Name: derivation of enriched oligodendrocyte cultures and oligodendrocyte/neuron myelinating co-cultures from post-natal murine tissues
Uploaded: 2011-08-21T12:00:00
Duration: 18 min 17 s
Description: Watch this Scientific Journal Video about Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues at JoVE.com

This project was funded by a grant from the Multiple Sclerosis Society of Canada to R.K. R.W.O is a recipient of a Studentship from the Multiple Sclerosis Society of Canada. S.D.R is a recipient of Post-Doctoral Fellowships from the Multiple Sclerosis Society of Canada and Canadian Institutes of Health Research.

<ol>
	<li>Avellana-Adalid, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+of+rat+oligodendrocyte+progenitors+into+proliferative+"oligospheres"+that+retain+differentiation+potential.">Expansion of rat oligodendrocyte progenitors into proliferative "oligospheres" that retain differentiation potential.</a> J Neurosci Res. 45, 558-570 (1996).</li><li>McCarthy, K. D., de Vellis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+separate+astroglial+and+oligodendroglial+cell+cultures+from+rat+cerebral+tissue.">Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue.</a> J Cell Biol. 85, 890-902 (1980).</li><li>Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+death+and+control+of+cell+survival+in+the+oligodendrocyte+lineage.">Cell death and control of cell survival in the oligodendrocyte lineage.</a> Cell. 70, 31-46 (1992).</li><li>Chan, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NGF+controls+axonal+receptivity+to+myelination+by+Schwann+cells+or+oligodendrocytes.">NGF controls axonal receptivity to myelination by Schwann cells or oligodendrocytes.</a> Neuron. 43, 183-1891 (2004).</li><li>Camara, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin-mediated+axoglial+interactions+initiate+myelination+in+the+central+nervous+system.">Integrin-mediated axoglial interactions initiate myelination in the central nervous system.</a> J Cell Biol. 185, 699-712 (2009).</li><li>Ishibashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes+promote+myelination+in+response+to+electrical+impulses.">Astrocytes promote myelination in response to electrical impulses.</a> Neuron. 49, 823-832 (2006).</li><li>Chen, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+rat+and+mouse+oligodendrocyte+precursor+cells.">Isolation and culture of rat and mouse oligodendrocyte precursor cells.</a> Nat Protoc. 2, 1044-1051 (2007).</li><li>Pedraza, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production,+characterization,+and+efficient+transfection+of+highly+pure+oligodendrocyte+precursor+cultures+from+mouse+embryonic+neural+progenitors.">Production, characterization, and efficient transfection of highly pure oligodendrocyte precursor cultures from mouse embryonic neural progenitors.</a> Glia. 56, 1339-1352 (2008).</li><li>Lewin, G. R., Ritter, A. M., Mendell, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+role+of+nerve+growth+factor+in+the+development+of+myelinated+nociceptors.">On the role of nerve growth factor in the development of myelinated nociceptors.</a> J Neurosci. 12, 1896-1905 (1992).</li><li>Greene, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+in+vitro+studies+on+the+nerve+growth+factor+(NGF)+requirement+of+neurons.+II.+Sensory+neurons.">Quantitative in vitro studies on the nerve growth factor (NGF) requirement of neurons. II. Sensory neurons.</a> Dev Biol. 58, 106-113 (1977).</li></ol>

No conflicts of interest declared.

This report describes a method for isolating murine OPCs for differentiation in OL-enriched cultures or OL/DRGN co-cultures. When cultured alone, the OPCs differentiate into MBP+ve OLs, producing myelin-like membranous sheets. When added to DRGN neurite beds, OLs enwrap the DRGN neurites with MBP+ve membrane. This model benefits the investigation of the complex underpinnings governing OL-mediated axonal ensheathment.
While of great value, the establishment of such cultures is technically challenging. In particular, demanding aspects include efficient tissue digestion/dissociation, maintenance of balanced culture media pH, and DRGN media changes. It is important to consider that the length of digestion, amount of tissue being digested and the amount of trituration affects the efficacy and end result of the tissue dissociation. It is not unusual for experienced researchers to obtain low cellular yields from dissociated nervous system tissues. In addition, murine OPCs tend to be sensitive to changes in the pH of the culture media, particularly under alkaline conditions. The maintenance of cultures at 8.5% CO2 aims to prevent this, since OPCs appear to better tolerate slightly acidic conditions over basic. With regards to feeding DRGNs, media changes must be performed quickly as to not desiccate the neurons, however, must be gentle as to not disrupt the developing neurite bed. Abrupt media changes may dislodge the neurite bed from the substrate, and likely result in its complete dissociation from the coverslip.
The potential merit of this model system greatly overshadows its technically demanding nature. One advantage of this system is the use of post-natal mice for cell culture derivation, circumventing the need to sacrifice breeding females to harvest embryonic tissue. Another advantage is the lack of requirement for growth factors (GFs) for the expansion of OPCs. Mixed glial cultures provide an environment that supports the propagation of OPCs, presumably due to the presence of astrocyte-derived trophic factors. Other methods, such as derivation via neurospheres7,8, rely on the mitogenic properties of GFs such as basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) for OPC expansion. Similarly, using postnatal (P5-10) mice for DRGNs avoids the requirement of supplementing the culture media with nerve growth factor (NGF), a neurotrophic factor required for the in vitro survival of embryonic DRGNs 9, 10. It is of interest to avoid using NGF as it negatively influences the myelinating capacity of OLs when cultured with DRGNs 4. Avoiding the use of GF-supplemented media also has economic benefits, as these reagents become costly when used on large scale.
Perhaps the most important benefit of this culture model is its derivation from mouse-only tissues, thus providing the opportunities to derive both OPCs and DRGNs from the wide variety of transgenic mouse lines. This allows for the study of both DRGN and/or OPC-specific properties that govern myelination. This will be especially important for elucidating the receptor/ligand interactions regulating OL-mediated myelination of axons. In all, this technique is of great value with regards to neuroscience research due to its applications towards understanding the molecular cues underlying myelination.

<table border="1">
	<tbody>
		<tr>
			<td>Product Name</td>
			<td>Company</td>
			<td>Product Number</td>
		</tr>
		<tr>
			<td>Dulbecco's Modified Eagle Medium (DMEM)</td>
			<td>Multicell</td>
			<td>319-005-CL</td>
		</tr>
		<tr>
			<td>Hank's Balanced Salt Solution (HBSS)</td>
			<td>Invitrogen</td>
			<td>14170-112</td>
		</tr>
		<tr>
			<td>Minimum Essential Media (MEM)</td>
			<td>GIBCO</td>
			<td>12360-038</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>GIBCO</td>
			<td>10091-148</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (Pen/Strep)</td>
			<td>GIBCO</td>
			<td>15140-122</td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Invitrogen</td>
			<td>35050-061</td>
		</tr>
		<tr>
			<td>Poly-l-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P2636</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A4503</td>
		</tr>
		<tr>
			<td>Human merosin purified protein (LN2)</td>
			<td>Millipore</td>
			<td>CC085</td>
		</tr>
		<tr>
			<td>Recombinant rat ciliary neurotrophic factor (CNTF)</td>
			<td>PeproTech</td>
			<td>450-50</td>
		</tr>
		<tr>
			<td>L-thyroxine</td>
			<td>Biochemika</td>
			<td>89430</td>
		</tr>
		<tr>
			<td>Holo-transferrin</td>
			<td>Sigma-Aldrich</td>
			<td>T0665</td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>GIBCO</td>
			<td>0080085-SA</td>
		</tr>
		<tr>
			<td>Bovine insulin</td>
			<td>Sigma-Aldrich</td>
			<td>I6634</td>
		</tr>
		<tr>
			<td>3,3',5-Triiodo-L-thyronine</td>
			<td>Sigma-Aldrich</td>
			<td>I6634</td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma-Aldrich</td>
			<td>P8783</td>
		</tr>
		<tr>
			<td>Putrescine</td>
			<td>Sigma-Aldrich</td>
			<td>P7505</td>
		</tr>
		<tr>
			<td>Sodium Selenite</td>
			<td>Sigma-Aldrich</td>
			<td>S5261</td>
		</tr>
		<tr>
			<td>5-Fluoro-2'-deoxyuridine (FuDR)</td>
			<td>Sigma-Aldrich</td>
			<td>F0503</td>
		</tr>
		<tr>
			<td>Papain solution</td>
			<td>Worthington</td>
			<td>LS003126</td>
		</tr>
		<tr>
			<td>DNaseI</td>
			<td>ROCHE</td>
			<td>1010159001</td>
		</tr>
		<tr>
			<td>L-cysteine</td>
			<td>Sigma-Aldrich</td>
			<td>C7352</td>
		</tr>
		<tr>
			<td>24-well tissue culture dishes</td>
			<td>Cellstar</td>
			<td>662-160</td>
		</tr>
		<tr>
			<td>T25-tissue culture flasks with vent cap</td>
			<td>Corning</td>
			<td>430639</td>
		</tr>
		<tr>
			<td>10 cm tissue culture dishes</td>
			<td>Corning</td>
			<td>430167</td>
		</tr>
		<tr>
			<td>10 cm petri dish</td>
			<td>Fisher Scientific</td>
			<td>0875713</td>
		</tr>
		<tr>
			<td>Collagenase A</td>
			<td>ROCHE</td>
			<td>103578</td>
		</tr>
		<tr>
			<td>CellTrics 50 &mu;m filter (optional)</td>
			<td>PARTEC</td>
			<td>04-004-2327</td>
		</tr>
		<tr>
			<td>Myelin Basic Protein (MBP) Antibody</td>
			<td>AbD Serotec</td>
			<td>MCA409S</td>
		</tr>
		<tr>
			<td>NG2 antibody</td>
			<td>Millipore</td>
			<td>AB5320</td>
		</tr>
		<tr>
			<td>Myelin-Associated Glycoprotein (MAG) antibody</td>
			<td>Millipore</td>
			<td>MAB1567</td>
		</tr>
		<tr>
			<td>2',3'-Cyclic-nucleotide 3'-phosphodiesterase (CNP) antibody</td>
			<td>Covance</td>
			<td>SMI-91R-100</td>
		</tr>
		<tr>
			<td>Actin pan Ab-5 antibody </td>
			<td>Fitzgerald</td>
			<td>10R-A106AX</td>
		</tr>
		<tr>
			<td>Neurofilament-200 (NF) antibody</td>
			<td>Sigma-Aldrich</td>
			<td>N4142</td>
		</tr>
		<tr>
			<td>Tubulin beta-3 chain (Tuj1) antibody</td>
			<td>Millipore</td>
			<td>MAB5544</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 goat anti-rabbit IgG (H+L) secondary antibody</td>
			<td>Invitrogen</td>
			<td>A11008</td>
		</tr>
		<tr>
			<td>Alexa Fluor 555 goat anti-mouse IgG (H+L) secondary antibody</td>
			<td>Invitrogen</td>
			<td>A21422</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 goat anti-rat (IgG) (H+L) secondary antibody</td>
			<td>Invitrogen</td>
			<td>A21247</td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG (H+L)-HRP conjugated secondary antibody</td>
			<td>BioRad</td>
			<td>170-6516</td>
		</tr>
		<tr>
			<td>Goat anti-rat IgG (H+L)-HRP conjugated secondary antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-2065</td>
		</tr>
		<tr>
			<td>4',6-diamidino-2-phenylindole (DAPI)</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
		</tr>
	</tbody>
</table>
Media Recipes
100X OL-Supplement* 
<table border="1">
	<tbody>
		<tr>
			<td>Ingredient</td>
			<td>Amount to add</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>1.02 g</td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>0.6 mg</td>
		</tr>
		<tr>
			<td>Putrescine</td>
			<td>161 mg</td>
		</tr>
		<tr>
			<td>Sodium Selenite</td>
			<td>0.05 mg</td>
		</tr>
		<tr>
			<td>3,3',5-Triiodo-L-thyronine</td>
			<td>4 mg</td>
		</tr>
	</tbody>
</table>
*Store at -80&deg;C in 250 &mu;L aliquots
OL media
<table border="1">
	<tbody>
		<tr>
			<td>Ingredient</td>
			<td>Amount to add</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>23.75 mL</td>
		</tr>
		<tr>
			<td>100X OL-Supplement</td>
			<td>250 &mu;L</td>
		</tr>
		<tr>
			<td>Bovine insulin (from 1 mg/mL stock)</td>
			<td>125 &mu;L</td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>250 &mu;L</td>
		</tr>
		<tr>
			<td>Holo-transferrin (from 33 mg/mL stock)</td>
			<td>37.5 &mu;L</td>
		</tr>
		<tr>
			<td>B27 Supplement</td>
			<td>500 &mu;L</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>125 &mu;L</td>
		</tr>
		<tr>
			<td>CNTF (from 50 ng/&mu;L stock)</td>
			<td>25 &mu;L</td>
		</tr>
	</tbody>
</table>
Mixed glial culture media (made up in DMEM)
<table border="1">
	<tbody>
		<tr>
			<td>Ingredient</td>
			<td>Final concentration </td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>10%</td>
		</tr>
		<tr>
			<td>Pen/Strep (0.33% from stock)</td>
			<td>33 units/mL Penicillin and 33 &mu;g/mL Streptomycin</td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>1%</td>
		</tr>
	</tbody>
</table>
DRGN media (made up in DMEM)
<table border="1">
	<tbody>
		<tr>
			<td>Ingredient</td>
			<td>Final concentration </td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>10%</td>
		</tr>
		<tr>
			<td>Pen/Strep (1% from stock)</td>
			<td>100 units/mL Penicillin and 100 &mu;g/mL Streptomycin</td>
		</tr>
	</tbody>
</table>
Digestion Solution Recipes:
OPC papain solution (made up in MEM)
<table border="1">
	<tbody>
		<tr>
			<td>Ingredient</td>
			<td>Final concentration </td>
		</tr>
		<tr>
			<td>Papain solution</td>
			<td>1.54 mg/mL</td>
		</tr>
		<tr>
			<td>L-cysteine</td>
			<td>360 &mu;g/mL</td>
		</tr>
		<tr>
			<td>DNaseI</td>
			<td>60 &mu;g/mL</td>
		</tr>
	</tbody>
</table>
DRG papain solution (made up in HBSS)
<table border="1">
	<tbody>
		<tr>
			<td>Ingredient</td>
			<td>Final concentration </td>
		</tr>
		<tr>
			<td>Papain</td>
			<td>1.54 mg/mL</td>
		</tr>
		<tr>
			<td>L-cysteine</td>
			<td>360 &mu;g/mL</td>
		</tr>
	</tbody>
</table>
DRG Collagenase A solution (made up in HBSS)
<table border="1">
	<tbody>
		<tr>
			<td>Ingredient</td>
			<td>Final concentration </td>
		</tr>
		<tr>
			<td>Collagenase A</td>
			<td>4 mg/mL</td>
		</tr>
	</tbody>
</table>

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.

Watch this Scientific Journal Video about Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues at JoVE.com

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

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 ...

derivation-enriched-oligodendrocyte-cultures-oligodendrocyteneuron

Research

JoVE Journal

Neuroscience

Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. For this protocol the freezing medium used is 10% dimethyl sulfoxide (DMSO) diluted in Hank's Buffered Salt Solution (HBSS). Blocks of cortical tissue are transferred to cryovials containing the freezing medium and slowly frozen at -1&deg;C/min in a rate-controlled freezing container. Post-thaw processing and dissociation of frozen tissue blocks consistently produced neuronal-enriched cultures which exhibited rapid neuritic growth during the first 5 days in culture and significant expansion of the neuronal network within 10 days. Immunocytochemical staining with the astrocytic marker glial fibrillary acidic protein (GFAP) and the neuronal marker beta-tubulin class III, revealed high numbers of neurons and astrocytes in the cultures. Generation of neural precursor cell cultures after tissue block dissociation resulted in rapidly expanding neurospheres, which produced large numbers of neurons and astrocytes under differentiating conditions. This simple cryopreservation protocol allows for the rapid, efficient, and inexpensive preservation of cortical brain tissue blocks, which grants increased flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

Here, we describe a method for efficient cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. This simple protocol provides flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly ...

Derivation of Glial Restricted Precursors from E13 mice

This is a protocol for derivation of glial restricted precursor (GRP) cells from the spinal cord of E13 mouse fetuses. These cells are early precursors within the oligodendrocytic cell lineage. Recently, these cells have been studied as potential source for restorative therapies in white matter diseases. Periventricular leukomalacia (PVL) is the leading cause of non-genetic white matter disease in childhood and affects up to 50% of extremely premature infants. The data suggest a heightened susceptibility of the developing brain to hypoxia-ischemia, oxidative stress and excitotoxicity that selectively targets nascent white matter. Glial restricted precursors (GRP), oligodendrocyte progenitor cells (OPC) and immature oligodendrocytes (preOL) seem to be key players in the development of PVL and are the subject of continuing studies. Furthermore, previous studies have identified a subset of CNS tissue that has increased susceptibility to glutamate excitotoxicity as well as a developmental pattern to this susceptibility. Our laboratory is currently investigating the role of oligodendrocyte progenitors in PVL and use cells at the GRP stage of development. We utilize these derived GRP cells in several experimental paradigms to test their response to select stresses consistent with PVL. GRP cells can be manipulated in vitro into OPCs and preOL for transplantation experiments with mouse PVL models and in vitro models of PVL-like insults including hypoxia-ischemia. By using cultured cells and in vitro studies there would be reduced variability between experiments which facilitates interpretation of the data. Cultured cells also allows for enrichment of the GRP population while minimizing the impact of contaminating cells of non-GRP phenotype.

This protocol outlines the derivation of Glial Restricted Precursors from fetal spinal cords and maintained in vitro either for transplantation or for the study of oligodendrocytic lineage.

This is a protocol for derivation of glial restricted precursor (GRP) cells from the spinal cord of E13 mouse fetuses. These cells are early precursors ...

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.

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 ...

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

Much information about the coupling of presynaptic ionic currents with the release of neurotransmitter has been obtained from invertebrate preparations, most notably the squid giant synapse1. However, except for the preparation described here, few vertebrate preparations exist in which it is possible to make simultaneous measurements of neurotransmitter release and presynaptic ionic currents. Embryonic Xenopus motoneurons and muscle cells can be grown together in simple culture medium at room temperature; they will form functional synapses within twelve to twenty-four hours, and can be used to study nerve and muscle cell development and synaptic interactions for several days (until overgrowth occurs). Some advantages of these co-cultures over other vertebrate preparations include the simplicity of preparation, the ability to maintain the cultures and work at room temperature, and the ready accessibility of the synapses formed2-4. The preparation has been used widely to study the biophysical properties of presynaptic ion channels and the regulation of transmitter release5-8. In addition, the preparation has lent itself to other uses including the study of neurite outgrowth and synaptogenesis9-12, molecular mechanisms of neurotransmitter release13-15, the role of diffusible messengers in neuromodulation16,17, and in vitro synaptic plasticity18-19.

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

This video demonstrates the procedures used to grow primary cultures of embryonic Xenopus nerve and muscle cells and the usefulness of this preparation for making simultaneous pre- and post-synaptic patch clamp recordings.

Much information about the coupling of  presynaptic ionic currents with the release of neurotransmitter has been obtained  from invertebrate preparations, ...

Preparation of Neuronal Co-cultures with Single Cell Precision

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms can be used to investigate a variety of questions, spanning developmental and functional neurobiology to infection and disease propagation. However, conventional systems require significant cellular inputs (many thousands per compartment), inadequate for studying low abundance cells, such as primary dopaminergic substantia nigra, spiral ganglia, and Drosophilia melanogaster neurons, and impractical for high throughput experimentation. The dense cultures are also highly locally entangled, with few outgrowths (&#60;10%) interconnecting the two cultures. In this paper straightforward microfluidic and patterning protocols are described which address these challenges: (i) a microfluidic single neuron arraying method, and (ii) a water masking method for plasma patterning biomaterial coatings to register neurons and promote outgrowth between compartments. Minimalistic neuronal co-cultures were prepared with high-level (&#62;85%) intercompartment connectivity and can be used for high throughput neurobiology experiments with single cell precision.

Protocols for single neuron microfluidic arraying and water masking for the in-chip plasma patterning of biomaterial coatings are described. Highly interconnected co-cultures can be prepared using minimal cell inputs.

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a promising target for intervention to improve functional regeneration. So far, no in vitro model exists that grants the possibility to examine functional recovery of propriospinal fibers. Therefore, a representative model that is based on two organotypic spinal cord sections of embryonic rat, cultured next to each other on multi-electrode arrays (MEAs) was developed. These slices grow and, within a few days in vitro, fuse along the sides facing each other. The design of the used MEAs permits the performance of lesions with a scalpel blade through this fusion site without inflicting damage on the MEAs. The slices show spontaneous activity, usually organized in network activity bursts, and spatial and temporal activity parameters such as the location of burst origins, speed and direction of their propagation and latencies between bursts can be characterized. Using these features, it is also possible to assess functional connection of the slices by calculating the amount of synchronized bursts between the two sides. Furthermore, the slices can be morphologically analyzed by performing immunohistochemical stainings after the recordings. 
Several advantages of the used techniques are combined in this model: the slices largely preserve the original tissue architecture with intact local synaptic circuitry, the tissue is easily and repeatedly accessible and neuronal activity can be detected simultaneously and non-invasively in a large number of spots at high temporal resolution. These features allow the investigation of functional regeneration of intraspinal connections in isolation in vitro in a sophisticated and efficient way.

Here we present a protocol that is based on spinal cord slices cultured on multi-electrode arrays to study functional regeneration of propriospinal connections in vitro.

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a ...

Derivation of Leptomeninges Explant Cultures from Postmortem Human Brain Donors

Even though great progress has been made in the clinical characterization of Parkinson&#39;s disease, several studies report that the diagnosis of Parkinson&#39;s disease is not pathologically confirmed in up to 25% of clinically diagnosed Parkinson&#39;s disease. Therefore, tissue collected from clinically diagnosed patients with idiopathic Parkinson&#39;s disease can have a high rate of misdiagnosis; hence in vitro studies from such tissues to study Parkinson&#39;s disease as a preclinical model can become futile.
By collecting postmortem human leptomeninges with a confirmed neuropathological diagnosis of Parkinson&#39;s disease and characterized by nigrostriatal cell loss and intracellular protein inclusions called Lewy bodies, one can be certain that clinically observed parkinsonism is not caused by another underlying disease process (e.g. tumor, arteriosclerosis).
This protocol presents the dissection and preparation of postmortem human leptomeninges for derivation of a meningeal fibroblast culture. This procedure is robust and has a high success rate. The challenge of the culture is sterility as the brain procurement is generally not performed under sterile conditions. Therefore, it is important to supplement the culture media with a cocktail of penicillin, streptomycin, and amphotericin B.
The derivation of meningeal fibroblasts from autopsy-confirmed cases with Parkinson&#39;s disease provides the foundation for in vitro modeling of Parkinson&#39;s disease. Meningeal fibroblasts appear 3-9 days after sample preparation and about 20-30 million cells can be cryopreserved in 6-8 weeks. The meningeal fibroblast culture is homogenous and the cells express fibronectin, a commonly used marker to identify meninges.

The leptomeninges explant culture protocol from human postmortem brain is a technically robust and simple way to derive fibronectin-positive meningeal fibroblasts within 6-8 weeks and cryopreserve approximately 20-30 million cells.

Even though great progress has been made in the clinical characterization of Parkinson&#39;s disease, several studies report that the diagnosis of ...

Neuron-Macrophage Co-cultures to Activate Macrophages Secreting Molecular Factors with Neurite Outgrowth Activity

There is strong evidence that macrophages can participate in the regeneration or repair of injured nervous system. Here, we describe a protocol in which macrophages are induced to produce conditioned medium (CM) that promotes neurite outgrowth. Adult dorsal root ganglion (DRG) neurons are acutely dissociated and plated. After the neurons are stably attached, peritoneal macrophages are co-cultured on a cell culture insert overlaid on the same well. Dibutyryl cyclic AMP (db-cAMP) is applied to the co-cultures for 24 h, after which the cell culture insert containing the macrophages is moved to another well to collect CM for 72 h. The CM from the co-cultures treated with db-cAMP, when applied to a separate adult DRG neuron culture, exhibits robust neurite outgrowth activity. The CM obtained from the db-cAMP-treated cultures consisting of single cell type alone, either DRG neuron or peritoneal macrophage, did not exhibit neurite outgrowth activity. This indicates that the interaction between neurons and macrophages is indispensable for the activation of macrophages secreting molecular factors with neurite outgrowth activity into CM. Thus, our co-culture paradigm will also be useful to study intercellular signaling in the neuron-macrophage interaction to stimulate the macrophages to be endowed with a pro-regenerative phenotype.

The current protocol presents experimental procedures to stimulate cultured macrophages to be endowed with capacity to release molecular factors that promote neurite outgrowth. Treatment of cAMP to the neuron-macrophage co-cultures induces the macrophages to produce conditioned medium that possesses strong neurite outgrowth activity.

There is strong evidence that macrophages can participate in the regeneration or repair of injured nervous system. Here, we describe a protocol in which ...

Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical conduction. Myelin alteration has been shown to occur in various neurological diseases, where it is associated with functional deficits. Here, we provide a detailed description of an ex vivo model consisting of mouse organotypic cerebellar slices, which can be maintained in culture for several weeks and further be labeled to visualize myelin.

Here we present a method to prepare organotypic slice cultures from mouse cerebellum and myelin sheath staining by immunohistochemistry suitable for investigating mechanisms of myelination and remyelination in the central nervous system.

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical ...