This work was supported by awards from the National Institute of Mental Health (R01MH110438) and National Institute of Neurological Disorders and Stroke (R01NS100808) to AV.

1. Culture and Storage of Human Periventricular Endothelial Cells
<ol>
	<li>Maintain human periventricular endothelial cells on basement membrane matrix-coated (see Table of Materials) 6-well plates in periventricular endothelial cell medium (E6 medium containing 50 ng/mL VEGF-A, 100 ng/mL FGF2 and 5 &#181;M GABA) at 37 &#176;C and 5% CO2. Change medium every alternate day.</li>
	<li>Thaw basement membrane matrix in 4 &#176;C, and make a 1:100 solution by diluting it in cold DMEM/F12 medium....

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABAergic+mechanisms+in+epilepsy.">GABAergic mechanisms in epilepsy.</a> Epilepsia. 42 (3), 8-12 (2001).</li><li>Datta, D., Subburaju, S., Kaye, S., Vasudevan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+forebrain+endothelial+cells+for+cell-based+therapy+of+neuropsychiatric+disorders.">Human forebrain endothelial cells for cell-based therapy of neuropsychiatric disorders.</a> Proceedings of 22nd Biennial Meeting of the International Society for Developmental Neuroscience. , (2018).</li><li>Bellin, M., Marchetto, M. C., Gage, F. H., Mummery, C. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+induced+pluripotent+stem+cells+for+modelling+neurodevelopmental+disorders.">Human induced pluripotent stem cells for modelling neurodevelopmental disorders.</a> Nature Reviews Neurology. 13 (5), 265-278 (2017).</li><li>Stubbs, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurovascular+congruence+during+cerebral+cortical+development.">Neurovascular congruence during cerebral cortical development.</a> Cerebral Cortex. 19 (1), 32-41 (2009).</li><li>Vissapragada, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bidirectional+crosstalk+between+periventricular+endothelial+cells+and+neural+progenitor+cells+promotes+the+formation+of+a+neurovascular+unit.">Bidirectional crosstalk between periventricular endothelial cells and neural progenitor cells promotes the formation of a neurovascular unit.</a> Brain Research. 1565, 8-17 (2014).</li><li>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Biology.+The+Transwell+Migration+Assay.">Cell Biology. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotaxis+under+agarose:+a+new+and+simple+method+for+measuring+chemotaxis+and+spontaneous+migration+of+human+polymorphonuclear+leukocytes+and+monocytes.">Chemotaxis under agarose: a new and simple method for measuring chemotaxis and spontaneous migration of human polymorphonuclear leukocytes and monocytes.</a> The Journal of Immunology. 115 (6), 1650-1656 (1975).</li><li>Zigmond, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ability+of+polymorphonuclear+leukocytes+to+orient+in+gradients+of+chemotactic+factors.">Ability of polymorphonuclear leukocytes to orient in gradients of chemotactic factors.</a> Journal of Cell Biology. 75 (2), 606-616 (1977).</li><li>Zicha, D., Dunn, G., Jones, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+chemotaxis+using+the+Dunn+direct-viewing+chamber.">Analyzing chemotaxis using the Dunn direct-viewing chamber.</a> Methods in Molecular Biology. 75, 449-457 (1997).</li><li>Kim, B. 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Here, we described three in vitro assays that together provide quantitative assessment of human periventricular endothelial cell-specific properties. These assays will be valuable in gaining mechanistic insights into the interaction of human periventricular endothelial cells with human interneurons. Experiments using ligands, inhibitors, or cells with gene-specific knockdown or overexpression will identify or validate molecular players that mediate endothelial cell-guided interneuron migration or long-distance migratory ...

Endothelial cells form the lining of blood vessels and mediate important functions that include maintenance of vessel wall permeability, regulation of blood flow, platelet aggregation, and formation of new blood vessels. In the brain, endothelial cells form part of a critical blood-brain-barrier that tightly controls exchange of materials between the brain and the bloodstream1. Our studies in the past decade have identified novel neurogenic roles of brain endothelial cells that have significant implications for brain development and behavior2,3,4,5. We have shown that the mouse embryonic forebrain is vascularized by two distinct subtypes of vessels, the pial vessels and the periventricular vessels, that differ in anatomy, origin, and developmental profile2. Endothelial cells lining these two vessel subtypes show distinct differences in their gene expression profiles. While pial endothelial cells mostly express genes related to inflammation and immune response, periventricular endothelial cells are uniquely enriched in expression of genes commonly associated with neurogenesis, neuronal migration, chemotaxis, and axon guidance3. Periventricular endothelial cells also house a novel GABA signaling pathway that is distinct from the traditional neuronal GABA signaling pathway5. Concomitant with its gene expression, periventricular endothelial cells were found to regulate migration and distribution of GABAergic interneurons in the developing neocortex. During embryonic development, periventricular endothelial cells undergo long-distance migration along a ventral-dorsal gradient to establish the periventricular vascular network2,3. This migratory route is mirrored a day later by interneurons. Migrating interneurons physically interact with the pre-formed periventricular vascular network and use it as a guiderail to reach their final destination in the neocortex. In addition to acting as a physical substrate, periventricular endothelial cells serve as the source of navigational cues for migrating neurons. Periventricular endothelial cell-secreted GABA guides interneuron migration and regulates their final distribution patterns4. Defects in interneuron migration and distribution are associated with neuropsychiatric disorders such as autism, epilepsy, schizophrenia and depression6,7,8,9,10. Therefore, study of periventricular endothelial cell functions and their influence on interneuron migration in human context becomes critical for addressing the pathogenesis of these disorders.
We have generated human periventricular-like endothelial cells from human embryonic stem cells in our laboratory11, using induced pluripotent stem cell (iPSC) technology12,13. To validate whether human periventricular endothelial cells faithfully mimic mouse periventricular endothelial cells, and to quantitatively assess their influence on interneuron migration, we developed three in vitro assays: a long-distance migration assay, a co-culture migration assay, and a chemo-attraction assay. Here we describe protocols for these assays in detail. All three assays are based on the usage of silicone culture inserts to create a small rectangular patch of cells (of fixed dimensions) surrounded by cell-free space. Migration distance is evaluated by measuring the distance between the final positions of cells from the border of the rectangular patch that has been outlined on day 0. In the long-distance migration assay, human periventricular endothelial cells are seeded as a patch in the center of a 35 mm dish, and the distances traveled by the cells over a long range of time are calculated. In the co-culture migration assay, human periventricular endothelial cells are co-seeded with human interneurons as one patch in a 35 mm dish. This setup allows examination of the effect of direct physical interactions of these two cell types on the rate of migration of interneurons. The chemo-attraction assay measures the migration of interneurons in response to chemo-attractive cues secreted by human periventricular endothelial cells. Interneurons are seeded as a rectangular patch, with human periventricular endothelial cells and control non-periventricular endothelial cells seeded as similar sized patches on either side. Each of the cell patches are separated by a cell-free gap of 500 &#181;m. Response of interneurons is assessed by quantifying the number of cells that have migrated towards periventricular endothelial cells compared to control non-periventricular endothelial cells.
These assays provide robust assessment of human periventricular endothelial cell functions and their influence on interneuron migration. The novel setup of long-distance assay and co-culture migration assay provides cell-free space in the range of centimeters (~1-1.5 cm) to allow detection of long-distance migration. A summary of the features of our assays compared to other popular assays is presented in Table 1. Collectively, the assays described here will serve as a platform for assessing &#34;diseased&#34; periventricular endothelial cells and interneurons generated from iPSCs of brain disorders like schizophrenia, autism or epilepsy. These assays can also be used to determine how different conditions (e.g. inhibitors, ligands, RNAi) affect cell migration. Finally, these assays can be optimized for other cell types to measure long-distance migration, chemo-attraction or cell-cell mediated migration.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Accutase dissociation solution</td>
			<td>Millipore Sigma</td>
			<td>SCR005</td>
			<td>Cell dissociation solution (for periventricular endothelial cells, step 1.4)</td>
		</tr>
		<tr>
			<td>Anti-human &#946;-Tubulin antibody</td>
			<td>Biolegend</td>
			<td>802001</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-human CD31 antibody</td>
			<td>Millipore Sigma</td>
			<td>CBL468</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti- MAP2 antibody</td>
			<td>Neuromics</td>
			<td>CH22103</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-active Caspase 3 antibody</td>
			<td>Millipore Sigma</td>
			<td>AB3623</td>
			<td></td>
		</tr>
		<tr>
			<td>Control human endothelial cells</td>
			<td>Cellular Dynamics</td>
			<td>R1022</td>
			<td></td>
		</tr>
		<tr>
			<td>Control endothelial Cells Medium Supplement</td>
			<td>Cellular Dynamics</td>
			<td>M1019</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryogenic vials</td>
			<td>Fisher Scientific</td>
			<td>03-337-7Y</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEMF/12 medium</td>
			<td>Thermofisher Scientific</td>
			<td>11320033</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>E6 medium</td>
			<td>Thermofisher Scientific</td>
			<td>A1516401</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF2</td>
			<td>Thermofisher Scientific</td>
			<td>PHG0261</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Thermofisher Scientific</td>
			<td>33016-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Freezing Container</td>
			<td>Thermofisher Scientific</td>
			<td>5100</td>
			<td></td>
		</tr>
		<tr>
			<td>GABA</td>
			<td>Sigma-Aldrich</td>
			<td>A2129</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Sigma-Aldrich</td>
			<td>Z359629</td>
			<td></td>
		</tr>
		<tr>
			<td>Human GABAergic neurons</td>
			<td>Cellular Dynamics</td>
			<td>R1013</td>
			<td></td>
		</tr>
		<tr>
			<td>Human GABAergic neurons base medium</td>
			<td>Cellular Dynamics</td>
			<td>M1010</td>
			<td></td>
		</tr>
		<tr>
			<td>Human GABAergic neuron Neural supplement</td>
			<td>Cellular Dynamics</td>
			<td>M1032</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma</td>
			<td>L2020</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>356230</td>
			<td>Basement membrane matrix</td>
		</tr>
		<tr>
			<td>Mounting Medium</td>
			<td>Vector laboratories</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr>
			<td>poly-L-ornithin</td>
			<td>Sigma</td>
			<td>p4957</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Thermofisher Scientific</td>
			<td>14190</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Thermofisher Scientific</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE</td>
			<td>Thermofisher Scientific</td>
			<td>12563011</td>
			<td>Cell dissociation solution (for GABAergic interneurons and endothelial cells, sections 3 and 4)</td>
		</tr>
		<tr>
			<td>VEGF-A</td>
			<td>Peprotech</td>
			<td>100-20</td>
			<td></td>
		</tr>
		<tr>
			<td>VascuLife VEGF Medium Complete Kit</td>
			<td>Lifeline Cell Technologies</td>
			<td>LL-0003</td>
			<td>Component of control human endothelial cell medium</td>
		</tr>
		<tr>
			<td>2-well silicone culture-Insert</td>
			<td>ibidi</td>
			<td>80209</td>
			<td></td>
		</tr>
		<tr>
			<td>3-well silicone culture-Insert</td>
			<td>ibidi</td>
			<td>80369</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm dish</td>
			<td>Corning</td>
			<td>430165</td>
			<td></td>
		</tr>
		<tr>
			<td>15-ml conical tube</td>
			<td>Fisher Scientific</td>
			<td>07-200-886</td>
			<td></td>
		</tr>
		<tr>
			<td>4% PFA solution</td>
			<td>Fisher Scientific</td>
			<td>AAJ19943K2</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culture plate</td>
			<td>Fisher Scientific</td>
			<td>14-832-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted phase contrast microscope</td>
			<td>Zeiss</td>
			<td>Zeiss Axiovert 40C</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent microscope</td>
			<td>Olympus</td>
			<td>FSX-100</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The steps to set up a one-well culture insert inside a 35 mm dish are shown in Figure 1. Long-distance migration assay and co-culture migration assay used a one-well insert to seed the desired number of cells in the center of a poly-L-ornithine/laminin coated 35 mm dish. On day 0, cells were present as a rectangular patch (Figure 2A,C). In day 0 images, the day 0 line could be easily identified by the sharp edge ...

Watch this Scientific Journal Video about Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons at JoVE.com

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

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 ...

This protocol describes three in vitro assays that collectively assess the functions of human periventricular endothelial cells and their interaction with human GABAergic interneurons. Results from these assays will provide critical insight into the link between periventricular endothelial cells and brain disorders. These assays are simple, low-cost and allow measurement of cell migration in the range of centimeters, which is not achieved by other existing assays.Defects in migration and distribution of GABAergic interneurons are associated with psychiatric disorders, such as autism, epilepsy, schizophrenia and depression. Therefore, it is critical to study the interaction of periventricular endothelial cells with GABAergic interneurons in human context to address the pathogenesis of these disorders. Begin by preparing human cells for the assay.Allow human periventricular endothelial cells to reach 70 to 80%confluency then dissociate them according to the manuscript's directions and count them using the trypan blue exclusion method. To prepare human GABAergic interneurons, warm cell dissociation solution and an aliquot of neuronal medium at 37 degrees celsius for 10 minutes. Then aspirate the medium from each well containing cells and wash them with one millimeter of PBS per well.Detach the cells by adding 0.5 milliliters of prewarmed dissociation solution per well and incubating them at 37 degrees celsius for five minutes. After the incubation, add one milliliter of neuronal medium per well and transfer the cell solution into a 15 milliliter conical tube, gently triturating to dissociate cell clumps. Centrifuge the cells at 380 times G for five minutes at room temperature, aspirate the supernatant and resuspend the cell pellet in one milliliter of neuronal medium.Then count the live cells using trypan blue exclusion. Prepare control human endothelial cells by warming the cell dissociation solution and an aliquot of endothelial medium at 37 degrees celsius, 10 minutes prior to use. Aspirate medium from each well and wash them with one milliliter of PBS per well.Detach the cells by adding 0.5 milliliters of the prewarmed dissociation solution to each well and incubating the plate at room temperature for five minutes. Then, add one milliliter of endothelial cell medium per well to neutralize the dissociation solution, and transfer the cells to a 15 milliliter conical tube. Centrifuge the cells at 200 times G for five minutes, aspirate the supernatant and resuspend the cells in one milliliter of endothelial cell medium.Then use the trypan exclusion method to count the cells. Prepare one well culture insert by cutting three sides of one well of a two well silicone culture insert with a sterile blade. Remove the insert with sterile tweezers and place it in the center of a poly-L-ornithine and laminin-coated dish, then press along the edge of the insert to fix it to the surface.Carefully turn the dish upside down to verify that the insert is firmly adhered, and mark the boundary of the insert compartment using a permanent black market with an ultra fine tip. Suspend human GABAergic interneurons in neuronal medium and human periventricular endothelial cells and endothelial cell medium at a concentration of 30, 000 per 70 microliters, then seed 70 microliters of the cell solution inside each one well culture insert. Add one milliliter of neuronal medium to the neuron dish to fill the area around the insert and prevent the coating from drying.Similarly, add one milliliter of endothelial cell medium to the periventricular endothelial cell dish. Check the cells under a microscope to verify that they are not leaking from the insert compartment, then incubate them at 37 degrees celsius and 5%carbon dioxide for 24 hours. After the incubation, check the cells again under the microscope to verify that they have properly attached.48 hours after seeding, gently remove the insert with sterile tweezers and check the cells under the microscope to verify that the cell layer remains undisturbed. Remove the medium from both the neuron and the periventricular endothelial cell dishes, and add one milliliter of fresh medium to each dish. Incubate the cells at 37 degrees celsius and 5%carbon dioxide for five days.Then, remove the medium, fix the cells with 4%PFA for 10 minutes and wash them three times with PBS. To perform the co-culture assay, suspend 30, 000 GABAergic interneurons and 30, 000 human periventricular endothelial cells in 70 microliters of co-culture medium, then seed this cell solution inside a one well insert compartment. After 48 hours, remove the insert.Incubate the co-culture at 37 degrees celsius and 5%carbon dioxide for five days. After the incubation, remove the medium, fix the cells with 4%PFA and wash them three times with PBS. To perform the chemo-attraction assay, place a three well culture insert in the center of a poly-L-ornithine and laminin-coated 35 millimeter dish, turn the dish upside down, and mark the boundary around the middle compartment of the insert using a permanent marker with an ultra fine tip.Seed 30, 000 GABAergic interneurons in 70 microliters of neuronal medium in the middle compartment, then seed 10, 000 periventricular endothelial cells and 10, 000 control endothelial cells and 70 microliters of their respective medium in the two outer compartments. Add one milliliter of co-culture medium along the side of the dish to prevent the coating on the dish from drying. After 48 hours, remove the insert and incubate the cells at 37 degrees celsius and 5%carbon dioxide for 36 hours.After the incubation, remove the medium, fix the cells and wash them three times with PBS. Long distance and co-culture migration assays were used to investigate the interactions between periventricular endothelial cells and GABAergic neurons. In the long distance migration assay, cells started out as a rectangular patch on day zero, but by 48 hours, they had migrated out into the cell-free space.Immunocytochemical staining with Anti-Active Caspase-3 antibody, a marker of apoptosis, showed no apoptotic signal in the seeded neurons. In the co-culture migration assay, when interneurons were co-seeded with human periventricular endothelial cells, neurons traveled farther distances compared to when interneurons were seeded alone or when co-seeded with control endothelial cells. In the chemo-attraction assay, a significantly higher number of interneurons migrated towards periventricular endothelial cells compared to control endothelial cells, confirming that GABAergic interneurons respond selectively to chemo-attractive cues secreted by periventricular endothelial cells.These assays can be modified using ligands, inhibitors or assay arenes to get mechanistic insights into interactions of human periventricular endothelial cells with human interneurons. They can also be used to study the interaction of human periventricular endothelial cells with other neural cells as reported by our group and others. When attempting this procedure, it is important to fix the insert firmly on the dish and keep it undisturbed throughout the assay, otherwise it may lead to cell leakage, cell detachment or misalignment of the insert with the drawn boundary.Our work has indicated cell autonomous role of human periventricular endothelial cells in pathogenesis of psychiatric disorders like schizophrenia, epilepsy and autism. These assays will therefore allow assessment of diseased periventricular endothelial cells derived from patients with these disorders using the IPSC technology.

migration-chemo-attraction-co-culture-assays-for-human-stem-cell

Research

JoVE Journal

Neuroscience

6.9K visualizações.  Harvard Medical School. Este protocolo descreve três ensaios in vitro que avaliam coletivamente as funções das células endoteliais periventriculares humanas e sua interação com interneurônios gabaérgicos humanos. Os resultados desses ensaios fornecerão uma visão crítica sobre a ligação entre células endoteliais periventriculares e distúrbios cerebrais. Estes ensaios são simples, de baixo custo e permitem a medição da migração celular na faixa de centímetros, o que não é alcançado por outros e...