Human iPSC-derived blood-brain barrier microvessels: validation of barrier function and endothelial cell behavior

Abstract

Microvessels of the blood-brain barrier (BBB) regulate transport into the brain. The highly specialized brain microvascular endothelial cells, a major component of the BBB, express tight junctions and efflux transporters which regulate paracellular and transcellular permeability. However, most existing models of BBB microvessels fail to exhibit physiological barrier function. Here, using (iPSC)-derived human brain microvascular endothelial cells (dhBMECs) within templated type I collagen channels we mimic the cylindrical geometry, cell-extracellular matrix interactions, and shear flow typical of human brain post-capillary venules. We characterize the structure and barrier function in comparison to non-brain-specific microvessels, and show that dhBMEC microvessels recapitulate physiologically low solute permeability and quiescent endothelial cell behavior. Transcellular permeability is increased two-fold using a clinically relevant dose of a p-glycoprotein inhibitor tariquidar, while paracellular permeability is increased using a bolus dose of hyperosmolar agent mannitol. Lastly, we show that our human BBB microvessels are responsive to inflammatory cytokines via upregulation of surface adhesion molecules and increased leukocyte adhesion, but no changes in permeability. Human iPSC-derived blood-brain barrier microvessels support quantitative analysis of barrier function and endothelial cell dynamics in quiescence and in response to biologically- and clinically-relevant perturbations.

Keywords: Blood-brain barrier; Brain microvascular endothelial cells; Induced pluripotent stem cells; Microvessels; Permeability; Tissue engineering.

Figure 1.

Fabrication, perfusion, and maintenance of human iPSC-derived blood-brain barrier microvessels. (A,B) Schematic illustrations of the side- and end-view of 150 μm diameter microvessel fabrication. Type I collagen and agarose is gelled around suspended wire. Wire removal results in a bare channel, which is sequentially treated with genipin and ECM proteins. 3D microvessels form following cell seeding. (C) Phase contrast images of sequential microvessel fabrication steps. (D) PDMS-based microfluidic chip. (E) Perfusion system comprised of tubing connecting inlet and outlet ports to an upper and lower media reservoir (Δh = 5 cm). (F) Shear stress for BC1 dhBMEC and HUVEC microvessels over six days. (G) Phase contrast images of dhBMEC microvessels constructed from various iPSC lines including BC1, iPS12, KW01, and AD6 on day two after seeding. (H,I) Fluorescence images of blood-brain barrier markers in BC1 dhBMEC microvessels on day 2: zona occluden-1 (ZO1), occludin, claudin-5, glucose transporter-1 (GLUT1), and P-glycoprotein (P-gp). Images shown are a 0.4 μm confocal z-slice of the bottom microvessel pole, or a cross-section of the microvessel. Nuclei visualized with DAPI (blue).

Figure 2.

Live-cell imaging of human iPSC-derived blood-brain barrier microvessels. (A) representative phase contrast images of a BC1-derived brain microvessel on days two, four and six under a wall shear stress of about 4 dyne cm^-2. (B) Phase contrast and fluorescence images of perfusion with Lucifer yellow, Rhodamine 123, and 10 kDa dextran at the microvessel midplane. (C,D) Phase contrast images at the top and bottom planes, respectively. (E) Representative fluorescence intensity for Lucifer yellow for a region of interest comprising both the microvessel and surrounding matrix: (i) Prior to perfusion of the dye. (ii) luminal filling where ΔI represents the increase in fluorescence intensity. (iii) Penetration of the dye into the surrounding matrix results in a linear increase in fluorescence intensity (dI/dt).

Figure 3.

Permeability of human iPSC-derived blood-brain barrier microvessels. Representative phase / fluorescence overlays of Lucifer yellow (LY), Rhodamine 123 (R123), and 10kDa dextran perfusion in (A) BC1 dhBMEC microvessels, and (B) HUVEC microvessels. t = 0 min represents the frame prior to initiation of luminal filling. Luminal filling occurred on average over 20 minutes for LY and R123, and 30 minutes for 10 kDa dextran. The rate of fluorescence change was determined over subsequent 20 minutes after luminal filling. (C-E) Permeability of LY, R123, and 10 kDa dextran in HUVEC and BC1 dhBMEC microvessels. (FH) Permeability of LY, R123, and 10kDa dextran in dhBMEC microvessels derived from multiple iPS cell lines (BC1, iPS12, KW01, AD6) on day two following seeding. N = 7 BC1 dhBMEC microvessels on day 2, N = 4 BC1 dhBMEC microvessels on day 4 and 6, N = 4 HUVEC microvessels across all timepoints, N = 3 iPSC12, KW01 and AD6 microvessels. *** p < 0.001.

Figure 4.

Endothelial cell turnover of human iPSC-derived blood-brain barrier microvessels. (A) Representative time-lapse images depicting cell division. Identification of cell division begins with alignment of DNA along the equatorial plate (t = 32 mins, white arrow), chromosomes can be identified pulling apart (t = 34 mins, white arrows), directly proceeding the formation of two daughter cells. (B) Representative time-lapse images depicting cell loss. Identification of cell loss begins with contraction of cell boundaries (t = 6 mins), followed by lysing of cell contents into the lumen associated with a breakdown of the cell membrane (t = 12 mins, white arrow). As the remaining portions of the cell envelope are shed into circulation the surrounding cells migrate to fill the space where the cell was removed. (C-E) Rates of proliferation, cell loss, and turnover on days 2, 4, and 6 for BC1 dhBMEC and HUVEC microvessels. (F) Cumulative proliferative and cell loss events during imaging of BC1 dhBMEC microvessels. N = 3 BC1 dhBMEC and HUVEC microvessels. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 5.

Endothelial cell motility of human iPSC-derived blood-brain barrier microvessels. (A) Root mean square (RMS) displacement. (B) Representative path for a dhBMEC (blue line) and HUVEC (black line) cell on day 2 following seeding. (C) Scatter plot showing the change in dhBMEC position over two hours normalized to the initial position for all cells tracked, the average cell displacement vector (x, y) = (1.16 μm, 0.22 μm). (D) Scatter plots of dhBMEC cell path and RMS displacement (dotted black line denotes average cell diameter). (E) Number of cell neighbors. (F) Fluorescence image showing representative six cell neighbors of dhBMEC microvessels. (G) The frequency of change in the number of cell neighbors. (H-I) Fluorescence images showing a stain for f-actin and basement membrane protein laminin α4 on the bottom pole of a microvessel on days 2 and 6. (J) Cross section of laminin α4 on days 2 and 6. N = 3 BC1 dhBMEC and HUVEC microvessels.

Figure 6.

Efflux inhibition of human iPSC-derived blood-brain barrier microvessels. (A) Phase / fluorescence overlays showing Rhodamine 123 (R123) permeability in BC1 dhBMEC microvessels treated with 2 μM tariquidar for 12 hours. (B) Comparison of BC1 dhBMEC microvessel permeability with and without tariquidar treatment. (C) The ratio of R123 permeability calculated only considering the ECM to including the ECM, lumen and intracellular compartments (P_ECM/P_total) in HUVEC and BC1 dhBMEC microvessels with and without tariquidar treatment. (D) Ratio of Lucifer yellow (LY) permeability to R123 permeability (P_LY / P_R123). (E) Cross section and pole confocal z-slice of R123 accumulation within HUVEC and BC1 dhBMEC microvessels with and without tariquidar treatment. (F) Phase contrast image of BC1 dhBMEC microvessels after tariquidar treatment. N = 3 BC1 dhBMEC microvessels exposed to tariquidar, compared to control BC1 dhBMEC microvessels. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 7.

Hyperosmolar blood-brain barrier opening of human iPSC-derived blood-brain barrier microvessels. (A) Phase contrast images at the pole of a BC1 dhBMEC microvessel following five minute mannitol exposure. t = 0 represents the frame prior to arrival of mannitol into the lumen. A vacuole formed following mannitol exposure is indicated by an arrow. Corresponding phase / fluorescence overlays for (B) Lucifer yellow (LY) and (C) 10 kDa dextran. Plumes of 10kDa dextran entering the ECM are marked with arrows. (D) Day two permeability increases with mannitol exposure. (E) Vacuoles per cell over two hours of imaging. (F) Fluorescence image showing f-actin phalloidin stain at the pole of a microvessel following mannitol exposure. N = 3 BC1 dhBMEC microvessels exposed to mannitol, compared to control BC1 dhBMEC microvessels. ** p < 0.01.

Figure 8.

Brain post-capillary venular model of leukocyte adhesion during inflammation. (A) Fluorescence images of the cross section of BC1 dhBMEC microvessels showing upregulation of surface adhesion markers (VCAM-1, ICAM-1) following 12-hour cytokine treatment with 10 ng mL⁻¹ TNF-α. (B) Permeability of Lucifer yellow (LY), Rhodamine 123 (R123), and 10 kDa dextran in BC1 dhBMEC microvessels following treatment with TNF-α. (C) Representative fluorescence images showing human peripheral blood mononuclear cells (PBMCs) adhered to the bottom pole of BC1 dhBMEC microvessels with and without cytokine activation. (D) Adhesion of PBMCs increases with TNF-α exposure. (E) Phase contrast image showing macroscopic breakdown of cytokine treated microvessels exposed to PBMCs within 24 hours. N = 3 BC1 dhBMEC microvessels exposed and not exposed to TNF-α. *** p < 0.001.