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

Abstract

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. 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. 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 pro-angiogenic 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. 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.

Keywords: Blood-brain barrier; Brain angiogenesis; Brain capillaries; Brain microvascular endothelial cells; Induced pluripotent stem cells; Microvascular tissue engineering.

Figure 1.. Three-dimensional iPSC model of brain angiogenesis.

(a) Schematic timeline of the differentiation of human induced pluripotent stem cell (hiPSC) into brain microvascular endothelial cells (dhBMECs) using sequential treatments with mTESR1, UM/F- and RA media (composition defined in Methods) over ten days on Matrigel-coated plates. (b) Phase contrast / epifluorescence overlays corresponding to steps shown in Fig. 1a. WTC iPSC line with RFP-tagged plasma membrane was used. (c) Schematic timeline of the bead angiogenesis assay showing multiplexed coating of beads with collagen IV (Cn IV) and fibronectin (Fn), seeding with dhBMECs, formation of blood-brain barrier (BBB) beads, embedding of BBB beads into extracellular matrix (ECM), and treatment with angiogenic stimuli. (d) Phase contrast / epifluorescence overlays corresponding to steps shown in Fig. 1c.

Figure 2.. Characterization of BBB beads: protein expression and function.

(a) Confluent monolayers of dhBMECs on 150 μm diameter beads express localized tight junction proteins (occludin and claudin-5), the glucose transporter-1 (GLUT1) nutrient transporter, the p-glycoprotein (Pgp) efflux pump, and endothelial markers (CD31). (b) BBB beads express receptors for basic fibroblast growth factor (FGFR2), vascular endothelial growth factor (VEGFR2), and wnt7a (GPR124). Beads were incubated in 200 μM Lucifer yellow (LY) for 3 hours. Three conditions were tested: (1) blank beads without LY, (2) blank beads with LY, and (3) beads with dhBMEC monolayers with LY. (c) Quantification of LY fluorescence in a circular region of interest (ROI) within the beads using confocal microscopy (shown is corresponding phase contrast image). (d) Comparison of normalized fluorescence across conditions (N = 3). (e) Normalized fluorescence images for each condition; beads with dhBMECs significantly restrict accumulation of LY. dhBMECs were generated from the plasma membrane (PM) RFP-tagged WTC iPSC line. *p < 0.05. ***p < 0.001.

Figure 3.. Influence of chemical factors on dhBMEC angiogenesis.

Three media conditions were tested: (1) basal media, (2) basal media + 20 ng mL⁻¹ bFGF, and (3) basal media + 20 ng mL⁻¹ bFGF + 50 ng mL⁻¹ VEGF + 50 ng mL⁻¹ wnt7a. Across these conditions, dhBMEC beads were embedded within 6 mg mL⁻¹ collagen I hydrogels. (a) Representative images of beads on day 2, 4 and 6 after embedding in hydrogels. Sprouts are marked with red asterisks. (b-d) Angiogenic fraction, sprout density and maximum sprout length quantified across conditions on day 6. (e) Plot of maximum sprout length over time for treatment with basal media + 20 ng mL⁻¹ bFGF + 50 ng mL⁻¹ VEGF + 50 ng mL⁻¹ wnt7a. (f) Confocal image of angiogenic processes at day 6 in basal media + 20 ng mL⁻¹ bFGF + 50 ng mL⁻¹ VEGF + 50 ng mL⁻¹ wnt7a. The image is a maximum intensity projection over a depth of 240 μm, with inset demonstrating a lumen-like structure. The dotted circle represents the border of the bead; the dotted line represents the location of the cross-section shown in the inset. Data obtained from N = 5 rounds of the bead assay from unique differentiations, with greater than 5 technical replicates per differentiation. * p < 0.05. ** p < 0.01.

Figure 4.. Influence of extracellular matrix components on dhBMEC angiogenesis.

Four ECM conditions were tested: (1) 6 mg mL⁻¹ collagen I, (2) 6 mg mL⁻¹ collagen I + 1.5 mg mL⁻¹ fibrin, (3) 6 mg mL⁻¹ collagen I + 1.5 mg mL⁻¹ Matrigel, (4) 6 mg mL⁻¹ collagen I + 0.5 mg mL⁻¹ fibronectin. Across these conditions, a combination of bFGF, VEGF and wnt7a were applied (media condition #3). (a) Representative images of dhBMEC beads on day 6 after embedding in hydrogels, across conditions. (b-d) Angiogenic fraction, sprout density, and maximum sprout length quantified across conditions on day 6. Data obtained from N = 5 rounds of the bead assay from unique differentiations, with greater than 5 technical replicates per differentiation. * p < 0.05.

Figure 5.. Influence of oxidative stress on dhBMEC angiogenesis.

dhBMEC beads were exposed to vehicle (H₂O), 10 μM, and 1 mM hydrogen peroxide (H₂O₂) after embedding into collagen I + Matrigel hydrogels supplemented with basal media. (a) Phase contrast images of angiogenic behavior across conditions on day 2. Sprouts are marked with red asterisks. (b) Day 2 sprout density across conditions. (c) Time course of angiogenic fraction across conditions. Data obtained from N = 4 rounds of the bead assay from unique differentiations, with greater than 5 technical replicates per differentiation. * p < 0.05, ** p < 0.01.

Figure 6.. Modeling angiogenesis from tissue-engineered brain microvessels.

(a-b) Schematic illustrations showing front and side views of model fabrication. Angiogenic factors are introduced after microvessel formation (one day after seeding BMECs) to promote sprouting. (c) Phase contrast and fluorescence image overlays of fabrication process. (d) Phase contrast and fluorescence image overlays of representative microvessels perfused with media conditions matching Figure 3. Early sprouts are marked with white asterisks.

Figure 7.. Hierarchical model of the BBB via angiogenesis between existing tissue-engineered brain microvessels.

(a-b) Front and side view schematics of hierarchical model of brain angiogenesis. (c) Flow system under to control perfusion of hierarchal model. (d) Phase contrast and fluorescence image overlays of hierarchical capillary network formation. After formation in basal media, microvessels are perfused at 2 dyne cm⁻² with 20 ng mL⁻¹ bFGF to promote anastomosis of sprouts. Anastomosed capillaries are visible after 3 days. (e) Hierarchical model perfused with 500 kDa dextran for one hour. No leakage of dye was observed indicating that capillaries were intact and preserved barrier function. (f) Confocal imaging of capillary lumen perfused with 500 kDa dextran. (g) Confocal imaging of glucose transporter-1 (GLUT1) nutrient transporter. Confocal images are shown at a specific z-plane, with the xz cross-section denoted as a dotted while line.