Abstract Body: Introduction: The tunica adventitia of human blood vessels homes stem/progenitor cells. Prior studies confirm that the adventitia resident progenitors participate in vascular pathologies such as atherosclerosis and vessel injury. However, primary challenge is to unravel the identity and function of these heterogenous cell population. To address this, an integrated single cell and spatial transcriptomics, and functional assays to identify adventitial progenitor cells within human blood vessels were performed. Our findings revealed the presence of Thrombomodulin (CD141/THBD) expressing adventitial progenitor subset with distinct proliferative and osteogenic capacity.
Methods and Results: Single-cell RNA sequencing of flow cytometry purified CD34⁺CD31^-CD45^-CD146^- human adipose derived adventitial progenitors were performed. Three clusters were identified that expressed adventitial cell marker, CD34, with no notable expression of smooth muscle, endothelial cell, or pericyte markers. Cell surface markers previously undescribed in adventitial cells were then investigated revealing the Thrombomodulin expressing subsets (THBD^High/Low). Combined spatial transcriptomics (n=4 vessels) and scRNA-seq showed the homogenous distribution of THBD^High cells in the adventitia of artery and vein . Increased expression of THBDwas associated with early pseudotime, making these cells primitive in progenitor hieracrchy . GO terms of THBD^High cell associated with proliferative and inflammatory responses while THBD^Low cells supported osteogenesis and angiogenesis. Curated KEGG pathways revealed major signaling mechanisms modulating these subsets. In situ immunofluorescence staining confirmed colocalization of CD34⁺CD141⁺ cells in adventitia. In vitro isolation and characterization of CD141^High/Low cells by flow cytometry confirmed CD141^High cells to be 4% (n=3 donors) of total adventitial cells and showed increased colony formation, proliferation with decreased osteogenic potential in comparison to CD141^Low expressing cells.
Conclusion: In summary, the present study highlights the presence of phenotypically, functionally, and spatially defined subsets of human adventitial progenitor cells based on expression of Thrombomodulin. Study resolves the complexity of human adventitial progenitor niche and with future implication on the role of these cells in vessel pathologies.