Key result
Magnetic CD144-based isolation achieved >95% purity of endocardial endothelial cells from pediatric congenital heart disease tissues, reproducibly preserving their phenotype and disease-specific susceptibility to endothelial-to-mesenchymal transition.
Why the study?
Endocardial fibroelastosis-derived endocardial endothelial cells (EdEECs) remain largely understudied in congenital heart disease due to lack of standardized isolation and purification protocols from pediatric tissue.
p-value: p=<0.001
A reproducible CD144-based magnetic isolation protocol successfully purifies human pediatric endocardial endothelial cells while preserving their endocardial phenotype and intrinsic disease-specific susceptibility to endothelial-to-mesenchymal transition.
Provides a reproducible in vitro platform to study endocardial fibroelastosis mechanisms; leaves open clinical translation in pediatric congenital heart disease.
Congenital heart defects (CHD) are characterized by pathological intracardiac blood flow, exposing the endocardium to disturbed shear stress. This contributes to the development of endocardial fibroelastosis (EFE), a restrictive fibrotic remodeling process associated with diastolic dysfunction. Despite the pathomechanistic role of the endocardium in CHD, EFE-derived endocardial endothelial cells (EdEECs) remain largely understudied. No standardized protocol exists for isolating and purifying them from human pediatric tissue. Tissue from N = 15 pediatric patients with (1) hypoplastic left heart syndrome, (2) pulmonary atresia with intact ventricular septum, and (3) pulmonary vein stenosis was included. Immunohistochemistry demonstrated endothelial-to-mesenchymal transition (EndMT). EdEECs were isolated from resected EFE tissue using CD144-based magnetic separation. Enrichment of the endocardial phenotype was evaluated by flow cytometry, immunocytochemistry and RT-qPCR. To mimic disease-like conditions, EdEECs were exposed to in vitro shear stress and transforming growth factor beta 1. EFE driven by fibrogenic activation of EECs through EndMT was observed in the left ventricle, right ventricle, and left atrium in patients with different CHDs. Magnetic CD144-based isolation provided a reliable, gentle approach to purify EdEECs from cardiac tissue, achieving > 95% purity while reproducibly preserving endocardial identity at both the protein and gene expression levels. Isolated EdEECs retained their intrinsic, disease-specific predisposition and vulnerability to EndMT under static, and high shear stress. Unlike healthy EECs (HEECs), physiological, low shear stress alone was not sufficient to restore the intrinsic fibrogenic vulnerability of EdEECs. EndMT-mediated EFE was observed across different CHD entities. A reproducible protocol for isolating and purifying EdEECs from EFE tissue preserves their endocardial phenotype and disease-specific characteristics. This provides a disease-mimicking in vitro model to study EdEEC-specific signaling pathways.
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Gaal et al. (2026) studied Congenital heart defects (hypoplastic left heart syndrome, pulmonary atresia, pulmonary vein stenosis) (n=15). Magnetic CD144-based cell isolation and in vitro disease modeling vs. Native unpurified cell cultures and healthy endocardial endothelial cells was evaluated on Enrichment and purity of the endocardial endothelial phenotype (p=<0.001). Magnetic CD144-based isolation achieved >95% purity of endocardial endothelial cells from pediatric congenital heart disease tissues, reproducibly preserving their phenotype and disease-specific susceptibility to endothelial-to-mesenchymal transition.
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