery at approximately 4 months after birth. This trial met its primary endpoint (safety through 1-year post-treatment). To assess whether Lomecel-B™ has survival benefits, all ELPIS patients ( n=10, 7 males, and 3 females) were enrolled in a multi-year follow-on study and compared to a retrospective control group which was identified by a relevant clinical HLHS database. Patients in both studies were assessed for up to 5 years (range 3.5 - 5.0 years post-treatment for ELPIS) for mortality, heart transplants, and stage III (Fontan) surgery. Outcomes were compared with long-term historical data from patients in the Single Ventricle Reconstruction (SVR) Trial receiving the same shunt type at Stage I (Norwood) operation. 5-year Kaplan-Meier survival was 100% in ELPIS patients with none requiring heart transplant. This compared to 81.6% (95% CI= [76.5, 87.0]) transplant-free survival in the SVR trial through 5 years post-Glenn surgery, and a 5.2% (95% CI= [2.0, 8.3]) heart transplantation rate (Figure 1). No stem cell related safety issues were reported. These findings support Lomecel-B™ as a potential adjunct to HLHS reconstruction surgery to improve clinical benefits and reduce the need for subsequent heart transplantation. Further long-term follow-up and controlled trials are both warranted and underway.

Small extracellular vesicles (sEVs) are promising for cell-based cardiac repair after myocardial infarction. These sEVs encapsulate potent cargo, including microRNAs (miRs), within a bilayer membrane that aids sEV uptake when administered to cells. However, despite their efficacy, sEV therapies are limited by inconsistencies in the sEV release from parent cells and variability in cargo encapsulation. Synthetic sEV mimics with artificial bilayer membranes allow for cargo control but suffer poor stability and rapid clearance when administered in vivo. Here, we developed an sEV-like vehicle (ELV) using an electroporation technique, building upon our previously published work, and investigated the potency of delivering electroporated ELVs with pro-angiogenic miR-126 both in vitro and in vivo to a rat model of ischemia-reperfusion. We show that electroporated miR-126+ ELVs improve tube formation parameters when administered to 2D cultures of cardiac endothelial cells and improve both echocardiographic and histological parameters when delivered to a rat left ventricle after ischemia reperfusion injury. This work emphasizes the value of using electroporated ELVs as vehicles for delivery of select miR cargo for cardiac repair.

Cardiac-derived c-kit+ progenitor cells (CPCs) are under investigation in the CHILD phase I clinical trial (NCT03406884) for the treatment of hypoplastic left heart syndrome (HLHS). The therapeutic efficacy of CPCs can be attributed to the release of extracellular vesicles (EVs). To understand sources of cell therapy variability we took a machine learning approach: combining bulk CPC-derived EV (CPC-EV) RNA sequencing and cardiac-relevant in vitro experiments to build a predictive model. We isolated CPCs from cardiac biopsies of patients with congenital heart disease (n = 29) and the lead-in patients with HLHS in the CHILD trial (n = 5). We sequenced CPC-EVs, and measured EV inflammatory, fibrotic, angiogeneic, and migratory responses. Overall, CPC-EV RNAs involved in pro-reparative outcomes had a significant fit to cardiac development and signaling pathways. Using a model trained on previously collected CPC-EVs, we predicted in vitro outcomes for the CHILD clinical samples. Finally, CPC-EV angiogenic performance correlated to clinical improvements in right ventricle performance.

ABSTRACTBackgroundHypoplastic left heart syndrome (HLHS) survival relies on surgical reconstruction for the right ventricle (RV) to provide systemic circulation. This leads to substantially increased loads on the RV, wall stress, maladaptive remodeling and dysfunction, which in turn can increase risk of death or transplantation.ObjectivesWe conducted a phase I multicenter trial to assess safety and feasibility of intra-operative MSC injection in HLHS patients to boost RV performance in the systemic position.MethodsAllogeneic MSCs were directly administered by intramyocardial injections during the second stage palliative operation. The primary endpoint was safety.ResultsTen patients received intramyocardial injections of allogeneic MSCs (Lomecel-B). No patients experienced major adverse cardiac events (MACE). All subjects were alive and transplant-free at 1 year following, and experienced growth comparable to healthy control historical data. Cardiac magnetic resonance imaging (CMR) revealed improving tricuspid regurgitant fraction (Baseline: 0.45±0.19; 6 mo.: 0.32±0.06; 12 mo.: 0.06±0.09), while global longitudinal strain (Baseline: -24.39±6.99; 6 mo.: -20.55±3.05, p > 0.05 vs baseline; 12 mo.: - 23.88±4.6, p>0.05 vs baseline) and RV ejection fraction (EF; baseline: 62.62±5.99; 6 mo.: 53.69±9.56; 12 mo.: 52.31±5.63: p=NS for change over time) were unchanged. Computational modeling identified 167 derived RNAs specific to circulating exosomes originating from transplanted MSCs corresponding to RVEF changes and identifying potential mechanistic underpinnings.ConclusionsIntramyocardial MSCs appear safe in HLHS patients, and may favorably affect RV performance. Circulating exosomes of transplanted MSC-specific provide novel insight into bioactivity. Conduct of a controlled phase trial is warranted and is underway.Condensed AbstractThe ELPIS phase I trial was designed to assess safety and feasibility of intramyocardial injection of allogeneic MSCs into the RV during second stage palliation of HLHS. There were no incidences of major adverse cardiac events (MACE) or other safety concerns, and there was a 100% transplant-free survival at 1-year follow-up, supporting the safety and feasibility of this approach. The ELPIS results are important for advancing MSC therapy for all ages and congenital heart conditions, and warrant further investigation in a controlled Phase II trial powered for efficacy.