Immune-mediated regeneration of cell-free vascular grafts in an ovine model

We have employed single cell RNA sequencing transcriptomics to characterize the cellular sub-types populating the H2R5 ATEV (Grafts) explanted at 12 weeks, from an Ovine (Sheep) model. The ATEVs were primarily reconstructed by the infiltrating myeloid-precursor cells/monocytes that differentiate under different physiological and mechanosensitive cues into macrophages, smooth muscle cells and endothelial cells. Cell specific markers (CSF1R, CD14, CD163, C1QA, PTPRC) for monocytes, (MYH11, ACTA2, MYL9, TAGLN) for Smooth muscle cells, (PECAM1, VWF, NOS3, CDH5, CLDN5) for endothelial cells were employed to determine the major cell populations in the ATEVs. Characterization of the immune-cell types populating the ATEVs hold immense potential in developing future generation grafts for cardiovascular applications. The ATEVs and the native carotid artery were explanted from the sheep after 12 weeks of implantation. The isolated tissues, maintained in EBM2 medium containing FBS, were first washed with PBS, then clamped with metal clippings and then perfused with Digestion buffer containing collagenase, Neutral protease, Elastase and DNAse1. Following incubation at 37°C for 20 minutes, the enzymatically digested cells were neutralized with FBS, passed through a 40 µ cell strainer and resuspended as single cells and analyzed using scRNA seq.