(630c) Modeling Diseased BBB through Directed Differentiation of Patient-Derived iPSCs into Brain Microvascular Endothelial Cells

The blood-brain barrier (BBB) forms the interface between the microvascular system and the brain and is responsible for maintaining homeostasis in the central nervous system. As such, disruption to the BBB is implicated in many neurological diseases, but whether the loss in BBB integrity is an upstream or downstream effect of the disease can be difficult to ascertain. We aimed to investigate this question for the disease cerebral childhood adrenoleukodystrophy (ccALD), which is an X-linked disease that affects males ages 3-10 and results in death within 3-5 years of clinical onset. ccALD is caused by mutations in the ABCD1 gene which codes for a transporter responsible for bringing very long chain fatty acids (VLCFAs) into the peroxisome for subsequent beta oxidation. Buildup of VLCFAs in the brain leads to rapid neurological deterioration in a subset of patients. We hypothesized that the BBB integrity of ccALD patients is inherently decreased compared to wild-type (WT) individuals. To model the BBB, we differentiated induced pluripotent stem cells (iPSCs) that originated from WT individuals and from ccALD patients into brain microvascular endothelial cells (BMECs) to compare their molecular and functional characteristics. The differentiated BMECs were characterized via PCR analyses and immunocytochemistry to confirm that the BMECs possess requisite BBB markers. Functional characteristics were quantified by the transendothelial electrical resistance (TEER), sodium fluorescein permeability, and P-glycoprotein activity. A stark functional difference was observed in the TEER of the BMECs. BMECs from ccALD patient iPSCs (ccALD-BMECs) displayed significantly reduced TEER (2590 ± 330 Ω·cm²; mean ± standard error) compared to that of the WT-BMECs (5000 ± 510 Ω·cm²). This result signifies that the barrier function of the ccALD-BMECs is inherently worse than that of the WT-BMECs. With this experimental evidence, we aimed to improve the barrier function of the ccALD-BMECs utilizing block copolymers. Block copolymers of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) have been demonstrated to be able to protect damaged cell membranes. Treatment of the ccALD-BMECs with a diblock copolymer of PEO and PPO (PPO-b-PEO) resulted in improvement of barrier function. We observed an approximate 60% increase in the TEER of ccALD-BMECs treated with PPO-b-PEO during development compared to the TEER of the control. Furthermore, we observed an approximate 20% increase in TEER of ccALD-BMECs treated with PPO-b-PEO post-development compared to the TEER of the control. In summary, we have provided evidence for functional differences between BBB cells derived from iPSCs that originated from ccALD and WT individuals. Furthermore, we have demonstrated that the deficient functionalities of the ccALD-BMECs can be improved by treatment with block copolymers.