Abstract Body (Do not enter title and authors here): Introduction: Turner syndrome (TS) is caused by the partial or complete absence of one sex chromosome and affects 1 in 2500 liveborn infants. Hypertension, bicuspid aortic valve, and thoracic aortic aneurysms are more prevalent in TS compared to the general population. We hypothesize that persistent abnormalities of endothelial-mesenchymal transition (endMT) in the embryo and adult mediated in part by abnormal responses to shear stress predispose to cardiovascular disease in TS. The objective of this study was to define functional and transcriptomic differences between euploid 46,XX and aneuploid 45,X induced pluripotent stem cell (iPSC)-derived endothelial cells (ECs) from adult donors with mosaic TS.

Methods: iPSCs were reprogrammed from peripheral blood mononuclear cells. Individual colonies were genotyped to identify isogenic euploid and aneuploid iPSC subclones, which were differentiated into ECs using standard methods. After selection for CD31 expression, baseline EC properties and response of EC monolayers to shear stress using a cone and plate viscometer system were characterized using fluorescence microscopy, qPCR, RNA sequencing, and functional assays.

Results: We generated 45,X, 46,X,iso(X)(q10) [iso], and 46,XX iPSCs from one mosaic donor. After confirming genomic integrity and pluripotency, we successfully differentiated multiple iPSC subclones of each karyotype into ECs. EC morphology, tube formation, and uptake of DiI-conjugated acetylated low-density lipoproteins were not significantly different between subclones, although 45,X ECs proliferated more slowly than 46,XX or iso ECs. Collagen gel contraction and migration by 45,X ECs was significantly higher than 46,XX or iso ECs at baseline and in response to exogenous TGF-β peptide. Comparative RNAseq and qRT-PCR analysis of 45,X and 46,XX ECs showed that VCAM1, SNAI1, ACTA2, TWIST1, and other endMT-associated genes were significantly upregulated at baseline in 45,X ECs and were only partially suppressed by shear stress, while expression of CDH2 was decreased in 45,X ECs. We also observed that 45,X ECs but not 46,XX or iso ECs underwent spontaneous endMT in static culture conditions.

Conclusion: Despite similar baseline EC phenotypes, the threshold for endMT activation appears to be lower in 45,X ECs compared to 46,XX or iso ECs. Dysregulation of endMT, possibly due to chronic derepression of SNAI1 expression, may contribute to congenital cardiovascular disease in TS.