Minimally Invasive Delivery of Synthetic Cardiac Stromal Cells Improves Heart Repair in Pigs

Introduction::
Myocardial infarction (MI) is the primary manifestation of cardiovascular events. Although cell therapeutics for cardiac regeneration are commonly used for tissue remodeling post-MI, their viability, retention, and cost have led researchers to explore acellular therapeutic approaches such as synthetic cardiac stromal cells (synCSCs). SynCSCs are generated by encapsulating secreted factors from isolated human cardiac stromal cells and have shown promising results for tissue repair after a heart attack. However, previous studies involved surgical placement of synCSCs embedded in scaffolds onto the surface of the heart, which carries a risk of surgical complications. Reducing the risks of surgical complications is crucial for translational research. Therefore, it would be beneficial to deliver synCSCs embedded in hyaluronic acid (HA) hydrogel using a minimally invasive approach such as intrapericardial cavity (iPC) injections. This preclinical study combined the benefits of an acellular cardiac regeneration therapeutic with an injectable delivery route in a large animal model. The results show that iPC injection of synCSC 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 synCSC embedded in HA hydrogel via iPC injection. Human cardiac stromal cell (CSC) factor-loaded synCSCs were fabricated according to previously described polymer encapsulation approaches [1]. HA hydrogel was prepared according to previous studies [2]. 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 conducted using MRI cine mode of the long and short axes, and left ventricle ejection fraction (LVEF) was calculated for pre-MI, 3 days post-MI, and 28 days post-MI. T2-weighted MRI scans were taken to measure the infarct area. 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 synCSC treatment. OsiriX DICOM Viewer was used to conduct MRI image analysis, while ImageJ and MATLAB were used to analyze histology and IHC images.


Results, Conclusions, and Discussions:: The iPC injection of synCSCs embedded in HA hydrogel improves cardiac function, enhances cardiomyocyte proliferation, and induces phenotype alteration in the pig MI model. T2-weighted MRI analysis depicts attenuated infarct size at Day 28 in the synCSC treatment group compared to both MI and MI + HA groups. The quantification of the infarct size exhibits a significant decrease in total MI area in the synCSC group. MRI cine mode analysis shows a significant recovery in LVEF on Day 28 compared to Day 3 post-MI for the synCSC group. Histology slides display more viable tissue in the synCSC group compared to the controls. The infarct wall thickness for the synCSC group is also significantly increased. Furthermore, the fibrotic percentage significantly decreased in the synCSC group. IHC analysis shows a significant increase in cardiomyocyte proliferation in the synCSC group indicated by both Ki67 and pH3 proliferation markers. SnRNA-seq elucidates immature cardiomyocytes with different gene expressions in the synCSC group.

This preclinical study aims to bridge the gap between laboratory research and clinical practice by utilizing a large animal model of pig myocardial infarction and a minimally invasive approach of intrapericardial cavity injection of synCSCs embedded in the hyaluronic acid hydrogel. The results of the synCSC treatment showed improved cardiac function, as indicated by elevated LVEF and attenuated infarct size. The increased wall thickness, decreased fibrotic percentage, and increased cardiomyocyte proliferation suggest the potential for cardiomyocyte regeneration and tissue repair in the synCSC treatment group. It is noteworthy that immature cardiomyocytes with different gene expressions were found in the synCSC group. The overexpression of genes involved with cell proliferation and angiogenesis was observed in the synCSC group, and gene expression analysis suggests the activation of anti-inflammatory protection in immature cardiomyocytes. Further snRNA-seq analyses are needed to uncover the molecular pathways activated by synCSC in the cardiac environment. The findings suggest that iPC injection of synCSC may offer a unique therapeutic option for patients suffering from MI, and phase I clinical trials are necessary to evaluate its safety and efficacy.


Acknowledgements (Optional): :

References (Optional): :
1. Huang, K. et al. An off-the-shelf artificial cardiac patch improves cardiac repair after myocardial infarction in rats and pigs. Science Translational Medicine 12. issn: 1946-6234 (Apr. 2020).




2. Zhu, D. et al. Minimally invasive delivery of therapeutic agents by hydrogel injection into the pericardial cavity for cardiac repair. Nature Communications 12, 1412. issn: 2041-1723 (Mar. 2021).