Establishment of a Human Blood-Brain Barrier Co-culture Model Mimicking the Neurovascular Unit Using Induced Pluri- and Multipotent Stem Cells

Abstract

In vitro models of the human blood-brain barrier (BBB) are highly desirable for drug development. This study aims to analyze a set of ten different BBB culture models based on primary cells, human induced pluripotent stem cells (hiPSCs), and multipotent fetal neural stem cells (fNSCs). We systematically investigated the impact of astrocytes, pericytes, and NSCs on hiPSC-derived BBB endothelial cell function and gene expression. The quadruple culture models, based on these four cell types, achieved BBB characteristics including transendothelial electrical resistance (TEER) up to 2,500 Ω cm² and distinct upregulation of typical BBB genes. A complex in vivo-like tight junction (TJ) network was detected by freeze-fracture and transmission electron microscopy. Treatment with claudin-specific TJ modulators caused TEER decrease, confirming the relevant role of claudin subtypes for paracellular tightness. Drug permeability tests with reference substances were performed and confirmed the suitability of the models for drug transport studies.

Keywords: blood-brain barrier (BBB) model; human induced pluripotent stem cells (hiPSCs); multipotent fetal neural stem cells (fNSCs); neurovascular unit in vitro.

Graphical abstract

Figure 1

In Vitro Characterization of Pluripotent and Multipotent Stem Cells (A–C, E–G, and I–K) Immunofluorescence staining of characteristic pluripotent stem cell (A–C) and NSC (E–G and I–K) markers. hiPSCs express OCT3/4 (A), SOX2 (B), and TRA1-81 (C), NSCs express SOX1 (E and I), SOX2 (F and J), and NESTIN (G and K). Cell nuclei were stained with DAPI in blue. (D, H, and L) Phase-contrast image of hiPSCs (D), NSCs differentiated from hiPSCs (H; hiPS-NSC) and NSCs isolated from fetal brain tissue (L; fNSC). Scale bars, 100 μm.

Figure 2

In Vitro Characterization of Differentiated BBB-Relevant Cell Types (A, E, I, and M) Phase-contrast image of BBB ECs differentiated from hiPSCs: hiPS-ECs (A), astrocytes differentiated from hiPSCs (hiPS-As) (E), human primary brain astrocytes (I), and pericytes (M). (B–D) Immunofluorescence staining of characteristic EC markers vWF (B), TJ-associated protein ZO1 (C), and glucose transporter GLUT1 (D). For further characterization, see also Figure S1. (F–H, J–L, and N–P) The astrocytic proteins GFAP (F and J) and S100β (G and K) were expressed by hiPS-A as well as by human primary brain astrocytes. Human primary brain pericytes were characterized by staining for αSMA (N) and PDGFRβ (O). Cell nuclei were stained with DAPI in blue. Fluorescence-activated cell sorting analysis of astrocytic protein GFAP (H and L) and pericyte marker PDGFRβ (P) reveals quantification of 53.8% GFAP-positive hiPS-As (H), 99.9% GFAP-positive primary astrocytes (L), and 85.2% primary pericytes. Scale bars, 100 μm.

Figure 3

Establishment of BBB Transwell Models, Tightness Characterization, Gene Expression, and Transporter Functionality (A) Schematic overview of BBB model establishment in transwell systems. At day −1, in total 5 × 10⁴ co-culture cells were seeded in the basolateral compartment. At day 0, 1 × 10⁶ hiPS-ECs/cm² were seeded on collagen IV-/fibronectin-coated transwell membranes (no direct contact with co-culture cells). At day 1, the growth factor concentration in the growth medium was reduced to stop hiPS-EC proliferation. The models were analyzed at day 2 by TEER measurement, qRT-PCR, western blot, electron microscopy, and transport studies. (B) Ten different variants of BBB models were established as transwell systems to investigate the impact of different co-culture cells on BBB integrity. TEER was measured at day 2 of co-culture and compared with hiPS-EC mono-culture models. TEER was most significantly increased by triple culture of hiPS-ECs with hiPS-NSCs and pericytes as well as by quadruple cultivation, indicated by the red box. Absolute TEER values are presented as means ± SD after data block-wise correction (n = 4); each biological replicate represents a new differentiation (^∗∗p < 0.01). (C) Overview of the minimal as well as the maximal measured TEER values of all ten variants of BBB models, representing the variabilities across the four independent biological experiments. Absolute TEER values are represented as measured raw data without manual data block-wise correction. (D) qRT-PCR analyses of efflux transporter ABCB1, glutamate transporter SLC1A1, glucose transporter SLC2A1, and occludin OCLN in hiPS-ECs of different co-culture BBB models shown as the change in gene expression compared with the hiPS-EC mono-culture model. Results are shown as means ± SD (n = 3–10); each biological replicate represents a new differentiation and co-culture experiment. Housekeeping genes for normalization were EEF1A1 and RPL6. The black horizontal line indicates an arbitrary threshold of 1.5-fold increase (^∗∗∗p < 0.001). (E) Permeability coefficients of the hiPS-ECs (PC_cell) after 15 min of transport of 100 μM rhodamine 123 compared between mono-cultures and quadruple cultures with and without 100 μM verapamil treatment. Results are shown as means ± SEM (n = 4–6); each biological replicate represents a new differentiation and co-culture experiment (^∗p < 0.05).

Figure 4

Expression of Major Tight Junction Proteins and Relevance of Claudins for Barrier Tightness (A) Western blot analysis of the TJ proteins (upper line) CLDN1 (22 kDa), CLDN4 (22 kDa), and CLDN5 (23 kDa) compared with mono-cultures (left lanes) and quadruple cultures (right lanes). α-Tubulin 52 kDa (lower line) was used in all blots as loading control. See also Figure S2 for further details. (B) Quantitative analysis of western blot results of the TJ proteins CLDN1, CLDN4, and CLDN5 shown as the change in protein expression compared with the hiPS-EC mono-culture models and hiPS-ECs of the quadruple cultures. (C) Effects of cCPE_Y306W/S313H, cCPE_wt, cCPE_Y306A/L315A proteins on TEER progression (%) of hiPSC-derived BBB monolayers normalized to the progression of controls. cCPE_wt binds with high affinity to CLDN3/4 and interacts with CLDN1, whereas cCPE_Y306W/S313H interacts strongly with CLDN5. The cCPE_Y306A/L315A control does not bind to claudins. Data are presented as means ± SD (n = 3–6); independent biological replicates (^∗p < 0.05, ^∗∗∗p < 0.001).

Figure 5

Ultrastructural Analysis of BBB Mono-culture and Quadruple Culture Models (A–D) Freeze-fracture EM analysis of the TJ ultrastructure of hiPS-ECs cultured without (A and B) or with (C and D) co-culture cells. Similar to brain microcapillary ECs in vivo, intramembranous TJ particles were found on the protoplasmic face (P face, PF) and exoplasmic face (E face, EF) of the plasma membrane. On the E face, TJ strands were detected as particles (black arrows) and particle-free grooves (white arrows). On the P face, TJ strands were detected as continuous strands (black arrowheads) and as beaded particles (white arrowheads). Mono-cultures (A and B) and quadruple cultures (C and D) showed variable although similar complex networks of meshes formed by branched strands with mixed P/E face association. Scale bars, 200 nm. (E–G) Transmission EM micrographs of the BBB models. Neighboring hiPS-ECs of both mono-cultures (E and F) and quadruple cultures (G) are connected by complex TJs constricting the paracellular space (black arrows). Furthermore, large desmosomes (macula adherens, black hash in E) anchored with intermediate filaments were detected as well as adhesion points (punctum adherens, black asterisk in G) anchored within the actin filament network. Scale bars, 200 nm.