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Method Article

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

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DOI:

10.3791/60295

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January 23rd, 2020

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1Department of Psychiatry, Harvard Medical School, 2Angiogenesis and Brain Development Laboratory, Division of Basic Neuroscience, McLean Hospital

In This Article

Summary

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.

Abstract

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.

Introduction

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 µm. 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 "diseased" 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.

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Protocol

1. Culture and Storage of Human Periventricular Endothelial Cells

  1. 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 µM GABA) at 37 °C and 5% CO2. Change medium every alternate day.
  2. Thaw basement membrane matrix in 4 °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 °C for at least 1 h before use.
  3. 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.
  4. Detach cells by adding 1 mL of cell dissociation solution (see Table of Materials) per well. Incubate at 37 °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.
  5. 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.
  6. 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 °C and 5% CO2.
  7. Store human periventricular endothelial cells by cryopreserving in freezing medium (90% periventricular endothelial cell medium and 10% DMSO).
    1. Dissociate and collect cells following steps 1.3 and 1.4 above. Count cells in the solution by the trypan blue exclusion method.
    2. 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.
    3. Dispense 1 mL of freezing medium plus cells per cryovial. Place the vials in isopropanol-filled chamber and cool overnight in -80 °C at 1 °C/min. Transfer vials to a liquid nitrogen tank on the next day for long term storage.

2. Preparation of Human Periventricular Endothelial Cells for Assay

  1. Allow human periventricular endothelial cells to reach 70%-80% confluency.
  2. Dissociate cells following steps 1.3 to 1.5 as described above. Count cells using the trypan blue exclusion method.

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's protocol.

  1. Thaw human GABAergic interneurons and culture them in 12-well plate for two weeks to a confluency of 70%-80%.
  2. On the day of the assay, warm cell dissociation solution (see Table of Materials) and an aliquot of neuronal medium at 37 °C for 10 min before use.
  3. Aspirate medium from each well containing the cells. Wash cells with 1 mL of sterile 1x PBS per well.
  4. Detach cells by adding 0.5 mL of pre-warmed dissociation solution per well and incubate at 37 °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.
  5. 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.

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's protocol. Fibronectin-coated plates were prepared following manufacturer's protocol.

  1. Thaw control human endothelial cells and culture them in 6-well plate to a confluency of 80%-90%.
  2. On the day of the assay, warm cell dissociation solution (see Table of Materials) and an aliquot of endothelial medium at 37 °C for 10 min before use.
  3. Aspirate the medium from each well containing the cells. Wash cells with 1 mL of sterile 1x PBS per well.
  4. 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.
  5. 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.

5. Preparation of One-well Culture Inserts

  1. Thaw 1 mg/mL laminin solution at room temperature or overnight at 4 °C.
  2. 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.
  3. Dilute 1 mg/mL laminin solution 1:300 in sterile water to a final concentration of 3.3 µg/mL immediately before use.
  4. 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.
  5. Add 1 mL of 3.3 µg/mL laminin solution to each dish and incubate at 37 °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 °C. Equilibrate the dishes in a 37 °C cell culture incubator before use.
  6. 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.
  7. 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.
  8. 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.
  9. Carefully turn the dish upside down to verify that the insert is firmly adhered.
  10. 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).

6. Long-distance Migration Assay

  1. Suspend human GABAergic interneurons at a concentration of 3 x 104 cells/70 µL of neuronal medium. Seed 70 µL of cell solution inside each one-well culture insert.
    NOTE: The seeding density of interneurons is as per manufacturer's recommendation.
  2. Suspend human periventricular endothelial cells at a concentration of 3 x 104 cells/70 µL of periventricular endothelial cell medium. Seed 70 µL 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.
  3. 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.
  4. Check under a microscope to verify that cells are not leaking from the insert compartment.
  5. Incubate cells for 24 h at 37 °C and 5% CO2. After 24 h incubation, check under microscope to verify that cells have attached properly and there is no overnight leak.
  6. 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).
  7. 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.
  8. Incubate cells for 5 days at 37 °C and 5% CO2. After 5 days, remove medium, fix cells with 4% PFA for 10 min, and wash 3x with 1x PBS.
  9. Stain neurons with anti-human β-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.

7. Co-culture Migration Assay

  1. Co-suspend 3 x 104 GABAergic interneurons and 3 x 104 human periventricular endothelial cells in 70 µL 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.
  2. Check under a microscope to verify that cells are not leaking from the insert compartment.
  3. 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.
  4. As a first control, seed 3 x 104 human GABAergic interneurons only in 70 µL of co-culture medium per one-well insert. Prepare an appropriate number of such dishes.
  5. As a second control, co-seed 3 x 104 GABAergic human interneurons with 3 x 104 control human endothelial cells in 70 µL of co-culture medium per one-well insert. Prepare an appropriate number of dishes.
  6. Incubate the dishes for 24 h at 37 °C and 5% CO2. After 24 h incubation, check under a microscope to verify that cells have attached properly and there is no leak.
  7. 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).
  8. Remove medium and add 1 mL of fresh co-culture medium.
    NOTE: Set aside an appropriate number of dishes for acquiring day 0 images.
  9. Incubate cells for 5 days at 37 °C and 5% CO2.
  10. After 5 days, remove medium, fix cells with 4% PFA for 10 min, and wash 3x with 1x PBS.
  11. Stain with anti-human β-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.

8. Chemo-attraction Assay

  1. Place a three-well culture insert in the center of a poly-L-ornithine/laminin coated 35 mm dish using sterile tweezers.
  2. Turn the dish upside down. Mark the boundary around the middle compartment of the insert using a permanent black marker with ultra-fine tip.
  3. Seed 3 x 104 human GABAergic interneurons in the middle compartment in 70 µL of neuronal medium (Figure 3A).
  4. Seed 104 human periventricular endothelial cells in 70 µL of periventricular endothelial cell medium and 104 control endothelial cells in 70 µL of control endothelial cell medium in the two outer compartments respectively (Figure 3A).
  5. 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.
  6. Check under microscope to verify that cells are not leaking from the insert compartment.
  7. Incubate cells for 24 h at 37 °C and 5% CO2. After 24 h incubation, check under a microscope to verify that cells have attached properly and there is no leak.
  8. 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).
  9. Remove medium and add 1 mL of fresh co-culture medium.
    NOTE: Set aside required number of dishes for day 0 images.
  10. Incubate cells for 36 h at 37 °C and 5% CO2. After 36 h, aspirate medium, fix cells with 4% PFA for 10 min, and wash 3x with 1x PBS.
  11. Stain human GABAergic interneurons with anti-human β-Tubulin or anti-human MAP2 antibody. At the end of the staining procedure, add 1 mL of antifade mounting medium in each dish.

9. Imaging and Data Analysis

  1. Place the immuno-stained assay dish under a microscope at 4X magnification.
  2. 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.
  3. Open the images in ImageJ. Calculate distance between each cell and the boundary mark (Figure 2D) using ImageJ.
  4. 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.

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Results

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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Discussion

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 ...

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Disclosures

Authors declare no conflict of interest.

Acknowledgements

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.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Accutase dissociation solutionMillipore SigmaSCR005Cell dissociation solution (for periventricular endothelial cells, step 1.4)
Anti-human β-Tubulin antibodyBiolegend802001
Anti-human CD31 antibodyMillipore SigmaCBL468
Anti- MAP2 antibodyNeuromicsCH22103
Anti-active Caspase 3 antibodyMillipore SigmaAB3623
Control human endothelial cellsCellular DynamicsR1022
Control endothelial Cells Medium SupplementCellular DynamicsM1019
Cryogenic vialsFisher Scientific03-337-7Y
DMEMF/12 mediumThermofisher Scientific11320033
DMSOSigma-AldrichD2650
E6 mediumThermofisher ScientificA1516401
FGF2Thermofisher ScientificPHG0261
FibronectinThermofisher Scientific33016-015
Freezing ContainerThermofisher Scientific5100
GABASigma-AldrichA2129
HemacytometerSigma-AldrichZ359629
Human GABAergic neuronsCellular DynamicsR1013
Human GABAergic neurons base mediumCellular DynamicsM1010
Human GABAergic neuron Neural supplementCellular DynamicsM1032
LamininSigmaL2020
MatrigelCorning356230Basement membrane matrix
Mounting MediumVector laboratoriesH-1200
poly-L-ornithinSigmap4957
PBSThermofisher Scientific14190
Trypan blueThermofisher Scientific15250061
TrypLEThermofisher Scientific12563011Cell dissociation solution (for GABAergic interneurons and endothelial cells, sections 3 and 4)
VEGF-APeprotech100-20
VascuLife VEGF Medium Complete KitLifeline Cell TechnologiesLL-0003Component of control human endothelial cell medium
2-well silicone culture-Insertibidi80209
3-well silicone culture-Insertibidi80369
35 mm dishCorning430165
15-ml conical tubeFisher Scientific07-200-886
4% PFA solutionFisher ScientificAAJ19943K2
6-well tissue culture plateFisher Scientific14-832-11
Inverted phase contrast microscopeZeissZeiss Axiovert 40C
Fluorescent microscopeOlympusFSX-100

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Human Periventricular Endothelial CellsGABAergic InterneuronsMigration AssayCo-Culture AssayChemo-Attraction AssayCell MigrationCell IsolationTrypan Blue ExclusionImmunocytochemical StainingNeuropsychiatric Disorders
Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons
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