Cell culture and iPSC differentiation
Control (CS06iCTR), PSEN1 (CS40iFAD) and PSEN2 (CS08iFAD) iPSC lines used in this study were acquired from the Cedars-Sinai iPSC core (Los Angeles, CA). The PSEN1 iPSC line was isolated from a 56-year old Caucasian male diagnosed with memory impairment and harbors an Ala246Glu mutation. The PSEN2 iPSC line was isolated from an 81-year old Caucasian female diagnosed with progressive dementia and harbors an Asn141Ile mutation.
Undifferentiated iPSCs were maintained in hESC-grade Matrigel® (Corning, Corning, NY) in presence of Essential 8 (E8) medium (Life Technologies, ThermoFisher, Waltham, MA) as previously described (24). iPSC differentiation into BMECs occurred following the differentiation protocol previously published by our lab (24). Briefly, cells were maintained in E8 for 5 days prior to differentiation, followed by 6 days in unconditioned medium [UM: Dulbecco’s modified Eagle’s medium/F12 with 15 mM HEPES (ThermoFisher), 20% knockout serum replacement (ThermoFisher), 1% non-essential amino acids (ThermoFisher), 0.5% Glutamax (ThermoFisher), and 0.1 mM b-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA)] and 2 days in EC+/+ [EC medium (ThermoFisher) supplemented with 1% platelet-poor derived serum (PDS, Alfa-Aesar, ThermoFisher, Haverhill, MA, USA), 20 ng/mL human recombinant basic fibroblast growth factor (Tocris, Abingdon, UK), and 10 µM retinoic acid (Sigma-Aldrich)]. At day 8 of differentiation, cells were enzymatically dissociated (Accutase®, Corning) and seeded on tissue culture plastic surfaces (TCPS) coated with collagen (isolated from human placenta, Sigma-Aldrich)/fibronectin (bovine plasma, Sigma-Aldrich) at concentrations of 80 µg/cm2 and 20 µg/cm2 respectively. At day 9 of differentiation, iPSC-derived BMECs were maintained in EC-/- medium [EC medium supplemented with 1% PDS] for 24 hours. Experiments were conducted at day 10 of differentiation.
Cells were quickly washed with ice-cold PBS and fixed in 4% paraformaldehyde (PFA, Electron Microscopy Sciences, Hatfield, PA, USA) and blocked for 30 minutes at room temperature (RT) in presence of PBS supplemented with 10% goat serum (ThermoFisher) supplemented with 0.2% Triton-X100 (Sigma). Cells were incubated overnight at 4ºC in primary antibodies targeting BCRP (1:100, Millipore, RRID: AB_11213795), claudin-5 (1:100, Life Technologies, RRID: AB_2533200), GLUT1 (1:100, ThermoFisher, AB_10979643), GLUT3 (1:100, ThermoFisher, AB_2809974), GLUT4 (1:100, ThermoFisher, AB_11153908), MRP1 (1:100, Millipore, RRID: AB_2143819), occludin (1:100, Life Technologies, AB_2533101), P-gp (1:50, ThermoFisher, AB_1233253) and ZO1 (1:100, RRID:AB_2533938) diluted in 10% goat serum (PBSG). Primary antibodies detection occurred by incubation with goat-anti mouse Alexa Fluor® 555-conjugated secondary antibody (Life Technologies) for 1 hour at room temperature. Cells were observed at 200X magnification (20X long-distance dry objective) and acquired using a Leica DMi-8 inverted epifluorescence microscope (Leica Microsystems, Wetzlar, Germany). Images were processed using ImageJ (Image J, NIH, Bethesda, MD). Relative fluorescence was quantified using the built-in function in ImageJ. Background fluorescence was subtracted from unlabeled cells incubated with the secondary antibody only.
TEER and permeability experiments
Barrier tightness was measured by assessing both transcellular electrical resistance (TEER) and fluorescein permeability (paracellular tracer). TEER was measured using an EVOHM STX2 chopstick electrode (World Precision Instruments, Sarasota, FL, USA). For each experiment, three measurements were performed for each insert, and the average resistance obtained was used to determine barrier function. Fluorescein permeability was assessed by incubating 10 µM sodium fluorescein (Sigma-Aldrich) in the donor (apical) chamber, with sampling in the donor (basolateral) chamber every 15 minutes for up to 60 minutes. Fluorescein permeability (Pe) was calculated using the clearance slopes obtained by extrapolation using the following formula:
Pt and Pf indicative of the clearance slopes of samples and blank (empty coated) filters, and S indicative of the insert surface area (cm2).
Drug uptake assay
Cells were incubated in the presence of 10µM Rhodamine 123 (P-gp substrate, Sigma), FL-BOPIDY (BCRP substrate, Sigma) or CM-DCFDA (MRP substrate, Sigma) for 1 hour at 37ºC followed by cell lysis using RIPA buffer (ThermoFisher). For assessing the contribution of efflux pump in the drug uptake, cells were pre-incubated for 1 hour in presence of 5µM cyclosporine A (CsA, P-gp inhibitor, Sigma), 1µM Ko143 (BCRP inhibitor, Sigma) or 10µM MK571 (MRPs inhibitor, Sigma) and maintained during the incubation with drug efflux substrate. Following incubation, cells were briefly washed with ice-cold PBS and lysed with RIPA buffer. Fluorescence in cell lysates was assessed using a SynergyMX2 ELISA plate reader (Bio-Tek, Winooski, VT, USA). Relative fluorescence units (RFU) were normalized against the total protein content and the protein levels were determined by bicinchoninic acid assay (BCA, ThermoFisher). Fluorescence values (expressed as relative fluorescence unit or RFU) obtained from cell lysates in the absence of inhibitor (named as controls) were normalized to the protein content and expressed as RFU/µg protein.
Glucose uptake assay
Cells were incubated in presence of [14C]-D-glucose (0.4µCi/mL) for 1 hour at 37ºC.
Following incubation, cells were briefly washed with ice-cold PBS and lysed with RIPA buffer. In experiments involving GLUT1 inhibition, cells were pre-incubated in presence of 10µM glucose transporter inhibitor II (Millipore-Sigma, Danvers, MA) for 1 hour prior incubation with glucose. Radioactivity in cell lysates was assessed using liquid scintillation cocktail (Scintisafe® 30%, ThermoFisher) and quantified with a Beckman-Coulter LS6500 (Beckman-Coulter, Brea, CA). Glucose uptake levels were normalized by the total amount of protein in samples.
Glycolytic flux and mitochondrial stress analysis
Glycolytic flux analysis was assessed using a Seahorse XFe-24 cell flux analyzer (Agilent Technologies, Santa Clara, CA). Cells were seeded at a density of 5 x 104 cells/well on custom-designed 24-well plates (Agilent Technologies) at day 8 of differentiation and allowed to grow for 48 hours. On the day of experiments, cell medium was replaced by glucose-free medium provided with the glycolytic stress test kit (Agilent) for 2 hours prior experiment. Cell medium was replaced once with glucose-free medium and initiated measurement. At 20 minutes of incubation, 10mM D-glucose was added in the incubation chamber, followed by the addition of 1µM of oligomycin at 40 minutes and finally addition of 100mM 2-deoxy-D-glucose (2-DG) at 60 minutes timepoint, with measurements occurring until the 90th minute timepoint.
JC-1 flow cytometry and live imaging
At day 10 of differentiation, cells were enzymatically dissociated with Accutase®. Cells were resuspended and centrifuged and resuspended in medium containing 5µM JC-1 dye (ThermoFisher) for 30 minutes at 37ºC. In experiments involving FCCP treatment, cells were simultaneously treated with 50nM FCCP. Following incubation with JC-1 dye, cells were washed by centrifugation and resuspension in 200µL PBS for flow cytometry analysis. In experiments involving acridine orange (AO), cells were maintained for 24 hours in EC-/- medium or in serum-free EC medium to induce serum starvation. Following such treatment, cells were centrifuged and stained with 1µg/mL acridine orange (AO, Sigma-Aldrich) dissolved in PBS and allowed to stain for 15 minutes, following the protocol of Thome and colleagues (25). Fluorescence detection in samples was performed using a FACSVerse® flow cytometer (BD Biosciences, San Jose, CA). Fluorescence PMTs were calibrated on unstained cells and set for the remaining of the experiments.
In experiments involving live imaging, cells were incubated in presence of 100µL of JC-1 reagent (Cayman Chemicals, Ann Arbor, MI) and incubated for 20 minutes at 37ºC/5% CO2. Live cells were observed under the Leica DMi-8 microscope, with the use of the 485/535nm for impaired cells and 540/570nm for healthy cells to detect JC-1 aggregates. The fluorimetric ratio of JC-1 was used as an indicator of mitochondrial dysfunction.
Lysosensor(R) live imaging
Live cells were incubated in presence of 1µM Lysosensor-Green DND 189 for 5 minutes, followed by a brief wash with ice-cold PBS and fixation with 4% paraformaldehyde. Cells were counterstained with 300nM DAPI solution and immediately processed for imaging under the Leica DMi-8 inverted fluorescence microscope at 20X.
Radical oxygen species assays
At day 10 of differentiation, intracellular radical oxygen species (ROS) was assessed using both a qualitative and quantitative approach.
The qualitative approach in assessing ROS production was performed using CellROX(R) assay (ThermoFisher Scientific). CellROX(R) reagent was added to live cells at a final concentration of 5µM to the medium and incubated for 30 minutes at 37°C/5% CO2. The medium was removed, and cells were quickly washed with PBS. Cells were observed at 20X magnification, and images were acquired using a Leica DMi-8 inverted fluorescence microscope (Leica Microsystems).
The quantitative approach in assessing ROS production was performed using a DCFDA-based cell kit (Cayman Chemicals), following the instruction provided by the vendor. DCFDA fluorescence was measured using SynergyMX2 ELISA plate reader (Bio-Tek). DCFDA fluorescence intensity in CTR90F BMECs was used as a baseline value for comparison to the PSEN BMECs.
To study the effect of mutation on cell viability, a LIVE/DEAD™ Viability/Cytotoxicity Kit (Invitrogen) was used. A calcein-AM/ethidium homodimer-1 (CaAM/EthD-1 staining) assay was performed based on the manufacturer’s protocol. Briefly, cells were stained by a dye solution consisted of 0.5ul of Calcein AM and 0.5ul of ethidium homodimer-1 per ml PBS. Cells were observed after 20min of incubation at 20X magnification, and fluorescence was acquired using a Leica DMi-8 inverted fluorescence microscope (Leica Microsystems, Wetzlar, Germany). Ethidium homodimer-1 enters into cells with compromised cell membranes and subsequently intercalates with nucleic acids. Thus, dead cells have red fluorescence. Calcein-AM penetrates living cell membranes and produces green fluorescence by cleavage of cytoplasmic esterase.
Ab1-40 and Ab1-42 ELISA
Cell conditioned medium from BMECs monolayers grown at Day 10 were collected and immediately frozen at -80ºC. Ab1-40 and 1-42 levels in supernatants were measured using their respective ELISA Quantikine(R) kits (R&D Systems, Minneapolis, MN).
Data are represented as mean ± S.D. from at least three independent experiments. Statistical analysis was performed using one-way analysis of the variance (ANOVA) using parametric (Dunnett) tests. Statistical analysis was performed using GraphPad Prism 8.0
(GraphPad Software, La Jolla, CA). A p-value lesser than 0.05 (P<0.05) was considered as indicative of a statistic difference between one or more groups.