obtaining a mixed oligodendrocyte and astrocyte culture from adult mouse neural stem cells

Obtaining a Mixed Oligodendrocyte and Astrocyte Culture From Adult Mouse Neural Stem Cells

For neural stem cell isolation from the subventricular zone from 2.5-month-old adult mice, place brains from four to five adult mice into a 50-milliliter tube of ice-cold HBSS and place one brain ventral side down in the rostrocaudal direction on a sterile aluminum foil-covered flask filled with -20 degrees Celsius overnight cold water. Use a razor blade to remove the olfactory bulbs, and to cut two to three one-millimeter thick coronal slices from the cortex to the optical chiasma. Place the slices on the cold surface in a ventro-dorsal position, and identify the corpus callosum and the two lateral ventricles. Using magnification, isolate the walls of the lateral ventricles, taking care not to include pieces of the corpus callosum, and place the isolated tissue in 5 to 10 milliliters of enzymatic dissociation buffer for a 15-minute incubation at 37 degrees Celsius.
At the end of the incubation, pipette the tissues at least 50 times before incubating the samples at 37 degrees Celsius for an additional 10 minutes. At the end of the incubation, neutralize the trypsin with 5 milliliters of standard culture medium and filter the tissue suspension through a 70-micron filter. Centrifuge the filtered solution for five minutes at 400 times g and resuspend the pellet in sucrose solution for a second centrifugation. At the end of the centrifugation, resuspend the pellet in bovine serum albumin washing solution for another centrifugation, then resuspend the pellet in standard culture medium for counting and plate the cells in an upright T25 or T45 flask.
For primary neurosphere differentiation, add basic fibroblast and endothelial growth factors to the neural stem cell culture every two days. When the neurospheres reach a 100 to 500-micron diameter, passage the cells by mechanical dissociation according to standard protocols.
For oligosphere differentiation, treat the cells with basic fibroblast growth factor and platelet-derived factor AA every two days. When the oligospheres reach a 100 to 150-micron diameter, dissociate the spheres by mechanical dissociation according to standard protocols, and seed the cell suspension at a 3,000 cell per square centimeter density onto Poly-D/L-Ornithine laminin coated plates. After three days, replace the supernatant of each culture with the same volume of oligodendrocyte differentiation medium.

obtaining-mixed-oligodendrocyte-astrocyte-culture-from-adult-mouse

Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Primary Neuronal Culture

EoE Sub Articles

eoe sub articles

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Solutions and reagents
<ol>
	<li>Prepare standard medium: DMEM/F12 GlutaMAX 1x; 8 mmol/L HEPES; 100 U/100 &#956;g Penicillin/Streptomycin (1% P/S); 1x B27; 1x N-2.</li>
	<li>Prepare neurosphere medium: add 10 ng/mL Basic Fibroblast Growth Factor (bFGF); 10 ng/mL Epidermal Growth Factor (EGF) to standard medium.</li>
	<li>Prepare oligosphere/oligodendrocyte precursor cell (OPC) medium: add 10 ng/mL bFGF; 10 ng/mL Platelet Derived Growth Factor-AA (PDGF-AA) to standard medium.</li>
	<li>Prepare oligodendrocyte differentiation medium: add 50 nM Triiodothyronine (T3); 10 ng/mL Ciliary Neurotropic Factor (CNTF); 1x N-acetyl-L-cysteine (NAC) to standard medium.</li>
	<li>Prepare sucrose solution: Hanks&#39; Balanced Salt Solution (HBSS), 0.3 g/mL sucrose.</li>
	<li>Prepare Bovine serum albumin (BSA) washing solution: Earle&#39;s Balanced Salt Solution (EBSS), 40 mg/mL BSA, 0.02 mL/l HEPES.</li>
	<li>Prepare enzymatic dissociation buffer: HBSS, 5.4 mg/mL D-glucose, 15 mmol/L HEPES, 1.33 mg/mL Trypsin, 0.7 mg/mL Hyaluronidase, 80 U/mL DNase.</li>
</ol>
2. Dissection and NSC isolation
NOTE: Adult neural stem cells (NSCs) were isolated from 2.5-month-old adult sub-ventricular zone (SVZ).
<ol>
	<li>Adult NSC cultures
	<ol>
		<li>Sacrifice animals by cervical dislocation.</li>
		<li>Collect brains from 4&#8211;5 mice in a 50 mL tube containing ice cold HBSS.</li>
		<li>Place the brain on a cold sterile surface. For this purpose, use a T-25 flask filled with water and placed at -20 &#176;C overnight. At the time of the experiment, cover the flask with sterile aluminum foil.</li>
		<li>Place the brain ventral side downwards, in rostro-caudal direction, and remove the olfactory bulbs using a razor blade.</li>
		<li>Using a razor blade, cut 2&#8211;3 coronal slices of 1 mm thickness, from the cortex to the optical chiasma.</li>
		<li>Place the slices on the cold surface in a ventro-dorsal position and identify the corpus callosum and the two lateral ventricles.</li>
		<li>Using magnifying glasses or a stereoscope, isolate the walls of the lateral ventricles, taking care not to carry pieces of the corpus callosum.</li>
		<li>Put the isolated tissue in the enzymatic dissociation buffer (5&#8211;10 mL) and incubate at 37 &#176;C for 15 min.</li>
		<li>Mix the solution, pipetting several times (at least 50), and incubate again at 37 &#176;C for 10 min.</li>
		<li>Neutralize the trypsin by adding 5 mL of standard culture medium and filter the solution using a 70 &#181;m filter.</li>
		<li>Centrifuge the filtered solution for 5 min at 400 x g.</li>
		<li>Resuspend the pellet in the sucrose solution and centrifuge for 10 min at&#160;&#160; 500 x g.</li>
		<li>Resuspend the pellet in BSA washing solution and centrifuge for 7 min at 400 x g.</li>
		<li>Resuspend the pellet in the standard culture medium. Count the cells and plate them in suspension at a density of 10&#8211;50 cells/&#956;L in a T-25 or T-45 flask containing 10&#8211;30 mL of neurosphere medium, kept in a vertical position to avoid cell adhesion.</li>
	</ol>
	</li>
</ol>
3. Primary neurospheres
<ol>
	<li>Add the growth factors (bFGF/EGF) every 2 days.</li>
	<li>Every 4&#8211;6 days (depending on cell density), change half of the medium as follows:
	<ol>
		<li>Transfer the entire cell suspension to a 15 or 50 mL tube.</li>
		<li>Centrifuge for 5 min at 400 x g.</li>
		<li>Remove half of the volume.</li>
		<li>Add the same amount of fresh medium, gently mix by pipetting, and add growth factors.</li>
	</ol>
	</li>
</ol>
4. Oligospheres
<ol>
	<li>When the neurospheres reach a diameter of 100&#8211;150 &#181;m, they are ready to be passed. To do so, transfer the entire cell suspension to a 15 or 50 mL tube, and centrifuge for 5 min at 400 x g.
	<ol>
		<li>Rapidly evaluate the diameter by taking pictures of the spheres using an inverted transmitted light microscope and opening them by ImageJ software.</li>
		<li>Click on the Analyze menu and from the Tools window, select Scale bar.</li>
		<li>Set 150 &#181;m as Width in microns and compare the scale bar with the spheres.</li>
	</ol>
	</li>
	<li>Remove the entire volume by inversion and resuspend the pellet in 180 &#181;L of fresh standard culture medium. Pipette 50 times to allow disaggregation of the spheres.</li>
	<li>Add 810 &#181;L of fresh standard culture medium, count the cells, and re-plate them as described for the neurospheres.</li>
	<li>Add bFGF/PDGF-AA 10 ng/mL every 2 days</li>
	<li>Every 4&#8211;6 days (depending on cell density), change half of the medium as follows:</li>
	<li>Transfer the entire cell suspension to a 15 or 50 mL tube.</li>
	<li>Centrifuge for 5 min at 400 x g.</li>
	<li>Remove half of the volume.</li>
	<li>Add the same amount of fresh medium, gently mix by pipetting, and add growth factors.</li>
</ol>
5. Plate coating
<ol>
	<li>Poly-D,L-ornithine/laminin coating: at least 2 days before plating the OPCs, add 50 &#181;g/mL poly-D,L-ornithine solution, diluted in PBS, to each well (40 &#181;L/well for 96- well plates) and incubate at RT overnight.</li>
	<li>The following day, remove the liquid and wash three times with distilled sterile water.</li>
	<li>Let the plates dry at RT overnight. The following day, add a laminin solution diluted in PBS (5 &#181;g/mL; 40 &#181;L/well for 96-well plates) and incubate for 2 h at 37&#176;C.</li>
</ol>
6. Cell seeding
<ol>
	<li>When the oligospheres reach a diameter of 100&#8211;150 &#181;m, they are ready to be dissociated and seeded on the poly-D,L-ornithine/laminin coated plates. To do so, transfer the entire cell suspension to a 15 or 50 mL tube, and centrifuge for 5 min at 400 x g.</li>
	<li>Remove the entire volume by inversion and resuspend the pellet in 180 &#181;L of fresh standard culture medium. Pipette 50 times to allow disaggregation of the spheres.</li>
	<li>Add 810 &#181;L of fresh standard culture medium and count the cells.</li>
	<li>Remove the laminin solution from the wells and plate the cells at 3,000 cell/cm2 density (100 &#181;L/well for 96-well plates)</li>
</ol>
7. OPC differentiation induction
<ol>
	<li>After 3 days, remove the entire medium and add the same volume of oligodendrocyte differentiation medium.</li>
	<li>Change half of the medium every 4 days and add fresh differentiation mix (T3/CNTF/NAC) every 2 days.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96-well plates - untreated</td>
			<td>NUNC</td>
			<td>267313</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement (100x)</td>
			<td>GIBCO</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Basic Fibroblast Growth Factor (bFGF)</td>
			<td>GIBCO</td>
			<td>PHG0024</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>Ciliary Neurotropic Factor (CNTF)</td>
			<td>GIBCO</td>
			<td>PHC7015</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM w/o glucose</td>
			<td>GIBCO</td>
			<td>A14430-01</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 GlutaMAX</td>
			<td>GIBCO</td>
			<td>31331-028</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase</td>
			<td>Sigma-Aldrich</td>
			<td>D5025-150KU</td>
			<td></td>
		</tr>
		<tr>
			<td>EBSS</td>
			<td>GIBCO</td>
			<td>14155-048</td>
			<td></td>
		</tr>
		<tr>
			<td>Epidermal Growth Factor (EGF)</td>
			<td>GIBCO</td>
			<td>PHG6045</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>GIBCO</td>
			<td>14170-088</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>GIBCO</td>
			<td>15630-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyaluronidase&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>H3884</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>GIBCO</td>
			<td>23017-051</td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetyl-L-cysteine</td>
			<td>Sigma-Aldrich</td>
			<td>A9165</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 supplement (50x)</td>
			<td>GIBCO</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS&#160;</td>
			<td>GIBCO</td>
			<td>70011-036</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin / Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr>
			<td>Platelet Derived Growth Factor (PDGF-AA)</td>
			<td>GIBCO</td>
			<td>PHG0035</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D,L-ornitine</td>
			<td>Sigma-Aldrich</td>
			<td>P4957</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma-Aldrich</td>
			<td>T142</td>
			<td></td>
		</tr>
		<tr>
			<td>Triiodothyronine&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>T2752-1G</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Baldassarro, V. A. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/61988">High-Content Screening Differentiation and Maturation Analysis of Fetal and Adult Neural Stem Cell-Derived Oligodendrocyte Precursor Cell Cultures.</a> J. Vis. Exp. (2021)
This video demonstrates a detailed protocol for isolating the lateral ventricle walls from the adult mouse brain and performing enzymatic tissue dissociation to obtain single cells. These cells are then subjected to specific culture conditions that selectively enhance the proliferation of neural stem cells and promote their differentiation into co-cultures of oligodendrocytes and astrocytes.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Solutions and reagents

	Prepare ...

Take a coronal mouse brain slice.
Identify the lateral ventricles, the C-shaped cavities in the brain, and isolate the ventricle walls.
Place the tissue in a buffer containing trypsin to degrade the extracellular matrix, releasing the cells.
Centrifuge and remove the supernatant. Resuspend the cells in a medium.
Plate the cells and incubate them vertically to maintain them in suspension.
Add growth factors that induce neural stem cell proliferation and aggregation to form three-dimensional neurospheres.
Harvest the neurospheres, centrifuge, and discard the supernatant.
Add fresh medium and mechanically dissociate the neurospheres.
Plate the cells and add a medium containing growth factors that induce neural stem cell differentiation into oligodendrocyte precursors.
The precursors proliferate and form oligospheres.
Mechanically dissociate the oligospheres to release the precursors.
Seed the precursors in extracellular matrix protein-coated wells to facilitate cellular attachment.
Add a medium containing specific hormones and neurotrophic factors to induce precursor maturation into oligodendrocytes and astrocytes.

31 Views. Uzyskiwanie mieszanej kultury oligodendrocytów i astrocytów z dorosłych komórek macierzystych nerwowych myszy

Video: Uzyskiwanie mieszanej kultury oligodendrocytów i astrocytów z dorosłych komórek macierzystych nerwowych myszy - Experiment

Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration in vertebrates. However, an in-depth analysis of the molecular mechanisms underlying zebrafish adult neurogenesis has been limited due to the lack of a reliable protocol for isolating and culturing neural adult stem/progenitor cells. Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from zebrafish whole brain or from the telencephalon, tectum and cerebellum regions of the adult zebrafish brain. The protocol involves, first the microdissection of zebrafish adult brain, then single cell dissociation and isolation of self-renewing multipotent neural stem/progenitor cells. The entire procedure takes eight days. Additionally, we describe how to manipulate gene expression in zebrafish neurospheres, which will be particularly useful to test the role of specific signaling pathways during adult neural stem/progenitor cell proliferation and differentiation in zebrafish.

Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from the whole brain or from either the telencephalic, tectal or cerebellar regions of the adult zebrafish brain. Additionally, we describe the procedure to manipulate gene expression in zebrafish neurospheres.

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration ...

JoVE Journal

Developmental Biology

Using Primary Neurosphere Cultures to Study Primary Cilia

The primary cilium is fundamentally important for the proliferation of neural stem/progenitor cells and for neuronal differentiation during embryonic, postnatal, and adult life. In addition, most differentiated neurons possess primary cilia that house signaling receptors, such as G-protein-coupled receptors, and signaling molecules, such as adenylyl cyclases. The primary cilium determines the activity of multiple developmental pathways, including the sonic hedgehog pathway during embryonic neuronal development, and also functions in promoting compartmentalized subcellular signaling during adult neuronal function. Unsurprisingly, defects in primary cilium biogenesis and function have been linked to developmental anomalies of the brain, central obesity, and learning and memory deficits. Thus, it is imperative to study primary cilium biogenesis and ciliary trafficking in the context of neural stem/progenitor cells and differentiated neurons. However, culturing methods for primary neurons require considerable expertise and are not amenable to freeze-thaw cycles. In this protocol, we discuss culturing methods for mixed populations of neural stem/progenitor cells using primary neurospheres. The neurosphere-based culturing methods provide the combined benefits of studying primary neural stem/progenitor cells: amenability to multiple passages and freeze-thaw cycles, differentiation potential into neurons/glia, and transfectability. Importantly, we determined that neurosphere-derived neural stem/progenitor cells and differentiated neurons are ciliated in culture and localize signaling molecules relevant to ciliary function in these compartments. Utilizing these cultures, we further describe methods to study ciliogenesis and ciliary trafficking in neural stem/progenitor cells and differentiated neurons. These neurosphere-based methods allow us to study cilia-regulated cellular pathways, including G-protein-coupled receptor and sonic hedgehog signaling, in the context of neural stem/progenitor cells and differentiated neurons.

The primary cilium is fundamentally important in neural progenitor cell proliferation, neuronal differentiation, and adult neuronal function. Here, we describe a method to study ciliogenesis and the trafficking of signaling proteins to cilia in neural stem/progenitor cells and differentiated neurons using primary neurosphere cultures.

The primary cilium is fundamentally important for the proliferation of neural stem/progenitor cells and for neuronal differentiation during embryonic, ...

Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it contributes to disease development and progression, particularly in the gray matter of the brain, remains largely unknown. The dysfunction of oligodendrocyte lineage cells is hallmarked by deficiencies in myelination and impaired self-renewal of oligodendrocyte precursor cells (OPCs). These two defects are caused at least in part by the disruption of interactions between neuron and oligodendrocytes along the buildup of pathology. OPCs give rise to myelinating oligodendrocytes during CNS development. In the mature brain cortex, OPCs are the major proliferative cells (comprising ~5% of total brain cells) and control new myelin formation in a neural activity-dependent manner. Such neuron-to-oligodendrocyte communications are significantly understudied, especially in the context of neurodegenerative conditions such as AD, due to the lack of appropriate tools. In recent years, our group and others have made significant progress to improve currently available protocols to generate functional neurons and oligodendrocytes individually from human pluripotent stem cells. In this manuscript, we describe our optimized procedures, including the establishment of a co-culture system to model the neuron-oligodendrocyte connections. Our illustrative results suggest an unexpected contribution from OPCs/oligodendrocytes to the brain amyloidosis and synapse integrity and highlight the utility of this methodology for AD research. This reductionist approach is a powerful tool to dissect the specific hetero-cellular interactions out of the inherent complexity inside the brain. The protocols we describe here are expected to facilitate future studies on oligodendroglial defects in the pathogenesis of neurodegeneration.

The neuron-glial interactions in neurodegeneration are not well understood due to inadequate tools and methods. Here, we describe optimized protocols to obtain induced neurons, oligodendrocyte precursor cells, and oligodendrocytes from human pluripotent stem cells and provide examples of the values of these methods in understanding cell-type-specific contributions in Alzheimer&#8217;s disease.

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it ...

Obtaining a Mixed Oligodendrocyte and Astrocyte Culture From Adult Mouse Neural Stem Cells

Transkrypcja

Obtaining a Mixed Oligodendrocyte and Astrocyte Culture From Adult Mouse Neural Stem Cells