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&#8242;,7&#8242;-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&#160;</td>
			<td>Corning</td>
			<td>&#160;8670</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning&#160;Costar Flat Bottom Cell Culture Plates (6-wells)</td>
			<td>Corning</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning&#160;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>&#160;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&#39; Balance Salt Solution with calcium and magnesium&#160;</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&#160;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

6.2K Views.  Massachusetts General Hospital. 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.

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

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The differentiation process is initiated by breaking the human PSCs into clumps which round up to form aggregates when the cells are placed in a suspension culture. The aggregates are then grown in hESC medium from days 1-4 to allow for spontaneous differentiation. During this time, the cells have the capacity to become any of the three germ layers. From days 5-8, the cells are placed in a neural induction medium to push them into the neural lineage. Around day 8, the cells are allowed to attach onto 6 well plates and differentiate during which time the neuroepithelial cells form. These neuroepithelial cells can be isolated at day 17. The cells can then be kept as neurospheres until they are ready to be plated onto coverslips. Using a basic medium without any caudalizing factors, neuroepithelial cells are specified into telencephalic precursors, which can then be further differentiated into dorsal telencephalic progenitors and glutamatergic neurons efficiently. Overall, our system provides a tool to generate human glutamatergic neurons for researchers to study the development of these neurons and the diseases which affect them.

This procedure yields telencephalic neurons by going through checkpoints which are similar to those observed during human development. The cells are allowed to spontaneously differentiate, are exposed to factors which push them towards the neural lineage, are isolated, and are plated onto coverslips to allow for terminal differentiation and maturation.

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The ...

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 ...

A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as &#8220;chopping&#8221; that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.

This protocol describes a novel mechanical chopping method that allows the expansion of spherical neural stem and progenitor cell aggregates without dissociation to a single cell suspension.&#160; Maintaining cell/cell contact allows rapid and stable growth for over 40 passages.

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson&#8217;s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson&#8217;s disease.

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

Isolation of Primary Murine Brain Microvascular Endothelial Cells

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional complex of tight and adherens junctions. Together with other cell-types such as astrocytes or pericytes, they form the neurovascular unit (NVU), which specifically regulates the interchange of fluids, molecules and cells between the peripheral blood and the CNS. Through this complex and dynamic system BMECs are involved in various processes maintaining the homeostasis of the CNS. A dysfunction of the BBB is observed as an essential step in the pathogenesis of many severe CNS diseases. However, specific and targeted therapies are very limited, as the underlying mechanisms are still far from being understood. 
Animal and in vitro models have been extensively used to gain in-depth understanding of complex physiological and pathophysiological processes. By reduction and simplification it is possible to focus the investigation on the subject of interest and to exclude a variety of confounding factors. However, comparability and transferability are also reduced in model systems, which have to be taken into account for evaluation. The most common animal models are based on mice, among other reasons, mainly due to the constantly increasing possibilities of methodology. In vitro studies of isolated murine BMECs might enable an in-depth analysis of their properties and of the blood-brain-barrier under physiological and pathophysiological conditions. Further insights into the complex mechanisms at the BBB potentially provide the basis for new therapeutic strategies.
This protocol describes a method to isolate primary murine microvascular endothelial cells by a sequence of physical and chemical purification steps. Special considerations for purity and cultivation of MBMECs as well as quality control, potential applications and limitations are discussed.

Brain microvascular endothelial cells (BMEC) are interconnected by specific junctional proteins forming a highly regulated barrier separating blood and the central nervous system (CNS), the so-called blood-brain-barrier (BBB). The isolation of primary murine brain microvascular endothelial cells, as discussed in this protocol, enables detailed in vitro studies of the BBB.

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional ...

Isolation of Primary Murine Skeletal Muscle Microvascular Endothelial Cells

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal muscles regulating the exchange of fluids and nutrients as well as the immune response against infectious agents by controlling immune cell migration. For these functions, MMEC form a functional &#34;myovascular unit&#34; (MVU), with further cell types, such as fibroblasts, pericytes and skeletal muscle cells. Consequently, a dysfunction of MMEC and therefore the MVU contributes to a vast variety of myopathies. However, regulatory mechanisms of MMEC in health and disease remain insufficiently understood and their elucidation precedes more specific treatments for myopathies. The isolation and in-depth investigation of primary MMEC functions in the context of the MVU might facilitate a better understanding of these processes.
This article provides a protocol to isolate primary murine MMEC of the skeletal muscle by mechanical and enzymatic dissociation including purification and culture maintenance steps.

Microvascular endothelial cells of skeletal muscles (MMEC) shape the inner wall of muscle capillaries and regulate both, exchange of fluids/molecules and migration of (immune) cells between muscle tissue and blood. Isolation of primary murine MMEC, as described here, enables comprehensive in vitro investigations of the &#34;myovascular unit&#34;.

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal ...

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have identified a special population of vascular cells, the periventricular endothelial cells, that play a critical role in the migration and distribution of forebrain GABAergic interneurons during embryonic development. This, coupled with their cell-autonomous functions, alludes to novel roles of periventricular endothelial cells in the pathology of neuropsychiatric disorders like schizophrenia, epilepsy, and autism. Here, we have described three different in vitro assays that collectively evaluate the functions of periventricular endothelial cells and their interaction with GABAergic interneurons. Use of these assays, particularly in a human context, will allow us to identify the link between periventricular endothelial cells and brain disorders. These assays are simple, low cost, and reproducible, and can be easily adapted to any adherent cell type.

We present three simple in vitro assays-the long-distance migration assay, the co-culture migration assay, and chemo-attraction assay-that collectively evaluate the functions of human stem cell derived periventricular endothelial cells and their interaction with GABAergic interneurons.

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have ...

Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it contributes to disease development and progression, particularly in the gray matter of the brain, remains largely unknown. The dysfunction of oligodendrocyte lineage cells is hallmarked by deficiencies in myelination and impaired self-renewal of oligodendrocyte precursor cells (OPCs). These two defects are caused at least in part by the disruption of interactions between neuron and oligodendrocytes along the buildup of pathology. OPCs give rise to myelinating oligodendrocytes during CNS development. In the mature brain cortex, OPCs are the major proliferative cells (comprising ~5% of total brain cells) and control new myelin formation in a neural activity-dependent manner. Such neuron-to-oligodendrocyte communications are significantly understudied, especially in the context of neurodegenerative conditions such as AD, due to the lack of appropriate tools. In recent years, our group and others have made significant progress to improve currently available protocols to generate functional neurons and oligodendrocytes individually from human pluripotent stem cells. In this manuscript, we describe our optimized procedures, including the establishment of a co-culture system to model the neuron-oligodendrocyte connections. Our illustrative results suggest an unexpected contribution from OPCs/oligodendrocytes to the brain amyloidosis and synapse integrity and highlight the utility of this methodology for AD research. This reductionist approach is a powerful tool to dissect the specific hetero-cellular interactions out of the inherent complexity inside the brain. The protocols we describe here are expected to facilitate future studies on oligodendroglial defects in the pathogenesis of neurodegeneration.

The neuron-glial interactions in neurodegeneration are not well understood due to inadequate tools and methods. Here, we describe optimized protocols to obtain induced neurons, oligodendrocyte precursor cells, and oligodendrocytes from human pluripotent stem cells and provide examples of the values of these methods in understanding cell-type-specific contributions in Alzheimer&#8217;s disease.

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it ...