Poster U6 - Minimally Invasive Delivery of Mesenchymal Stem Cell-derived Exosomes Improves Heart Repair in Pigs

Introduction: Ischemic heart disease remains the leading cause of mortality worldwide. Myocardial infarction (MI) is the primary manifestation of cardiovascular events. An emerging body of literature has suggested that the therapeutic effect of mesenchymal stem cells (MSCs) is mainly driven by the paracrine effects of the MSC cellular contents. Exosome is credited to be one of the most important mediators of cellular communications due to their ability to encapsulate and transport nucleic acids and proteins from one cell to another. Application of these therapeutics requires an open thoracotomy which is inherently associated with high morbidity. Translation of this MSC-derived exosome (MSC-Exo) into clinical praxis, however, demands establishing a safe and effective route of delivery. Therefore, it is beneficial to deliver MSC-Exo embedded in hyaluronic acid (HA) hydrogel using a minimally invasive approach such as intrapericardial cavity (iPC) injections. This preclinical study combined the benefits of stem cell-derived nanotherapeutics with an injectable delivery route in a large animal model. The results show that iPC injection of MSC-Exo embedded in HA hydrogel on a pig model of MI significantly improved cardiac function and cardiomyocyte (CM) proliferation. Interestingly, immature cardiomyocytes of a different phenotype emerged in the therapeutic group. These findings provide an unprecedented dataset to facilitate the clinical translation of cardiac tissue regeneration therapeutics.

Materials and

Methods: The purpose of this study is to explore the therapeutic effects of MSC-Exo embedded in HA hydrogel via iPC injection. Human MSC were treated with serum-free media for 2 days. Conditioned media were collected. Centrifugated was used to remove cellular debris. The supernatant was subjected to ultrafiltration using a 100 kDa ultrafilter (Merck Millipore) to concentrate EVs, centrifuged at 3500g, and finally filtered through a 0.22 μm filter. 0.1 g HA was dissolved in 10 mL deionized water (DI) water and stirred for 30 min. A pig MI model was created using coronary balloon occlusion, and Yorkshire male pigs underwent video-assisted thoracoscopic surgery for iPC injections. Cardiac function studies were measured using an echocardiogram, and left ventricle ejection fraction (LVEF) was calculated for pre-MI, 3 days post-MI, and 28 days post-MI. Histology and immunohistochemistry (IHC) analyses were performed on heart tissue samples collected at endpoints to investigate CM proliferation. Smooth muscle cells were stained with mouse anti–α-SA, while rabbit anti-Ki67 and rabbit anti-Histone H3 were used to stain for cell proliferation. DAPI with mounting media was applied to the samples. Single nucleus RNA-sequencing (snRNA-sq) analysis is performed to identify key gene features and phenotype changes that may be affected by the MSC-Exo treatment. ImageJ and MATLAB were used to analyze histology and IHC images.

Results, Conclusions, and Discussions: The intrapericardial cavity (iPC) injection of MSC-Exo encapsulated in HA hydrogel demonstrated significant enhancements in cardiac function, cardiomyocyte proliferation, and phenotype alteration in a porcine myocardial infarction (MI) model. Notably, echocardiogram analysis revealed a substantial improvement in left ventricular ejection fraction (LVEF) by Day 28 post-MI compared to Day 3 in the MSC-Exo group. Histological examination indicated greater tissue viability in the MSC-Exo group alongside a marked increase in infarct wall thickness and a significant decrease in fibrotic tissue percentage. Immunohistochemistry (IHC) analysis underscored a notable increase in cardiomyocyte proliferation, as evidenced by heightened levels of both Ki67 and pH3 proliferation markers. Moreover, single-nucleus RNA sequencing (snRNA-seq) unveiled the presence of immature cardiomyocytes with distinct gene expressions within the MSC-Exo group. This preclinical investigation aimed to bridge the translational gap between laboratory research and clinical application by employing a large animal model of pig MI and utilizing a minimally invasive approach of iPC injection of MSC-Exo embedded in hyaluronic acid hydrogel. Results demonstrated improved cardiac function, marked by increased LVEF and reduced infarct size in the MSC-Exo treatment group. The observed augmentation in infarct wall thickness, reduction in fibrotic tissue, and heightened cardiomyocyte proliferation suggest the potential for cardiomyocyte regeneration and tissue repair facilitated by MSC-Exo therapy. Remarkably, the identification of immature cardiomyocytes with altered gene expressions highlights the therapeutic potential of MSC-Exo in orchestrating cellular responses conducive to cardiac repair. Gene expression profiling indicated the upregulation of genes associated with cell proliferation, angiogenesis, and anti-inflammatory pathways in immature cardiomyocytes following MSC-Exo treatment. Further elucidation of molecular pathways activated by MSC-Exo in the cardiac microenvironment warrants additional snRNA-seq analyses. These findings collectively advocate for the potential of iPC injection of MSC-Exo as a promising therapeutic avenue for patients afflicted with MI. Consequently, phase I clinical trials are imperative to assess the safety and efficacy of MSC-Exo therapy in clinical settings.

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