1Regenerative Medicine Program, Ottawa Hospital Research Institute, 2Department of Cellular and Molecular Medicine, University of Ottawa, 3Department of Pharmacological Sciences, Stony Brook University, 4Department of Medicine, University of Ottawa
O'Meara, R. W., Ryan, S. D., Colognato, H., Kothary, R. Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues. J. Vis. Exp. (54), e3324, doi:10.3791/3324 (2011).
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 & 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.
The mice used in this work were cared for according to Canadian Council on Animal Care (CCAC) guidelines. Ethical approval for experiments conducted was obtained from the University of Ottawa Animal Care Committee under protocol number OGH-119.
1. Dissection – neonatal mouse cortex for OPC extraction
2. Dissociation of neonatal cortices and maintenance of mixed glial cultures
Note: The introduction of bubbles into the cell suspension should be avoided during all of the following steps.
Note: Under-trituration will result in poor dissociation of the tissue, whereas over-trituration will negatively impact on cell viability. It is important to not introduce bubbles into the solution, as this will severely impact cell viability.
3. DRGN isolation
Note: To produce OL/DRGNs co-cultures, DRGNs should be established the day after mixed glial culture generation. Both culture types are grown independently, and combined after 9-10 days.
Note: Many contaminating cells will have strongly adhered to the Petri dish, thereby enriching your cell suspension for DRGNs.
4. Purification of OPCs from mixed glial cultures for establishment of OL-enriched cultures or OL/DRGN co-cultures
5. Processing of cultures for immunofluorescence microscopy
6. Whole cell protein extraction from OL-enriched cultures
7. SDS-PAGE analysis on enriched-OL culture protein
8. Representative Results:
In this protocol, OPCs are expanded on an astrocyte monolayer within a mixed glial culture. This mixed glial culture is derived from P0-P2 neonatal mouse cortex. At day 1 in vitro (DIV1), the mixed glial culture contains cells with varying morphologies as seen by phase contrast microscopy (Fig 2a). At DIV3, an astrocyte monolayer begins to form on the base of the flask, and at DIV8, OPCs can be clearly observed on the monolayer surface. At DIV9, the proliferating OPCs have reached sufficient density to be purified by overnight high-speed orbital shaking. Once the purification process has been completed, the result is an OPC-enriched cell population. At DIV1-post purification, OPCs have simple morphology, extending few processes (Fig 2b). At DIV3 post purification, cells have extended a complex meshwork of processes, reminiscent of immature OLs. At DIV6 post purification, the purified OLs have flattened and projected leaflet-like membrane structures. This morphological development is typical of the in vitro maturation of OLs.
Immunofluorescence microscopy indicates the purified cells are of OL-lineage (Fig 3a). Seeded OPCs initially express chondroitin sulfate proteoglycan (NG2), and develop into myelin-associated glycoprotein (MAG) positive immature OLs within three days post seeding (Fig 3b). At DIV6, many OLs express myelin basic protein (MBP), and possess typical mature OL morphology. Percent OL-lineage cells were quantified at different time points to determine the purity of the OL-enriched cultures (Fig 3c). At DIV1 post purification, cultures are 50±14% NG2+ve OPCs, with no MAG+ve or MBP+ve OLs. This indicates the purified OL-lineage cells are in the precursor stage at seeding time, with negligible numbers of differentiated OLs. At DIV3, many OLs have differentiated into MAG+ve cells (24±5.9%) while some retain the precursor phenotype, and remain NG2+ (13±8.0%). At DIV3, a small proportion of MAG+ve cells (3.2 1.2%) are also expressing MBP. At DIV6, 20±5.9% of OLs are MAG+ve while 12±7.3% persist as NG2+ve OPCs. In addition, 21±9.3% of cells within the culture are MBP+ve OLs at this time point. SDS-PAGE analysis shows the graded expression of 2'3'-cyclic-nucleotide 3'-phosphodiesterase (CNP) and MBP over the 6 day culture period, further demonstrating the ability of OPCs in culture to terminally differentiate into mature OLs (Fig 3d). Collectively, these data establishes this method as a means of producing an OL-enriched culture system suitable for the study of OL maturation from OPCs.
This protocol also describes methods to establish OL/DRGN co-cultures using murine-only tissue sources. However, in order to produce the co-culture, DRGNs must first be cultured alone to produce an adequate neurite network. These post-natal murine neuron cultures are grown for 9 days in low serum media with 10 μM FuDR supplementation to prevent the proliferation of contaminating fibroblasts and glial cells. Over the course of 9 days in vitro, isolated DRGNs produce a dense neurite bed (Fig 4a). This neurite bed is immunopositive for the neuronal markers neurofilament 200 (NF) and Tuj1 (Fig 4b). At this point, purified OPCs may be added to the neurite beds, and cultured for an additional 6 days to produce myelinating co-cultures.
At DIV6 of OL/DRGN co-culture, many MBP+ve OLs can be observed among the NF+ve DRGN neurites (Fig 5a). Upon closer examination, OLs are evidenced to make contact with numerous DRGN neurites, often ensheathing them with an MBP+ve membrane (Fig 5b,c).
Figure 1. Dissection microscope images of particular aspects of neonatal mouse cortex and DRG isolation. (a) Dorsal view of a freshly extracted neonatal mouse brain. The dotted lines indicate the area where incisions must be made to facilitate the removal of the meningeal layer. (b) Ventral view of brain, dotted lines indicate the area where the cortex meets the ventral diencephalon. Deep incisions must be made along the dotted lines to aid the isolation of the cortices. (c-c') Visual depiction of how to pry the cortex away from the remainder of the brain. (d) A freshly isolated P5-P10 mouse spine prior to trimming away excess muscle and bone (d'). (e) Location of DRGs within the spinal column. (f) The approximate number of DRGs that should be isolated from one mouse. (g) A DRG with long roots that require trimming prior to enzymatic digestion. The dotted line indicates the region where the roots should be trimmed. (g') DRG post root-trimming.
Figure 2. OPCs are expanded within a mixed glial culture, purified, and subsequently differentiated as an OL-enriched culture. (a) Phase contrast images of mixed glial cultures at different stages of development. At DIV1, cells appear round with few flattened cells. Stratification of mixed glial culture begins at DIV3, where astrocytes form a uniform monolayer at the base of the flask, upon which OPCs proliferate. Many OPCs are seen at DIV8 (arrows) adhered to the surface of the astrocyte monolayer. (b) Once purified from the mixed glial culture, DIV1 OPCs have extended only a few processes. At DIV3, cells have extended many processes, reminiscent of intermediate-stage OLs. At DIV6, flattened OLs (asterisk) appear to have produced membranous sheets (dashed line). Scale bars, 50 μm.
Figure 3. Characterization of OL-enriched culture. (a) Confocal images of isolated OLs at different stages of development. NG2+ve OPCs have simple morphology, whereas MAG+ve OLs possess multiple arborous processes. MBP+ve OLs have extended membranous myelin-like sheets. Scale bar, 50 μm. (b) Purified OL-lineage cells originate as OPCs, and differentiate into MAG+ve, MBP+ve OLs over 6 DIV. At DIV1, all OPCs are NG2+ve, while none are MAG+ve or MBP+ve. At DIV3, MAG+ve and few MBP+ve OLs are now evident. The majority of OLs are MAG and MBP+ve at DIV6, with few remaining NG2+ve OPCs. Scale bar, 100 μm. (c) Mean values ± SD of the percent OL-lineage cells at different stages of development over 6 DIV. At DIV1, all OL-lineage cells are NG2+ve, accounting for 50±14% of total cells within the culture. At DIV3 and DIV6, OL-lineage cells respectively account for 36±6.8% and 32±8.4% of total cells, consisting of varying proportions of NG2+ve, MAG+ve and MBP+ve OLs. (d) SDS-PAGE performed on protein derived from enriched OL-cultures demonstrating the graded expression of OL-markers CNP and MBP over the 6 DIV culture period.
Figure 4. Characterization of the DRGN culture pre-OPC seeding. (a) Phase contrast images of DRGNs over the 9 DIV culture period pre-OPC seeding. DRGNs originate as large-bodied cells with few processes, and produce an increasingly complex neurite network. Scale bar, 100 μm. (b) Confocal images of DRGN cultures fixed at DIV9 (pre-OPC seeding) and stained for neuron-specific markers Tuj1 and NF200. DRGNs have produced a neurite network upon which OPCs may be seeded to produce OL/DRGN myelinating co-cultures. Scale bar, 50 μm.
Figure 5. OLs co-cultured with DRGNs result in OL-mediated wrapping of DRGN neurites with MBP+ve membrane. (a) A 4-field confocal image montage of a DIV6 OL/DRGN co-culture. Many MBP+ve OLs can be seen interacting with the underlying DRGN neurite bed. Scale bar, 100 μm. (b) A magnified confocal view of MBP+ve OLs wrapping multiple DRGN neurites. Scale bar, 50 μm. (c) Digital magnification of the region denoted in (b) where a DRGN neurite is being wrapped with OL membrane. Scale bar, 25 μm.
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.
No conflicts of interest declared.
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.
|Dulbecco's Modified Eagle Medium (DMEM)||Multicell||319-005-CL|
|Hank's Balanced Salt Solution (HBSS)||Invitrogen||14170-112|
|Minimum Essential Media (MEM)||GIBCO, by Life Technologies||12360-038|
|Fetal Bovine Serum (FBS)||GIBCO, by Life Technologies||10091-148|
|Penicillin-Streptomycin (Pen/Strep)||GIBCO, by Life Technologies||15140-122|
|Bovine Serum Albumin (BSA)||Sigma-Aldrich||A4503|
|Human merosin purified protein (LN2)||EMD Millipore||CC085|
|Recombinant rat ciliary neurotrophic factor (CNTF)||PeproTech Inc||450-50|
|B27 supplement||GIBCO, by Life Technologies||0080085-SA|
|Papain solution||Worthington Biochemical||LS003126|
|24-well tissue culture dishes||Cellstar||662-160|
|T25-tissue culture flasks with vent cap||Corning||430639|
|10 cm tissue culture dishes||Corning||430167|
|10 cm petri dish||Fisher Scientific||0875713|
|Collagenase A||Roche Group||103578|
|CellTrics 50 μm filter (optional)||PARTEC||04-004-2327|
|Myelin Basic Protein (MBP) Antibody||AbD Serotec||MCA409S|
|NG2 antibody||EMD Millipore||AB5320|
|Myelin-Associated Glycoprotein (MAG) antibody||EMD Millipore||MAB1567|
|2',3'-Cyclic-nucleotide 3'-phosphodiesterase (CNP) antibody||Covance||SMI-91R-100|
|Actin pan Ab-5 antibody||Fitzgerald||10R-A106AX|
|Neurofilament-200 (NF) antibody||Sigma-Aldrich||N4142|
|Tubulin beta-3 chain (Tuj1) antibody||EMD Millipore||MAB5544|
|Alexa Fluor 488 goat anti-rabbit IgG (H+L) secondary antibody||Invitrogen||A11008|
|Alexa Fluor 555 goat anti-mouse IgG (H+L) secondary antibody||Invitrogen||A21422|
|Alexa Fluor 647 goat anti-rat (IgG) (H+L) secondary antibody||Invitrogen||A21247|
|Goat anti-mouse IgG (H+L)-HRP conjugated secondary antibody||Bio-Rad||170-6516|
|Goat anti-rat IgG (H+L)-HRP conjugated secondary antibody||Santa Cruz Biotechnology, Inc.||SC-2065|
*Store at -80 °C in 250 μL aliquots
Mixed glial culture media (made up in DMEM)
DRGN media (made up in DMEM)
Digestion Solution Recipes:
OPC papain solution (made up in MEM)
DRG papain solution (made up in HBSS)
DRG Collagenase A solution (made up in HBSS)