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 &#38; 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.

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 &#956;M with rho-associated protein kinase inhibitor (ROCKi) Y-27632, and 10% (v/v) dimethyl sulfide (DMSO), in cryopreserved vials in liquid nitrogen at -160 &#176;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
<ol>
	<li>Dilute (1:50) growth factor reduced basement membrane matrix purified from Engelbreth-Holm-Swarm tumor in Dulbecco&#39;s Modified Eagle Medium (DMEM) without phenol red.</li>
	<li>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 &#176;C for at least 1 hour.</li>
</ol>
2. iPSC maintenance
NOTE: The maximum confluency per well in a 6-well flat-bottom plate is ~1.2 x 106 cells.
<ol>
	<li>Thaw cryopreserved iPSCs into SCM with 10 &#956;M Y-27632 and plate onto a 6-well plate coated with diluted growth factor reduced basement membrane matrix.</li>
	<li>Maintain iPSCs in SCM with 10 &#956;M Y-27632 for the first 24 hours after thawing. Switch to fresh medium after 24 hours.</li>
	<li>Maintain iPSCs in SCM until cells reach 80-90% confluency before passaging.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>To passage, wash the cells with Hanks&#8217; Balanced Salt Solution (HBBS). Then, incubate the cells with non-enzymatic ethylenediaminetetraacetic acid (EDTA) (see material list) for 5 minutes at 37 &#176;C.
	<ol>
		<li>Use a cell scraper to gently lift off the cells. Collect cells in fresh SCM.</li>
		<li>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 &#956;M Y-27632, and 10% DMSO (v/v) in cryopreserved vials in liquid nitrogen at temperature of -160 &#176;C.</li>
	</ol>
	</li>
</ol>
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.
<ol>
	<li>Wash iPSCs once with Dulbecco&#8217;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 &#176;C to yield a single cell suspension.</li>
	<li>Collect cells and centrifuge at 300 x g (relative centrifugal force) for 5 minutes at room temperature. Resuspend cell pellets in SCM containing 10 &#956;M Y-27632.</li>
	<li>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 &#956;M Y-27632 for 24 hours.</li>
	<li>Initiate differentiation after 24 hours by changing SCM to E6 medium. Change E6 medium daily for the next 4 days.</li>
	<li>On day 4 of differentiation, replace E6 medium with hESFM supplemented with diluted (1:200) B27 supplement, 20 ng/mL bFGF, and 10 &#956;M RA. Do not change this medium for the next 48 hours.</li>
	<li>Prepare 200 mL of hESFM with diluted (1:200) B27, mix 1 mL of 50x concentrated B27 supplement to 199 mL of hESFM.</li>
	<li>Prepare 20 ng/mL of bFGF by reconstituting 50 &#956;g of bFGF in 250 &#956;L of Tris buffer (5 mM Tris, pH 7.6, 150 mM NaCl) to make 200 &#956;g/mL stock solution. Prepare 200 mL of hESFM containing 20 ng/mL bFGF by mixing 20 &#956;L of 200 &#956;g/mL bFGF with 200 mL of hESFM.</li>
	<li>Prepare 10 &#956;M 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 &#956;M RA by mixing 200 &#956;L of 10&#956;M RA in 200 mL hESFM.</li>
</ol>
4. Coating collagen IV (COL4) and fibronectin (FN) Matrix for Purification of iPSC-Derived BMEC
<ol>
	<li>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.
	<ol>
		<li>Allow FN to dissolve for at least 30 minutes at 37 &#176;C and the COL4 to dissolve at room temperature.</li>
	</ol>
	</li>
	<li>Dilute FN stock solution in sterile water to a final concentration of 100 &#956;g/mL and COL4 stock solution to a final concentration of 400 &#956;g/mL.</li>
	<li>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 &#956;g/mL COL4 and 100 &#956;g/mL FN.</li>
	<li>Incubate plates for a minimum of 2 hours or overnight at 37 &#176;C; for Transwell filtered plates, a minimum of 4 hours is recommended.</li>
</ol>
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.
<ol>
	<li>On day 6 of differentiation, wash cells twice with DPBS. Incubate with 1 mL of enzymatic EDTA for at least 15 minutes at 37 &#176;C until a single cell suspension is obtained.</li>
	<li>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 &#956;M RA.</li>
	<li>Seed cells onto plates coated with a mixture of 400 &#956;g/mL COL4 and 100 &#956;g/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.</li>
	<li>Seed undifferentiated iPSCs from the same cell line onto COL4/FN coated 12-transwell filtered plate as negative control for TEER analysis.</li>
	<li>After 24 hours of sub-culturing, change medium to hESFM with B27 supplement only. No medium changes are needed after this step.</li>
</ol>
6. Sprouting assay
<ol>
	<li>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 &#956;L/cm2 of basement membrane matrix.</li>
	<li>Treat these cells with hESFM with diluted (1:200) B27 and 40 ng/mL of vascular endothelial growth factor A (VEGFA165).</li>
	<li>Observe cells every 24 hours and change the medium every two days.</li>
</ol>
7. Immunocytochemistry (ICC)
NOTE: ICC is carried out on 24-well flat-bottom plates.
<ol>
	<li>After 48 hours of sub-culturing (day 8), wash cells twice with DPBS. Fix cells with 4% paraformaldehyde (PFA) for 20 minutes.</li>
	<li>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).</li>
	<li>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&#176;C.
	<ol>
		<li>Rinse cells once with DPBS and then wash five times for 5 minutes per wash with DPBS.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>Following this incubation, add Hoechst 33342 trihydrochloride trihydrate diluted (1:1000) in DPBS for 10 minutes.
	<ol>
		<li>Remove Hoechst 33342 solution and rinse once with DPBS and wash four times with DPBS for 5 minutes per wash.</li>
	</ol>
	</li>
	<li>Visualize cells on fluorescence microscopes to look for expression and localization of cell makers.</li>
</ol>
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).
<ol>
	<li>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.
	<ol>
		<li>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.</li>
		<li>Switch the power on and calibrate the ohm meter as recommended by the manufacturer.</li>
		<li>Plug in the chopstick electrodes and rinse electrodes with 70% ethanol followed by DPBS.</li>
		<li>Place the shorter end electrode into the trans-well insert (the apical chamber) and the longer end into the basolateral chamber.</li>
		<li>First measure a blank well that is coated with COL4/FN only. Then measure the other wells.</li>
		<li>Quickly rinse chopstick electrodes with 70% ethanol followed by DPBS when measuring different conditions (i.e. measuring different cell lines).</li>
	</ol>
	</li>
	<li>After all measurements (in &#937;) 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.</li>
	<li>Average the triplicate TEER values (in &#937;) from the blank well and subtract this average value from each raw TEER value by condition.
	<ol>
		<li>Average the subtracted values and multiply them by 1.12 cm2 (the surface area of the 12-transwell insert).</li>
		<li>Use transformed values from step 6 to generate the graph showing TEER value and standard errors for each day of TEER measurement.</li>
	</ol>
	</li>
</ol>
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).
<ol>
	<li>After 48 hours of sub-culturing (day 8), incubate cells with 10 &#956;M Valspodar (ABCB1 inhibitor) or 10 &#956;M MK571 (ABCC1 inhibitor) for 1 hour at 37 &#176;C.
	<ol>
		<li>Prepare 10 mM Valspodar stock by dissolving 5 mg of powder (1214.64 g/mol) in 412 &#956;L of DMSO and dilute to working concentration of 10 &#956;M. For example, to make 10 mL of hESFM with 10 &#956;M Valspodar, mix 10 &#956;L of 10 mM Valspodar stock with 10 mL of hESFM.</li>
		<li>Prepare 10 mM MK571 stock by dissolving 5 mg powder (537.07 g/mol) in 931 &#956;L and dilute to working concentration of 10 &#956;M. For example, to make 10 mL of hESFM with 10 &#956;M MK571, mix 10 &#956;L of 10 mM MK571 stock with 10 mL of hESFM.</li>
	</ol>
	</li>
	<li>After 1 hour, incubate cells with 10 &#956;M rhodamine 123 (ABCB1 substrate) or 10 &#956;M 2&#39;,7&#39;-dichlorodihydrofluorescein diacetate (H2DCFDA, ABCC1 substrate) with or without their respective inhibitors for 1 hour at 37 &#176;C.
	<ol>
		<li>Prepare 10 mM rhodamine 123 by dissolving 10 mg of powder (380.82 g/mol) in 875 &#956;L of DMSO and dilute to working concentration of 10 &#956;M. For example, to make 10 mL of hESFM with 10 &#956;M rhodamine 123, mix 10 &#956;L of 10 mM rhodamine 123 stock with 10 mL of hESFM.</li>
		<li>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 &#956;M. For example, to make 10 mL of hESFM with 10 &#956;M rhodamine 123, mix 10 &#956;L of 10 mM rhodamine 123 stock with 10 mL of hESFM.</li>
	</ol>
	</li>
	<li>Wash cells twice with 0.5 mL of DPBS and lyse using DPBS containing 5% Triton-X (v/v).</li>
	<li>Measure fluorescence of the lysed cells using a multi-plate or microplate reader (see material list).
	<ol>
		<li>Set fluorescent plate reader instrument to 485 nanometer excitation and 530 nanometer emission and measure fluorescence at these wavelengths.</li>
		<li>For wells not used in the transporter assay, wash cells twice with DPBS before fixing them with 4% PFA for cell nuclei quantification.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>Count nuclei using Fiji and normalize fluorescence values on a per-cell basis to these counts.
	<ol>
		<li>Calculate average accumulation of fluorescence by subtracting raw fluorescence accumulation value for each condition from its respective blank value.</li>
		<li>Average subtracted values for each condition.</li>
		<li>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.</li>
		<li>Use normalized values to generate a graphical representation for each inhibitor condition and perform any necessary statistical analysis.</li>
	</ol>
	</li>
</ol>
10. Passaging, Expanding, and Cryopreserving BMECs
<ol>
	<li>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.</li>
	<li>Coat a new 12-Transwell filtered plate and a 24-well flat-bottom plate with 400 &#956;g/mL COL4 and 100 &#956;g/mL FN and incubate for 4 hours.</li>
	<li>On day 10, wash cells with DPBS and incubate with 1 mL of enzymatic EDTA for at least 15 minutes at 37&#176;C until a single cell suspension is obtained.</li>
	<li>Collect cells via centrifugation at 300 x g for 5 minutes at room temperature.
	<ol>
		<li>To cryopreserve these cells, resuspend cell pellets with fresh hESFM with 30% Fetal Bovine Serum (FBS) and 10% DMSO.</li>
		<li>Store iPSC-derived BMECs in cryopreserved vials in an isopropanol container for the first 24 hours at -80 &#176;C, then place in liquid nitrogen for long-term storage at -160 &#176;C.</li>
		<li>To passage these cells, resuspend cell pellets with fresh hESFM supplemented with diluted (1:200) B27.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>On day 11, measure TEER by following steps listed in Step 8.</li>
	<li>On day 12, perform ICC by following steps listed in Step 9.</li>
	<li>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.
	<ol>
		<li>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 &#956;M RA and 10 &#956;M Y-27632.</li>
		<li>After 24 hours, switch medium to hESFM supplemented with diluted (1:200) B27 and 10 &#956;M Y-27632 without RA.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

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...

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,&#160;and extracellular matrix20,29,30,31,32&#160;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&#160;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&#160;to study BBB function. However, many of these models failed to attain desirable BBB phenotypes, such a&#160;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 &#62;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 (~2000&#937; x cm2)37&#160;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&#160;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&#160;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&#8217;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.

<table><tbody><tr><td>2&amp;prime;,7&amp;prime;-dichlorodihydrofluorescein diacetate</td><td>Sigma Aldrich</td><td>D6883-50MG</td><td></td></tr><tr><td>Accutase</td><td>Sigma Aldrich</td><td>A6964-100mL</td><td></td></tr><tr><td>Alexa Fluor 488 Donkey anti-Mouse IgG</td><td>Life Technologies</td><td>A-21202</td><td></td></tr><tr><td>Alexa Fluor 555 Donkey anti-Rabbit IgG</td><td>Life Technologies</td><td>A-31572</td><td></td></tr><tr><td>B27 Supplement</td><td>Thermo Fisher Scientific</td><td>17504044</td><td></td></tr><tr><td>CD31 (PECAM-1) (89C2) Mouse mAb</td><td>Cell Signaling</td><td>3528S</td><td></td></tr><tr><td>CLDN5 (Claudin-5)</td><td>Thermo Fisher Scientific</td><td>35-2500</td><td></td></tr><tr><td>Collagen IV from human placenta</td><td>Sigma Aldrich</td><td>C5533-5mg</td><td></td></tr><tr><td>Corning 2 mL Internal Threaded Polypropylene Cryogenic Vial&amp;nbsp;</td><td>Corning</td><td>&amp;nbsp;8670</td><td></td></tr><tr><td>Corning&amp;nbsp;Costar Flat Bottom Cell Culture Plates (6-wells)</td><td>Corning</td><td>353046</td><td></td></tr><tr><td>Corning&amp;nbsp;Falcon Flat Bottom Cell Culture Plates (24-wells)</td><td>Corning</td><td>353047</td><td></td></tr><tr><td>Corning Transwell Multiple Well Plate with Permeable Polyester Membrane Inserts (12-wells)</td><td>Corning</td><td>3460</td><td></td></tr><tr><td>Countess slides</td><td>Thermo Fisher Scientific</td><td>C10228</td><td></td></tr><tr><td>DMEM/F12 (without phenol red)</td><td>Thermo Fisher Scientific</td><td>&amp;nbsp;A1413202</td><td></td></tr><tr><td>DMSO</td><td>Sigma Aldrich</td><td>D2438-50mL</td><td></td></tr><tr><td>Donkey serum</td><td>Sigma Aldrich</td><td>D9663-10ML</td><td></td></tr><tr><td>DPBS (+/+)</td><td>Gibco/Thermo Fisher Scientific</td><td>14040-117</td><td></td></tr><tr><td>Epithelial Volt/Ohm (TEER) Meter (EVOM2) STX2</td><td>World Precision Instruments</td><td>N/A</td><td></td></tr><tr><td>Essential 6 Medium (Thermo Fisher)</td><td>Thermo Fisher Scientific</td><td>A1516401</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Sigma Aldrich</td><td>F2442</td><td></td></tr><tr><td>Fibronectin</td><td>Sigma Aldrich</td><td>F2006-2mg</td><td></td></tr><tr><td>Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix</td><td>Thermo Fisher Scientific</td><td>A1413202</td><td></td></tr><tr><td>Hanks&#039; Balance Salt Solution with calcium and magnesium&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>24020-117</td><td></td></tr><tr><td>Hoechst 33342, Trihydrochloride, Trihydrate</td><td>Thermo Fisher Scientific</td><td>H3570</td><td></td></tr><tr><td>Human endothelial serum-free medium</td><td>Thermo Fisher Scientific</td><td>11111044</td><td></td></tr><tr><td>InCell Analyzer 6000</td><td>General Electric</td><td>N/A</td><td></td></tr><tr><td>Invitrogen&amp;nbsp;Countess Automated Cell Counter</td><td>Thermo Fisher Scientific</td><td>N/A</td><td></td></tr><tr><td>MK-571</td><td>Sigma Aldrich</td><td>M7571-5MG</td><td></td></tr><tr><td>NutriStem</td><td>Stemgent</td><td>01-0005</td><td></td></tr><tr><td>Occludin</td><td>Thermo Fisher Scientific</td><td>33-1500</td><td></td></tr><tr><td>Paraformaldehyde 16%</td><td>Electron Microscopy Services</td><td>15710</td><td></td></tr><tr><td>Perkin Elmer Envision 2103 multi-plate Reader</td><td>Perkin Elmer</td><td>N/A</td><td></td></tr><tr><td>Recombinant Human VEGF 165</td><td>Peprotech</td><td>100-20</td><td></td></tr><tr><td>Recombinant Human FGF-basic (154 a.a.)</td><td>Peprotech</td><td>100-18B</td><td></td></tr><tr><td>Retinoic acid</td><td>Sigma Aldrich</td><td>R2625-100MG</td><td></td></tr><tr><td>Rhodamine 123</td><td>Sigma Aldrich</td><td>83702-10MG</td><td></td></tr><tr><td>SLC2A1 (GLUT-1)</td><td>ThermoFisher</td><td>PA1-21041</td><td></td></tr><tr><td>SOX17</td><td>Cell Signaling</td><td>81778S</td><td></td></tr><tr><td>TJP-1 (ZO-1)</td><td>ThermoFisher</td><td>PA5-28869</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma Aldrich</td><td>T8787-50ML</td><td></td></tr><tr><td>Trypan Blue Stain (0.4%) for use with the Countess Automated Cell Counter</td><td>Thermo Fisher Scientific</td><td>T10282</td><td></td></tr><tr><td>Valspodar (Sigma) (cyclosporin A)</td><td>Sigma Aldrich</td><td>SML0572-5MG</td><td></td></tr><tr><td>Versene solution</td><td>Thermo Fisher Scientific</td><td>15040066</td><td></td></tr><tr><td>Y-27632 dihydrochloride (ROCK inhibitor)</td><td>Tocris/Thermo Fisher Scientific</td><td>1254</td><td></td></tr></tbody></table>

derivation, expansion, cryopreservation and characterization of brain microvascular endothelial cells from human induced pluripotent stem cells

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 &#937; 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.

BMEC Differentiation 
A few critical steps in this protocol should be&#160;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&#160;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...

Watch this Scientific Journal Video about Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells at JoVE.com

Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells

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.

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models ...

derivation-expansion-cryopreservation-characterization-brain

Research

JoVE Journal

Neuroscience

6.3K Views.  Massachusetts General Hospital. Dit protocol beschrijft een aangepaste methode om microvasculaire endotheelcellen van de hersenen af te leiden, uit te breiden en cryopreserveren, verkregen door het differentiëren van door de mens geïnduceerde pluripotente stamcellen, en om de eigenschappen van de bloed-hersenbarrière in een ex vivo model te bestuderen.

Video: Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The blood-brain barrier (BBB) consisting of EC, astrocyte end-feet, pericytes and the basal membrane is responsible for the protection and homeostasis of the brain parenchyma. In vitro BBB models are common tools to study the structure and function of the BBB at the cellular level. A considerable number of different in vitro BBB models have been established for research in different laboratories to date. Usually, the cells are obtained from bovine, porcine, rat or mouse brain tissue (discussed in detail in the review by Wilhelm et al. 1). Human tissue samples are available only in a restricted number of laboratories or companies 2,3. While primary cell preparations are time consuming and the EC cultures can differ from batch to batch, the establishment of immortalized EC lines is the focus of scientific interest.
Here, we present a method for establishing an immortalized brain microvascular EC line from neonatal mouse brain. We describe the procedure step-by-step listing the reagents and solutions used. The method established by our lab allows the isolation of a homogenous immortalized endothelial cell line within four to five weeks. The brain microvascular endothelial cell lines termed cEND 4 (from cerebral cortex) and cerebEND 5 (from cerebellar cortex), were isolated according to this procedure in the F&ouml;rster laboratory and have been effectively used for explanation of different physiological and pathological processes at the BBB. Using cEND and cerebEND we have demonstrated that these cells respond to glucocorticoid- 4,6-9 and estrogen-treatment 10 as well as to pro-infammatory mediators, such as TNFalpha 5,8. Moreover, we have studied the pathology of multiple sclerosis 11 and hypoxia 12,13 on the EC-level. The cEND and cerebEND lines can be considered as a good tool for studying the structure and function of the BBB, cellular responses of ECs to different stimuli or interaction of the EC with lymphocytes or cancer cells.

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

This method describes how to isolate and immortalize microvascular endothelial cells from mouse brain. We describe a step-by-step protocol starting from the homogenization of brain tissue, digestion steps, seeding and immortalization of the cells. Usually, it takes about five weeks to obtain a homogenous, immortalized microvascular endothelial cell line.

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The ...

An In Vitro 3D Model and Computational Pipeline to Quantify the Vasculogenic Potential of iPSC-Derived Endothelial Progenitors

Induced pluripotent stem cells (iPSCs) are a patient-specific, proliferative cell source that can differentiate into any somatic cell type. Bipotent endothelial progenitors (EPs), which can differentiate into the cell types necessary to assemble mature, functional vasculature, have been derived from both embryonic and induced pluripotent stem cells. However, these cells have not been rigorously evaluated in three-dimensional environments, and a quantitative measure of their vasculogenic potential remains elusive. Here, the generation and isolation of iPSC-EPs via fluorescent-activated cell sorting are first outlined, followed by a description of the encapsulation and culture of iPSC-EPs in collagen hydrogels. This extracellular matrix (ECM)-mimicking microenvironment encourages a robust vasculogenic response; vascular networks form after a week of culture. The creation of a computational pipeline that utilizes open-source software to quantify this vasculogenic response is delineated. This pipeline is specifically designed to preserve the 3D architecture of the capillary plexus to robustly identify the number of branches, branching points, and the total network length with minimal user input.

Endothelial progenitors derived from induced pluripotent stem cells (iPSC-EPs) have the potential to revolutionize cardiovascular disease treatments and to enable the creation of more faithful cardiovascular disease models. Herein, the encapsulation of iPSC-EPs in three-dimensional (3D) collagen microenvironments and a quantitative analysis of these cells&#8217; vasculogenic potential are described.

Induced pluripotent stem cells (iPSCs) are a patient-specific, proliferative cell source that can differentiate into any somatic cell type. Bipotent ...

Developmental Biology

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

Generation of a Human iPSC-Based Blood-Brain Barrier Chip

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the blood that can disrupt delicate brain function. As such, the BBB is a major obstacle to the delivery of therapeutics to the CNS. Accumulating evidence suggests that the BBB plays a key role in the onset and progression of neurological diseases. Thus, there is a tremendous need for a BBB model that can predict penetration of CNS-targeted drugs as well as elucidate the BBB&#39;s role in health and disease.
We have recently combined organ-on-chip and induced pluripotent stem cell (iPSC) technologies to generate a BBB chip fully personalized to humans. This novel platform displays cellular, molecular, and physiological properties that are suitable for the prediction of drug and molecule transport across the human BBB. Furthermore, using patient-specific BBB chips, we have generated models of neurological disease and demonstrated the potential for personalized predictive medicine applications. Provided here is a detailed protocol demonstrating how to generate iPSC-derived BBB chips, beginning with differentiation of iPSC-derived brain microvascular endothelial cells (iBMECs) and resulting in mixed neural cultures containing neural progenitors, differentiated neurons, and astrocytes. Also described is a procedure for seeding cells into the organ chip and culturing of the BBB chips under controlled laminar flow. Lastly, detailed descriptions of BBB chip analyses are provided, including paracellular permeability assays for assessing drug and molecule permeability as well as immunocytochemical methods for determining the composition of cell types within the chip.

The blood-brain barrier (BBB) is a multicellular neurovascular unit tightly regulating brain homeostasis. By combining human iPSCs and organ-on-chip technologies, we have generated a personalized BBB chip, suitable for disease modeling and CNS drug penetrability predictions. A detailed protocol is described for the generation and operation of the BBB chip.

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the ...

Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial cells, which line blood vessel lumens, from human pluripotent stem cells is crucial for a multitude of tissue engineering and regeneration applications. In vivo, primordial endothelial cells are derived from the mesodermal lineage and are specified toward specific subtypes, including arterial, venous, capillary, hemogenic, and lymphatic. Hemogenic endothelial cells are of particular interest because, during development, they give rise to hematopoietic stem and progenitor cells, which then generate all blood lineages throughout life. Thus, creating a system to generate hemogenic endothelial cells in vitro would provide an opportunity to study endothelial-to-hematopoietic transition, and may lead to ex vivo production of human blood products and reduced reliance on human donors. While several protocols exist for the derivation of progenitor and primordial endothelial cells, generation of well-characterized hemogenic endothelial cells from human stem cells has not been described. Here, a method for the derivation of hemogenic endothelial cells from human embryonic stem cells in approximately 1 week is presented: a differentiation protocol with primitive streak cells formed in response to GSK3&#946; inhibitor (CHIR99021), then mesoderm lineage induction mediated by bFGF, followed by primordial endothelial cell development promoted by BMP4 and VEGF-A, and finally hemogenic endothelial cell specification induced by retinoic acid. This protocol yields a well-defined population of hemogenic endothelial cells that can be used to further understand their molecular regulation and endothelial-to-hematopoietic transition, which has the potential to be applied to downstream therapeutic applications.

Presented here is a simple protocol for the directed differentiation of hemogenic endothelial cells from human pluripotent stem cells in approximately 1 week.

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial ...

Generating Human Brain Organoids with Resident Microglia Using iPSC-Derived HPCs

Derivation of a Human Brain Organoid with Microglia Development

Three-dimensional (3D) brain organoid cultures derived from induced pluripotent stem cells (iPSC) provide an important alternative in vitro tool&#160;for studying human brain development and pathogenesis of neurological diseases. However, the lack of incorporation of microglia in the human brain organoids is still a major hurdle for 3D models of neuroinflammation. Current approaches include either the incorporation of fully differentiated microglia into mature brain organoids or the induction of microglial differentiation from the early stage of iPSC-derived embryoid bodies (EBs). The first approach misses the stage when microglial differentiation interacts with the adjacent neural environment, and the later approach is technically challenging, resulting in inconsistency among the final organoids in terms of the quantity and quality of microglia. To model brain organoids with microglia to study the early interactions between microglial and neuronal development, highly pure hematopoietic progenitor cells (HPC) differentiated from human iPSCs were incorporated&#160;into iPSC-derived EBs to make brain organoids. Using immunostaining and single-cell RNA sequencing (sc-RNA-seq) analysis, we confirmed that HPCs were incorporated into the 3D organoids, which eventually developed into brain organoids with both microglia and neurons. Compared to brain organoids without HPCs, this approach produces significant microglial incorporation in the brain organoids. This novel 3D organoid model, which consists of both microglial and neural development properties, can be used to study the early interactions between innate immune and nervous system development and potentially as a model for neuroinflammation and neuroinfectious disorders.

We present a protocol to generate a human brain organoid with resident microglia by incorporating Induced pluripotent stem cell (iPSC)-derived hematopoietic progenitor cells (HPCs) into organoid development.

Three-dimensional (3D) brain organoid cultures derived from induced pluripotent stem cells (iPSC) provide an important alternative in vitro tool&#160;for ...

Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells

Meningococcal meningitis is a life-threatening infection that occurs when Neisseria meningitidis (meningococcus, Nm) can gain access to the central nervous system (CNS) by penetrating highly specialized brain endothelial cells (BECs). As Nm is a human-specific pathogen, the lack of robust in vivo model systems makes study of the host-pathogen interactions between Nm and BECs challenging and establishes a need for a human based model that mimics native BECs. BECs possess tighter barrier properties when compared to peripheral endothelial cells characterized by complex tight junctions and elevated trans-endothelial electrical resistance (TEER). However, many in vitro models, such as primary BECs and immortalized BECs, either lack or rapidly lose their barrier properties after removal from the native neural microenvironment. Recent advances in human stem-cell technologies have developed methods for deriving brain-like endothelial cells from induced pluripotent stem-cells (iPSCs) that better phenocopy BECs when compared to other in vitro human models. The use of iPSC-derived BECs (iPSC-BECs) to model Nm-BEC interaction has the benefit of using human cells that possess BEC barrier properties, and can be used to examine barrier destruction, innate immune activation, and bacterial interaction. Here we demonstrate how to derive iPSC-BECs from iPSCs in addition to bacterial preparation, infection, and sample collection for analysis.

Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells

The protocol described here highlights the major steps in the differentiating induced pluripotent stem-cell derived brain-like endothelial cells, the preparation of Neisseria meningitidis for infection, and sample collection for other molecular analyses.

Meningococcal meningitis is a life-threatening infection that occurs when Neisseria meningitidis (meningococcus, Nm) can gain access to the central ...

Immunology and Infection

Differentiation of Human Induced Pluripotent Stem Cells to Brain Microvascular Endothelial Cell-Like Cells with a Mature Immune Phenotype

Blood-brain barrier (BBB) dysfunction is a pathological hallmark of many neurodegenerative and neuroinflammatory diseases affecting the central nervous system (CNS). Due to the limited access to disease-related BBB samples, it is still not well understood whether BBB malfunction is causative for disease development or rather a consequence of the neuroinflammatory or neurodegenerative process. Human induced pluripotent stem cells (hiPSCs) therefore provide a novel opportunity to establish in vitro BBB models from healthy donors and patients, and thus to study disease-specific BBB characteristics from individual patients. Several differentiation protocols have been established for deriving brain microvascular endothelial cell (BMEC)-like cells from hiPSCs. Consideration of the specific research question is mandatory for the correct choice of the respective BMEC-differentiation protocol. Here, we describe the extended endothelial cell culture method (EECM), which is optimized to differentiate hiPSCs into BMEC-like cells with a mature immune phenotype, allowing the study of immune cell-BBB interactions. In this protocol, hiPSCs are first differentiated into endothelial progenitor cells (EPCs) by activating Wnt/&#946;-catenin signaling. The resulting culture, which contains smooth muscle-like cells (SMLCs), is then sequentially passaged to increase the purity of endothelial cells (ECs) and to induce BBB-specific properties. Co-culture of EECM-BMECs with these SMLCs or conditioned medium from SMLCs allows for the reproducible, constitutive, and cytokine-regulated expression of EC adhesion molecules. Importantly, EECM-BMEC-like cells establish barrier properties comparable to primary human BMECs, and due to their expression of all EC adhesion molecules, EECM-BMEC-like cells are different from other hiPSC-derived in vitro BBB models. EECM-BMEC-like cells are thus the model of choice for investigating the potential impact of disease processes at the level of the BBB, with an impact on immune cell interaction in a personalized fashion.

Here, we describe a protocol, the extended endothelial cell culture method (EECM), that allows differentiation of pluripotent stem cells to brain microvascular endothelial cell (BMEC)-like cells. These cells show endothelial cell adhesion molecule expression and are thus a human blood-brain barrier model suitable to study immune cell interactions in vitro.

Blood-brain barrier (BBB) dysfunction is a pathological hallmark of many neurodegenerative and neuroinflammatory diseases affecting the central nervous ...

Bioengineering

hiPSC-Derived Vascular Organoids for Drug Screening and Disease Modeling

Vascular Organoid Generation from Human-Induced Pluripotent Stem Cells

Vascular organoids derived from human induced pluripotent stem cells (hiPSCs) recapitulate the cell type diversity and complex architecture of human vascular networks. This three-dimensional (3D) model holds substantial potential for vascular pathology modeling and in vitro drug screening. Despite recent advances, a key technical challenge remains in reproducibly generating organoids with consistent quality, which is crucial for downstream assays and applications. Here, a modified protocol is presented that improves both the homogeneity and reproducibility of vascular organoid generation. The modified protocol incorporates the use of microwells and the CEPT cocktail (chroman 1, emricasan, polyamines, and the integrated stress response inhibitor, trans-ISRIB) to improve embryoid body formation and cell survival. Differentiated, mature vascular organoids generated using this protocol are characterized by whole-mount 3D immunofluorescence microscopy to analyze their morphology and complex vasculature. This protocol enables the production of high-quality vascular organoids in a scalable manner, potentially facilitating their use in disease modeling and drug screening applications.

Author Spotlight: Improving Reproducibility in Vascular Organoids Using ROCK Inhibitors and Microwell Confinement

This protocol presents a method for generating vascular organoids using human pluripotent stem cells.

Vascular organoids derived from human induced pluripotent stem cells (hiPSCs) recapitulate the cell type diversity and complex architecture of human ...

Generating Brain Endothelial Cells from Induced Pluripotent Stem Cells

Source: Endres, L. M., et al., <a href="https://app.jove.com/v/61400">Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells.</a> J. Vis. Exp. (2020)
This video demonstrates a protocol to induce the differentiation of induced pluripotent stem cells into brain endothelial cells.

1. Preparation of materials required for iPSC culture and BEC differentiation.

	Matrix coating of tissue culture (TC) plastic for IMR90-4 iPSC culture
	
	...

Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells

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