Four induced pluripotent stem cell (iPSC) sources were used in this work: juvenile-onset HD iPSCs with 180 CAG repeats (HD180) , isogenic CRISPR-corrected controls of HD180 with 18 CAG repeats (HD-corrected) , non-isogenic adult onset HD iPSCs with 50 CAG repeats (HD50) (from NINDS cell repository #NN0003930), and non-isogenic control iPSCs with 21 CAG repeats (HD21) (from Allen Cell Institute #AICS-0023). The two isogenic cells were provided by the Pouladi Lab at the National University of Singapore. Details of each cell line are summarized in Table S1. Cell culture was performed at 37 ˚C and 5% CO2. iBMECs were differentiated from iPSCs using protocols developed in the Searson Group [13, 14]. Briefly, iPSC colonies were formed by seeding iPSCs singularized with Accutase (Invitrogen) at 10,000 cells cm-2 (additional seeding densities outlined below) and growing in mTeSR1 or TeSR-E8 (Stem Cell Technologies) for three days on six-well plates. Colonies were then treated with UM/F- media (DMEM/F12, 20% knockout serum replacement, 1% non-essential amino acids, 0.5% GlutaMAX, and 0.836 µM beta-mercaptoethanol) for six days, and endothelial media (human endothelial cell serum-free medium, 1% human platelet poor derived serum, 2 ng mL-1 bFGF, and 10 mM all-trans retinoic acid) for two days. iPSC media was switched daily using a media volume of 2 mL; UM/F- and endothelial media were switched daily using a media volume of 1 mL. At various stages of the differentiation, viable cells were manually counted on a hemacytometer based on Trypan blue (Corning) exclusion. At the end of the differentiation, cells were singularized using a 30-minute treatment with Accutase. Adherent cells were isolated by sub-culture on a plate coated overnight with 50 µg mL-1 human placental collagen IV (Sigma) and 25 µg mL-1 fibronectin from human plasma (Sigma). This process was conducted for one hour in endothelial media supplemented with 1% penicillin-streptomycin (ThermoFisher Scientific) and 10 µM ROCK inhibitor Y27632 (ATCC). Following subculture, the monolayer of adherent cells was washed with phosphate-buffered saline (PBS; ThermoFisher) and then singularized using a 10-minute treatment with Accutase. Viable cells were seeded onto collagen IV and fibronectin-coated surfaces at 0.33 x 106 cells cm-2. For the first 24 h of culture, the media matched that of subculture but was then replaced with basal media (human endothelial cell serum-free medium, 1% human platelet poor derived serum, and 1% penicillin-streptomycin). HD180 and HD-corrected iPSCs were confirmed to be isogenic using a PowerPlex 18D kit (Promega). A PCR based MycoDtect kit (Greiner Bio-One) was used to confirm absence of mycoplasma.
Beyond the differentiation scheme presented above, differentiation variables were adjusted to determine effects on differentiation outcomes and iBMEC phenotype. These variables included: (1) initial iPSC seeding density, (2) transwell seeding density, (3) removal of the sub-culture step before seeding for experiments, (4) media volume used during differentiation, and (5) use of a serum-free medium alternative during differentiation and transwell culture. To test the effect of initial seeding density on differentiation outcomes, hiPSCs were passed using the technique described previously, but seeded at densities of 5, 10, 20, 30, 40 x 103 cells cm-2 in parallel on Matrigel-coated plates. To test the effect of transwell seeding density on the barrier function of iBMECs, the cells were harvested using the technique described previously, and seeded on transwells at the densities of 0.33 and 1 x 106 cells cm-2 (3-fold difference in density) without the use of a sub-culture purification step. To determine the effect of the media volume used during differentiation, cells were grown in either 1 or 2 mL of UM/F- and RA media throughout the duration of the differentiation. To determine the effect of performing a serum-free differentiation, the 1% human platelet poor derived serum in endothelial media used in the final two days on the differentiation and during transwell culture was replaced with 1% B-27 Supplement (ThermoFisher), as previously demonstrated .
Quantitative polymerase chain reaction (qPCR)
Quantitative PCR (qPCR) was conducted on an Applied Biosystems StepOnePlus Real-Time PCR system using TaqMan® probes. Confluent monolayers of iBMECs two days after seeding on 6-well plates were washed with PBS and lysed using RLT buffer (Qiagen RNeasy Mini Kit). Fold changes between HD180 and HD-corrected were calculated using the comparative CT method (ΔΔCt) normalizing to ACTB expression (housekeeping gene). The following genes and primers were used: CLDN5 (#Hs00533949), OCLN (#Hs00170162), SLC2A1 (#Hs00892681), ABCB1 (#Hs00184500), PECAM1 (#Hs00169777), CDH5 (#Hs00901463), ACTB (#Hs01060665).
iBMECs were seeded at 250,000 cells cm-2 on borosilicate cover glass slides and cultured for two days using media outlined above. iBMECs were then washed with PBS, fixed with methanol for 15 min, and blocked with 10% goat serum (Cell Signaling Technology) or 10% donkey serum (Millipore Sigma) supplemented with 0.3% Triton X-100 (Millipore Sigma) in PBS for 30 min. Primary antibodies are summarized in Table S2. Cells were treated with Alexa Flour-647 and Alexa Flour-488 conjugated secondary antibodies (Life Technologies) diluted 1:200 in blocking buffer for 45 min at room temperature. To localize nuclei, cells were treated with 1 µg mL-1 DAPI (ThermoFisher). Images were acquired at 40× magnification using a swept field confocal microscope (Bruker).
Two biological replicates were analyzed of: HD180 iPSCs, HD180 iBMECs, HD-corrected iPSCs, and HD-corrected iBMECs. iPSCs were harvested prior to differentiation in UM/F- media, while iBMECs were harvested as confluent monolayers two days following subculture on collagen IV and fibronectin-coated tissue-culture plates. To harvest total RNA, cells were lysed using RLT buffer (Qiagen) supplemented with β-mercaptoethanol (Sigma) and then RNA isolated using a RNeasy Mini Kit (Qiagen) with DNase I digestion (Qiagen). All sequenced samples had RNA integrity numbers above 9.7, as measured by an Agilent 2100 bioanalyzer. Total RNA was subjected to oligo (dT) capture and enrichment, and the resulting mRNA fraction was used to construct cDNA libraries. Approximately 20 million paired end 150 bp reads were collected per sample using Illumina NovoSeq (performed by Novogene). Alignment and quantification to reference genome (GRCh38) was performed using Rsubread (Version 2.0.1) . Normalization (rlog transformed), visualization, and differential analysis was performed using DESeq2 (v1.28.1) . Differentially expressed genes (DEGs) were determined using the Wald test with Benjamini-Hochberg correction (adjusted p values < 0.05 was considered statistically significant). Pathway enrichment analysis (Hallmark gene sets) was conducted on DEGs using Enrichr, with built in statistical analysis used at a adjusted p-value cutoff of 0.05 . Raw data are deposited at NCBI.
Barrier function measurements
Transendothelial electrical resistance (TEER; Ω cm2) was recorded using an EndOhm (World Precision Instruments) as previously reported . Measurements were performed on 6.5 mm Transwells with a 0.4 µm pore polyester membrane insert (Corning). TEER values were corrected for the resistance of the transwell insert without cells. iBMECs were seeded at a density of 0.33–1.00 x 106 cm-2 onto transwells in endothelial media as previously described. After 24 hours, medium was switched to basal medium and daily recordings were collected for ten days.
At day two (and sometimes day ten), the permeability of 200 µM Lucifer yellow (ThermoFisher), 2 µM 10 kDa dextran (ThermoFisher), 10 µM rhodamine 123 (ThermoFisher), and 25 mM d-glucose (Sigma) across BMEC monolayers was measured using previously reported protocols . The following excitation and emission settings were utilized on a Synergy™ H4 microplate reader (Biotek): Lucifer yellow (428 nm / 545 nm), 10 kDa dextran (647 nm / 667 nm), rhodamine 123 (495 nm / 525 nm). Glucose transport was quantified using a glucose colorimetric detection kit (ThermoFisher Scientific), utilizing absorbance measurements at 560 nm. Concentrations of all compounds were determined from calibration curves based on serial dilution of each compound spanning four orders of magnitude. The apparent permeability of each compound was calculated as P = (dC/dt)(V)(1/A)(1/C0), where dC/dt is the slope of cumulative concentration, V is the volume of the receiving compartment (i.e. basolateral or apical chamber), A is the area of the monolayer, and C0 is the dosed concentration of solute . For rhodamine 123, efflux ratios were calculated as the ratio of basolateral-to-apical and apical-to-basolateral permeability normalized to 10 kDa dextran (a non-efflux substrate). Biological replicates of permeability measurements were averaged across at least two transwells (technical replicates).
Responsiveness to chemical perturbation
A bead angiogenesis assay was conducted as previously reported . Briefly, 150 µm diameter Cytodex™ 3 microcarrier beads (GE Healthcare) were coated with collagen IV and fibronectin and then seeded with singularized iBMECs in endothelial media supplemented with 1% penicillin-streptomycin and 10 µM ROCK inhibitor. After 80 min, with gentle agitation every 20 min, the beads were washed to remove non-adherent cells and then cultured on a shaker at 100 rpm for 24 h. Next, beads were embedded within 6 mg mL-1 neutralized rat tail type I collagen (Corning) and treated with basal media with and without 50 ng mL-1 recombinant human VEGF-165 (VEGF; Biolegend). After three days in culture, the sprout density (# bead-1) was manually counted from phase contrast images across at least 8 beads (technical replicates) for each condition.
For oxidative and osmotic stress experiments, iBMECs were seeded onto transwells as previously described and exposed to hydrogen peroxide (H2O2) or mannitol (for 10 mins) after 48 h. To avoid the need for a media switch, 5 µL of concentrated H2O2 freshly prepared in sterile water was added to the apical chamber of transwells to achieve final concentrations of 0.2–1 mM. TEER was recorded daily after exposure. As mannitol only induces BBB opening near its concentration limit, the medium was changed to basal medium with 1.4 M mannitol for 10 min, and then switched back to basal medium. TEER was recorded immediately before treatment, immediately after treatment, one hour later and one day later. To visualize reactive oxygen species and actin cytoskeleton, some transwells were treated with 50 µM CellROX® Green Reagent (Invitrogen), AlexaFluor647 phalloidin (Invitrogen), and DAPI solution (Thermo Scientific) for 30 min at 37 ˚C after one day of exposure to 0.6 mM H2O2.
Tissue-engineered BBB model
Tissue-engineered BBB microvessels were fabricated as previously reported . iBMECs were sub-cultured for one hour and then detached using Accutase before seeding into 150 µm diameter channels patterned in 7 mg mL-1 type I collagen. Prior to seeding, the collagen matrix was cross-linked with 20 mM genipin to increase stiffness and then the channel surface was coated in collagen IV and fibronectin to promote cell adhesion. Cells were seeded into microvessels and cultured for 30 min under no flow to facilitate adhesion, then microvessels were perfused at ~ 1 dyne cm-2 shear stress for the remainder of experimentation. After formation of confluent monolayers, microvessels were perfused with 200 µM Lucifer yellow for one hour, with images collected every two minutes. From the plot of fluorescence over time, permeability was calculated as previously reported . The turnover of iBMECs in microvessels was calculated from phase contrast images acquired simultaneously with fluorescence images. Cell loss and cell proliferation events were manually counted on the top plane of the microvessel as previously reported . From counts of cell loss and proliferation events, values were normalized to total number of cells in the imaging plane and to time, with final units of % h-1. Net microvessel turnover was calculated as the difference in rate between proliferation and loss (% h-1).
THP-1 (ATCC® TIB-202™) is a human leukemia monocytic cell line . THP-1s were grown in suspension with RPMI-1640 Medium (Sigma) supplemented with 10% fetal bovine serum (Sigma) and 1% penicillin-streptomycin. Before use, cells were labeled with 5 µM Calcein AM (ThermoFisher) for 15 min, and then resuspended at 1 x 106 cells mL-1 in basal media. Microvessels were perfused with THP-1s under low shear stress (~ 0.2 dyne cm-2) for 10 min, and then washed out using higher shear stress (~ 2 dyne cm-2). Adherent immune cells were manually counted along the length of microvessels using fluorescence microscopy.
All statistical analysis was performed using Prism ver. 8 (GraphPad). Metrics are presented as mean ± SEM (standard error of the mean). A student’s unpaired t-test (two-tailed with unequal variance) was used for comparison of two groups, and an analysis of variance (ANOVA) for comparison of three or more groups. Number of biological replicates are reported in figure legends. P-values were multiplicity adjusted using a Tukey test. Differences were considered statistically significant for p < 0.05, with thresholds of * p < 0.05, ** p < 0.01, and *** p < 0.001.