The presented work was supported by the Ministe&#768;re de L&#8217;Education Nationale, de L&#8217;Enseignement Supe&#769;rieur et de la Recherche and INSERM. We gratefully acknowledge the BICeL- Campus Scientific City Facility for access to instruments and technical advices. We gratefully acknowledge Jean-Pascal Gimeno, Soulaimane Aboulouard and Isabelle Fournier for the Mass spectrometry assistance. We gratefully acknowledge Tanina Arab, Christelle van Camp, Francoise le Marrec-Croq, Jacopo Vizioli and Pierre-Eric Sauti&#232;re for their strong contribution to the scientific and technical developments.

1. Primary Culture of Microglia/Macrophages
<ol>
	<li>Primary culture of microglia
	<ol>
		<li>Culture commercial rat primary microglia (2 x 106 cells) (see the Table of Materials) in Dulbecco&#39;s modified Eagle medium (DMEM) supplemented with 10% exosome-free serum, 100 U/mL of penicillin, 100 &#181;g/mL streptomycin, and 9.0 g/L glucose at 37 &#176;C and 5% CO2.</li>
		<li>Collect the conditioned medium after a 48 h culture and proceed to the isolation of EVs.</li>
	</ol>
	</li>
	<li>Primary culture of macrophages
	<ol>
		<li>Culture commercial rat primary macrophages (1 x 106 cells) in the medium provided by the manufacturer (see the Table of Materials) at 37 &#176;C and 5% CO2, with exosome-free serum.</li>
		<li>Collect the conditioned medium after a 24 h culture and proceed to the isolation of EVs.</li>
	</ol>
	</li>
</ol>
2. Isolation of EVs
<ol>
	<li>Pre-isolation of EVs from conditioned medium
	<ol>
		<li>Transfer the conditioned culture medium from microglia or macrophage cultures (steps 1.1.2 or 1.2.2) into a conical tube.</li>
		<li>Centrifuge at 1,200 x g for 10 min at room temperature (RT) to pellet the cells.</li>
		<li>Transfer the supernatant into a new conical tube. Centrifuge at 1,200 x g for 20 min at RT to eliminate apoptotic bodies.</li>
		<li>Transfer the supernatant into a 10.4 mL polycarbonate tube and transfer the tube into a 70.1 Ti rotor. Ultracentrifuge at 100,000 x g for 90 min at 4 &#176;C to pellet the EVs.</li>
		<li>Discard the supernatant and resuspend the pellet containing EVs in 200 &#181;L of 0.20 &#181;m filtered phosphate buffer saline (PBS).</li>
	</ol>
	</li>
	<li>Isolation of EVs
	<ol>
		<li>Preparation of the home-made size exclusion chromatography column (SEC)
		<ol>
			<li>Empty a glass chromatography column (length: 26 cm; diameter: 0.6 cm) (see the Table of Materials), wash and sterilize it.</li>
			<li>Place a 60 &#181;m filter at the bottom of the column.</li>
			<li>Stack the column with cross-linked agarose gel filtration base matrix to create a stationary phase of a 0.6 cm diameter and a 20 cm height.</li>
			<li>Rinse the phase with 50 mL of 0.20 &#181;m filtered PBS. Store at 4 &#176;C to be used later if necessary.</li>
		</ol>
		</li>
		<li>Place the resuspended EV pellet on top of the stationary phase of the SEC column.</li>
		<li>Collect 20 sequential fractions of 250 &#181;L while continuing to add 0.20 &#181;m filtered PBS on the top of stationary phase to prevent drying of the column. Store the fractions at -20 &#176;C if necessary. 
		NOTE: A longer storage than one week can be performed at -80 &#176;C to maintain the EV integrity for molecular analysis.</li>
	</ol>
	</li>
	<li>Matrix assisted laser desorption ionization (MALDI) mass spectrometry analysis of the SEC fractions
	<ol>
		<li>Proceed to the EV isolation as described in section 2.2.</li>
		<li>Resuspend the EV pellet with 200 &#181;L of peptide calibration mix solution (see the Table of Materials).</li>
		<li>Proceed to EV collection as described in sections 2.2.1 to 2.2.3.</li>
		<li>Completely dry the fraction with a vacuum concentrator.</li>
		<li>Reconstitute the fractions with 10 &#181;L of 0.1% trifluoroacetic acid (TFA).</li>
		<li>Mix 1 &#181;L of reconstituted fraction with 1 &#181;L of &#945;-cyano-4-hydroxycinnamic acid (HCCA) matrix on a MALDI polished steel target plate.</li>
		<li>Analyze all fractions with a MALDI mass spectrometer.</li>
		<li>Analyze generated spectra with dedicated software (see Table of Materials).</li>
	</ol>
	</li>
</ol>
3. Characterization of EVs
<ol>
	<li>Nanoparticle tracking analysis (NTA) 
	NOTE: The NTA analysis is performed with a nanoparticle tracking analysis instrument (see the Table of Materials) and an automated syringe pump.
	<ol>
		<li>Make a dilution (range of 1:50 to 1:500) of each SEC fraction from step 2.2.3 with 0.20 &#181;m filtered PBS.</li>
		<li>Vortex the solution to eliminate EV aggregates.</li>
		<li>Put the diluted solution in a 1 mL syringe and place it in the automated syringe pump.</li>
		<li>Adjust Camera setting to screen gain level (3) and camera level (13).</li>
		<li>Click on Run and launch the following script.
		<ol>
			<li>Load the sample in analysis chamber (infusion rate: 1,000 for 15 s).</li>
			<li>Decrease and stabilize speed flow for video recording (infusion rate: 25 for 15 s). Capture three consecutive 60 s videos of the particle flow.</li>
			<li>Adjust the camera level (13) and the detection threshold (3) before videos analysis. Click on Settings| Ok to start the analysis and click on Export when the analysis is done.</li>
		</ol>
		</li>
		<li>Between each fraction analysis, wash with 1 mL of 0.20 &#181;m filtered PBS.</li>
	</ol>
	</li>
	<li>Electron microscopy (EM) analysis
	<ol>
		<li>Isolate EVs as described in section 2. 
		NOTE: Sterile conditions are not required.</li>
		<li>Repeat section 3.1 to quantify EVs. 
		NOTE: Only positive fractions will be used for EM analysis.</li>
		<li>Use a 50 kDa centrifugal filter (see Table of Materials) to concentrate EV-containing SEC fractions.</li>
		<li>Resuspend concentrated EVs in 30 &#181;L of 2% paraformaldehyde (PFA).</li>
		<li>Load 10 &#181;L of the sample onto a carbon-coated copper grid.</li>
		<li>Incubate for 20 min in a wet environment.</li>
		<li>Repeat steps 3.2.5 and 3.2.6 for a good absorption of the sample on the grid.</li>
		<li>Transfer the grid into a drop of 1% glutaraldehyde in PBS for 5 min at RT.</li>
		<li>Wash the sample with ultrapure water several times.</li>
		<li>Contrast the sample for 10 min on ice with a mixture of 4% uranyl acetate and 2% methylcellulose (1:9, v/v). Remove excess of the mixture using a filter paper.</li>
		<li>Dry the sample and observe it under a transmission electron microscope at 200 kV (see the Table of Materials).</li>
	</ol>
	</li>
	<li>Western blot analysis
	<ol>
		<li>Protein extraction
		<ol>
			<li>Repeat the steps 3.2.1 to 3.2.3 to isolate and concentrate EVs.</li>
			<li>Mix 50 &#181;L of RIPA buffer (150 mM sodium chloride [NaCl], 50 mM Tris, 5 mM Ethylene glycol-bis(2-aminoethylether)-N,N,N&#8242;,N&#8242;-tetraacetic acid [EGTA], 2 mM Ethylenediamine-tetraacetic acid [EDTA], 100 mM sodium fluoride [NaF], 10 mM sodium pyrophosphate, 1% Nonidet P-40, 1 mM Phenylmethanesulfonyl fluoride [PMSF], 1x protease inhibitor) with the EV sample (25 &#181;L resulting from the EV concentration on filter) for 5 min on ice to extract proteins.</li>
			<li>Sonicate for 5 s (amplitude: 500 W; frequency: 20 kHz), 3 times on ice.</li>
			<li>Remove vesicular debris by centrifugation at 20,000 x g for 10 min, at 4 &#176;C.</li>
			<li>Collect the supernatant and measure the protein concentration with Bradford protein assay method.</li>
		</ol>
		</li>
		<li>Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting
		<ol>
			<li>Mix protein extracts (30 &#181;g) with 5c Laemmli sample buffer (v/v).</li>
			<li>Load the protein mix on a 12% polyacrylamide gel.</li>
			<li>Migrate the proteins in the gel with TGS buffer (25 mM Tris pH 8.5, 192 mM Glycine, and 0.1% SDS), at 70 V for 15 min and 120 V for 45 min.</li>
			<li>Transfer the proteins onto nitrocellulose membrane with semi-dry system at 230 V for 30 min.</li>
			<li>Saturate the membrane for 1 h at RT with blocking buffer (0.05% Tween 20 w/v, 5% milk powder w/v in 0.1 M PBS, pH 7.4).</li>
			<li>Incubate the membrane overnight at 4 &#176;C with mouse monoclonal anti-human heat-shock protein 90 (HSP90) antibody diluted in blocking buffer (1:100).</li>
			<li>Wash the membrane three times with PBS-Tween (PBS, 0.05% Tween 20 w/v) for 15 min.</li>
			<li>Incubate the membrane for 1 h at RT with goat horseradish peroxidase-conjugated anti-mouse IgG secondary antibody diluted in blocking buffer (1:10,000). 
			NOTE: A negative control is performed using secondary antibody alone.</li>
			<li>Repeat the washing step (step 3.3.2.7).</li>
			<li>Reveal the membrane with enhanced chemiluminescence (ECL) western blotting substrate kit (see Table of Materials).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Proteomic analysis
	<ol>
		<li>Protein extraction and in-gel digestion
		<ol>
			<li>Repeat the steps 3.2.1 to 3.2.3 to isolate and concentrate the EVs. Repeat step 3.3.1 for EV protein extraction.</li>
			<li>Perform protein migration in the stacking gel of a 12% polyacrylamide gel.</li>
			<li>Fix the proteins in the gel with coomassie blue for 20 min at RT.</li>
			<li>Excise each colored gel piece and cut it into small pieces of 1 mm3.</li>
			<li>Wash the gel pieces successively with 300 &#181;L of each solutions: ultrapure water for 15 min, 100% acetonitrile (ACN) for 15 min, 100 mM ammonium bicarbonate (NH4HCO3) for 15 min, ACN:100 mM NH4HCO3 (1:1, v/v) for 15 min, and 100% ACN for 5 min with continuous stirring.</li>
			<li>Dry completely gel pieces with vacuum concentrator.</li>
			<li>Perform protein reduction with 100 &#181;L of 100 mM NH4HCO3 containing 10 mM dithiothreitol for 1 h at 56&#176;C.</li>
			<li>Perform protein alkylation with 100 &#181;L of 100 mM NH4HCO3 containing 50 mM iodoacetamide for 45 min in the dark at RT.</li>
			<li>Wash the gel pieces successively with 300 &#181;L of each solution: 100 mM of NH4HCO3 for 15 min, ACN:20 mM NH4HCO3 (1:1, v/v) for 15 min, and 100% ACN for 5 min with a continuous stirring.</li>
			<li>Completely dry the gel pieces with a vacuum concentrator.</li>
			<li>Perform protein digestion with 50 &#181;L of trypsin (12.5 &#181;g/mL) in 20 mM NH4HCO3 overnight at 37 &#176;C.</li>
			<li>Extract the digested proteins from the gel with 50 &#181;L of 100% ACN for 30 min at 37 &#176;C and then 15 min at RT with continuous stirring.</li>
			<li>Repeat the following extraction procedures twice: 50 &#181;L of 5% TFA in 20 mM NH4HCO3 solution for 20 min with continuous stirring.</li>
			<li>Add 100 &#181;L of 100% ACN for 10 min with continuous stirring.</li>
			<li>Dry digested proteins with a vacuum concentrator and resuspend in 20 &#181;L of 0.1% TFA.</li>
			<li>Desalt the sample using a 10 &#181;L pipette tip with C18 reverse phase media for desalting and concentrating peptides (see the Table of Materials) and elute peptides with ACN:0.1% formic acid (FA) (80:20, v/v).</li>
			<li>Completely dry the sample with a vacuum concentrator and resuspend in 20 &#181;L of ACN:0.1% FA (2:98, v/v) for liquid chromatography tandem mass spectrometry (LC-MS/MS).</li>
		</ol>
		</li>
		<li>LC-MS/MS Analysis
		<ol>
			<li>Load the digested peptide into the LC-MS/MS instrument and perform sample and data analysis according to parameters described in detail elsewhere42.</li>
		</ol>
		</li>
		<li>Raw data analysis
		<ol>
			<li>Process mass spectrometry data to identify proteins and compare identified proteins of each sample with a quantitative proteomics software package using standard parameters.</li>
			<li>Export, using standard parameters, the list of exclusive and over-represented proteins from EV positive samples in a software predicting protein networks and biological processes.</li>
			<li>Compare the list of identified proteins in the fractions with the top 100 EV markers from the Exocarta open access database (see the Table of Materials).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4. Functional EVs Effects Assay
<ol>
	<li>Neurite outgrowth assay on PC-12 cell line
	<ol>
		<li>Culture PC12 cell line in complete DMEM medium (2 mM L-glutamine, 10% fetal horse serum (FHS), 5% fetal bovine serum (FBS), 100 UI/mL penicillin, 100 &#181;g/mL streptomycin). 
		NOTE: The sera are exosome free in the whole procedure.</li>
		<li>Add a cover glass (all wells) to a 24-well plate; coat the plate with poly-D-lysine (0.1 mg/mL) and seed 260,000 cells/well.</li>
		<li>Incubate the cells at 37 &#176;C under 5% CO2.</li>
		<li>After 24 h of incubation, change the medium to DMEM differentiation medium (DMEM with 2 mM L-glutamine, 0.1% FHS, 100 UI/mL penicillin, 100 &#181;g/mL streptomycin) with 1 x 106 microglia EVs (from step 1.1.2). 
		NOTE: Control condition is performed without microglia EVs in the differentiation medium.</li>
		<li>At day 4 after seeding, load all wells with 100 &#181;L of complete DMEM medium.</li>
		<li>At day 7 after seeding, fix the cells with 4% PFA for 20 min at RT and rinse three times (10 min each) with PBS.</li>
		<li>Stain the cells with rhodamine-conjugated phalloidin for 30 min at 4 &#176;C and rinse 3 times (10 min each) with PBS.</li>
		<li>Stain the cells with diluted Hoechst 33342 (1:10,000) for 30 min at RT and rinse 3 times (10 min each) with PBS.</li>
		<li>Mount the cover glass on a slide with fluorescent mounting medium (see Table of Materials).</li>
		<li>Analyze the slide with a confocal microscope, take 5 random images of each slide.</li>
		<li>Measure neurite outgrowth using an automated quantification software (total neurite length) as described in detail elsewhere43.</li>
	</ol>
	</li>
	<li>Neurite outgrowth on rat primary neurons
	<ol>
		<li>Coat an 8-well glass slide with poly-D-lysine (0.1 mg/mL) and laminin (20 &#181;g/mL).</li>
		<li>Culture rat commercial primary neurons in appropriate culture medium (see Table of Materials) by plating 50,000 cells per well and incubating the cells at 37 &#176;C under 5% CO2 for 48 h. 
		NOTE: The sera are exosome-free in the whole procedure.</li>
		<li>Add 1 x 106 microglia EVs to neuron culture medium and incubate at 37 &#176;C under 5% CO2 for 48 h more. 
		NOTE: Control condition is performed without microglia EVs in neuron culture medium.</li>
		<li>Follow steps 4.1.6 to 4.1.9 to fix and stain the cells.</li>
		<li>Follow steps 4.1.10 and 4.1.11 to analyze the slide.</li>
	</ol>
	</li>
	<li>Glioma cell invasion
	<ol>
		<li>Resuspend C6 rat glioma cells in complete DMEM medium (DMEM with 10% FBS, 2 mM L-glutamine, 1x antibiotics) containing 5% collagen at a final concentration of 8,000 cells in 20 &#181;L. 
		NOTE: The sera are exosome-free in the whole procedure.</li>
		<li>Place 5 mL of PBS AT the bottom of a 60 mm tissue culture dish. Invert the lid and deposit drops of 20 &#181;L (8,000 cells) of cell suspension onto the bottom of the lid.</li>
		<li>Invert the lid onto the PBS-filled bottom chamber and incubate the plate at 37 &#176;C and 5% CO2 for 72 h until cell spheroids are formed.</li>
		<li>Add 1 x 108 macrophage EVs (from step 1.2.2) to a 2.2 mg/mL collagen mixture (2 mL of bovine collagen type I solution [3 mg/mL] with 250 &#181;L of 10x minimum essential medium (MEM) and 500 &#181;L of 0.1 M sodium hydroxide). 
		NOTE: Control condition is performed without macrophage EVs in the collagen mixture.</li>
		<li>Distribute the collagen mixture containing EVs in a 24-well plate for embedding cell spheroids.</li>
		<li>Implant the newly formed cell spheroids at the center of each well.</li>
		<li>Incubate the plate for 30 min at 37&#176;C and 5% CO2 to solidify the gel.</li>
		<li>Thereafter, overlay 400 &#181;L of complete DMEM medium on the collagen matrix in each well.</li>
		<li>Incubate the complete system for a total of 6 days at 37 &#176;C and 5% CO2. 
		NOTE: Cell invasion out of the spheroid is monitored by digital photography using an inverted light microscope using a 4x/0.10 N.A. objective.</li>
		<li>Acquire images of each well every day.</li>
		<li>Process images and quantify invasion of cell spheroid areas using the software as previously described in detail42. 
		NOTE: Invasion and spheroid areas were normalized for each day to the invasion and spheroid areas measured on day 0.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

The central nervous system (CNS) is a complex tissue in which cell-to-cell communication regulates normal neuronal functions necessary for homeostasis30. EVs are now widely studied and described as important molecular cargos for cell-to-cell communication31. They specifically deliver a cocktail of mediators to recipient cells thereby affecting their functions in healthy and pathological conditions32. Recent studies indicate that EVs play a crucial ro...

Many neuropathologies are related to the neuro-inflammatory state which is a complex mechanism that is increasingly considered, but still poorly understood because the immune processes are diverse and depend upon the cell environment. Indeed, the CNS disorders do not systematically involve the same activation signals and immune cell populations and thus the pro- or anti-inflammatory responses are difficult to evaluate as causes or consequences of pathologies. The brain resident macrophages called &#8220;microglia&#8221; appear to be at the interface between the nervous and immune systems1. Microglia have a myeloid origin and are derived from the yolk sac during primitive hematopoiesis to colonize the brain, whereas peripheral macrophages are derived from the fetal liver during definitive hematopoiesis to become peripheral macrophages2. The microglia cells communicate with neurons and neuron-derived glial cells such as astrocytes and oligodendrocytes3. Several recent studies have demonstrated that microglia are involved in neuronal plasticity during CNS development and adult tissue homeostasis, and also in the inflammatory state associated with neurodegenerative diseases4,5. Otherwise, the integrity of the blood brain barrier can be compromised in other CNS pathologies. The immune responses, especially in the glioblastoma multiforme cancer, are not supported only by microglia cells as the blood brain barrier is reorganized through angiogenic processes and the presence of lymphatic vessels6,7. Therefore, a large bone marrow-derived macrophages (BMDMs) infiltration occurs in the brain tumor throughout tumor-dependent angiogenesis mechanisms8. The cancer cells exert a significant influence on infiltrated BMDMs leading to immunosuppressive properties and tumor growth9. Thus the communication between the immune cells and their brain microenvironment is difficult to understand as the cell origin and activation signals are diverse10,11. It is thus interesting to apprehend the functions of immune cell-associated molecular signatures in physiological conditions. In this regard, the cell-cell communication between immune cells and cell microenvironment can be studied through the release of extracellular vesicles (EVs).
The EVs are being studied more and more in the regulation of immune functions in healthy as well as pathological conditions12,13. Two populations, exosomes and microvesicles, can be taken into account. They present different biogenesis and size ranges. The exosomes are vesicles of ~30&#8211;150 nm diameter and are generated from the endosomal system and secreted during fusion of multivesicular bodies (MVBs) with the plasma membrane. The microvesicles are about 100&#8211;1,000 nm in diameter and are generated by an outward budding from the cell plasma membrane14. Because the exosome versus microvesicle discrimination is still difficult to realize according to the size and molecular patterns, we will only use the term EVs in the present report. The EV-associated communication in the CNS represents an ancestral mechanism since studies showed their involvement in invertebrate species including nematodes, insects or annelids15,16. Moreover, the results showing that EVs can communicate with cells from different species demonstrate this mechanism to be a key-lock system, based first on surface-molecule recognition between vesicles and recipient cells and then allowing the uptake of mediators16,17. Indeed, the EVs contain many molecules like proteins (e.g., enzymes, signal transduction, biogenesis factor), lipids (e.g., ceramide, cholesterol) or nucleic acids (e.g., DNA, mRNA or miRNAs) acting as direct or indirect regulators of the recipient cell activities14. That is why methodological studies were also performed on immune cells to isolate EVs and fully characterize their protein signatures18,19.
The earliest studies demonstrated the release of exosomes from primary cultured rat microglia as an inducible mechanism following a Wnt3a- or serotonin-dependent activation20,21. Functionally in the CNS, microglia-derived EVs regulate the synaptic vesicle release by presynaptic terminals in neurons contributing to the control of the neuronal excitability22,23. Microglia-derived EVs could also propagate cytokines-mediated inflammatory response in large brain regions24,25. Importantly, the diverse ligands for toll-like receptor family might activate specific productions of EVs in the microglia26. For example, in vitro studies show that LPS-activated microglia BV2 cell lines produce differential EV contents including pro-inflammatory cytokines27. Therefore, the functional diversity of immune cell subpopulations in the CNS, microglia and infiltrating BMDMs, might be evaluated through their own EV populations including the EV impact on recipient cells and the identification of EV contents.
We previously described methods to evaluate the functional properties of microglia- and BMDM-derived EVs after their isolation16,19. In the present report, we propose to independently evaluate the effect of microglia-derived EVs on neurite outgrowth, and the effect of macrophage-derived EVs on the control of glioma cell aggregates. This study also proposes a wide proteomic analysis of the EV fractions in order to validate the EV isolation procedure as well as identify the biologically active protein signatures. The beneficial effects and the molecular deciphering of EV contents could help their possible manipulation and use as therapeutic agents in brain disorders.

<table>
<tbody>
<tr>
<td>12% Mini-PROTEAN&nbsp;TGX Precast Protein Gels</td>
<td>Bio-rad</td>
<td>4561045EDU</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Acetonitrile</td>
<td>Fisher Chemicals</td>
<td>A955-1</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Amicon 50 kDa centrifugal filter</td>
<td>Merck</td>
<td>UFC505024</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Ammonium bicarbonate</td>
<td>Sigma-Aldrich</td>
<td>9830</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>HSP90 &alpha;/&beta; antibody (RRID: AB_675659)</td>
<td>Santa-cruz</td>
<td>sc-13119</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>B27 Plus supplement</td>
<td>Gibco</td>
<td>A3582801</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>BenchMixer V2 Vortex Mixer</td>
<td>Benchmark Scientific</td>
<td>BV1003</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Bio-Rad Protein Assay Dye Reagent Concentrate (Bradford)</td>
<td>Bio-Rad</td>
<td>5000006</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>C18 ZipTips</td>
<td>Merck Millipore</td>
<td>ZTC18S096</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>C6 rat glioma cell</td>
<td>ATCC</td>
<td>ATCC CCL-107</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Canonical tubes</td>
<td>Sarstedt</td>
<td>62.554.002</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Centrifuge</td>
<td>Eppendorf</td>
<td>5804000010</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>CO2 Incubator</td>
<td>ThermoFisher</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Confocal microscope LSM880</td>
<td>Carl Zeiss</td>
<td>LSM880</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Cover glass</td>
<td>Marienfeld</td>
<td>111580</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Culture Dish (60 mm)</td>
<td>Sarstedt</td>
<td>82.1473</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Dithiothreitol</td>
<td>Sigma-Aldrich</td>
<td>43819</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>DMEM</td>
<td>Gibco</td>
<td>41966029</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>EASY-nLC 1000 Liquid Chromatograph</td>
<td>ThermoFisher</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Electron microscope JEM-2100</td>
<td>JEOL</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Ethylene glycol-bis(2-aminoethylether)-N,N,N&prime;,N&prime;-tetraacetic acid</td>
<td>Sigma-Aldrich</td>
<td>03777-10G</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Ethylenediaminetetraacetic acid</td>
<td>Sigma-Aldrich</td>
<td>ED-100G</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Exo-FBS</td>
<td>Ozyme</td>
<td>EXO-FBS-50A-1</td>
<td>Exosome depleted FBS</td>
</tr>
<tr>
<td>ExoCarta database (top 100 proteins of Evs)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>http://www.exocarta.org/</td>
</tr>
<tr>
<td>Fetal Bovine Serum</td>
<td>Gibco</td>
<td>16140071</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Fetal Horse Serum</td>
<td>Biowest</td>
<td>S0960-500</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Filtropur S 0.2</td>
<td>Sarstedt</td>
<td>83.1826.001</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Fisherbrand&nbsp;Q500 Sonicator with Probe</td>
<td>Fisherbrand</td>
<td>12893543</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>FlexAnalysis</td>
<td>Brucker</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Fluorescence mounting medium</td>
<td>Agilent</td>
<td>S3023</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Formic Acid</td>
<td>Sigma-Aldrich</td>
<td>695076</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Formvar-carbon coated copper grids</td>
<td>Agar scientific Ltd</td>
<td>AGS162-3</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Glucose</td>
<td>Sigma-Aldrich</td>
<td>G8769</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Glutaraldehyde</td>
<td>Sigma-Aldrich</td>
<td>340855</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Hoechst 33342</td>
<td>Euromedex</td>
<td>17535-AAT</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Idoacetamide</td>
<td>Sigma-Aldrich</td>
<td>I1149</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>InstantBlue Coomassie Protein Stain</td>
<td>Expedeon</td>
<td>ISB1L</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Invert light microscope CKX53</td>
<td>Olympus</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>L-glutamine</td>
<td>Gibco</td>
<td>25030-024</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>LabTek II 8 wells</td>
<td>&nbsp;Nunc</td>
<td>154534</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Laemmli 2X</td>
<td>Bio-Rad</td>
<td>1610737</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Laminin</td>
<td>Corning</td>
<td>354232</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>MaxQuant software (proteins identification software)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>https://maxquant.net/maxquant/</td>
</tr>
<tr>
<td>MBT Polish stell</td>
<td>Brucker</td>
<td>8268711</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>MEM 10X</td>
<td>Gibco</td>
<td>21090-022</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Methylcellulose</td>
<td>Sigma-Aldrich</td>
<td>M6385-100G</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>MiliQ water</td>
<td>Merck Millipore</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Milk</td>
<td>Regilait</td>
<td>REGILAIT300</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Mini PROTEAN Vertical Electrophoresis Cell</td>
<td>Bio-Rad</td>
<td>1658000FC</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>MonoP FPLC column</td>
<td>GE Healthcare</td>
<td>&nbsp;</td>
<td>no longer available</td>
</tr>
<tr>
<td>Nanosight NS300</td>
<td>Malvern Panalytical</td>
<td>NS300</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>NanoSight NTA software v3.2</td>
<td>Malvern Panalytical</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>NanoSight syringe pump</td>
<td>Malvern Panalytical</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Neurobasal</td>
<td>Gibco</td>
<td>21103-049</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Nitrocellulose membrane</td>
<td>GE Healthcare</td>
<td>10600007</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Nonidet P-40</td>
<td>Fluka</td>
<td>56741</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Nunc multidish 24 wells</td>
<td>ThermoFisher</td>
<td>82.1473</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Paraformaldehyde</td>
<td>Electro microscopy Science</td>
<td>15713</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>PC-12 cell line</td>
<td>ATCC</td>
<td>ATCC CRL-1721</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Penicillin-Streptomycin</td>
<td>Gibco</td>
<td>15140-122</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Peptide calibration mix</td>
<td>LaserBio Labs</td>
<td>C101</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L)</td>
<td>Jackson ImmunoResearch</td>
<td>115-035-003</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Perseus software (Processing of identified proteins)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>https://maxquant.net/perseus/</td>
</tr>
<tr>
<td>Phalloidin-tetramethylrhodamine conjugate</td>
<td>Santa-cruz</td>
<td>sc-362065</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Phenylmethanesulfonyl fluoride</td>
<td>Sigma-Aldrich</td>
<td>78830</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Phosphate Buffer Saline</td>
<td>Invitrogen</td>
<td>14190094</td>
<td>no calcium, no magnesium</td>
</tr>
<tr>
<td>pluriStrainer M/ 60 &micro;m</td>
<td>pluriSelect</td>
<td>43-50060</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Poly-D-lysine</td>
<td>Sigma-Aldrich</td>
<td>P6407</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Polycarbonate centrifuge tubes</td>
<td>Beckman Coulter</td>
<td>355651</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Protease Inhibitor</td>
<td>Sigma-Aldrich</td>
<td>S8830-20TAB</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>PureCol</td>
<td>Cell Systems</td>
<td>5005</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Q-Exactive mass spectrometer</td>
<td>ThermoFisher</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>rapifleX mass spectrometer</td>
<td>Brucker</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Rat cortical neurons</td>
<td>Cell Applications</td>
<td>R882N-20</td>
<td>Cell origin : Derived from cerebral cortices of day 18 embryonic Sprague Dawley rat brains</td>
</tr>
<tr>
<td>Rat Macrophage &amp; Microglia Culture Medium</td>
<td>Cell Applications</td>
<td>R620K-100</td>
<td>Cell orgin : Normal healthy Rat bone marrow</td>
</tr>
<tr>
<td>Rat primary macrophages</td>
<td>Cell Applications</td>
<td>R8818-10a</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Rat primary microglia</td>
<td>Lonza</td>
<td>RG535</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Sepharose CL-2B</td>
<td>GE Healthcare</td>
<td>17014001</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Sequencing Grade Modified Trypsin</td>
<td>Promega</td>
<td>V5111</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Slide</td>
<td>Dustsher</td>
<td>100204</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Sodium Chloride</td>
<td>Scharlau</td>
<td>SO0227</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Sodium Dodecyl Sulfate</td>
<td>Sigma-Aldrich</td>
<td>L3771</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Sodium Fluoride</td>
<td>Sigma-Aldrich</td>
<td>S7920-100G</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Sodium hydroxide</td>
<td>Scharlab</td>
<td>SO0420005P</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Sodium pyrophosphate</td>
<td>Sigma-Aldrich</td>
<td>S6422-100G</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>SpeedVac Vacuum Concentrator</td>
<td>ThermoFisher</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>String software (functional protein association networks)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>https://string-db.org/</td>
</tr>
<tr>
<td>SuperSignal West Dura extended Duration Substrate</td>
<td>ThermoFisher</td>
<td>34075</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Syringe 1.0 mL</td>
<td>Terumo</td>
<td>8SS01H1</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Trans-Blot SD Semi-Dry Transfer cell</td>
<td>Bio-Rad</td>
<td>1703940</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Trifluoroacetic acid</td>
<td>Sigma-Aldrich</td>
<td>T6508</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Tris</td>
<td>Interchim</td>
<td>UP031657</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Tris-Glycine</td>
<td>Euromedex</td>
<td>EU0550</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Tween 20</td>
<td>Sigma-Aldrich</td>
<td>P2287</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Ultracentrifuge</td>
<td>Beckman Coulter</td>
<td>A95765</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Ultracentrifuge Rotor 70.1 Ti</td>
<td>Beckman Coulter</td>
<td>342184</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Uranyl acetate</td>
<td>Agar Scientific Ltd</td>
<td>AGR1260A</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Whatman filter paper</td>
<td>Sigma-Aldrich</td>
<td>WHA10347510</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>&alpha;-Cyano-4-hydroxycinnamic acid</td>
<td>Sigma-Aldrich</td>
<td>C2020-25G</td>
<td>&nbsp;</td>
</tr>
</tbody>
</table>

characterization of immune cell-derived extracellular vesicles and studying functional impact on cell environment

The neuroinflammatory state of the central nervous system (CNS) plays a key role in physiological and pathological conditions. Microglia, the resident immune cells in the brain, and sometimes the infiltrating bone marrow-derived macrophages (BMDMs), regulate the inflammatory profile of their microenvironment in the CNS. It is now accepted that the extracellular vesicle (EV) populations from immune cells act as immune mediators. Thus, their collection and isolation are important to identify their contents but also evaluate their biological effects on recipient cells. The present data highlight chronological requirements for EV isolation from microglia cells or blood macrophages including the ultracentrifugation and size-exclusion chromatography (SEC) steps. A non-targeted proteomic analysis permitted the validation of protein signatures as EV markers and characterized the biologically active EV contents. Microglia-derived EVs were also functionally used on primary culture of neurons to assess their importance as immune mediators in the neurite outgrowth. The results showed that microglia-derived EVs contribute to facilitate the neurite outgrowth in vitro. In parallel, blood macrophage-derived EVs were functionally used as immune mediators in spheroid cultures of C6 glioma cells, the results showing that these EVs control the glioma cell invasion in vitro. This report highlights the possibility to evaluate the EV-mediated immune cell functions but also understand the molecular bases of such a communication. This deciphering could promote the use of natural vesicles and/or the in vitro preparation of therapeutic vesicles in order to mimic immune properties in the microenvironment of CNS pathologies.

One of the main challenges to attributing biologicals effects to extracellular vesicles (EVs) is the ability to isolate the EVs from the whole culture medium. In this report, we present a method using ultracentrifugation (UC) and size-exclusion chromatography (SEC) which is coupled to the large-scale analysis of protein signatures to validate EV markers and identify bioactive compounds. The macrophage- or microglia-derived EVs were isolated from the conditioned medium after a 24 h or 48 h culture respectively (<strong cl...

Watch this Scientific Journal Video about Characterization of Immune Cell-derived Extracellular Vesicles and Studying Functional Impact on Cell Environment at JoVE.com

Characterization of Immune Cell-derived Extracellular Vesicles and Studying Functional Impact on Cell Environment

The present report highlights chronological requirements for extracellular vesicle (EV) isolation from microglia or blood macrophages. Microglia-derived EVs were evaluated as regulators of the neurite outgrowth while blood macrophage-derived EVs were studied in the control of C6 glioma cell invasion in in vitro assays. The goal is to better understand these EV functions as immune mediators in specific microenvironments.

The neuroinflammatory state of the central nervous system (CNS) plays a key role in physiological and pathological conditions. Microglia, the resident ...

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Neuroscience

6.7K Views.  Univ. Lille, INSERM. The present report highlights chronological requirements for extracellular vesicle (EV) isolation from microglia or blood macrophages. Microglia-derived EVs were evaluated as regulators of the neurite outgrowth while blood macrophage-derived EVs were studied in the control of C6 glioma cell invasion in in vitro assays. The goal is to better understand these EV functions as immune mediators in specific microenvironments.

Video: Characterization of Immune Cell-derived Extracellular Vesicles and Studying Functional Impact on Cell Environment

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression (SD), as a means to develop novel therapeutic targets for episodic and chronic migraine. SD is the likely cause of migraine aura and migraine pain. It is a paroxysmal loss of neuronal function triggered by initially increased neuronal activity, which slowly propagates within susceptible brain regions. Normal brain function is exquisitely sensitive to, and relies on, coincident low-level immune signaling. Thus, neural immune signaling likely affects electrical activity of SD, and therefore migraine. Pain perception studies of SD in whole animals are fraught with difficulties, but whole animals are well suited to examine systems biology aspects of migraine since SD activates trigeminal nociceptive pathways. However, whole animal studies alone cannot be used to decipher the cellular and neural circuit mechanisms of SD. Instead, in vitro preparations where environmental conditions can be controlled are necessary. Here, it is important to recognize limitations of acute slices and distinct advantages of hippocampal slice cultures. Acute brain slices cannot reveal subtle changes in immune signaling since preparing the slices alone triggers: pro-inflammatory changes that last days, epileptiform behavior due to high levels of oxygen tension needed to vitalize the slices, and irreversible cell injury at anoxic slice centers. 
In contrast, we examine immune signaling in mature hippocampal slice cultures since the cultures closely parallel their in vivo counterpart with mature trisynaptic function; show quiescent astrocytes, microglia, and cytokine levels; and SD is easily induced in an unanesthetized preparation. Furthermore, the slices are long-lived and SD can be induced on consecutive days without injury, making this preparation the sole means to-date capable of modeling the neuroimmune consequences of chronic SD, and thus perhaps chronic migraine. We use electrophysiological techniques and non-invasive imaging to measure neuronal cell and circuit functions coincident with SD. Neural immune gene expression variables are measured with qPCR screening, qPCR arrays, and, importantly, use of cDNA preamplification for detection of ultra-low level targets such as interferon-gamma using whole, regional, or specific cell enhanced (via laser dissection microscopy) sampling. Cytokine cascade signaling is further assessed with multiplexed phosphoprotein related targets with gene expression and phosphoprotein changes confirmed via cell-specific immunostaining. Pharmacological and siRNA strategies are used to mimic and modulate SD immune signaling.

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

Migraine and its transformation to chronic migraine are immense healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression in hippocampal slice cultures, as a means to develop novel therapeutic targets.

Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options.  We seek to define how neural immune ...

Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, cell-based model that produces functioning synapses and undergoes physiological responses to intoxication. Here we describe a simple and robust method to efficiently differentiate murine embryonic stem cells (ESCs) into defined lineages of synaptically active, networked neurons. Following an 8 day differentiation protocol, mouse embryonic stem cell-derived neurons (ESNs) rapidly express and compartmentalize neurotypic proteins, form neuronal morphologies and develop intrinsic electrical responses. By 18 days after differentiation (DIV 18), ESNs exhibit active glutamatergic and &#947;-aminobutyric acid (GABA)ergic synapses and emergent network behaviors characterized by an excitatory:inhibitory balance. To determine whether intoxication with CNTs functionally antagonizes synaptic neurotransmission, thereby replicating the in vivo pathophysiology that is responsible for clinical manifestations of botulism or tetanus, whole-cell patch clamp electrophysiology was used to quantify spontaneous miniature excitatory post-synaptic currents (mEPSCs) in ESNs exposed to tetanus neurotoxin (TeNT) or botulinum neurotoxin (BoNT) serotypes /A-/G. In all cases, ESNs exhibited near-complete loss of synaptic activity within 20 hr. Intoxicated neurons remained viable, as demonstrated by unchanged resting membrane potentials and intrinsic electrical responses. To further characterize the sensitivity of this approach, dose-dependent effects of intoxication on synaptic activity were measured 20 hr after addition of BoNT/A. Intoxication with 0.005 pM BoNT/A resulted in a significant decrement in mEPSCs, with a median inhibitory concentration (IC50) of 0.013 pM. Comparisons of median doses indicate that functional measurements of synaptic inhibition are faster, more specific and more sensitive than SNARE cleavage assays or the mouse lethality assay. These data validate the use of synaptically coupled, stem cell-derived neurons for the highly specific and sensitive detection of CNTs.

A custom protocol is described to differentiate mouse ES cells into defined populations of highly pure neurons exhibiting functioning synapses and emergent network behavior. Electrophysiological analysis demonstrates the loss of synaptic transmission following exposure to botulinum neurotoxin serotypes /A-/G and tetanus neurotoxin.

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

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 ...

Monitoring the Effect of Osmotic Stress on Secretory Vesicles and Exocytosis

Amperometry recording of cells subjected to osmotic shock show that secretory cells respond to this physical stress by reducing the exocytosis activity and the amount of neurotransmitter released from vesicles in single exocytosis events. It has been suggested that the reduction in neurotransmitters expelled is due to alterations in membrane biophysical properties when cells shrink in response to osmotic stress and with assumptions made that secretory vesicles in the cell cytoplasm are not affected by extracellular osmotic stress. Amperometry recording of exocytosis monitors what is released from cells the moment a vesicle fuses with the plasma membrane, but does not provide information on the vesicle content before the vesicle fusion is triggered. Therefore, by combining amperometry recording with other complementary analytical methods that are capable of characterizing the secretory vesicles before exocytosis at cells is triggered offers a broader overview for examining how secretory vesicles and the exocytosis process are affected by osmotic shock. We here describe how complementing amperometry recording with intracellular electrochemical cytometry and transmission electron microscopy (TEM) imaging can be used to characterize alterations in secretory vesicles size and neurotransmitter content at chromaffin cells in relation to exocytosis activity before and after exposure to osmotic stress. By linking the quantitative information gained from experiments using all three analytical methods, conclusions were previously made that secretory vesicles respond to extracellular osmotic stress by shrinking in size and reducing the vesicle quantal size to maintain a constant vesicle neurotransmitter concentration. Hence, this gives some clarification regarding why vesicles, in response to osmotic stress, reduce the amount neurotransmitters released during exocytosis release. The amperometric recordings here indicate this is a reversible process and that vesicle after osmotic shock are refilled with neurotransmitters when placed cells are reverted into an isotonic environment.

Osmotic stress affects exocytosis and the amount of neurotransmitter released during this process. We demonstrate how combining electrochemical methods together with transmission electron microscopy can be used to study the effect of extracellular osmotic pressure on exocytosis activity, vesicle quantal size, and the amount of neurotransmitter released during exocytosis.

Amperometry recording of cells subjected to osmotic shock show that secretory cells respond to this physical stress by reducing the exocytosis activity ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...

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 ...

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.

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 ...

Subretinal Transplantation of Human Embryonic Stem Cell-Derived Retinal Tissue in a Feline Large Animal Model

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and Leber Congenital Amaurosis (LCA) cause progressive and debilitating vision loss. There is an unmet need for therapies that can restore vision once photoreceptors have been lost. Transplantation of human pluripotent stem cell (hPSC)-derived retinal tissue (organoids) into the subretinal space of an eye with advanced RD brings retinal tissue sheets with thousands of healthy mutation-free photoreceptors and has a potential to treat most/all blinding diseases associated with photoreceptor degeneration with one approved protocol. Transplantation of fetal retinal tissue into the subretinal space of animal models and people with advanced RD has been developed successfully but cannot be used as a routine therapy due to ethical concerns and limited tissue supply. Large eye inherited retinal degeneration (IRD) animal models are valuable for developing vision restoration therapies utilizing advanced surgical approaches to transplant retinal cells/tissue into the subretinal space. The similarities in globe size, and photoreceptor distribution (e.g., presence of macula-like region area centralis) and availability of IRD models closely recapitulating human IRD would facilitate rapid translation of a promising therapy to the clinic. Presented here is a surgical technique of transplanting hPSC-derived retinal tissue into the subretinal space of a large animal model allowing assessment of this promising approach in animal models.

Presented here is a surgical technique for transplanting human pluripotent stem cell (hPSC)-derived retinal tissue into the subretinal space of a large animal model.

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and ...

High-Content Screening Differentiation and Maturation Analysis of Fetal and Adult Neural Stem Cell-Derived Oligodendrocyte Precursor Cell Cultures

The main hurdle in developing drug screening techniques for assessing the efficacy of therapeutic strategies in complex diseases is striking a balance between in vitro simplification and recreating the complex in vivo environment, along with the main aim, shared by all screening strategies, of obtaining robust and reliable data, highly predictive for in vivo translation.
In the field of demyelinating diseases, the majority of drug screening strategies are based on immortalized cell lines or pure cultures of isolated primary oligodendrocyte precursor cells (OPCs) from newborn animals, leading to strong biases due to the lack of age-related differences and of any real pathological condition or complexity.
Here we show the setup of an in vitro system aimed at modeling the physiological differentiation/maturation of neural stem cell (NSC)-derived OPCs, easily manipulated to mimic pathological conditions typical of demyelinating diseases. Moreover, the method includes isolation from fetal and adult brains, giving a system which dynamically differentiates from OPCs to mature oligodendrocytes (OLs) in a spontaneous co-culture which also includes astrocytes. This model physiologically resembles the thyroid hormone-mediated myelination and myelin repair process, allowing the addition of pathological interferents which model disease mechanisms. We show how to mimic the two main components of demyelinating diseases (i.e., hypoxia/ischemia and inflammation), recreating their effect on developmental myelination and adult myelin repair and taking all the cell components of the system into account throughout, while focusing on differentiating OPCs.
This spontaneous mixed model, coupled with cell-based high-content screening technologies, allows the development of a robust and reliable drug screening system for therapeutic strategies aimed at combating the pathological processes involved in demyelination and at inducing remyelination.

We describe the production of mixed cultures of astrocytes and oligodendrocyte precursor cells derived from fetal or adult neural stem cells differentiating into mature oligodendrocytes, and in vitro modeling of noxious stimuli. The coupling with a cell-based high-content screening technique builds a reliable and robust drug screening system.

The main hurdle in developing drug screening techniques for assessing the efficacy of therapeutic strategies in complex diseases is striking a balance ...