Fig 1.
Time course of the differentiation of the hiPSCs and a schematic representation of the hydrogel model on transwell filters.
HiPSCs were maintained in mTeSR1 medium. When they had reached an optimum density of 3x104/cm2 they were switched to unconditioned (UC) medium for 6 days, followed by serum free endothelial cell (EC) medium containing B27, human basic fibroblast growth factor (hbFGF) and retinoic acid (RA) for 2 days. Strongly adherent cells were isolated by 1hour incubation on collagen IV/fibronectin-coated plates in Hanks balanced salt solution (HBSS). Adherent, endothelial-like cells were plated onto a 1cm2 transwell insert that had a 0.3ml collagen I hydrogel and a fibronectin/collagen IV surface layer, prepared 24hours previously. The cells were cultured in EC medium for 24-48hours before use, and the inserts were usable for up to 7 days.
Table 1.
Primary antibodies.
Fig 2.
Immunofluorescence micrographs of differentiated (d10) endothelial-like cells showing junctional expression of ZO-1, occludin, claudin-5 and VE-cadherin, weak surface expression of PECAM and granular expression of von Willebrand factor in the cytoplasm (green).
The cell nuclei were stained with DAPI (blue). Scale bars = 50μm. Scale bar on VWF = 20μm.
Fig 3.
Transmission electron microscopy images of astrocytes in a collagen hydrogel with differentiated endothelial-like cells on the surface.
A. A transverse section with the apical surface of the endothelium at the top. B. Transverse section showing the basal surface of the endothelial cells. Scale bars = 1μm.
Fig 4.
Trans endothelial electrical resistance of 5 hydrogel cultures and 1 matched culture on 0.4μm pore-size filter only (no gel) measured for 54hrs after culture set up, using a Cellzscope.
Each trace represents the resistance from 1 hydrogel or transwell, measured at 1 minute intervals.
Table 2.
TEER values of differentiated cultures.
Table 3.
TEER values of hydrogel BBB models during transport assays.
Table 4.
Permeability of hydrogel cultures to paracellular tracers.
Fig 5.
Transcytosis of TfR-binding peptides to the lower chamber of hydrogel cultures at 6 hours after application to the apical compartment.
Values are expressed as the concentration in the basal chamber, combined data from 2 independent experiments (n = 7) determined by fluorimetry. Data was analysed by one way ANOVAR using Welche’s correction for different SD values (P< 0.0001). Subsequent analysis compared TfR binding peptides with Pep-R1 by Tukey’s test. **** P<0.0001, *** P< 0.001, * P<0.05.
Fig 6.
Activity of ABCB1 measured with Rho-123, comparing transfer across hydrogel cultures at 6hrs, in the absence or presence of the inhibitor zosuquidar.
Mean ± SEM, n = 4, P = 0.0024 by one way t-test. Activity of ABCG2 measured with mitoxantrone, comparing transport at 24hrs across cells on filters, in the absence or presence of its inhibitor Ko143. Mean ± SEM, n = 6, P = 0.062.