A novel human induced pluripotent stem cell blood-brain barrier model: Applicability to study antibody-triggered receptor-mediated transcytosis

We have developed a renewable, scalable and transgene free human blood-brain barrier model, composed of brain endothelial cells (BECs), generated from human amniotic fluid derived induced pluripotent stem cells (AF-iPSC), which can also give rise to syngeneic neural cells of the neurovascular unit. These AF-iPSC-derived BECs (i-BEC) exhibited high transendothelial electrical resistance (up to 1500 Ω cm2) inducible by astrocyte-derived molecular cues and retinoic acid treatment, polarized expression of functional efflux transporters and receptor mediated transcytosis triggered by antibodies against specific receptors. In vitro human BBB models enable pre-clinical screening of central nervous system (CNS)-targeting drugs and are of particular importance for assessing species-specific/selective transport mechanisms. This i-BEC human BBB model discriminates species-selective antibody- mediated transcytosis mechanisms, is predictive of in vivo CNS exposure of rodent cross-reactive antibodies and can be implemented into pre-clinical CNS drug discovery and development processes.


DNA isolation and methylation assay
DNA was isolated from 1.0 x10 6 AF cells and iPSC cells using the Quick gDNA Miniprep (Zymo Research), according to the manufacturer's protocol. The EZ DNA Methylation gold Kit (Zymo Research) was used to modify unmethylated cytosine residues to thymidine. Briefly, the conversion of approximately 300 ng of genomic DNA was carried out in a thermal cycler at 98°C for 10 min, 64°C for 2.5 hr and then stored at 4°C for up to 20 hr. The reaction was then followed by a clean-up, desulfonation and the bisulfite-modified DNA was eluted and immediately used for PCR amplification. Briefly, 2 µl of bisulfite converted DNA was added to a solution containing 1X MSB PCR buffer (16.6 mM ammonium sulfate, 67 mM Tris, pH 8.8, 6.7 mM MgCl 2 , 10 mM 2-mercaptoethanol), 2.5 µl of DMSO, dNTPs (1.25 mM each), primers (1 µM each) and 2.5 U Platinum Taq DNA Polymerase High Fidelity in a final volume of 50 µl (Life Technologies). The primers were specific for bisulfite-converted DNA only (Supplementary Table 2). The PCR reactions were carried out in a Biorad DNA Engine Dyad thermal cycler using the following protocol: 1 cycle of denaturation at 95 °C for 3 min, 35 cycles of amplification at 95 °C for 1 min, 55-58 °C for 1 min, 72 °C for 1 min and 1 cycle of extension at 72 °C for 10 min. PCR products were loaded onto a 2% agarose gel and the amplicon band was excised and purified using the Geneclean Spin Kit (MP Biomedicals), as per the manufacturer's instructions. Zero Blunt TOPO PCR Cloning Kit (Life Technologies) was used for cloning the PCR product obtained and clones were PCR screened with M13 primers (Supplementary Table 2

Microarray printing, cDNA synthesis and hybridization
A BBB specific oligonucleotide set of 434 oligos (Eurofins) was printed, in house, onto Nexterion E epoxysilane coated slides (Applied Microarrays) with a Nano-plotter NP2.1(GeSiM) at 50% humidity and a temperature of 8°C. The slides were transferred to a 42°C oven and incubated at 80% humidity overnight then stored in a desiccator (Bel-Art) until ready for use.
cDNA was prepared according to the 3DNA array detection 900 kit recommendations (Genisphere), using 1µg of total RNA from each sample and Superscript II Reverse Transcriptase (Life Technologies). The slides were washed and blocked according to the manufacturer's recommendations, spun dry at 200 x g for 5 min and placed in a SlideBooster Microarray Hybridisation Station SB401 (Beckman Coulter). The cDNA hybridization mix was pipetted on top of the slides and then covered with Lifter Slips (Thermo Scientific) and the hybridization reaction was carried out at 42 °C overnight. The slides were then washed in 2X SSC, 0.2% SDS; 2X SSC and 0.2XSSC each for 15 min at room temperature on a shaker then spun dry. The 3DNA capture reagents were prepared, as per manufacturer's protocol, then pipetted onto the slides in the SlideBooster Hybridization Station, as previously described and incubated at 42 °C for 4 hr. The slides were then washed as described above and scanned immediately with the GenePix scanner 4200A (Molecular Devices), as per the manufacturer's protocol and the data was analyzed using the GenePix Pro 6.1 software (Molecular Devices).
Individual gene intensity values for the genes assessed are listed in Supplementary Table 4.

Permeability coefficient (Pe) calculations
Permeability coefficient (Pe) is a parameter that is determined in an in vitro or ex vivo transport assay system that represents the permeability of a compound across the cell monolayer only. For this calculation, the barrier effect of the cell membrane/insert alone (empty inserts) is subtracted from the overall barrier effect of the cells plus membrane (inserts with cells). Permeability coefficients are calculated from each insert separately and from the pooled data of three inserts in each experiment. Clearance volumes (µl) are calculated as previously described 49 : representing permeability x surface area product (PS; µl/min), are calculated using linear regression analysis. The PS value for endothelial monolayer (PSe) is calculated from: PSc+m is the slope of the clearance curve for cell monolayer and membrane together, and PSm is the slope of the clearance curve of membrane alone (ie. no cells). The permeability coefficient (Pe; cm/min) for the endothelial cell monolayer is calculated by dividing PSe by surface area of the membrane (0.9 cm 2 for our inserts).