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. Coat each well of a 6-well plate with 1 mL of matrix solution. Incubate plates at 37 &#176;C for at least 1 h before use.</li>
	<li>Allow human periventricular endothelial cells to reach a confluency of 80%-90%. Aspirate medium from the well. Wash the wells once with 1 mL of sterile 1x PBS per well.</li>
	<li>Detach cells by adding 1 mL of cell dissociation solution (see Table of Materials) per well. Incubate at 37 &#176;C for 5 min. After 5 min, add 1 mL of periventricular endothelial cell medium. Transfer the cell solution into a 15 mL conical tube. 
	NOTE: We use Accutase for cell dissociation here, as opposed to TrypLE in sections 3 and 4.</li>
	<li>Centrifuge cells at 500 x g for 5 min at room temperature, aspirate the supernatant and resuspend the cell pellet in 1 mL of periventricular endothelial cell medium.</li>
	<li>Count live cells using the trypan blue exclusion method. Seed cells in fresh matrix-coated plates at a density of 1.2 x 105 cells/cm2. Incubate at 37 &#176;C and 5% CO2.</li>
	<li>Store human periventricular endothelial cells by cryopreserving in freezing medium (90% periventricular endothelial cell medium and 10% DMSO).
	<ol>
		<li>Dissociate and collect cells following steps 1.3 and 1.4 above. Count cells in the solution by the trypan blue exclusion method.</li>
		<li>Centrifuge cells at 500 x g for 5 min at room temperature. Aspirate the supernatant and resuspend the cell pellet at 5 x 106 cells/ mL of freezing medium.</li>
		<li>Dispense 1 mL of freezing medium plus cells per cryovial. Place the vials in isopropanol-filled chamber and cool overnight in -80 &#176;C at 1 &#176;C/min. Transfer vials to a liquid nitrogen tank on the next day for long term storage.</li>
	</ol>
	</li>
</ol>
2. Preparation of Human Periventricular Endothelial Cells for Assay
<ol>
	<li>Allow human periventricular endothelial cells to reach 70%-80% confluency.</li>
	<li>Dissociate cells following steps 1.3 to 1.5 as described above. Count cells using the trypan blue exclusion method.</li>
</ol>
3. Preparation of Human GABAergic Interneurons for Assay
NOTE: Human induced pluripotent stem cell (iPSC)-derived GABAergic interneurons and the neuronal medium were commercially purchased (see Table of Materials). The neurons are generated by differentiating a human fibroblast-derived iPSC line following a protocol developed by the manufacturer. The cells were thawed and cultured according to manufacturer&#39;s protocol.
<ol>
	<li>Thaw human GABAergic interneurons and culture them in 12-well plate for two weeks to a confluency of 70%-80%.</li>
	<li>On the day of the assay, warm cell dissociation solution (see Table of Materials) and an aliquot of neuronal medium at 37 &#176;C for 10 min before use.</li>
	<li>Aspirate medium from each well containing the cells. Wash cells with 1 mL of sterile 1x PBS per well.</li>
	<li>Detach cells by adding 0.5 mL of pre-warmed dissociation solution per well and incubate at 37 &#176;C for 5 min. Add 1 mL of neuronal medium per well. Transfer cell solution into a 15 mL conical tube. Gently triturate to dissociate cell clumps.</li>
	<li>Centrifuge cells at 380 x g for 5 min at room temperature, aspirate the supernatant and resuspend the cell pellet in 1 mL of neuronal medium. Count live cells using the trypan blue exclusion method.</li>
</ol>
4. Preparation of Control Human Endothelial Cells for Assay
NOTE: Control human iPSC-derived endothelial cells and endothelial cell medium were commercially purchased (Table of Materials). These endothelial cells are generated by differentiating a human fibroblast-derived iPSC line to endothelial fate following a protocol developed by the manufacturer. The cells were thawed and cultured on Fibronectin substrate according to manufacturer&#39;s protocol. Fibronectin-coated plates were prepared following manufacturer&#39;s protocol.
<ol>
	<li>Thaw control human endothelial cells and culture them in 6-well plate to a confluency of 80%-90%.</li>
	<li>On the day of the assay, warm cell dissociation solution (see Table of Materials) and an aliquot of endothelial medium at 37 &#176;C for 10 min before use.</li>
	<li>Aspirate the medium from each well containing the cells. Wash cells with 1 mL of sterile 1x PBS per well.</li>
	<li>Detach cells by adding 0.5 mL of pre-warmed dissociation solution per well. Incubate at room temperature for 5 min. Add 1 mL of endothelial cell medium per well to neutralize the dissociation solution. Transfer cell solution into a 15 mL conical tube.</li>
	<li>Centrifuge cells at 200 x g for 5 min at room temperature. Aspirate supernatant and resuspend the cell pellet in 1 mL of endothelial cell medium. Count live cells using the trypan blue exclusion method.</li>
</ol>
5. Preparation of One-well Culture Inserts
<ol>
	<li>Thaw 1 mg/mL laminin solution at room temperature or overnight at 4 &#176;C.</li>
	<li>Coat an appropriate number of 35 mm dishes with 0.01% poly-L-ornithine solution (1 mL per dish). Incubate the dishes in room temperature for at least 1 h.</li>
	<li>Dilute 1 mg/mL laminin solution 1:300 in sterile water to a final concentration of 3.3 &#181;g/mL immediately before use.</li>
	<li>Completely aspirate poly-L-ornithine from each dish. Rinse each dish thoroughly 3x with sterile water and aspirate completely to avoid poly-L-ornithine-induced cell toxicity.</li>
	<li>Add 1 mL of 3.3 &#181;g/mL laminin solution to each dish and incubate at 37 &#176;C overnight or at least 1 h. Remove laminin solution from the dish immediately before use. 
	NOTE: Alternatively, store the laminin-containing dishes in 4 &#176;C. Equilibrate the dishes in a 37 &#176;C cell culture incubator before use.</li>
	<li>Cut three sides of one well of a two-well silicone culture insert (Figure 1A) using a sterile blade to generate a one-well insert (Figure 1B). 
	NOTE: Keep the two-well insert firmly attached to the surface of the original packaging while cutting to ensure a smooth cut and protect the adhesiveness of the insert.</li>
	<li>Aspirate laminin solution from the dishes. 
	NOTE: Do not wash the dishes with sterile PBS or water after laminin incubation. Wet surfaces will prevent tight adhesion of the culture insert.</li>
	<li>Remove one-well insert with sterile tweezers and place it in the center of the poly-L-ornithine/laminin coated dish. Press along the edges of the insert to fix it to the surface of the dish.</li>
	<li>Carefully turn the dish upside down to verify that the insert is firmly adhered.</li>
	<li>Keep the dish upside down and mark the boundary of the insert compartment using a permanent black marker with an ultra-fine tip (Figure 1C).</li>
</ol>
6. Long-distance Migration Assay
<ol>
	<li>Suspend human GABAergic interneurons at a concentration of 3 x 104 cells/70 &#181;L of neuronal medium. Seed 70 &#181;L of cell solution inside each one-well culture insert. 
	NOTE: The seeding density of interneurons is as per manufacturer&#39;s recommendation.</li>
	<li>Suspend human periventricular endothelial cells at a concentration of 3 x 104 cells/70 &#181;L of periventricular endothelial cell medium. Seed 70 &#181;L of cell solution inside each one-well culture insert. 
	NOTE: The number of human periventricular endothelial cells seeded at a 1:1 ratio with the number of seeded neurons.</li>
	<li>Add 1 mL of neuronal medium in neuron dish to fill the area around the insert and prevent the coating from drying. Similarly, add 1 mL of periventricular endothelial cell medium in periventricular endothelial cell dish. 
	NOTE: Add medium slowly along the edge of the dish so that the insert is not disturbed.</li>
	<li>Check under a microscope to verify that cells are not leaking from the insert compartment.</li>
	<li>Incubate cells for 24 h at 37 &#176;C and 5% CO2. After 24 h incubation, check under microscope to verify that cells have attached properly and there is no overnight leak.</li>
	<li>After 48 h of seeding, gently remove the insert using a sterile tweezer. Check under the microscope to verify that the cell layer remains undisturbed (day 0).</li>
	<li>Remove medium from the neuron dish and add 1 mL of fresh neuronal medium. Similarly, remove medium from the periventricular endothelial cell dish and add 1 mL of fresh periventricular endothelial cell medium. 
	NOTE: Set aside a required number of dishes and fix with 4% PFA for day 0 images.</li>
	<li>Incubate cells for 5 days at 37 &#176;C and 5% CO2. After 5 days, remove medium, fix cells with 4% PFA for 10 min, and wash 3x with 1x PBS.</li>
	<li>Stain neurons with anti-human &#946;-Tubulin or anti-human MAP2 antibody, and endothelial cells with anti-human CD31 antibody. At the end of immunostaining, add 1 mL of antifade mounting medium to each dish.</li>
</ol>
7. Co-culture Migration Assay
<ol>
	<li>Co-suspend 3 x 104 GABAergic interneurons and 3 x 104 human periventricular endothelial cells in 70 &#181;L of co-culture medium (50% periventricular maintenance medium without GABA and 50% neuronal medium). Seed this cell solution inside the one-well insert compartment. Prepare an appropriate number of such assay dishes. 
	NOTE: GABA was not added in the co-culture medium to exclude the effect of exogenous GABA on migration.</li>
	<li>Check under a microscope to verify that cells are not leaking from the insert compartment.</li>
	<li>Slowly add 1 mL of co-culture medium along the side of the dish to prevent the coating from drying. 
	NOTE: Add medium slowly along the edge of the dish so that the insert is not disturbed.</li>
	<li>As a first control, seed 3 x 104 human GABAergic interneurons only in 70 &#181;L of co-culture medium per one-well insert. Prepare an appropriate number of such dishes.</li>
	<li>As a second control, co-seed 3 x 104 GABAergic human interneurons with 3 x 104 control human endothelial cells in 70 &#181;L of co-culture medium per one-well insert. Prepare an appropriate number of dishes.</li>
	<li>Incubate the dishes for 24 h at 37 &#176;C and 5% CO2. After 24 h incubation, check under a microscope to verify that cells have attached properly and there is no leak.</li>
	<li>After 48 h of seeding, gently remove the insert using a sterile tweezer. Check under the microscope to verify that the cell layer is not disturbed (day 0).</li>
	<li>Remove medium and add 1 mL of fresh co-culture medium. 
	NOTE: Set aside an appropriate number of dishes for acquiring day 0 images.</li>
	<li>Incubate cells for 5 days at 37 &#176;C and 5% CO2.</li>
	<li>After 5 days, remove medium, fix cells with 4% PFA for 10 min, and wash 3x with 1x PBS.</li>
	<li>Stain with anti-human &#946;-Tubulin or anti-human MAP2 antibody to label the neurons. At the end of immunostaining, add 1 mL of antifade mounting medium to each dish.</li>
</ol>
8. Chemo-attraction Assay
<ol>
	<li>Place a three-well culture insert in the center of a poly-L-ornithine/laminin coated 35 mm dish using sterile tweezers.</li>
	<li>Turn the dish upside down. Mark the boundary around the middle compartment of the insert using a permanent black marker with ultra-fine tip.</li>
	<li>Seed 3 x 104 human GABAergic interneurons in the middle compartment in 70 &#181;L of neuronal medium (Figure 3A).</li>
	<li>Seed 104 human periventricular endothelial cells in 70 &#181;L of periventricular endothelial cell medium and 104 control endothelial cells in 70 &#181;L of control endothelial cell medium in the two outer compartments respectively (Figure 3A).</li>
	<li>Add 1 mL of co-culture medium (50% periventricular maintenance medium without GABA and 50% neuronal medium) along the side of the dish to prevent the coating on the dish from drying.</li>
	<li>Check under microscope to verify that cells are not leaking from the insert compartment.</li>
	<li>Incubate cells for 24 h at 37 &#176;C and 5% CO2. After 24 h incubation, check under a microscope to verify that cells have attached properly and there is no leak.</li>
	<li>After 48 h of seeding, gently remove the insert using a sterile tweezer. Check under the microscope to verify that the cell layer is not disturbed (day 0).</li>
	<li>Remove medium and add 1 mL of fresh co-culture medium. 
	NOTE: Set aside required number of dishes for day 0 images.</li>
	<li>Incubate cells for 36 h at 37 &#176;C and 5% CO2. After 36 h, aspirate medium, fix cells with 4% PFA for 10 min, and wash 3x with 1x PBS.</li>
	<li>Stain human GABAergic interneurons with anti-human &#946;-Tubulin or anti-human MAP2 antibody. At the end of the staining procedure, add 1 mL of antifade mounting medium in each dish.</li>
</ol>
9. Imaging and Data Analysis
<ol>
	<li>Place the immuno-stained assay dish under a microscope at 4X magnification.</li>
	<li>Keep one long-edge of the rectangular boundary (made in step 5.10 above) in the field of view. Take images of cells in the cell-free space adjacent to that boundary. Acquire images along the right long-edge and the left long-edge of the rectangular boundary (Figure 2B). 
	NOTE: Cells positioned diagonally with respect to the rectangle are not considered due to ambiguity in selecting the short- or long-edge as starting mark. Also, numbers of cells migrating across the short-edge are often significantly fewer (possibly due to lesser number of starting cells along the short-edge) and are not considered.</li>
	<li>Open the images in ImageJ. Calculate distance between each cell and the boundary mark (Figure 2D) using ImageJ.</li>
	<li>To assess migration in terms of cell numbers, set a specific distance from the boundary in the acquired image in ImageJ. Count the number of cells that are present within this distance. Calculate average number, standard deviation, and statistical significance using appropriate software.</li>
</ol>

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Authors declare no conflict of interest.

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 ...

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

Research

JoVE Journal

Neuroscience

6.9K Views.  Harvard Medical School. 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.

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

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA Receptors

Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA (&#947;-aminobutyric acid) is the predominant inhibitory neurotransmitter in the brain. It is released from the presynaptic terminals of inhibitory neurons within highly specialized intercellular junctions known as synapses, where it binds to GABAA receptors (GABAARs) present at the plasma membrane of the synapse-receiving, postsynaptic neurons. Activation of these GABA-gated ion channels leads to influx of chloride resulting in postsynaptic potential changes that decrease the probability that these neurons will generate action potentials. 
During development, diverse types of inhibitory neurons with distinct morphological, electrophysiological and neurochemical characteristics have the ability to recognize their target neurons and form synapses which incorporate specific GABAARs subtypes. This principle of selective innervation of neuronal targets raises the question as to how the appropriate synaptic partners identify each other. 
To elucidate the underlying molecular mechanisms, a novel in vitro co-culture model system was established, in which medium spiny GABAergic neurons, a highly homogenous population of neurons isolated from the embryonic striatum, were cultured with stably transfected HEK293 cell lines that express different GABAAR subtypes. Synapses form rapidly, efficiently and selectively in this system, and are easily accessible for quantification. Our results indicate that various GABAAR subtypes differ in their ability to promote synapse formation, suggesting that this reduced in vitro model system can be used to reproduce, at least in part, the in vivo conditions required for the recognition of the appropriate synaptic partners and formation of specific synapses. Here the protocols for culturing the medium spiny neurons and generating HEK293 cells lines expressing GABAARs are first described, followed by detailed instructions on how to combine these two cell types in co-culture and analyze the formation of synaptic contacts.

Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA Receptors

The molecular mechanisms that co-ordinate the formation of inhibitory GABAergic synapses during ontogeny are largely unknown. To study these processes,we have developed a co-culture model system which incorporates embryonic medium spiny GABAergic neurons cultured together with stably transfected human embryonic kidney 293 (HEK293) cells expressing functional GABAA receptors.

Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA ...

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair.

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) are structures containing the sensory neurons of the peripheral nervous system. When dissociated, they can be co-cultured with SC-like adipose-derived stem cells (ASC), providing a valuable model to study in vitro nerve regeneration and myelination, mimicking the in vivo environment at the injury site.

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the ...

Enumeration of Neural Stem Cells Using Clonal Assays

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. NSCs and neural progenitors (NPs) are commonly cultured in vitro as neurospheres. This protocol describes in detail how to determine the NSC frequency in a given cell population under clonal conditions. The protocol begins with the seeding of the cells at a density that allows for the generation of clonal neurospheres. The neurospheres are then transferred to chambered coverslips and differentiated under clonal conditions in conditioned medium, which maximizes the differentiation potential of the neurospheres. Finally, the NSC frequency is calculated based on neurosphere formation and multipotency capabilities. Utilities of this protocol include the evaluation of candidate NSC markers, purification of NSCs, and the ability to distinguish NSCs from NPs. This method takes 13 days to perform, which is much shorter than current methods to enumerate NSC frequency.

Neural stem cells (NSCs) refer to cells&#160;which can self-renew and differentiate into the three neural lineages. Here, we describe a protocol to determine NSC frequency in a given cell population using neurosphere formation and differentiation under clonal conditions.

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. ...

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions in newborn infants. While mosquitos are the main route of viral transmission, it has also been shown to spread via sexual contact and vertical mother-to-fetus transmission. In this latter case of transmission, due to the unique viral tropism of ZIKV, the virus is believed to predominantly target the neural progenitor cells (NPCs) of the developing brain.
Here a method for modeling ZIKV infection, and the resulting microcephaly, that occur when human cerebral organoids are exposed to live ZIKV is described. The organoids display high levels of virus within their neural progenitor population, and exhibit severe cell death and microcephaly over time. This three-dimensional cerebral organoid model allows researchers to conduct species-matched experiments to observe and potentially intervene with ZIKV infection of the developing human brain. The model provides improved relevance over standard two-dimensional methods, and contains human-specific cellular architecture and protein expression that are not possible in animal models.

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

This protocol describes a technique used to model Zika virus infection of the developing human brain. Using wildtype or engineered stem cell lines, researchers may use this technique to uncover the various mechanisms or treatments that may affect early brain infection and resulting microcephaly in Zika virus-infected embryos.

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions ...

Compartmentalization of Human Stem Cell-Derived Neurons within Pre-Assembled Plastic Microfluidic Chips

Use of microfluidic devices to compartmentalize cultured neurons has become a standard method in neuroscience. This protocol shows how to use a pre-assembled multi-compartment chip made in a cyclic olefin copolymer (COC) to compartmentalize neurons differentiated from human stem cells. The footprint of these COC chips are the same as a standard microscope slide and are equally compatible with high resolution microscopy. Neurons are differentiated from human neural stem cells (NSCs) into glutamatergic neurons within the chip and maintained for 5 weeks, allowing sufficient time for these neurons to develop synapses and dendritic spines. Further, we demonstrate multiple common experimental procedures using these multi-compartment chips, including viral labeling, establishing microenvironments, axotomy, and immunocytochemistry.

This protocol demonstrates the use of compartmentalized microfluidic chips, injection molded in a cyclic olefin copolymer to cultured neurons differentiated from human stem cells. These chips are preassembled and easier-to-use than traditional compartmentalized poly(dimethylsiloxane) devices. Multiple common experimental paradigms are described here, including viral labeling, fluidic isolation, axotomy, and immunostaining.

Use of microfluidic devices to compartmentalize cultured neurons has become a standard method in neuroscience. This protocol shows how to use a ...

Post-differentiation Replating of Human Pluripotent Stem Cell-derived Neurons for High-content Screening of Neuritogenesis and Synapse Maturation

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model system to explore disease mechanisms and carry out high content screening (HCS) to interrogate compound libraries or identify gene mutation phenotypes. However, with human cells the transition from NPC to functional, mature neuron requires several weeks. Synapses typically start to form after 3 weeks of differentiation in monolayer culture, and several neuron-specific proteins, for example the later expressing pan-neuronal marker NeuN, or the layer 5/6 cerebral cortical neuron marker CTIP2, begin to express around 4-5 weeks post-differentiation. This lengthy differentiation time can be incompatible with optimal culture conditions used for small volume, multi-well HCS platforms. Among the many challenges are the need for well-adhered, uniformly distributed cells with minimal cell clustering, and culture procedures that foster long-term viability and functional synapse maturation. One approach is to differentiate neurons in a large volume format, then replate them at a later time point in HCS-compatible multi-wells. Some main challenges when using this replating approach concern reproducibility and cell viability, due to the stressful disruption of the dendritic and axonal network. Here we demonstrate a detailed and reliable procedure for enzymatically resuspending human induced pluripotent stem cell (hiPSC)-derived neurons after their differentiation for 4-8 weeks in a large-volume format, transferring them to 384-well microtiter plates, and culturing them for a further 1-3 weeks with excellent cell survival. This replating of human neurons not only allows the study of synapse assembly and maturation within two weeks from replating, but also enables studies of neurite regeneration and growth cone characteristics. We provide examples of scalable assays for neuritogenesis and synaptogenesis using a 384-well platform.

This protocol describes a detailed procedure for resuspending and culturing human stem cell derived neurons that were previously differentiated from neural progenitors in vitro for multiple weeks. The procedure facilitates imaging-based assays of neurites, synapses, and late-expressing neuronal markers in a format compatible with light microscopy and high-content screening.

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model ...

Analyzing Mitochondrial Transport and Morphology in Human Induced Pluripotent Stem Cell-Derived Neurons in Hereditary Spastic Paraplegia

Neurons have intense demands for high energy in order to support their functions. Impaired mitochondrial transport along axons has been observed in human neurons, which may contribute to neurodegeneration in various disease states. Although it is challenging to examine mitochondrial dynamics in live human nerves, such paradigms are critical for studying the role of mitochondria in neurodegeneration. Described here is a protocol for analyzing mitochondrial transport and mitochondrial morphology in forebrain neuron axons derived from human induced pluripotent stem cells (iPSCs). The iPSCs are differentiated into telencephalic glutamatergic neurons using well-established methods. Mitochondria of the neurons are stained with MitoTracker CMXRos, and mitochondrial movement within the axons are captured using a live-cell imaging microscope equipped with an incubator for cell culture. Time-lapse images are analyzed using software with &#34;MultiKymograph&#34;, &#34;Bioformat importer&#34;, and &#34;Macros&#34; plugins. Kymographs of mitochondrial transport are generated, and average mitochondrial velocity in the anterograde and retrograde directions is read from the kymograph. Regarding mitochondrial morphology analysis, mitochondrial length, area, and aspect ratio are obtained using the ImageJ. In summary, this protocol allows characterization of mitochondrial trafficking along axons and analysis of their morphology to facilitate studies of neurodegenerative diseases.

Impaired mitochondrial transport and morphology are involved in various neurodegenerative diseases. The presented protocol uses induced pluripotent stem cell-derived forebrain neurons to assess mitochondrial transport and morphology in hereditary spastic paraplegia. This protocol allows characterization of mitochondrial trafficking along axons and analysis of their morphology, which will facilitate the study of neurodegenerative disease.

Neurons have intense demands for high energy in order to support their functions. Impaired mitochondrial transport along axons has been observed in human ...

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.

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 ...

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 ...