This research was supported by the Intramural Research Program at the National Institutes of Health, NINDS. The authors would like to thank all the members of the Laboratory of Molecular Medicine and Neuroscience, Rick Dreyfuss for helping with microscopy and Pamela C. Sieving for helping with the editing.

Note: For routine culturing of neural progenitor and oligodendrocytic lineage cells, incubation is done at 37 &deg;C in a humidified 5% CO2 atmosphere. Every 2 days, the medium is replaced using 50 to 100% of fresh medium if culture is 40-70% confluent. At the time of near confluency, the cultures are passaged at 2-2.5e6/T75 flask usually on a weekly schedule.
1. Preparing the Coated Flask
<ol>
	<li>To prepare coated flasks dilute 5 mg of poly-D-lysine (PDL) in 100 ml of deionized water (DI-water), then coat T75 flasks with 50 &mu;g/ml of PDL at room temperature (RT) in the dark. After 1&frac12; hr, aspirate the PDL and rinse the flasks once with DI-water. Let the flasks dry before seeding the cells (Table 1).</li>
</ol>
2. Starting Differentiation of Neural Progenitor Cells into Progenitor-derived Oligodendrocytes (PDO)
The protocol to isolate human CNS progenitor cells is described in a previous publication and it is not part of this protocol1. Human CNS progenitor cells were isolated from the telencephalon of an 8-week gestation fetal brain, obtained in accordance with NIH guidelines.
<ol>
	<li>Grow multipotential population of neural progenitor cells on PDL coated flasks in neurobasal medium supplemented with 25 ng/ml of bFGF and 20 ng/ml epidermal growth factor (EGF) (Figure 1A). Do not use them if higher than passage 11 (p11).</li>
	<li>Start to differentiate the neural progenitor cells when the culture is approximately 70% confluent.</li>
	<li>Make complete differentiation oligodendrocyte medium using serum-free DMEM/HAMS F12 1:1 medium supplemented with bovine serum albumin, L-glutamine, gentamicin, N2 components, T3, Shh, NT-3, bFGF, and PDGF-AA (Table 1). This medium will be defined as oligo medium with growth factors (oligo medium + GF).</li>
	<li>Remove progenitor media from the flasks, rinse cells once with phosphate-buffered saline (PBS), then add oligo medium + GF (details described in 2.3) to start oligodendrocyte differentiation. Only cells committed to the oligodendrocytic lineage will grow (Figure 1B). Culture the cells in this medium for 3 weeks.</li>
	<li>Every week, when the cells are at 90-95% confluence, passage them using trypsin (0.05%)-EDTA (0.1%) (4 ml for a T75 flask). Transfer the entire medium from the cells to be passaged into a 50 ml conical tube; this conditioned medium will be used to quench the trypsin. Make sure not to dry out the cells. Then add trypsin.</li>
	<li>Incubate cells in trypsin for 5 min at RT, gently tapping the flask several times to help cell detachment.</li>
	<li>Quench the trypsin by adding 4-5 ml of conditioned medium to the flask with the detached cells.</li>
	<li>Transfer the medium and the cells to the same 50 ml conical tube. Centrifuge the medium and the cells at 1,200 rpm (~300 x g) for 5 min at RT.</li>
	<li>Aspirate the supernatant, resuspend the cell pellet, gently tipping the tube, and then add fresh oligo medium + GF . Gently pipette medium and cells up and down to make a uniform cell suspension.</li>
	<li>Count the cells, and transfer 2.5e6 into each new PDL-coated T75 flask.</li>
</ol>
3. Final Step in the Differentiation of Oligodendrocytes
<ol>
	<li>After 3 weeks of culture in oligo medium + GF, when the cells are at 70-80% confluence, start the final process of differentiation (Figure 1C).</li>
	<li>Prepare medium in the same way as the oligo medium + GF but without adding the four GF: Shh, NT-3, bFGF and PDGF-AA. This medium without the four GF will be defined as oligo medium - GF.</li>
	<li>Aspirate the oligo medium + GF from the flask, rinse once with PBS and then add a total volume of 16 ml of medium, of which &frac34; (12 ml) is oligo medium - GF and &frac14; (4 ml) is PDN conditioned medium, for 6-10 days). The use of conditioned medium from PDN is not critical to the differentiation process or to the survival of the cells. We in fact observed that only direct contact of PDO with PDN cells in co-culture experiments had an effect on the length of the PDO survival. The survival time in oligo medium - GF could be prolonged to two or three weeks if the PDO cells are co-cultured with PDN cells. The protocol to differentiate human neural progenitor cells into PDN is described in a previous publication and it is not part of this protocol1. Growth factor withdrawal from the culture medium results in progressively differentiated culture of cells with multiple processes (Figure 1D).</li>
</ol>
4. Flow Cytometry Assay
The flow cytometry assay compares the acquisition of oligodendrocyte markers during the differentiation process in relationship with those of their parental population.
<ol>
	<li>Use neural progenitors as control. Seed PDO cells at 2.5x106 cells in two T75 flasks coated with poly-D-lysine in oligo medium + GF.</li>
	<li>A week later, replace medium with fresh oligo medium + GF in flasks as a control culture for cell viability, while other flasks had oligo medium - GF.</li>
	<li>To dissociate PDO cells in both media use 20 U/ml of papain and not trypsin. This is because papain has a gentler effect on the cells during the dissociation preserving the cellular morphology.</li>
	<li>Add 5 ml of Earle's Balanced Salt Solution (EBSS) to a papain vial. Place the vial at 37 &deg;C for 10 min or until the papain is completely dissolved and the solution appears clear. This is the papain solution used to dissociate the cells.</li>
	<li>Add 0.5 ml of EBSS to a Deoxyribonuclease I (DNase) vial (Table 1). Mix gently. Add 0.25 ml of this solution to the vial of dissolved papain. This preparation contains a final concentration of approximately 20 U/ml papain and 0.005% DNase.</li>
	<li>Place papain solution (as described above) on the cells and incubate at 37 &deg;C for 15-30 min; monitor the detachment of the cells using a microscope. Stop the reaction with 5 ml of medium with or without GF and centrifuge at 1,200 rpm for 7 min.</li>
	<li>Transfer cell pellets from both flasks into two different 5 ml polypropylene tubes and wash them with normal physiological medium (NPM: 145 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 10 mM Hepes and 10 mM glucose) supplemented with 1 mg/ml bovine serum albumin (NPM+BSA).</li>
	<li>Perform surface immunostaining at 4 &deg;C for 20 min using specific and appropriately titrated primary antibodies for A2B5, O4, GalC (Table 2).</li>
	<li>Wash cells with NPM+BSA at 4 &deg;C for 5 min and incubate them with specific secondary antibodies at 4 &deg;C for 20 min (Table 2).</li>
	<li>To stain intracellular antigens including nestin, glial fibrillary acidic protein (GFAP), class III &beta;-tubulin, and myelin basic protein (MBP) (Table 2), fix cells in 2% paraformaldehyde (PFA) for 20 min at RT and permeabilize with cold 70% ethanol for 15 min at -20 &deg;C.</li>
	<li>Wash cells with 1x PBS and incubate with specific secondary antibodies (Table 2).</li>
	<li>To discriminate between intact cells and subcellular debris during flow cytometry analysis, stain cells with 4,6-diamidino-2-phenyllindole, dihydrochloride (DAPI) (Life Technologies, Grand Island, NY).</li>
	<li>In optimizing the above protocol, we performed all the appropriate control experiments to confirm the specificity of each immunoreagent. Briefly, for directly labeled antibodies, the appropriate negative control was a directly conjugated antibody of the same immunoglobulin isotype class (or subclass) and fluorochrome conjugate as the primary antibody (i.e., the isotype control). For indirect immunostaining of primary antibodies, the appropriate negative control was the secondary antibody conjugated to the fluorochrome of interest that specifically targeted the immunoglobulin class (or subclass) of the host in which the primary antibody was generated. When primary antibodies from multiple hosts (mouse, rabbit, chicken) were used together, we used the secondary antibody targeting the primary antibody of each host and each secondary antibody used was typically cross-adsorbed against the immunoglobulins from the other hosts to minimize cross-host immunoreactivity. The positive controls included direct or indirect immunostaining of cell preparations known to express the antigens of interest using either direct or indirect immunoreactions with the primary antibodies specific for these antigens. The above optimization methods were carried out once for each type of immunostaining experiments. Control immunoreactions revealed no significant cross-epitope immunoreactivity among primary and secondary antibodies. Flow cytometry was performed on uniformly suspended fluorescently labeled cells using a FACVantage SE cell sorter (BD Biosciences, San Jose, CA) equipped with three lasers, which provide excitation wavelengths tuned to 488 nm, 647 nm, and a broad UV (351-364 nm) (Figure 2).</li>
</ol>
5. Immunofluorescence Assay
Note: For immunofluorescence experiments, PDO are plated in oligo medium + GF or oligo medium - GF at 2.5e5 in 6 well plates coated with PDL. The cells are fixed in 2% PFA at various time points after withdrawal of growth factors . The antibody stain is performed on plastic 6 well plates instead of glass coverslips.
<ol>
	<li>Remove media and add the 2% PFA. Incubate for 10 min at RT. Discard PFA. Wash cells 3 times with PBS, 5 min each time. Be careful not to dry out the cells.</li>
	<li>Permeabilize cells for intracellular markers using 0.25% triton solution in PBS for 10 min at RT.</li>
	<li>To prevent non-specific binding, block cells for 10 min with HHG (1 mM HEPES buffer, 2% horse serum, and 10% goat serum in Hanks' balanced salt solution). Dilute specific primary antibodies (Table 2) to a pre-determined concentration in HHG. Place diluted antibodies onto cells. Incubate 1 hr at RT on the shaker or at 4 &deg;C overnight.</li>
	<li>Wash cells 3 times with PBS, for 5 min each time. Dilute appropriate secondary antibodies (Table 2) coupled with a fluorochrome to a pre-determined concentration in HHG. Place the mixture of diluted secondary antibodies onto cells. Incubate 1 hr at RT on a shaker in the dark.</li>
	<li>Wash cells 3 times with PBS, for 5 min each time.</li>
	<li>To avoid photobleaching add 10 &mu;l of Prolong Gold with DAPI to the cells and place a glass coverslip on top of them. Visualize labeled cells using an Axiovert 200M fluorescence microscope (Zeiss, Thornwood, NY) (Figure 3).</li>
</ol>

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No conflicts of interest declared.

This protocol describes how to derive fetal oligodendrocytes from primary human neural progenitor cells and characterize their phenotype using both flow cytometry and immunofluorescence staining. The expansion and growth of neural progenitors from fetal CNS has been very well described1-4. However, obtaining sufficient human oligodendrocytes for in vitro experimentation remains difficult, even though it is possible to identify and isolate glial precursors from adult human white matter5-13</s...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Final concentration</td>
 </tr>
 <tr>
 <td>DME/HAMS F12 1:1</td>
 <td>Omega Scientist</td>
 <td>DM-251</td>
 <td>1X</td>
 </tr>
 <tr>
 <td>Bovine Albumin</td>
 <td>Sigma</td>
 <td>A9418</td>
 <td>1%</td>
 </tr>
 <tr>
 <td>Gentamicin</td>
 <td>Quality Biologicals</td>
 <td>120-098-031</td>
 <td> 50 &mu;g/ml</td>
 </tr>
 <tr>
 <td>L-Glutamine</td>
 <td>Quality Biologicals</td>
 <td>118-084-061</td>
 <td>2 mM</td>
 </tr>
 <tr>
 <td>T3</td>
 <td>Sigma</td>
 <td>T2877</td>
 <td>3 nM</td>
 </tr>
 <tr>
 <td>N2 Components</td>
 <td>Gibco BRL</td>
 <td>17502</td>
 <td>1:100</td>
 </tr>
 <tr>
 <td>NT-3</td>
 <td>PeproTech Inc</td>
 <td>450-03</td>
 <td>2 ng/ml</td>
 </tr>
 <tr>
 <td>Shh</td>
 <td>R&amp;D System</td>
 <td>1314-SH/CF</td>
 <td>2 ng/ml</td>
 </tr>
 <tr>
 <td>bFGF</td>
 <td>PeproTech Inc</td>
 <td>100-18B</td>
 <td>20 ng/ml</td>
 </tr>
 <tr>
 <td>PDGF-AA</td>
 <td>PeproTech Inc</td>
 <td>100-13A</td>
 <td>10 ng/ml</td>
 </tr>
 <tr>
 <td>PDL</td>
 <td>Sigma</td>
 <td>P6407</td>
 <td>50 &mu;g/m</td>
 </tr>
 <tr>
 <td>PFA</td>
 <td>Electron Microscopy Sciences</td>
 <td>15712</td>
 <td>2%</td>
 </tr>
 <tr>
 <td>Trypsin</td>
 <td>Quality Biologicals</td>
 <td> 118-087-721</td>
 <td></td>
 </tr>
 <tr>
 <td>Papain</td>
 <td>Worthington</td>
 <td>LK003178</td>
 <td>20 U/ml</td>
 </tr>
 <tr>
 <td>DNase vials</td>
 <td>Worthington</td>
 <td>LK003172</td>
 <td>0.005%</td>
 </tr>
 <tr>
 <td>EBSS</td>
 <td>Worthington</td>
 <td>LK003188</td>
 <td></td>
 </tr>
 <tr>
 <td>ProLong Gold with DAPI</td>
 <td>Invitrogen</td>
 <td>P36931</td>
 <td></td>
 </tr>
 <tr>
 	<td></td>
 <td></td>
 <td></td>
 <td>Table 1. Reagents.</td>
 </tr>
 <tr>
 	<td></td>
 <td></td>
 <td></td>
 <td>
 <table border="1">
 <tbody>
 <tr>
 <td>Primary Abs(all at 1 &mu;g/ml)</td>
 <td></td>
 <td>Species Isotype</td>
 <td>Source </td>
 <td>Secondary Abs</td>
 </tr>
 <tr>
 <td colspan="5">Flow Cytometry</td>
 </tr>
 <tr>
 <td>A2B5-Biotin </td>
 <td>Mouse </td>
 <td> IgM </td>
 <td>Gift J. Nielson </td>
 <td> Streptavidin PETR, Invitrogen, CA </td>
 </tr>
 <tr>
 <td>O4FITC </td>
 <td>Mouse </td>
 <td> IgM </td>
 <td> Gift from J. Nielson</td>
 <td></td>
 </tr>
 <tr>
 <td>A2B5 </td>
 <td>Mouse </td>
 <td>IgM </td>
 <td>Millipore, MA </td>
 <td>g&alpha;m IgM-FITC, Invitrogen, CA</td>
 </tr>
 <tr>
 <td>O4 </td>
 <td>Mouse </td>
 <td>IgM</td>
 <td> Millipore, MA </td>
 <td> g&alpha;m IgM-FITC, Invitrogen, CA</td>
 </tr>
 <tr>
 <td>GalC </td>
 <td>Mouse </td>
 <td> IgG3</td>
 <td> Millipore, MA </td>
 <td> g&alpha;m IgG3-PE, Southern Biotech, AL</td>
 </tr>
 <tr>
 <td>MBP</td>
 <td>Chicken </td>
 <td>IgY </td>
 <td> Millipore, MA </td>
 <td> d&alpha;ck IgY-AMCA, Jackson Immu., PA </td>
 </tr>
 <tr>
 <td>Nestin </td>
 <td>Mouse </td>
 <td>IgG1 </td>
 <td> Messam et al. 200028</td>
 <td>g&alpha;m IgG1-PECy5, Invitrogen, CA</td>
 </tr>
 <tr>
 <td>GFAP </td>
 <td> Rabbit </td>
 <td>IgG </td>
 <td> Millipore, MA </td>
 <td> g&alpha;rb IgG-PE, Jackson Immu, PA </td>
 </tr>
 <tr>
 <td>&beta;III tubulin </td>
 <td> Mouse </td>
 <td>IgG2 </td>
 <td>Covance, CA </td>
 <td> g&alpha;m IgG2a-PETR, Invitrogen, CA</td>
 </tr>
 <tr>
 <td colspan="6">Immunocytochemistry</td>
 </tr>
 <tr>
 <td>&beta;III tubulin (1:1,500) </td>
 <td>Mouse </td>
 <td> IgG2a </td>
 <td> Covance, CA </td>
 <td> g&alpha;m IgG2a-FITC, Invitrogen, CA (1:1,000)</td>
 </tr>
 <tr>
 <td>GFAP 
 (1:1,000) </td>
 <td>Rabbit </td>
 <td> IgG </td>
 <td> Millipore, MA </td>
 <td>g&alpha;rb IgG-FITC, Jackson Immu, PA 
 (1:500)</td>
 </tr>
 <tr>
 <td>MBP 
 (1:50)</td>
 <td>Chicken </td>
 <td> IgY </td>
 <td> Millipore, MA </td>
 <td>d&alpha;ck IgY-FITC, Jackson Immu, PA 
 (1:100) </td>
 </tr>
 <tr>
 <td>O4 
 (1:100) </td>
 <td>Mouse </td>
 <td> IgM </td>
 <td> Millipore, MA </td>
 <td> g&alpha;m IgM-AF546, Invitrogen, CA 
 (1:100)</td>
 </tr>
 <tr>
 <td>GalC 
 (1:10) </td>
 <td>Rabbit </td>
 <td>IgG </td>
 <td>Millipore, MA </td>
 <td>g&alpha;m IgM-AF750, Invitrogen, CA 
 (1:100)</td>
 </tr>
 </tbody>
</table>
Table 2. Antibodies (Abs) used for flow cytometry and immunocytochemistry assays. Antibody conjugates: PE, phycoerythrin; PETR, phycoerythrin Texas Red; AMCA, amino-methyl-coumarin-acetate; Cy, cyanine; FITC, fluorescein isothiocyanate. Ig: immunoglobulin. AF: Alexa Fluor; g&#x03B1;m: goat anti-mouse; g&#x03B1;rb: goat anti-rabbit; d&#x03B1;ck: donkey anti-chicken.</td>
 </tr>
 </tbody>
</table>

progenitor-derived oligodendrocyte culture system from human fetal brain

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell lineage development. In vitro expansion of neural progenitors from fetal CNS tissue has been well characterized. Despite the identification and isolation of glial progenitors from adult human sub-cortical white matter and development of various culture conditions to direct differentiation of fetal neural progenitors into myelin producing oligodendrocytes, acquiring sufficient human oligodendrocytes for in vitro experimentation remains difficult. Differentiation of galactocerebroside+ (GalC) and O4+ oligodendrocyte precursor or progenitor cells (OPC) from neural precursor cells has been reported using second trimester fetal brain. However, these cells do not proliferate in the absence of support cells including astrocytes and neurons, and are lost quickly over time in culture. The need remains for a culture system to produce cells of the oligodendrocyte lineage suitable for in vitro experimentation.
Culture of primary human oligodendrocytes could, for example, be a useful model to study the pathogenesis of neurotropic infectious agents like the human polyomavirus, JCV, that in vivo infects those cells. These cultured cells could also provide models of other demyelinating diseases of the central nervous system (CNS). Primary, human fetal brain-derived, multipotential neural progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons (progenitor-derived neurons, PDN) and astrocytes (progenitor-derived astrocytes, PDA) This study shows that neural progenitors can be induced to differentiate through many of the stages of oligodendrocytic lineage development (progenitor-derived oligodendrocytes, PDO). We culture neural progenitor cells in DMEM-F12 serum-free media supplemented with basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF-AA), Sonic hedgehog (Shh), neurotrophic factor 3 (NT-3), N-2 and triiodothyronine (T3). The cultured cells are passaged at 2.5e6 cells per 75cm flasks approximately every seven days. Using these conditions, the majority of the cells in culture maintain a morphology characterized by few processes and express markers of pre-oligodendrocyte cells, such as A2B5 and O-4. When we remove the four growth factors (GF) (bFGF, PDGF-AA, Shh, NT-3) and add conditioned media from PDN, the cells start to acquire more processes and express markers specific of oligodendrocyte differentiation, such as GalC and myelin basic protein (MBP). We performed phenotypic characterization using multicolor flow cytometry to identify unique markers of oligodendrocyte.

It is very important to start the differentiation process from a 70%-80% confluent neural progenitor cell culture (Figure 1A). Many cells will die out after changing the culture medium from progenitor to oligo medium since it includes specific growth factors. This indicates that the growth of neural progenitor cells not committed to an oligodendrocytic phenotype will not be supported by the new medium (Figure 1B). Incubation in oligo medium + GF for one week resulted in an interm...

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Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Primary, human fetal brain-derived, multipotential progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons and astrocytes. This work shows that neural progenitors can be induced to differentiate through stages of the oligodendrocytic lineage by conditioning with select growth factors.

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell ...

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Neuroscience

14.8K Views.  National Institute of Neurological Disorders and Stroke, National Institutes of Health. Primary, human fetal brain-derived, multipotential progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons and astrocytes. This work shows that neural progenitors can be induced to differentiate through stages of the oligodendrocytic lineage by conditioning with select growth factors.

Video: Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. 
Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications for understanding the pathogenesis of demyelinating diseases such as Multiple Sclerosis (MS). Cellular development is commonly studied with primary cell culture models. Primary cell culture facilitates the evaluation of a given cell type by providing a controlled environment, free of the extraneous variables that are present in vivo. While OL cultures derived from rats have provided a vast amount of insight into OL biology, similar efforts at establishing OL cultures from mice has been met with major obstacles. Developing methods to culture murine primary OLs is imperative in order to take advantage of the available transgenic mouse lines. 
Multiple methods for extraction of OPCs from rodent tissue have been described, ranging from neurosphere derivation, differential adhesion purification and immunopurification 1-3. While many methods offer success, most require extensive culture times and/or costly equipment/reagents. To circumvent this, purifying OPCs from murine tissue with an adaptation of the method originally described by McCarthy &amp; 
de Vellis 2 is preferred. This method involves physically separating OPCs from a mixed glial culture derived from neonatal rodent cortices. The result is a purified OPC population that can be differentiated into an OL-enriched culture. This approach is appealing due to its relatively short culture time and the unnecessary requirement for growth factors or immunopanning antibodies. 
While exploring the mechanisms of OL development in a purified culture is informative, it does not provide the most physiologically relevant environment for assessing myelin sheath formation. Co-culturing OLs with neurons would lend insight into the molecular underpinnings regulating OL-mediated myelination of axons. For many OL/neuron co-culture studies, dorsal root ganglion neurons (DRGNs) have proven to be the neuron type of choice. They are ideal for co-culture with OLs due to their ease of extraction, minimal amount of contaminating cells, and formation of dense neurite beds. While studies using rat/mouse myelinating xenocultures have been published 4-6, a method for the derivation of such OL/DRGN myelinating co-cultures from post-natal murine tissue has not been described. Here we present detailed methods on how to effectively produce such cultures, along with examples of expected results. These methods are useful for addressing questions relevant to OL development/myelinating function, and are useful tools in the field of neuroscience.

This article describes methods to derive enriched populations of murine oligodendrocyte precursor cells (OPCs) in primary culture, which differentiate to produce mature oligodendrocytes (OLs). In addition, this report describes techniques to produce murine myelinating co-cultures by seeding mouse OPCs onto a neurite bed of mouse dorsal root ganglion neurons (DRGNs).

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

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

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

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

A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as &#8220;chopping&#8221; that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.

This protocol describes a novel mechanical chopping method that allows the expansion of spherical neural stem and progenitor cell aggregates without dissociation to a single cell suspension.&#160; Maintaining cell/cell contact allows rapid and stable growth for over 40 passages.

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the ...

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or postmitotic neurons. Later, some progenitors switch to a gliogenic fate, adding to the astrocyte and oligodendrocyte populations. Using time-lapse video-microscopy of primary cerebral cortex cell cultures, it is possible to study the cellular and molecular mechanisms controlling the mode of cell division and cell cycle parameters of progenitor cells. Similarly, the fate of postmitotic cells can be examined using cell-specific fluorescent reporter proteins or post-imaging immunocytochemistry. More importantly, all these features can be analyzed at the single-cell level, allowing the identification of progenitors committed to the generation of specific cell types. Manipulation of gene expression can also be performed using viral-mediated transfection, allowing the study of cell-autonomous and non-cell-autonomous phenomena. Finally, the use of fusion fluorescent proteins allows the study of symmetric and asymmetric distribution of selected proteins during division and the correlation with daughter cells fate. Here, we describe the time-lapse video-microscopy method to image primary cerebral cortex murine cells for up to several days and analyze the mode of cell division, cell cycle length and fate of newly generated cells. We also describe a simple method to transfect progenitor cells, which can be applied to manipulate genes of interest or simply label cells with reporter proteins.

Live imaging is a powerful tool to study cellular behaviors in real time. Here, we describe a protocol for time-lapse video-microscopy of primary cerebral cortex cells that allows a detailed examination of the phases enacted during the lineage progression from primary neural stem cells to differentiated neurons and glia.

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

Generation of Oligodendrocytes and Oligodendrocyte-Conditioned Medium for Co-Culture Experiments

In the central nervous system, oligodendrocytes are well-known for their role in axon myelination, that accelerates the propagation of action potentials through saltatory conduction. Moreover, an increasing number of reports suggest that oligodendrocytes interact with neurons beyond myelination, notably through the secretion of soluble factors. Here, we present a detailed protocol allowing purification of oligodendroglial lineage cells from glial cell cultures also containing astrocytes and microglial cells. The method relies on overnight shaking at 37 &#176;C, which allows selective detachment of the overlying oligodendroglial cells and microglial cells, and the elimination of microglia by differential adhesion. We then describe the culture of oligodendrocytes and production of oligodendrocyte-conditioned medium (OCM). We also provide the kinetics of OCM treatment or oligodendrocytes addition to purified hippocampal neurons in co-culture experiments, studying oligodendrocyte-neuron interactions.

Herein, we display an efficient method for the purification of oligodendrocytes and production of oligodendrocyte-conditioned medium that can be used for co-culture experiments.

In the central nervous system, oligodendrocytes are well-known for their role in axon myelination, that accelerates the propagation of action potentials ...

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine neonates for the isolation of glial subpopulations. The purpose of the procedure is to dissociate microglia, oligodendrocyte progenitor cells (OPCs), and astrocytes from cortical, cerebellar, brainstem, and spinal cord tissue to facilitate further in vitro analysis. The CNS region isolation procedures allow for the determination of regional heterogeneity among glia in multiple cell culture systems. Rapid CNS region isolation is performed, followed by the mechanical removal of meninges to prevent meningeal cell contamination of glia. This protocol combines gentle tissue dissociation and plating on a specified matrix designed to preserve cell integrity and adherence. Isolating mixed glia from multiple CNS regions provides a comprehensive analysis of potentially heterogenous glia while maximizing the use of individual experimental animals. Additionally, following dissociation of regional tissue, mixed glia are further divided into multiple cell types including microglia, OPCs, and astrocytes for use in either single cell type, cell culture plate inserts, or co-culture systems. Overall, the demonstrated techniques provide a comprehensive protocol of broad applicability for careful dissection of four individual CNS regions from murine neonates and includes methods for the isolation of three individual glia cell types to examine regional heterogeneity in any number of in vitro cell culture systems or assays.

Here we present a protocol for in vitro isolation of multiple glial cell populations from a mouse CNS. This method allows for the segregation of regional microglia, oligodendrocyte precursor cells, and astrocytes to study the phenotypes of each in a variety of culture systems.

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine ...

Obtaining Human Microglia from Adult Human Brain Tissue

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining homeostasis, and during infection or injury. Several independent research groups have highlighted the central role that microglia play in autoimmune diseases, autoinflammatory syndromes and cancers. The activation of microglia in some neurological diseases may directly participate in pathogenic processes. Primary microglia are a powerful tool to understand the immune responses in the brain, cell-cell interactions and dysregulated microglia phenotypes in disease. Primary microglia mimic in vivo microglial properties better than immortalized microglial cell lines. Human adult microglia exhibit distinct properties as compared to human fetal and rodent microglia. This protocol provides an efficient method for isolation of primary microglia from adult human brain. Studying these microglia can provide critical insights into cell-cell interactions between microglia and other resident cellular populations in the CNS including, oligodendrocytes, neurons and astrocytes. Additionally, microglia from different human brains may be cultured for characterization of unique immune responses for personalized medicine and a myriad of therapeutic applications.

This protocol is an efficient, cost effective and robust method of isolating primary microglia from live, adult, human brain tissue. Isolated primary human microglia can serve as a tool for studying cellular processes in homeostasis and disease.

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining ...

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 ...