34 Generating Human Brain Microvascular Endothelial Cells Derived From Inducible Pluripotent Stem Cells
Bokai K. Zhang and J David Symons
Faculty Mentor: J David Symons (Nutrition and Integrative Physiology, University of Utah)
The mechanistic relationship between aging and worse outcomes of acute ischemic stroke (AIS) is unclear. In pilot studies, we find that 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 us to hypothesize that cerebral ECs from older individuals vs. adults 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 first seeding at a cell density of 15,800 cells/cm² (Day 1) and treating with E6 media (Days 2-5), followed by human endothelial serum-free medium containing basic fibroblast growth factor (bFGF), retinoic acid (RA), and B27 supplements (Day 6-8). Next, iPSCs were transferred to collagen IV/fibronectin-coated plates, and RA and bFGF were withdrawn for 24 hours, (Day 9) thereby completing the iPSC-BMEC transition procedure.[1][2] iPSC-BMECs were first validated through morphological analysis and cell viability assays, followed by assessments of key BMEC characteristics. Specifically, we evaluated: (i) tube formation capacity; (ii) tight junction barrier integrity; (iii) expression of glucose transporter isoform 1 and claudin-5; and (iv) insulin-stimulated nitric oxide generation. iPSC-BMECs exhibited a spindle-shaped, cobblestone-like morphology, maintained high viability 48 hours post-seeding, and demonstrated paracellular barrier integrity comparable to immortalized BMECs. However, the insulin-stimulated nitric oxide production and tube formation assays require further optimization. The next step is to reprogram iPSCs from PBMCs of adult and older volunteers into BMECs to test the hypothesis that cerebral EC autophagy is repressed by aging.
Figure 1. The figures show cell images at days 1, 4, 6, 8, and 9 of the iPSC-BMEC transition procedure.
Acknowledgement
Bokai Zhang was funded by an American Heart Association Institutional Award for Undergraduate Student Training (25IAUST1374429). This program is a partner of the Office of Undergraduate Research at the University of Utah.
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