(507d) Exploring the Role of Pericytes in Blood-Brain Barrier Maintenance Using hiPSC-Derived 2D and 3D in Vitro models

Objective: Pericytes of the blood-brain barrier (BBB) are embedded within basement membrane between microvascular endothelial cells (BMECs) and astrocyte end-feet, and are believed to modulate vascular permeability. Despite the direct cell-cell contact observed in vivo, most in vitro BBB models introduce an artificial membrane that separates pericytes from BMECs. In this study, we investigated the effects of pericytes on BMEC barrier function across a range of in vitro platforms with varied spatial orientations and levels of cell-cell contact. We also examined the effects of pericytes on barrier function under conditions designed to mimic cerebral ischemia.

Methods: We differentiated RFP-pericytes and GFP-BMECs from human induced pluripotent stem cells (hiPSCs) and monitored transendothelial electrical resistance (TEER) across BMECs on transwell inserts while pericytes were either directly co-cultured on the membrane, indirectly co-cultured in the basolateral chamber, or embedded in a collagen I gel formed on the transwell membrane. We also incorporated pericytes into a tissue-engineered microvessel model of the BBB and measured pericyte motility and microvessel permeability. We then exposed these models to oxygen and/or glucose deprivation (OGD) conditions in order to recapitulate key aspects of cerebral ischemia and explore the timing and extent of BBB disruption.

Results: We found that BMEC monolayers did not require co-culture with pericytes to achieve physiological TEER values (> 1500 Ω cm²). However, under stressed conditions where TEER values for BMEC monolayers were reduced, indirectly co-cultured hiPSC-derived pericytes or conditioned media restored optimal TEER. In the microvessel model, we observed direct pericyte-BMEC contact, abluminal pericyte localization, and physiologically-low Lucifer yellow permeability comparable to that of BMEC microvessels. When subjected to hypoxic conditions, BMECs cultured in pericyte-conditioned media maintained a significantly tighter barrier than non-conditioned media.

Conclusions: We demonstrated that monocultured BMECs do not require co-culture to achieve physiological TEER, but that suboptimal TEER in stressed monolayers can be increased through co-culture with hiPSC-derived pericytes or conditioned media, suggesting we may be able to identify factors capable of restoring and preserving human BBB barrier function after ischemic injury. We also developed the first BBB microvessel model using exclusively hiPSC-derived BMECs and pericytes, which could be used to examine vascular dysfunction in the human CNS.