Presentation description

The mechanistic relationship between aging and worse outcomes of acute ischemic stroke (AIS) is unclear. In pilot studies we find AIS-induced upregulation of cerebral endothelial cell (EC) autophagy is repressed in older vs. adult mice. If EC autophagy affords intrinsic cytoprotection, we reasoned that adult mice with EC-specific depletion of autophagy should display worse AIS outcomes and pilot data are supportive. These findings inspire the hypothesis that cerebral ECs from older vs. adult individuals display repressed indices of autophagy. The focus of my summer project is to develop and optimize the differentiation of induced pluripotent stem cells (iPSCs) derived from human peripheral blood mononuclear cells (PBMCs) into brain microvascular endothelial cells (BMECs). As a first step, iPSCs obtained from a healthy adult female were differentiated into BMECs by treating with E6 media (4 days) followed by human endothelial serum free medium containing basic fibroblast growth factor (bFGF), retinoic acid (RA), and B27 supplements (2 days). Next, iPSCs were transferred to collagen IV/fibronectin-coated plates, and RA and bFGF were withdrawn, thereby completing the iPSC-BMEC transition procedure. At present, studies are ongoing to confirm the BMEC phenotype. For example, after completing initial morphological analyses and cell viability assays, we will determine: (i) tube formation capacity; (ii) tight junction barrier integrity; (iii) glucose transporter isoform 1, claudin-5, and VE-cadherin protein expression; and (iv) insulin-stimulated nitric oxide generation. We hypothesize that results from each assay will be similar between BMECs and commercially available immortalized human brain microvascular endothelial cells, whereas human embryonic kidney cells will be used as a negative control. If successful, we aim to reprogram iPSCs from PBMCs of adult and older volunteers into BMECs, to test the hypothesis that autophagy is repressed by aging.