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Transcriptional and functional profiling identifies inflammation and endothelial to mesenchymal transition as potential drivers for phenotypic heterogeneity within a cohort of Endothelial Colony Forming Cells.

BACKGROUND: Endothelial colony forming cells (ECFCs) derived from patients can be used to investigate pathogenic mechanisms of vascular diseases like Von Willebrand Disease. Considerable phenotypic heterogeneity has been observed between ECFC clones derived from healthy donors. This heterogeneity needs to be well understood in order to use ECFCs as endothelial models for disease. Therefore, we aim to determine phenotypic and gene expression differences between control ECFCs.

METHODS: A total of 34 ECFC clones derived from 16 healthy controls were analyzed. The transcriptome of a selection of ECFC clones (n=15) was analyzed by bulk RNA sequencing and gene set enrichment analysis. Gene expression was measured in all ECFC clones by qPCR. Phenotypic profiling and migration speed of the ECFCs was done using confocal microscopy, followed by automated quantification of cell morphometrics and migration speed.

RESULTS: Through hierarchical clustering of RNA expression profiles, we could distinguish two major clusters within the ECFC cohort. Major differences were associated with proliferation and migration in cluster 1, and inflammation and endothelial to mesenchymal transition in cluster 2. Phenotypic profiling showed significantly more and smaller ECFCs in cluster 1 which contained more and longer Weibel-Palade bodies (WPBs). Migration speed in cluster 1 was also significantly higher.

CONCLUSION: We observed a range of different RNA expression patterns between ECFC clones mostly associated with inflammation and clear differences in WPB count and structure. We developed a qPCR panel which can be used for the characterization of ECFC clones which is essential for the correct analysis of pathogenic mechanisms in vascular disorders.

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