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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This protocol details an adapted method to derive, expand, and cryopreserve brain microvascular endothelial cells obtained by differentiating human induced pluripotent stem cells, and to study blood brain barrier properties in an ex vivo model.

Abstract

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models for studying blood-brain barrier (BBB) function. This modified protocol provides detailed steps to derive, expand, and cryopreserve BMECs from human iPSCs using a different donor and reagents than those reported in previous protocols. iPSCs are treated with essential 6 medium for 4 days, followed by 2 days of human endothelial serum-free culture medium supplemented with basic fibroblast growth factor, retinoic acid, and B27 supplement. At day 6, cells are sub-cultured onto a collagen/fibronectin matrix for 2 days. Immunocytochemistry is performed at day 8 for BMEC marker analysis using CLDN5, OCLN, TJP1, PECAM1, and SLC2A1. Western blotting is performed to confirm BMEC marker expression, and absence of SOX17, an endodermal marker. Angiogenic potential is demonstrated with a sprouting assay. Trans-endothelial electrical resistance (TEER) is measured using chopstick electrodes and voltohmmeter starting at day 7. Efflux transporter activity for ATP binding cassette subfamily B member 1 and ATP binding cassette subfamily C member 1 is measured using a multi-plate reader at day 8. Successful derivation of BMECs is confirmed by the presence of relevant cell markers, low levels of SOX17, angiogenic potential, transporter activity, and TEER values ~2000 Ω x cm2. BMECs are expanded until day 10 before passaging onto freshly coated collagen/fibronectin plates or cryopreserved. This protocol demonstrates that iPSC-derived BMECs can be expanded and passaged at least once. However, lower TEER values and poorer localization of BMEC markers was observed after cryopreservation. BMECs can be utilized in co-culture experiments with other cell types (neurons, glia, pericytes), in three-dimensional brain models (organ-chip and hydrogel), for vascularization of brain organoids, and for studying BBB dysfunction in neuropsychiatric disorders.

Introduction

Blood-Brain Barrier Function
The blood-brain barrier (BBB) forms a boundary that limits movement of substances from the blood to the brain. The BBB is comprised of brain microvascular endothelial cells (BMECs) that form a monolayer lining the vasculature. BMECs, together with astrocytes, neurons, pericytes, microglia, and extracellular matrix, form the neurovascular unit. BMECs have a tightly regulated paracellular structure that allows the BBB to maintain high trans-endothelial electrical resistance (TEER), which limits passive diffusion and serves as an indicator of barrier integrity1,2. BMECs also have proteins that assist with transcellular movement such as endocytosis, transcytosis, and transmigration, as well as extravasation of leukocytes during an immune response3. BMECs rely on influx and efflux transporters for nourishment and removal of waste products, in order to maintain a homeostatic balance in the brain3. For example, solute carrier family 2 member 1 (SLC2A1) is an influx transporter responsible for the movement of glucose across the BBB4, while efflux transporters such as the ATP binding cassette subfamily B member 1 (ABCB1) and the ATP binding cassette subfamily C member 1 (ABCC1) are responsible for returning substrates back into the blood stream3,5,6,7. ABCB1 substrates include morphine, verapamil4, and antipsychotics such as olanzapine and risperidone8, while the ABCC1 transporter has a variety of substrates including sulfate conjugates, vincristine, and glucuronide conjugates4.

Application of BBB Models in Psychiatric Disorders
BBB dysfunction has been implicated in a number of neurological and psychiatric disorders, including schizophrenia and bipolar disorder9,10. Recently, iPSC-derived ex vivo cellular models are being utilized to interrogate the cellular and molecular underpinnings of psychiatric disorders, but these models currently do not take into account the potential role played by the neurovasculature11,12,13. It is hypothesized that peripheral inflammatory cytokines circulating in the blood can adversely impact the BBB14,15,16,17, but there is also evidence for paracellular18,19,20,21,22, transcellular23,24,25,26,27,28,29, and extracellular matrix20,29,30,31,32 abnormalities contributing to BBB dysfunction. Disruption of the BBB can result in the contents of the blood entering the brain parenchyma and activating astrocytes and/or microglia to release proinflammatory cytokines, which in turn initiate an inflammatory response33 that can have detrimental effects on the brain34. BMECs are the primary component of the BBB and examining the structure and function of these cells can enhance the understanding of BBB dysfunction in neurological and psychiatric disorders.

Alternative BMEC Models
Prior to the development of efficient protocols for deriving BMECs from iPSCs1,6,35,36, researchers had employed immortalized BMECs37 to study BBB function. However, many of these models failed to attain desirable BBB phenotypes, such a physiological range of TEER values38,39. Utilizing iPSCs has the advantage of retaining the genetic background of the individual from which the cells are derived. Scientists are actively working on establishing iPSC-derived ex vivo microenvironment models that recapitulate the structure and function of the human brain. Researchers have developed methods to derive BMECs that are structurally and physiologically similar to BMECs found in vivo. Methods for obtaining purified populations of iPSC-derived BMECs require a number of different steps with protocols being optimized in the last few years1,6,35,36. Generally, iPSC-derived BMECs are cultured in Essential 6 (E6) medium for 4 days, followed by 2 days in human endothelial serum-free medium (hESFM) supplemented with basic fibroblast growth factor (bFGF), retinoic acid (RA), and B27 supplement. The cells are then cultured on a collagen IV (COL4) and fibronectin (FN) matrix to obtain >90% homogeneous BMECs1.

The identity of BMECs are confirmed by immunofluorescence showing the co-expression of BMEC proteins including platelet-endothelial cell adhesion molecule-1 (PECAM1), SLC2A1, and tight junction proteins such as tight junction protein 1 (TJP1), occludin (OCLN), and claudin-5 (CLDN5)6. Sprouting assays have been used to confirm the angiogenic potential of iPSC-derived BMECs.6 The BBB integrity of BMECs is evaluated by the presence of physiologic in vitro TEER values (~2000Ω x cm2)37 and measurable activity for efflux transporters such as ABCB1 and ABCC11,6,36. Recent methodological advances by the Lippmann group have led to iPSC-derived BMEC protocols with reduced experimental variability and enhanced reproducibility1. However, it is not known whether they can be expanded and passaged beyond the sub-culturing stage. Our modified protocol aims to address this issue by passaging iPSC-derived BMECs beyond day 8 and assessing whether they can be further expanded to retain BBB properties after cryopreservation. While no studies have described passaging of iPSC-derived BMECs, a protocol exists for BMEC cryopreservation that retains physiologic BBB properties after undergoing a freeze-thaw cycle40. However, it is not known post-cryopreservation BMECs can be passaged and retain BBB properties.

BMECs derived from iPSCs using the Lippmann protocol have been utilized to model BBB disruption in neurological disorders such as Huntington’s disease7. Such iPSC-derived BMECs have also been used to investigate the effects of bacterial infection such as Neisseria meningitidis or Group B Streptococcus on disruption of blood-CSF barrier and BBB respectively41,42. Also, using iPSC-derived BMECs from 22q deletion syndrome patients with schizophrenia, researchers observed an increase in intercellular adhesion molecule-1 (ICAM-1), a major adhesion molecule in BMECs that assist with recruitment and extravasation of leukocytes into the brain43. Taken together, these studies demonstrate the utility of iPSC-derived BMECs for studying BBB disruption in complex neuropsychiatric disorders.

Protocol

Human iPSCs were reprogrammed from the fibroblasts of healthy donors using a protocol approved by the Institutional Review Boards of Massachusetts General Hospital and McLean Hospital, and characterized as described in previous studies44,45,46.

NOTE: Briefly, fibroblasts were reprogrammed to iPSC via mRNA-based genetic reprogramming47. The iPSCs were maintained in stem cell medium (SCM) (see material list) and stored at a density of ~1.2 x 102 cells/mL with 1 mL of SCM, 10 μM with rho-associated protein kinase inhibitor (ROCKi) Y-27632, and 10% (v/v) dimethyl sulfide (DMSO), in cryopreserved vials in liquid nitrogen at -160 °C. All of the following procedures below are carried out in a biosafety cabinet unless stated otherwise.

1. Basement membrane matrix dilution and plate coating

  1. Dilute (1:50) growth factor reduced basement membrane matrix purified from Engelbreth-Holm-Swarm tumor in Dulbecco's Modified Eagle Medium (DMEM) without phenol red.
  2. Coat cell culture plates with the appropriate amount of diluted basement membrane matrix (i.e., 6-well plate = 1mL, 12-well plate = 0.5 mL) and incubate these plates at 37 °C for at least 1 hour.

2. iPSC maintenance

NOTE: The maximum confluency per well in a 6-well flat-bottom plate is ~1.2 x 106 cells.

  1. Thaw cryopreserved iPSCs into SCM with 10 μM Y-27632 and plate onto a 6-well plate coated with diluted growth factor reduced basement membrane matrix.
  2. Maintain iPSCs in SCM with 10 μM Y-27632 for the first 24 hours after thawing. Switch to fresh medium after 24 hours.
  3. Maintain iPSCs in SCM until cells reach 80-90% confluency before passaging.
    1. Calculate how many iPSCs will be needed for differentiation by multiplying desired density for differentiation (15,600 cells/cm2) by the surface area of the well. For a 6-well flat-bottom plate, multiply 15,600 cells/cm2 by 9.6 cm2 for a total of 149,760 cells/well.
  4. To passage, wash the cells with Hanks’ Balanced Salt Solution (HBBS). Then, incubate the cells with non-enzymatic ethylenediaminetetraacetic acid (EDTA) (see material list) for 5 minutes at 37 °C.
    1. Use a cell scraper to gently lift off the cells. Collect cells in fresh SCM.
    2. Plate cells onto cell culture plates coated with diluted SCM and maintain cells as described in step 2.3 or store them at ~1.2 x 106 cells/mL in 1 mL of SCM, 10 μM Y-27632, and 10% DMSO (v/v) in cryopreserved vials in liquid nitrogen at temperature of -160 °C.

3. Differentiation of iPSCs to BMECs

NOTE: Non-enzymatic EDTA separates cells into clumps. Enzymatic EDTA (see Table of Materials) separates cells into single cell suspension. Retinoic acid (RA) should be protected from light.

  1. Wash iPSCs once with Dulbecco’s Phosphate Buffer Saline (DPBS). Incubate with enzymatic EDTA (1 mL for 6-well plate, 0.5 mL for 12-well plate, and 0.25 mL for 24-well plate) for approximately 5 minutes at 37 °C to yield a single cell suspension.
  2. Collect cells and centrifuge at 300 x g (relative centrifugal force) for 5 minutes at room temperature. Resuspend cell pellets in SCM containing 10 μM Y-27632.
  3. Determine cell density using Trypan Blue and automated cell counter or a hemocytometer device. Plate cells at a density of 15,600 cells/cm2 or 149,760 cells/well of a 6-well flat-bottom plate (with a surface area of 9.6 cm2/well) in SCM containing 10 μM Y-27632 for 24 hours.
  4. Initiate differentiation after 24 hours by changing SCM to E6 medium. Change E6 medium daily for the next 4 days.
  5. On day 4 of differentiation, replace E6 medium with hESFM supplemented with diluted (1:200) B27 supplement, 20 ng/mL bFGF, and 10 μM RA. Do not change this medium for the next 48 hours.
  6. Prepare 200 mL of hESFM with diluted (1:200) B27, mix 1 mL of 50x concentrated B27 supplement to 199 mL of hESFM.
  7. Prepare 20 ng/mL of bFGF by reconstituting 50 μg of bFGF in 250 μL of Tris buffer (5 mM Tris, pH 7.6, 150 mM NaCl) to make 200 μg/mL stock solution. Prepare 200 mL of hESFM containing 20 ng/mL bFGF by mixing 20 μL of 200 μg/mL bFGF with 200 mL of hESFM.
  8. Prepare 10 μM RA by first making a 40 mg/mL of RA stock solution by adding 2.5 mL of DMSO to 100 mg of RA powder. Dilute this concentration to 3 mg/mL to make 10 mM stock solution. Prepare 200 mL of hESFM containing 10 μM RA by mixing 200 μL of 10μM RA in 200 mL hESFM.

4. Coating collagen IV (COL4) and fibronectin (FN) Matrix for Purification of iPSC-Derived BMEC

  1. Add 2 mL of sterile water to 2 mg of FN to make 1 mg/mL FN stock solution. Add 5 mL of sterile water to 5 mg of COL4 to make a 1 mg/mL COL4 stock solution.
    1. Allow FN to dissolve for at least 30 minutes at 37 °C and the COL4 to dissolve at room temperature.
  2. Dilute FN stock solution in sterile water to a final concentration of 100 μg/mL and COL4 stock solution to a final concentration of 400 μg/mL.
  3. Coat the desired plates (6-well plate = 1 mL of COL4/FN solution, 12-well plate = 0.5 mL, 24-well plate= 0.25 mL, and 12-transwell filtered plate = 0.25 mL) with the mixture of 400 μg/mL COL4 and 100 μg/mL FN.
  4. Incubate plates for a minimum of 2 hours or overnight at 37 °C; for Transwell filtered plates, a minimum of 4 hours is recommended.

5. Sub-culture and purification of iPSC-Derived BMECs

NOTE: Incubation with enzymatic EDTA may take longer than 15 minutes depending on the confluency of the cells on day 6 of differentiation.

  1. On day 6 of differentiation, wash cells twice with DPBS. Incubate with 1 mL of enzymatic EDTA for at least 15 minutes at 37 °C until a single cell suspension is obtained.
  2. Collect cells via centrifugation at 300 x g for 5 minutes at room temperature. Resuspend cell pellets with fresh hESFM with diluted (1:200) B27 supplement, 20 ng/mL bFGF, and 10 μM RA.
  3. Seed cells onto plates coated with a mixture of 400 μg/mL COL4 and 100 μg/mL FN. Seed cells using a ratio of 1 well of a 6-well plate to 3 wells of a 12-well plate, 3 wells of a 12-transwell filtered plate, or 6 wells of a 24-well plate.
  4. Seed undifferentiated iPSCs from the same cell line onto COL4/FN coated 12-transwell filtered plate as negative control for TEER analysis.
  5. After 24 hours of sub-culturing, change medium to hESFM with B27 supplement only. No medium changes are needed after this step.

6. Sprouting assay

  1. Collect Day 8 iPSC-derived BMECs and seed them at 100,000 cells/well onto a 24-well flat-bottom plate freshly coated with 200 μL/cm2 of basement membrane matrix.
  2. Treat these cells with hESFM with diluted (1:200) B27 and 40 ng/mL of vascular endothelial growth factor A (VEGFA165).
  3. Observe cells every 24 hours and change the medium every two days.

7. Immunocytochemistry (ICC)

NOTE: ICC is carried out on 24-well flat-bottom plates.

  1. After 48 hours of sub-culturing (day 8), wash cells twice with DPBS. Fix cells with 4% paraformaldehyde (PFA) for 20 minutes.
  2. Wash cells three times with DPBS, 5 minutes per wash. Pre-block cells for 1 hour at room temperature in DPBS with 5% donkey serum and 0.3% Triton X-100 (v/v).
  3. Incubate with primary antibodies: mouse anti-human-PECAM1 (1:100, stock 0.5 mg/mL), rabbit anti-human-TJP1 (1:200, stock 0.53 mg/mL), mouse anti-human-CLDN5 (1:200, stock 0.5 mg/mL), mouse anti-human-OCLN (1:200, stock 0.5 mg/mL), and rabbit anti-human-SLC2A1(1:100, stock 0.2 mg/mL) in DPBS containing 5% donkey serum overnight at 4°C.
    1. Rinse cells once with DPBS and then wash five times for 5 minutes per wash with DPBS.
  4. Incubate cells with secondary antibodies: donkey-anti-rabbit Alexa Fluor 555 (1:200) and donkey-anti-mouse 488 (1:200) in DPBS containing 5% donkey serum for 1 hour.
  5. Following this incubation, add Hoechst 33342 trihydrochloride trihydrate diluted (1:1000) in DPBS for 10 minutes.
    1. Remove Hoechst 33342 solution and rinse once with DPBS and wash four times with DPBS for 5 minutes per wash.
  6. Visualize cells on fluorescence microscopes to look for expression and localization of cell makers.

8. TEER Measurement and Analysis

NOTE: Corning 12-Transwell filtered plates are equipped with filters consisting of 1.12 cm2 polyethylene terephthalate membranes and 0.4 micrometer pores. TEER measurements are obtained in technical (3 per well) and biological replicates (3 wells per cell line and/or condition).

  1. 24 hours after sub-culturing (day 7), measure TEER using chopstick electrodes and a voltohmmeter every 24 hours. Refer to voltohmmeter user manual for specific instructions on obtaining measurements.
    1. To measure TEER, charge the voltohmmeter instrument the night before. Lightly wipe the instrument and chopstick electrodes with 70% ethanol before placing them in the safety hood.
    2. Switch the power on and calibrate the ohm meter as recommended by the manufacturer.
    3. Plug in the chopstick electrodes and rinse electrodes with 70% ethanol followed by DPBS.
    4. Place the shorter end electrode into the trans-well insert (the apical chamber) and the longer end into the basolateral chamber.
    5. First measure a blank well that is coated with COL4/FN only. Then measure the other wells.
    6. Quickly rinse chopstick electrodes with 70% ethanol followed by DPBS when measuring different conditions (i.e. measuring different cell lines).
  2. After all measurements (in Ω) have been recorded, rinse chopstick electrodes with 70% ethanol and then sterile water. Gently wipe electrode and let it air dry in the safety hood.
  3. Average the triplicate TEER values (in Ω) from the blank well and subtract this average value from each raw TEER value by condition.
    1. Average the subtracted values and multiply them by 1.12 cm2 (the surface area of the 12-transwell insert).
    2. Use transformed values from step 6 to generate the graph showing TEER value and standard errors for each day of TEER measurement.

9. Efflux Transporter Activity and Analysis

NOTE: Efflux transporter activity assay is performed on a 24-well flat-bottom plate. Efflux transporters of interest include ABCB1 and ABCC1. It is recommended that each condition should be performed in triplicate with control wells (i.e. blank wells without the respective inhibitors).

  1. After 48 hours of sub-culturing (day 8), incubate cells with 10 μM Valspodar (ABCB1 inhibitor) or 10 μM MK571 (ABCC1 inhibitor) for 1 hour at 37 °C.
    1. Prepare 10 mM Valspodar stock by dissolving 5 mg of powder (1214.64 g/mol) in 412 μL of DMSO and dilute to working concentration of 10 μM. For example, to make 10 mL of hESFM with 10 μM Valspodar, mix 10 μL of 10 mM Valspodar stock with 10 mL of hESFM.
    2. Prepare 10 mM MK571 stock by dissolving 5 mg powder (537.07 g/mol) in 931 μL and dilute to working concentration of 10 μM. For example, to make 10 mL of hESFM with 10 μM MK571, mix 10 μL of 10 mM MK571 stock with 10 mL of hESFM.
  2. After 1 hour, incubate cells with 10 μM rhodamine 123 (ABCB1 substrate) or 10 μM 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA, ABCC1 substrate) with or without their respective inhibitors for 1 hour at 37 °C.
    1. Prepare 10 mM rhodamine 123 by dissolving 10 mg of powder (380.82 g/mol) in 875 μL of DMSO and dilute to working concentration of 10 μM. For example, to make 10 mL of hESFM with 10 μM rhodamine 123, mix 10 μL of 10 mM rhodamine 123 stock with 10 mL of hESFM.
    2. Prepare 10 mM H2DCFDA by dissolving 50 mg of powder (487.29 g/mol) in 10.26 mL of DMSO and dilute to working concentration of 10 μM. For example, to make 10 mL of hESFM with 10 μM rhodamine 123, mix 10 μL of 10 mM rhodamine 123 stock with 10 mL of hESFM.
  3. Wash cells twice with 0.5 mL of DPBS and lyse using DPBS containing 5% Triton-X (v/v).
  4. Measure fluorescence of the lysed cells using a multi-plate or microplate reader (see material list).
    1. Set fluorescent plate reader instrument to 485 nanometer excitation and 530 nanometer emission and measure fluorescence at these wavelengths.
    2. For wells not used in the transporter assay, wash cells twice with DPBS before fixing them with 4% PFA for cell nuclei quantification.
  5. Incubate cells with Hoechst 33342 trihydrochloride trihydrate diluted (1:1000) in DPBS for 10 minutes. Image multiple visual fields in each well to calculate average cell nuclei counts using fluorescence microscopes.
  6. Count nuclei using Fiji and normalize fluorescence values on a per-cell basis to these counts.
    1. Calculate average accumulation of fluorescence by subtracting raw fluorescence accumulation value for each condition from its respective blank value.
    2. Average subtracted values for each condition.
    3. Divide average values from step 9.6.1 by the average cell counts. Use these values to normalize fluorescence values on a per-cell basis.
    4. Use normalized values to generate a graphical representation for each inhibitor condition and perform any necessary statistical analysis.

10. Passaging, Expanding, and Cryopreserving BMECs

  1. Replenish day 8 BMEC cultures with fresh hESFM supplemented with diluted (1:200) B27 and allow cells to expand for two more days on the COL4/FN matrix.
  2. Coat a new 12-Transwell filtered plate and a 24-well flat-bottom plate with 400 μg/mL COL4 and 100 μg/mL FN and incubate for 4 hours.
  3. On day 10, wash cells with DPBS and incubate with 1 mL of enzymatic EDTA for at least 15 minutes at 37°C until a single cell suspension is obtained.
  4. Collect cells via centrifugation at 300 x g for 5 minutes at room temperature.
    1. To cryopreserve these cells, resuspend cell pellets with fresh hESFM with 30% Fetal Bovine Serum (FBS) and 10% DMSO.
    2. Store iPSC-derived BMECs in cryopreserved vials in an isopropanol container for the first 24 hours at -80 °C, then place in liquid nitrogen for long-term storage at -160 °C.
    3. To passage these cells, resuspend cell pellets with fresh hESFM supplemented with diluted (1:200) B27.
  5. Seed cells onto coated plates prepared in Step 10.2. Seed cells using a ratio of 1 well of a 6-well plate to 3 wells of a 12-transwell filtered plate and to 6 wells of a 24-well plate. Allow cells to grow and expand for 24 hours.
  6. On day 11, measure TEER by following steps listed in Step 8.
  7. On day 12, perform ICC by following steps listed in Step 9.
  8. To thaw cryopreserved BMECs, place the cryopreserved vials in a warm water or bead bath at 37oC. Then transfer the thawed BMECs to 5 mL of hESFM supplemented with diluted (1:200) B27.
    1. Collect cells via centrifugation at 300 x g for 5 minutes at room temperature. Resuspend the cells in hESFM supplemented with diluted (1:200) B27, 10 μM RA and 10 μM Y-27632.
    2. After 24 hours, switch medium to hESFM supplemented with diluted (1:200) B27 and 10 μM Y-27632 without RA.

Results

BMEC Differentiation
A few critical steps in this protocol should be followed precisely (Figure 1). E6 medium use on day 1 is important, since it is often used for deriving neuroectoderm lineage from iPSCs within a relatively short period of time yielding reproducible results across multiple cell lines36. Another important step is on day 4 of differentiation, where E6 medium should be switched to hESFM with diluted (1:200) B27, 20 ng/m...

Discussion

Modifications and Troubleshooting

In this protocol, we made some modifications in using a commonly used extracellular matrix and cell culture media during iPSC culturing for derivation of BMECs (Figure 1). These changes did not impact the ability to derive BMECS from human iPSCs as described in the Lippmann protocol1. An iPSC line from a different healthy donor was used to demonstrate that this modified protocol shows resul...

Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported by a National Institute of Mental Health Biobehavioral Research Awards for Innovative New Scientists (BRAINS) Award R01MH113858 (to R.K.), a National Institutes of Health Award KL2 TR002542 (PL). a National Institute of Mental Health Clinical Scientist Development Award K08MH086846 (to R.K.), a Sydney R Baer Jr Foundation Grant (to P.L.) the Doris Duke Charitable Foundation Clinical Scientist Development Award (to R.K.), the Ryan Licht Sang Bipolar Foundation (to R.K.), the Phyllis & Jerome Lyle Rappaport Foundation (to R.K.), the Harvard Stem Cell Institute (to R.K.) and by Steve Willis and Elissa Freud (to R.K.). We thank Dr. Annie Kathuria for her critical reading and feedback on the manuscript.

Materials

NameCompanyCatalog NumberComments
2′,7′-dichlorodihydrofluorescein diacetateSigma AldrichD6883-50MG
AccutaseSigma AldrichA6964-100mL
Alexa Fluor 488 Donkey anti-Mouse IgGLife TechnologiesA-21202
Alexa Fluor 555 Donkey anti-Rabbit IgGLife TechnologiesA-31572
B27 SupplementThermo Fisher Scientific17504044
CD31 (PECAM-1) (89C2) Mouse mAbCell Signaling3528S
CLDN5 (Claudin-5)Thermo Fisher Scientific35-2500
Collagen IV from human placentaSigma AldrichC5533-5mg
Corning 2 mL Internal Threaded Polypropylene Cryogenic Vial Corning 8670
Corning Costar Flat Bottom Cell Culture Plates (6-wells)Corning353046
Corning Falcon Flat Bottom Cell Culture Plates (24-wells)Corning353047
Corning Transwell Multiple Well Plate with Permeable Polyester Membrane Inserts (12-wells)Corning3460
Countess slidesThermo Fisher ScientificC10228
DMEM/F12 (without phenol red)Thermo Fisher Scientific A1413202
DMSOSigma AldrichD2438-50mL
Donkey serumSigma AldrichD9663-10ML
DPBS (+/+)Gibco/Thermo Fisher Scientific14040-117
Epithelial Volt/Ohm (TEER) Meter (EVOM2) STX2World Precision InstrumentsN/A
Essential 6 Medium (Thermo Fisher)Thermo Fisher ScientificA1516401
Fetal Bovine Serum (FBS)Sigma AldrichF2442
FibronectinSigma AldrichF2006-2mg
Geltrex LDEV-Free Reduced Growth Factor Basement Membrane MatrixThermo Fisher ScientificA1413202
Hanks' Balance Salt Solution with calcium and magnesium Thermo Fisher Scientific24020-117
Hoechst 33342, Trihydrochloride, TrihydrateThermo Fisher ScientificH3570
Human endothelial serum-free mediumThermo Fisher Scientific11111044
InCell Analyzer 6000General ElectricN/A
Invitrogen Countess Automated Cell CounterThermo Fisher ScientificN/A
MK-571Sigma AldrichM7571-5MG
NutriStemStemgent01-0005
OccludinThermo Fisher Scientific33-1500
Paraformaldehyde 16%Electron Microscopy Services15710
Perkin Elmer Envision 2103 multi-plate ReaderPerkin ElmerN/A
Recombinant Human VEGF 165Peprotech100-20
Recombinant Human FGF-basic (154 a.a.)Peprotech100-18B
Retinoic acidSigma AldrichR2625-100MG
Rhodamine 123Sigma Aldrich83702-10MG
SLC2A1 (GLUT-1)ThermoFisherPA1-21041
SOX17Cell Signaling81778S
TJP-1 (ZO-1)ThermoFisherPA5-28869
Triton X-100Sigma AldrichT8787-50ML
Trypan Blue Stain (0.4%) for use with the Countess Automated Cell CounterThermo Fisher ScientificT10282
Valspodar (Sigma) (cyclosporin A)Sigma AldrichSML0572-5MG
Versene solutionThermo Fisher Scientific15040066
Y-27632 dihydrochloride (ROCK inhibitor)Tocris/Thermo Fisher Scientific1254

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