A Biomimetic Leaflet Scaffold for Aortic Valve Remodeling.

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.

Myocardial Matrix Hydrogels Mitigate Negative Remodeling and Improve Function in Right Heart Failure Model.

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.

Circulating T cell specific extracellular vesicle profiles in cardiac allograft acute cellular rejection.

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.

The Effect of Parent Cell Type on Small Extracellular Vesicle-Derived Vehicle Functionality.

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.

Statistical modeling of extracellular vesicle cargo to predict clinical trial outcomes for hypoplastic left heart syndrome.

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.

Customized Loading of microRNA-126 to Small Extracellular Vesicle-Derived Vehicles Improves Cardiac Function after Myocardial Infarction.

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.

Mitochondrial citrate carrier SLC25A1 is a dosage-dependent regulator of metabolic reprogramming and morphogenesis in the developing heart.

The developing mammalian heart undergoes an important metabolic shift from glycolysis toward mitochondrial oxidation, such that oxidative phosphorylation defects may present with cardiac abnormalities. Here, we describe a new mechanistic link between mitochondria and cardiac morphogenesis, uncovered by studying mice with systemic loss of the mitochondrial citrate carrier SLC25A1. Slc25a1 null embryos displayed impaired growth, cardiac malformations, and aberrant mitochondrial function. Importantly, Slc25a1 haploinsufficient embryos, which are overtly indistinguishable from wild type, exhibited an increased frequency of these defects, suggesting Slc25a1 dose-dependent effects. Supporting clinical relevance, we found a near-significant association between ultrarare human pathogenic SLC25A1 variants and pediatric congenital heart disease. Mechanistically, SLC25A1 may link mitochondria to transcriptional regulation of metabolism through epigenetic control of PPARγ to promote metabolic remodeling in the developing heart. Collectively, this work positions SLC25A1 as a novel mitochondrial regulator of ventricular morphogenesis and cardiac metabolic maturation and suggests a role in congenital heart disease.

Knockdown of deleterious miRNA in progenitor cell-derived small extracellular vesicles enhances tissue repair in myocardial infarction.

Small extracellular vesicles (sEVs) play a critical role in cardiac cell therapy by delivering molecular cargo and mediating cellular signaling. Among sEV cargo molecule types, microRNA (miRNA) is particularly potent and highly heterogeneous. However, not all miRNAs in sEV are beneficial. Two previous studies using computational modeling identified miR-192-5p and miR-432-5p as potentially deleterious in cardiac function and repair. Here, we show that knocking down miR-192-5p and miR-432-5p in cardiac c-kit+ cell (CPC)-derived sEVs enhances the therapeutic capabilities of sEVs in vitro and in a rat in vivo model of cardiac ischemia reperfusion. miR-192-5p- and miR-432-5p-depleted CPC-sEVs enhance cardiac function by reducing fibrosis and necrotic inflammatory responses. miR-192-5p-depleted CPC-sEVs also enhance mesenchymal stromal cell-like cell mobilization. Knocking down deleterious miRNAs from sEV could be a promising therapeutic strategy for treatment of chronic myocardial infarction.

Intramyocardial cell-based therapy with Lomecel-B during bidirectional cavopulmonary anastomosis for hypoplastic left heart syndrome: the ELPIS phase I trial.

Aims: Hypoplastic left heart syndrome (HLHS) survival relies on surgical reconstruction of the right ventricle (RV) to provide systemic circulation. This substantially increases the RV load, wall stress, maladaptive remodelling, and dysfunction, which in turn increases the risk of death or transplantation. Methods and results: We conducted a phase 1 open-label multicentre trial to assess the safety and feasibility of Lomecel-B as an adjunct to second-stage HLHS surgical palliation. Lomecel-B, an investigational cell therapy consisting of allogeneic medicinal signalling cells (MSCs), was delivered via intramyocardial injections. The primary endpoint was safety, and measures of RV function for potential efficacy were obtained. Ten patients were treated. None experienced major adverse cardiac events. All were alive and transplant-free at 1-year post-treatment, and experienced growth comparable to healthy historical data. Cardiac magnetic resonance imaging (CMR) suggested improved tricuspid regurgitant fraction (TR RF) via qualitative rater assessment, and via significant quantitative improvements from baseline at 6 and 12 months post-treatment (P < 0.05). Global longitudinal strain (GLS) and RV ejection fraction (EF) showed no declines. To understand potential mechanisms of action, circulating exosomes from intramyocardially transplanted MSCs were examined. Computational modelling identified 54 MSC-specific exosome ribonucleic acids (RNAs) corresponding to changes in TR RF, including miR-215-3p, miR-374b-3p, and RNAs related to cell metabolism and MAPK signalling. Conclusion: Intramyocardially delivered Lomecel-B appears safe in HLHS patients and may favourably 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.Trial registration number NCT03525418.

Computational analysis of serum-derived extracellular vesicle miRNAs in juvenile sheep model of single stage Fontan procedure.

Patients with single ventricle heart defects requires a series of staged open-heart procedures, termed Fontan palliation. However, while lifesaving, these operations are associated with significant morbidity and early mortality. The attendant complications are thought to arise in response to the abnormal hemodynamics induced by Fontan palliation, although the pathophysiology underlying these physicochemical changes in cardiovascular and other organs remain unknown. Here, we investigated the microRNA (miRNA) content in serum and serum-derived extracellular vesicles (EVs) by sequencing small RNAs from a physiologically relevant sheep model of the Fontan operation. The differential expression analysis identified the enriched miRNA clusters in (1) serum vs. serum-derived EVs and (2) pre-Fontan EVs vs. post-Fontan EVs. Metascape analysis showed that the overexpressed subset of EV miRNAs by Fontan procedure target liver-specific cells, underscoring a potentially important pathway involved in the liver dysfunction that occurs as a consequence of Fontan palliation. We also found that post-Fontan EV miRNAs were associated with senescence and cell death, whereas pre-Fontan EV miRNAs were associated with stem cell maintenance and epithelial-to-mesenchymal transition. This study shows great potential to identify novel circulating EV biomarkers from Fontan sheep serum that may be used for the diagnosis, prognosis, and therapeutics for patients that have undergone Fontan palliation.

Single-Cell RNA Sequencing Reveals Distinct Cardiac-Derived Stromal Cell Subpopulations.

Human cardiac-derived c-kit+ stromal cells (CSCs) have demonstrated efficacy in preclinical trials for the treatment of heart failure and myocardial dysfunction. Unfortunately, large variability in patient outcomes and cell populations remains a problem. Previous research has demonstrated that the reparative capacity of CSCs may be linked to the age of the cells: CSCs derived from neonate patients increase cardiac function and reduce fibrosis. However, age-dependent differences between CSC populations have primarily been explored with bulk sequencing methods. In this work, we hypothesized that differences in CSC populations and subsequent cell therapy outcomes may arise from differing cell subtypes within donor CSC samples. We performed single-cell RNA sequencing on four neonatal CSC (nCSC) and five child CSC (cCSC) samples. Subcluster analysis revealed cCSC-enriched clusters upregulated in several fibrosis- and immune response-related genes. Module-based analysis identified upregulation of chemotaxis and ribosomal activity-related genes in nCSCs and upregulation of immune response and fiber synthesis genes in cCSCs. Further, we identified versican and integrin alpha 2 as potential markers for a fibrotic cell subtype. By investigating differences in patient-derived CSC populations at the single-cell level, this research aims to identify and characterize CSC subtypes to better optimize CSC-based therapy and improve patient outcomes.

Comparative efficacy and mechanism of action of cardiac progenitor cells after cardiac injury.

Successful cell therapy requires cells to resist the hostile ischemic myocardium, be retained to continue secreting cardioprotective growth factors/exosomes, and resist immunological host responses. Clinically relevant stem/progenitor cells in a rodent model of acute myocardial infarction (MI) demonstrated that neonatal cardiac mesenchymal stromal cells (nMSCs) provide the most robust cardiac functional recovery. Transplanted nMSCs significantly increased the number of tissue reparative macrophages and regulatory T-cells and decreased monocyte-derived inflammatory macrophages and neutrophils in the host myocardium. mRNA microarray and single-cell analyses combined with targeted depletion studies established CD47 in nMSCs as a key molecule responsible for cell retention in the myocardium through an antiphagocytic mechanism regulated by miR34a-5p. Gain and loss-of-function studies demonstrated that miR34a-5p also regulated the production of exosomes and cardioprotective paracrine factors in the nMSC secretome. In conclusion, miR34a-5p and CD47 play an important role in determining the composition of nMSCs' secretome and immune evasion, respectively.

Characterization of decellularized left and right ventricular myocardial matrix hydrogels and their effects on cardiac progenitor cells.

Congenital heart defects are the leading cause of right heart failure in pediatric patients. Implantation of c-kit+ cardiac-derived progenitor cells (CPCs) is being clinically evaluated to treat the failing right ventricle (RV), but faces limitations due to reduced transplant cell survival, low engraftment rates, and low retention. These limitations have been exacerbated due to the nature of cell delivery (narrow needles) and the non-optimal recipient microenvironment (reactive oxygen species (ROS)). Extracellular matrix (ECM) hydrogels derived from porcine left ventricular (LV) myocardium have emerged as a potential therapy to treat the ischemic LV and have shown promise as a vehicle to deliver cells to injured myocardium. However, no studies have evaluated the combination of an injectable biomaterial, such as an ECM hydrogel, in combination with cell therapy for treating RV failure. In this study we characterized LV and RV myocardial matrix (MM) hydrogels and performed in vitro evaluations of their potential to enhance CPC delivery, including resistance to forces experienced during injection and exposure to ROS, as well as their potential to enhance angiogenic paracrine signaling. While physical properties of the two hydrogels are similar, the decellularized LV and RV have distinct protein signatures. Both materials were equally effective in protecting CPCs against needle forces and ROS. CPCs encapsulated in either the LV MM or RV MM exhibited similar enhanced potential for angiogenic paracrine signaling when compared to CPCs in collagen. The RV MM without cells, however, likewise improved tube formation, suggesting it should also be evaluated as a potential standalone treatment.

Encapsulation of Pediatric Cardiac-Derived C-Kit+ Cells in Cardiac Extracellular Matrix Hydrogel for Echocardiography-Directed Intramyocardial Injection in Rodents.

Pediatric cardiac-derived c-kit+ cell therapies represent an innovative approach for cardiac tissue repair that have demonstrated promising improvements in recent studies and offer multiple benefits, such as easy isolation and autologous transplant. However, concerns about failure of engraftment and transient paracrine effects have thus far limited their use. To overcome these issues, an appropriate cell delivery vehicle such as a cardiac extracellular matrix (cECM) hydrogel can be utilized. This naturally derived biomaterial can support embedded cells, allowing for local diffusion of paracrine factors, and provide a healthy microenvironment for optimal cellular function. This protocol focuses on combining cardiac-derived c-kit+ cells and a cECM hydrogel to prepare a minimally invasive, dual therapeutic for in vivo delivery. We also outline a detailed method for ultrasound-guided intramyocardial injection of cell-laden hydrogels in a rodent model. Additional steps for labeling cells with a fluorescent dye for in vivo cell tracking are provided.

Autologous Cardiac Stem Cell Injection in Patients with Hypoplastic Left Heart Syndrome (CHILD Study).

Mortality in infants with hypoplastic left heart syndrome (HLHS) is strongly correlated with right ventricle (RV) dysfunction. Cell therapy has demonstrated potential improvements of RV dysfunction in animal models related to HLHS, and neonatal human derived c-kit+ cardiac-derived progenitor cells (CPCs) show superior efficacy when compared to adult human cardiac-derived CPCs (aCPCs). Neonatal CPCs (nCPCs) have yet to be investigated in humans. The CHILD trial (Autologous Cardiac Stem Cell Injection in Patients with Hypoplastic Left Heart Syndrome) is a Phase I/II trial aimed at investigating intramyocardial administration of autologous nCPCs in HLHS infants by assessing the feasibility, safety, and potential efficacy of CPC therapy. Using an open-label, multicenter design, CHILD investigates nCPC safety and feasibility in the first enrollment group (Group A/Phase I). In the second enrollment group, CHILD uses a randomized, double-blinded, multicenter design (Group B/Phase II), to assess nCPC efficacy based on RV functional and structural characteristics. The study plans to enroll 32 patients across 4 institutions: Group A will enroll 10 patients, and Group B will enroll 22 patients. CHILD will provide important insights into the therapeutic potential of nCPCs in patients with HLHS.Clinical Trial Registration https://clinicaltrials.gov/ct2/home NCT03406884, First posted January 23, 2018.

Comparative computational RNA analysis of cardiac-derived progenitor cells and their extracellular vesicles.

Stem/progenitor cells, including cardiac-derived c-kit+ progenitor cells (CPCs), are under clinical evaluation for treatment of cardiac disease. Therapeutic efficacy of cardiac cell therapy can be attributed to paracrine signaling and the release of extracellular vesicles (EVs) carrying diverse cargo molecules. Despite some successes and demonstrated safety, large variation in cell populations and preclinical/clinical outcomes remains a problem. Here, we investigated this variability by sequencing coding and non-coding RNAs of CPCs and CPC-EVs from 30 congenital heart disease patients and used machine learning methods to determine potential mechanistic insights. CPCs retained RNAs related to extracellular matrix organization and exported RNAs related to various signaling pathways to CPC-EVs. CPC-EVs are enriched in miRNA clusters related to cell proliferation and angiogenesis. With network analyses, we identified differences in non-coding RNAs which give insight into age-dependent functionality of CPCs. By taking a quantitative computational approach, we aimed to uncover sources of CPC cell therapy variability.

Using computational methods to design patient-specific electrospun cardiac patches for pediatric heart failure.

Autologous cardiac cell therapy is a promising treatment for combating the right ventricular heart failure (RVHF) that can occur in patients with congenital heart disease (CHD). However, autologous cell therapies suffer from low cell retention following injection and patient-to-patient variability in cell quality. Here, we demonstrate how computational methods can be used to identify mechanisms of cardiac-derived c-Kit+ cell (CPC) reparative capacity and how biomaterials can be designed to improve cardiac patch performance by engaging these mechanisms. Computational modeling revealed the integrin subunit αV (ITGAV) as an important mediator of repair in CPCs with inherently low reparative capacity (CPCslow). We could engage ITGAV on the cell surface and improve reparative capacity by culturing CPCs on electrospun polycaprolactone (PCL) patches coated with fibronectin (PCL + FN). We tested CPCs from 4 different donors and found that only CPCslow with high ITGAV expression (patient 956) had improved anti-fibrotic and pro-angiogenic paracrine secretion on PCL + FN patches. Further, knockdown of ITGAV via siRNA led to loss of this improved paracrine secretion in patient 956 on PCL + FN patches. When implanted in rat model of RVHF, only PCL + FN + 956 patches were able to improve RV function, while PCL +956 patches did not. In total, we demonstrate how cardiac patches can be designed in a patient-specific manner to improve in vitro and in vivo outcomes.

A mathematical nutrition model adequately predicts beef and dairy cow intake and biological efficiency.

The beef cow-calf sector accounts for 70% of feed consumed and greenhouse gases emitted for the beef industry, but there is no straightforward method to measure biological efficiency in grazing conditions. The objective of this study was to evaluate a mathematical nutrition model to estimate the feed intake and biological efficiency of mature beef cows. Data from dams (N = 160) and their second and third progeny (312 pairs) were collected from 1953 through 1980. Individual feed intake was measured at 28-d intervals year-round for dams and during 240-d lactation for progeny. Body weights of progeny were measured at 28-d intervals from birth to weaning, and of dams at parturition and weaning each production cycle. The milk yield of dams was measured at 14-d intervals. Dam metabolizable energy intake (DMEI) and milk energy yield (MEL) of each cow were predicted using the Cattle Value Discovery System beef cow (CVDSbc) model for each parity. Biological efficiency (Mcal/kg) was computed as the ratio of observed or predicted DMEI to observed calf weaning weight (PWW). Pearson correlation coefficients were computed using corr.test function and model evaluation was performed using the epiR function in R software. Average (SD) dam weight, PWW, DMEI, and observed MEL were 527 (86) kg, 291 (47) kg, 9584 (2701) Mcal/production cycle, and 1029 (529) Mcal, respectively. Observed and predicted DMEI (r = 0.93 and 0.91), and observed and predicted MEL (r = 0.58 and 0.59) were positively correlated for progeny 2 and 3, respectively. The CVDSbc model under-predicted DMEI (mean bias [MB] = 1,120 ± 76 Mcal, 11.7% of observed value) and MEL (MB = 30 ± 25 Mcal, 2.9% of observed value). Observed and predicted progeny feed intake were not correlated (r = 0.01, P-value = 0.79). Observed and predicted biological efficiency were positively correlated (r = 0.80 and 0.80, P-value ≤ 0.05) for parity 2 and 3, respectively, and the CVDSbc model under-predicted biological efficiency by 11% (MB = 3.59 ± 0.25 Mcal/kg). The CVDSbc provides reasonable predictions of feed intake and biological efficiency of mature beef cows, but further refinement of the relationship between calf feed intake and milk yield is recommended to improve predictions. Mathematical nutrition models can assist in the discovery of the biological efficiency of mature beef cows.

In vivo evaluation of bioprinted cardiac patches composed of cardiac-specific extracellular matrix and progenitor cells in a model of pediatric heart failure.

Pediatric patients with congenital heart defects (CHD) often present with heart failure from increased load on the right ventricle (RV) due to both surgical methods to treat CHD and the disease itself. Patients with RV failure often require transplantation, which is limited due to lack of donor availability and rejection. Previous studies investigating the development and in vitro assessment of a bioprinted cardiac patch composed of cardiac extracellular matrix (cECM) and human c-kit + progenitor cells (hCPCs) showed that the construct has promise in treating cardiac dysfunction. The current study investigates in vivo cardiac outcomes of patch implantation in a rat model of RV failure. Patch parameters including cECM-inclusion and hCPC-inclusion are investigated. Assessments include hCPC retention, RV function, and tissue remodeling (vascularization, hypertrophy, and fibrosis). Animal model evaluation shows that both cell-free and neonatal hCPC-laden cECM-gelatin methacrylate (GelMA) patches improve RV function and tissue remodeling compared to other patch groups and controls. Inclusion of cECM is the most influential parameter driving therapeutic improvements, with or without cell inclusion. This study paves the way for clinical translation in treating pediatric heart failure using bioprinted GelMA-cECM and hCPC-GelMA-cECM patches.

Designing a 3D Printing Based Auxetic Cardiac Patch with hiPSC-CMs for Heart Repair.

Myocardial infarction is one of the largest contributors to cardiovascular disease and reduces the ability of the heart to pump blood. One promising therapeutic approach to address the diminished function is the use of cardiac patches composed of biomaterial substrates and cardiac cells. These patches can be enhanced with the application of an auxetic design, which has a negative Poisson's ratio and can be modified to suit the mechanics of the infarct and surrounding cardiac tissue. Here, we examined multiple auxetic models (orthogonal missing rib and re-entrant honeycomb in two orientations) with tunable mechanical properties as a cardiac patch substrate. Further, we demonstrated that 3D printing based auxetic cardiac patches of varying thicknesses (0.2, 0.4, and 0.6 mm) composed of polycaprolactone and gelatin methacrylate can support induced pluripotent stem cell-derived cardiomyocyte function for 14-day culture. Taken together, this work shows the potential of cellularized auxetic cardiac patches as a suitable tissue engineering approach to treating cardiovascular disease.