Single-Cell Transcriptomic Profiling and Characterization of Endothelial Progenitor Cells: New Approach for Finding Novel Markers

Abstract

Background: Endothelial progenitor cells (EPCs) are promising candidates for the cellular therapy of peripheral arterial & cardiovascular diseases. However, hitherto there is no specific marker(s) defining precisely EPCs. Herein, we are proposing a new in-silico approach for finding novel EPCs markers. Methods: we assembled five groups of chosen EPCs-related genes/factors using Pubmed literature & gene ontology databases. This shortened database of EPCs factors was fed into publically-published transcriptome matrix to compare their expression between endothelial colony-forming cells (ECFCs), HUVECs and two adult endothelial cell types (ECs) from skin and adipose tissue. Further, the database was used for functional enrichment on mouse phenotype database and protein-protein interaction network analyses. Moreover, we built a digital matrix of healthy donors' PBMCs (33 thousand single cell transcriptomes) and analyzed the expression of these EPCs factors Results: Transcriptome analyses showed that BMP2,4 & ephrinB2 were exclusively highly expressed in EPCs; the expression of neuropilin-1 & VEGF-C were significantly higher in EPCs & HUVECs compared with other ECs; Notch 1 was highly expressed in EPCs & skin-ECs; MIR21 was highly expressed in skin-ECs; PECAM-1 were significantly higher in EPCs & adipose ECs. Moreover, functional enrichment of EPCs-related genes on mouse phenotype and STRING protein database has revealed significant relations between chosen EPCs factors and endothelial & vascular functions, development and morphogenesis, where ephrinB2, BMP2 and BMP4 were highly expressed in EPCs and were connected to abnormal vascular functions. Single cell RNA-sequencing analyses has revealed that among the EPCs regulated markers in transcriptome analyses: i-ICAM1 & Endoglin were weekly expressed in the monocyte compartment of peripheral blood; ii-CD163 & CD36 were highly expressed in CD14+ monocyte compartment whereas CSF1R was highly expressed in CD16+ monocyte compartment; iii-L-selectin & IL6R were globally expressed in the lymphoid/myeloid compartments; iv-interestingly, PLAUR/UPAR & NOTCH2 were highly expressed in both CD14+ & CD16+ monocytic compartments. Conclusions: The current study has identified novel EPCs markers that could be used for better characterization of EPCs sub-population in adult peripheral blood and subsequent usage of EPCs for various cell therapy and regenerative medicine applications.