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

Abstract

Background: During brain development, chemical cues released by developing neurons, cellular signaling with pericytes, and mechanical cues within the brain extracellular matrix (ECM) promote angiogenesis of brain microvascular endothelial cells (BMECs). Angiogenesis is also associated with diseases of the brain due to pathological chemical, cellular, and mechanical signaling. Existing in vitro and in vivo models of brain angiogenesis have key limitations. Methods: Here, we develop a high-throughput in vitro blood-brain barrier (BBB) bead assay of brain angiogenesis utilizing 150 μm diameter beads coated with induced pluripotent stem-cell (iPSC)derived human BMECs (dhBMECs). After embedding the beads within a 3D matrix, we introduce various chemical cues and extracellular matrix components to explore their effects on angiogenic behavior. Based on the results from the bead assay, we generate a multi-scale model of the human cerebrovasculature within perfusable three-dimensional tissue-engineered blood-brain barrier microvessels. Results: A sprouting phenotype is optimized in confluent monolayers of dhBMECs using chemical treatment with vascular endothelial growth factor (VEGF) and wnt ligands, and the inclusion of proangiogenic ECM components. As a proof-of-principle that the bead angiogenesis assay can be applied to study pathological angiogenesis, we show that oxidative stress can exert concentration-dependent effects on angiogenesis. Finally, we demonstrate the formation of a hierarchical microvascular model of the human blood-brain barrier displaying key structural hallmarks. Conclusions: We develop two in vitro models of brain angiogenesis: the BBB bead assay and the tissue-engineered BBB microvessel model. These platforms provide a tool kit for studies of physiological and pathological brain angiogenesis, with key advantages over existing two-dimensional models. Background Brain angiogenesis is a multistage process by which new capillaries sprout from existing blood vessels. The culmination of brain angiogenesis during development results in a 600 km network of capillaries forming the blood-brain barrier (BBB) [1]. Brain microvascular endothelial cells (BMECs),

Three-dimensional microenvironment regulates gene expression, function, and tight junction dynamics of iPSC-derived blood-brain barrier microvessels

“…The angiogenic response was compared across three conditions (microvessels, microvessels cocultured with pericytes, and a bead assay) by measuring the total length of sprouts per unit area of endothelium. As previously observed, iBMECs were responsive to growth factors, including bFGF [48] (Fig. 3C).…”

“…Genipin crosslinking was not used for these studies as the increased stiffness suppresses angiogenic sprouting. As a static control, we lined microspheres with iBMECs and embedded them within hydrogels as previous reported [48]. After 48 h treatment with 20 ng mL -1 bFGF (R&D Systems), sprouts were manually counted in ImageJ, where "sprout length" represents the total length of sprouts per unit area of endothelium.…”

Commentary on human pluripotent stem cell-based blood–brain barrier models

“…Most relevant to this commentary, induction and maintenance of endothelial character with a corresponding reduction of epithelial character continues to be a goal. Factors such as hypoxia [19], shear stress [9,19], and three dimensional architecture [10,28,29] have been suggested to increase vascular character, and other models based on induction of BBB character in generic hiPSC-derived ECs are beginning to emerge [32]. Despite these advances, we believe it is prudent to exercise caution when utilizing hPSC-derived BMEC-like cells for studies where the endothelial phenotype is crucial.…”

“…We have observed VEGF-dependent network formation in hPSC-derived BMECs using in vitro Matrigel assays [ 1 ], and VEGF has been shown to regulate PLVAP expression in a three-dimensional BBB model that contains hPSC-derived BMECs [ 10 ]. hPSC-derived BMECs also respond to shear; we have observed sprouting-like behavior after culturing these cells in engineered hydrogel matrices under constant perfusion [ 28 ], and others have observed similar phenotypes in hydrogels in response to growth factors and other stimuli such as oxidative stress [ 29 ]. In addition, application of shear after differentiation also led to activation of cholesterol metabolism, proliferation, and angiogenesis transcriptional pathways compared to static controls [ 9 ].…”

Brain Barriers and brain fluids research in 2020 and the fluids and barriers of the CNS thematic series on advances in in vitro modeling of the blood–brain barrier and neurovascular unit

“…A plethora of new models have been developed (too many to review fully here). For example, Nishihara et al [163] used what they term an extended endothelial culture method for human iPSCs which allows the study of immune cell interactions with BMEC-like cells and Linville et al [164] have developed a novel 3-D model using human iPSCs to study human brain angiogenesis. Also, brain organoids were made from human embryonic stem cells that form blood vessel-like structures (Ham et al [165]) and Ahn et al [166] developed a microphysiological human BBB platform that allows 3D tracking of nanoparticles in the vascular and perivascular spaces.…”