S18 - Single-Cell Resolution Mapping of Pericyte Contributions to Cardiac Organoids: Building a Better Cardiac Regeneration Platform

Introduction: : Cardiovascular diseases (CVDs) are the leading cause of death worldwide. Due to the lack of regenerative capacity of adult hearts, cell-based therapeutics have been investigated to regain cardiac function post-myocardial infarction (MI). Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) hold significant promise for treatment of infarcted hearts. However, the current approach of intramyocardial injection of dissociated hPSC-CMs suffers from low cell survival, moderate functional recovery, arrhythmogenic risk, and poor scalability.

Materials and

Methods: : We performed scRNA-seq on isogenic cardiac organoids five days post-sequential seeding. Organoids were dissociated with TrypLE (Gibco) for 40 minutes, then counted to ensure proper dissociation without significant cell death. Libraries were prepared according to the 10X protocol, and 4 libraries (2 3-cell type cardiac organoids, 2 4-cell type cardiac organoids) were sent to Azenta Life Sciences for sequencing. To align reads to the reference human genome GRCh38, Cell Ranger (10X Genomics) was used. Default parameters were used to align reads and count unique molecular identifiers (UMIs) in order to create gene-cell expression matrices. The Python package Scanpy (version 1.10.2) was used to perform downstream scRNA-seq analysis. Datasets were filtered for low-quality cells, removing any cells with greater than 25% mitochondrial reads, less than 200 genes, and less than 3 cells. Datasets were preprocessed by cutting off outlying data, using an upper limit of 98% and lower limit of 2%, while normalizing each cell to 10,000 UMI and performing a log transformation to reduce expression differences across libraries. Unsupervised clustering analysis and visualization via Uniform Manifold Approximation and Projection (UMAP) was performed to visualize. Canonical markers for cardiomyocytes, cardiac fibroblasts, endothelial cells, and pericytes were used to annotate clusters. Additionally, saved anndata (.h5ad) objects after pre-processing, cluster annotation, and harmony integration, and translated them into R for Seurat use, so that we could use powerful packages built for R, such as CellChat, NicheNet, and DecoupleR.

Results, Conclusions, and Discussions:: The addition of hPSC-PCs promoted cardiac organoid survival in hypoxic conditions, enhanced sarcomere organization of organoid cardiomyocytes, and increased hPSC-EC expression. To further elucidate mechanisms of action in in vitro observations, we conducted single cell RNA-seq (scRNA-seq) on cardiac organoids without (3-cell type) and with (4-cell type) hPSC-PCs. A novel population of PDGFRB+/RGS5+ pericytes were identified in 4-cell type cardiac organoids via scRNA-seq. Importantly, an increase of PECAM1+ hPSC-ECs were identified upon the addition of hPSC-PCs, supporting vasculature stabilization findings in vitro. Downstream analyses, including NicheNet ligand-receptor inference, Pseudo bulk transcription factor inference, and CellChat pathway-specific cellular crosstalk allowed deep insight into multiple mechanisms of hPSC-PC-induced enhancement of cardiac organoid survival. hPSC-PC addition increased angiogenesis via upregulation of BHLHE40 and VEGF signaling, HIF-1α modulation of fibrosis, and BMP and MEF2C-mediated cardiomyocyte maturation. These findings highlight the importance of including hPSC-PCs within cardiac organoids, as they promote survival as a cellular therapeutic, further increasing cardiac organoid efficacy in cardiac recovery with a lower dose of cells.

Acknowledgements and/or References (Optional)::