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 med...

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 ...

BMECs generated from iPSC provide new experimental models for studying blood brain barrier function and dysfunction. We have used this protocol to examine whether BMECs retain their barrier properties after expansion and cryopreservation. This technique enables the generation of disease-specific BMECs from patients with disorders in which blood-brain barrier disruption plays a role in the pathophysiology of disease.To induce BMEC differentiation from human iPSCs, wash the confluent iPSC culture with DPBS one time before incubating the cells with the appropriate volume of enzymatic EDTA for approximately five minutes at 37 degrees Celsius. When a single cell suspension has been obtained collect the cells by centrifugation and resuspend the pellet in an appropriate volume of stem cell medium supplemented with 10 micromolar ROCK inhibitor for counting. Dilute the cells to 1.56 times 10 to the fourth cells per cubic centimeter concentration and seed two milliliters of cells into individual wells of a six-well flat bottom.After 24 hours in the cell culture incubator replace the supernatant in each well with E6 medium and return the plate to the incubator for four more days. To purify the iPSC-derived BMECs, coat the subculturing plates with the appropriate volume of a freshly prepared collagen IV and fibronectin solution per well and incubate the plates for a minimum of two hours at 37 degrees Celsius. On day six of differentiation collect the cells by centrifugation and resuspend the pellets in the appropriate volume of fresh human endothelial serum-free medium supplemented with B27, basic fibroblasts growth factor, and retinoic acid.For TEER analysis, plate the differentiated cells into each well of a collagen and fibronectin coated 12-transwell filtered plate. For efflux transporter activity analysis, plate the cells into each well of a collagen and fibronectin coated 24-well flat bottom plate. After 24 hours of subculture, replace the supernatant in each well with fresh human endothelial serum-free medium supplemented only with B27 supplement.To measure the blood brain barrier properties of the BMECs using TEER, charge the TEER instrument overnight and lightly wipe the instrument and chopstick electrodes with 70%ethanol before placing them into the safety hood. Switch on the power and calibrate the ohm meter according to the manufacturer's specifications. Plug in the electrodes and rinse them one time with 70%ethanol and one time with DPBS.Place the shorter end of the electrode into the atypical chamber of the insert and the longer end into the basolateral chamber of a blank well. After measuring the negative control response, quickly rinse the electrodes as demonstrated and measure the first experimental well. After all of the measurements have been recorded rinse chopstick electrodes again, gently wipe the electrodes and let them air dry in the safety hood.To assess the efflux transporter activity of the cells, on day eight of culturing treat the appropriate number of wells with a 10 micromolar efflux transporter inhibitor solution and place in an incubator for one hour at 37 degrees Celsius. At the end of the incubation, treat the cells with a 10 micromolar efflux transporter substrate solution for one hour in the incubator. At the end of the incubation, wash each well twice with 500 microliters of DPBS per well before lysing the cells with 500 microliters DPBS supplemented with 5%Triton-X per well.After five minutes, use a microplate reader to measure the fluorescence of the lysed cells at a 485 nanometer excitation and a 530 nanometer emission. For wells not used in the transporter assay, wash the cells twice with DPBS before fixing with 4%paraformaldehyde per cell nuclei quantification. After one day of culture in E6 medium, the cell morphology is similar to that of iPSCs.By day four, the cells are visibly distinct from iPSCs and cover most of the well. Two days of culture in human endothelial serum-free medium induces an elongated and cobblestone appearance in the cells. At day eight, individual cells form a mostly large cobblestone pattern.A sprouting assay can be performed to demonstrate the angiogenic potential of iPSC-derived BMECs. This leads to the development of tube-like structures after three days of growth factor treatment. The iPSC-derived BMECs generated using this protocol co-expressed tight junction proteins expressed in endothelial cells in the brain, lung, liver, and kidney, as well as vascular endothelial markers expressed at the blood brain barrier.As observed, 48 hours after subculture and medium change iPSC-derived BMECs demonstrate TEER values within the range described for iPSC-derived BMECs that were co-cultured with rat primary astrocytes. Efflux transporter activity was also observed at this stage. After cryopreservation, TEER measurements are lower compared to freshly derived BMECs.Western blot analysis reveals reduced levels of tight junction markers, such as occludin, while immunocytochemistry shows freckled and or frayed patterns of tight junction proteins. A too high or too low iPSC density will affect the differentiation efficiency and impact the development of appropriate blood brain barrier properties. This method enables the investigation of barrier properties cellular signaling and transporter activity using individualized BMECs to model the physiology and pathophysiology of the blood-brain barrier function.

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6.1K Views.  Massachusetts General Hospital. BMECs generated from iPSC provide new experimental models for studying blood brain barrier function and dysfunction. We have used this protocol to examine whether BMECs retain their barrier properties after expansion and cryopreservation. This technique enables the generation of disease-specific BMECs from patients with disorders in which blood-brain barrier disruption plays a role in the pathophysiology of disease.To induce BMEC differentiation from human iPSCs, wash the confluent iPS...