A HUMANIZED MODEL OF BLOOD-BRAIN BARRIER TO INVESTIGATE IMMUNE CELLS INFILTRATION IN MULTIPLE SCLEROSIS: TOWARD A PERSONALIZED APPROACH

The blood-brain barrier (BBB) is a highly specialized barrier composed of cerebral endothelial cells (ECs) surrounded by pericytes and astrocytic endfeet forming the neurovascular unit (NVU). Progressive BBB breakdown and infiltration of autoreactive leukocytes into the CNS are central features in the pathogenesis of neuroinflammatory disorders, such as Multiple Sclerosis (MS). With the aim of developing a biomimetic humanized NVU in vitro model for addressing MS pathogenic molecular mechanisms and to characterize the phenotype of specific T cells subsets transmigrating across the BBB in MS, we exploited circulating endothelial colony forming cells (ECFCs) isolated from the peripheral blood of MS patients and healthy subjects as a source of primary endothelial cells. Our model consists of a contact co-culture of primary ECs and human astrocytes growing on opposite sides of a matrix-coated permeable membrane, where astrocytes had been purified from the peripheral area of surgical samples of patients undergoing cerebral tumor resection. Autologous lymphocytes are used to perform patient-specific transmigration studies across the BBB model. The frequency of appearance of ECFCs colonies was significantly increased in MS naïve patients compared to both healthy controls and follow-up patients that underwent pharmacological therapies. The endothelial identity of cultured ECFCs was characterized by polychromatic immunophenotyping, confirming their surface expression of the endothelial epitopes CD31, CD146, CD144, CD105, CD106, CD54, CD34, CXCR4 and GLUT1. Confocal microscopy confirmed ECFCs’ endothelial markers expression and their ability to uptake acetylated low-density lipoprotein. ECFCs isolated from MS patients, expanded and cultured in vitro under pro-inflammatory conditions, upregulated endothelial activation markers and adhesion molecules and, reflecting ex vivo the fingerprint of the subjects from which they originated. Also, ECFCs integrated into the NVU in vitro