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><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 &amp;beta;-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 ...

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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

7.0K Views.  Harvard Medical School. Dit protocol beschrijft drie in vitro tests die gezamenlijk de functies van menselijke periventriculaire endotheelcellen en hun interactie met menselijke GABAergic interneurons beoordelen. Resultaten van deze testen zullen kritisch inzicht geven in het verband tussen periventriculaire endotheelcellen en hersenaandoeningen. Deze tests zijn eenvoudig, goedkoop en maken het mogelijk om celmigratie in het bereik van centimeters te meten, wat niet wordt bereikt door andere bestaande tests.G...

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

Migratory Behavior of Cells Generated in Ganglionic Eminence Cultures

Migration of cells is a common process that leads to the development and maturation of the vertebrate central nervous system (Hatten, '99). The cerebral cortex consists of two basic neuronal types: excitatory and inhibitory. These cells arise in distinct areas and migrate into the cortex along different routes (Pearlman et al., '98). Inhibitory interneurons migrate tangentially from subcortical sources, mostly from different regions of the ganglionic eminences (Gelman et al., '09; Xu et al., '04). Their movement requires precise spatiotemporal control imposed by environmental cues, to allow for the establishment of proper cytoarchitecture and connectivity in the cerebral cortex (Caviness &amp; Rakic, '78; Hatten, '90; Rakic, '90). To study the migratory behavior of cells generated in proliferative zones of the ganglionic eminences (GE) in newborn ferrets in vitro we used a 3 dimensional culture arrangement in a BD Matrigel Matrix. The culture setup consisted of two GE explants and a source of tested proteins extracted from the cerebral cortex and adsorbed on fluorescent latex Retrobeads IX positioned between the explants (Hasling et al., '03; Riddle et al., '97). After 2-3 days of culture, the cells start to appear at the edge of the explant showing a propensity to leave the tissue in a radial direction. Live imaging allowed observation of migratory patterns without the necessity of labeling or marking the cells. When exposed to fractions of the protein extract obtained from isochronic ferret cortex, the GE cells displayed different behaviors as judged by quantitative kinetic analysis of individual moving cells.

Time lapse imaging of 3D tissue culture allows studying migratory behavior of individual cells originating from ganglionic eminence in reaction to fractionated protein extract from cerebral cortex.

Migration of cells is a common process that leads to the development and maturation of the vertebrate central nervous system (Hatten, '99). The cerebral ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

Cancer Research

Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is largely due to the difficulty with which one can create a cell-compatible and steady microenvironment using conventional microfabrication techniques and materials. We address this problem via the introduction of a novel microfabrication material, agarose gel, as the base material for the microfluidic device. Agarose gel is highly malleable, and permeable to gas and nutrients necessary for cell survival, and thus an ideal material for cell-based assays. We have shown previously that agarose gel based devices have been successful in studying bacterial and neutrophil cell migration2. In this report, three parallel microfluidic channels are patterned in an agarose gel membrane of about 1mm thickness. Constant flows with media/buffer are maintained in the two side channels using a peristaltic pump. Cells are maintained in the center channel for observation. Since the nutrients and chemicals in the side channels are constantly diffusing from the side to center channel, the chemical environment of the center channel is easily controlled via the flow along the side channels. Using this device, we demonstrate that the movement of neural stem cells can be monitored optically with ease under various chemical conditions, and the experimental results show that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells. Motility of neural stem cells is an important biomarker for assessing cells aggressiveness, thus tumorigenic factor3. Deciphering the mechanism underlying NSC motility will yield insight into both disorders of neural development and into brain cancer stem cell invasion.

We demonstrate that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells(NSCs) using a novel agarose gel based microfluidic device. This technology can be readily adaptable to other mammalian cell systems where cell sources are scarce, such as human neural stem cells, and the turn around time is critical.

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is ...

Biology

Real-Time Monitoring of Human Glioma Cell Migration on Dorsal Root Ganglion Axon-Oligodendrocyte Co-Cultures

Glioblastoma is one of the most aggressive human cancers due to extensive cellular heterogeneity and the migration properties of hGCs. In order to better understand the molecular mechanisms underlying glioma cell migration, an ability to study the interaction between hGCs and axons within the tumor microenvironment is essential. In order to model this cellular interaction, we developed a mixed culture system consisting of hGCs and dorsal root ganglia (DRG) axon-oligodendrocyte co-cultures. DRG cultures were selected because they can be isolated efficiently and can form the long, extensive projections which are ideal for migration studies of this nature. Purified rat oligodendrocytes were then added on purified rat DRG axons and induced to myelinate. After confirming the formation of compact myelin, hGCs were finally added to the co-culture and their interactions with DRG axons and oligodendrocytes was monitored in real-time using time-lapse microscopy. Under these conditions, hGCs form tumor-like aggregate structures that express GFAP and Ki67, migrate along both myelinated and non-myelinated axonal tracks and interact with these axons through the formation of pseudopodia. Our ex vivo co-culture system can be used to identify novel cellular and molecular mechanisms of hGC migration and could potentially be used for in vitro drug efficacy testing.

Here we present an ex-vivo mixed monolayer culture system for the study of human glioma cell (hGC) migration in real-time. This model provides the ability to observe interactions between hGCs and both myelinated and non-myelinated axons within a compartmentalized chamber.

Glioblastoma is one of the most aggressive human cancers due to extensive cellular heterogeneity and the migration properties of hGCs. In order to better ...

Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite significant advances in the genetics of these tumors, how GBM cells invade the healthy brain parenchyma is not well understood. Notably, it has been shown that GBM cells invade the peritumoral space via different routes; the main interest of this paper is the route along white matter tracts (WMTs). The interactions of tumor cells with the peritumoral nervous cell components are not well characterized. Herein, a method has been described that evaluates the impact of neurons on GBM cell invasion. This paper presents an advanced co-culture in vitro assay that mimics WMT invasion by analyzing the migration of GBM stem-like cells on neurons. The behavior of GBM cells in the presence of neurons is monitored by using an automated tracking procedure with open-source and free-access software. This method is useful for many applications, in particular, for functional and mechanistic studies as well as for analyzing the effects of pharmacological agents that can block GBM cell migration on neurons.

Here, we present an easy-to-use co-culture assay to analyze glioblastoma (GBM) migration on patterned neurons. We developed a macro in FiJi software for easy quantification of GBM cell migration on neurons, and observed that neurons modify GBM cell invasive capacity.

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

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

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

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

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 ...

Assessing Interneuron Response to Chemoattractive Cues from Periventricular Endothelial Cells

Source: Datta, D., et al. <a href="https://app.jove.com/v/60295">Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons.</a> J. Vis. Exp. (2020).
This video shows a method to test interneuron migration in response to chemoattractive cues from periventricular endothelial cells (PVECs). Interneurons are placed in a central compartment, with PVECs and control cells on either side. Increased migration towards PVECs indicates a positive response to their chemoattractive signals.

1. Chemo-attraction Assay

	Place a three-well culture insert in the center of a poly-L-ornithine/laminin-coated 35 mm dish using sterile tweezers.
	Turn ...

JoVE Encyclopedia of Experiments

Neuropathology

Developmental and Pediatric Disorders

Establishing a Co-Culture of Interneurons with Periventricular Endothelial Cells

Source: Datta, D., et al. <a href="https://app.jove.com/v/60295">Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons.</a> J. Vis. Exp. (2020)&#160;
This video demonstrates a technique for establishing a co-culture of interneurons with perivascular endothelial cells. The cells are added to a one-well insert placed inside a culture dish. After the cells adhere to the dish, the insert is removed, allowing the cells to proliferate and interact and form a co-culture.

1. Culture and Storage of Human Periventricular Endothelial Cells

	Maintain human periventricular endothelial cells on basement membrane matrix-coated ...

Neuronal Culture Techniques

Co-culture and Interaction Studies

Co-Culturing Glioblastoma Cells on Patterned Neurons: A Technique to Monitor the Migration of Glioblastoma Cells on Rat Hippocampal Neurons In Vitro

Source: Guyon, J. et al. <a href="https://www.jove.com/video/62213" target="_blank">Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions.</a> J. Vis. Exp. (2021)
This video describes a co-culture assay of glioblastoma cells on micropatterned neurons, which mimic the myelinated neuronal tracts invasion by the glioblastoma stem-like cells as they migrate on neurons in the peritumoral spaces. This assay can help analyze the effects of pharmacological drugs that inhibit glioblastoma cells&#39; migration on neurons in a glioma.

Source: Guyon, J. et al. Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions. J. Vis. Exp. 
...

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

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