Role of iPSC-derived pericytes on barrier function of iPSC-derived brain microvascular endothelial cells in 2D and 3D

Abstract

Background Pericytes of the blood–brain barrier (BBB) are embedded within basement membrane between brain microvascular endothelial cells (BMECs) and astrocyte end-feet. 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. Methods We differentiated RFP-pericytes and GFP-BMECs from 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 then incorporated pericytes into a tissue-engineered microvessel model of the BBB and measured pericyte motility and microvessel permeability. Results We found that BMEC monolayers did not require co-culture with pericytes to achieve physiological TEER values (> 1500 Ω cm 2 ). However, under stressed conditions where TEER values for BMEC monolayers were reduced, indirectly co-cultured hiPSC-derived pericytes restored optimal TEER. Conversely, directly co-cultured pericytes resulted in a decrease in TEER by interfering with BMEC monolayer continuity. 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. In addition, pericyte motility decreased during the first 48 h of co-culture, suggesting progression towards pericyte stabilization. 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. 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. Electronic supplementary material The online version of this article (10.1186/s12987-019-0136-7) contains supplementary material, which is available to authorized users.

Three-dimensional induced pluripotent stem-cell models of human brain angiogenesis

“…Recent reports of an isogenic multicellular iPSC-based BBB transwell assay provide the foundations for building more complex angiogenesis models [66]. Additionally, we previously incorporated iPSC-derived pericytes into a 3D microvessel BBB model, showing that they do not significantly alter barrier properties [67]. Future work is required to examine how iPSC-derived pericytes and other cells of the BBB may alter angiogenesis in vitro.…”

Engineering Brain-Specific Pericytes from Human Pluripotent Stem Cells

“…However, coculture with PCs restored optimal TEER under stressed conditions. 41 In our study, we found that coculturing MSCs with endothelial spheroids and cortical spheroids promoted the expression of BBB-related genes such as GLUT-1 (gene encoding glucose transporter 1) and BCRP (gene encoding breast cancer resistance protein). 42 Similarly, a protocol was described for coculture of primary endothelial cells, PCs, and astrocytes as 3D organoids that reproduce some of the BBB properties, including the expression of tight junctions, drug efflux pumps, and molecular transporters.…”

“…This method generated the cells expressing CD105, NG2, PDGFRb, CD146, CD73, and CD44 with a 30% yield. 41,106,107 Similarly, a-minimal essential medium plus 10% serum followed by VEGF and SB431542 (the TGF-b superfamily type I activin receptor-like kinase inhibitor) in endothelial cell culture medium produced cells expressing NG2, PDGFRb, CD73, and CD44, which were different from vSMCs. 108 The derived cells were capable of secreting ECM proteins, such as collagens and elastin, as well as ECM remodeling proteins MMP2, 9, 14.…”

Recent advances in human iPSC-derived models of the blood–brain barrier

“…More recent work has focused on differentiating iPSCs into the additional cells of the NVU and combining these with iBMECs for fully iPSC-derived BBB models. Similar to results with primary cells, co-culture of iBMECs with iPSC-derived cells of the NVU can also raise TEER values and improve barrier function [26][27][28][29][30][31], however these additional cells are not required for iBMECs to achieve physiological TEER levels and may only improve barrier properties under suboptimal starting conditions or stress [32]. These studies have initiated a personalized approach to BBB modeling, which will likely provide new insights into genetic-based neurological diseases that may involve cell-cell interactions of the NVU.…”