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Oligodendrocyte–spinal cord explant co-culture: An in vitro model for the study of myelination
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available at www.sciencedirect.com
www.elsevier.com/locate/brainres
Research Report
Oligodendrocyte–spinal cord explant co-culture: An in vitro model for the study of myelination Zhifang Chen, Zhengwen Ma, Yanxia Wang, Ying Li, Hezuo Lü, Saili Fu, Qin Hang, Pei-Hua Lu⁎ Department of Neurobiology, Shanghai Jiaotong University School of Medicine, 280 South Chongqing Road, Shanghai 200025, P.R. China
A R T I C LE I N FO
AB S T R A C T
Article history:
The in vitro models developed to investigate the growth and myelination of axons, such as
Accepted 24 October 2009
dorsal root ganglion (DRG)–Schwann cell co-culture, DRG–oligodendrocyte co-culture and
Available online 30 October 2009
central nervous system (CNS) neuron–oligodendrocyte co-culture, have provided an effective way to reveal the mechanisms that underlie the interaction between neurons
Keywords:
and myelin-forming cells. In order to better understand the complex process of myelination
Spinal cord explant
during CNS development and spinal cord repair, we established a rat spinal cord neuron–
Neurite
oligodendrocyte co-culture model. In this co-culture system, the spinal cord explants were
Oligodendrocyte
used as the source of neurons, and the oligodendrocytes were induced from GFP-
Myelin
oligodendrocyte precursor cells (GFP-OPCs). The results showed that the GFP-
GFP
oligodendrocytes that differentiated from GFP-OPCs in co-culture attached to the neurites
Co-culture
growing out from the spinal cord explants and formed myelin structures. As the oligodendrocytes expressed GFP, and the neuron somas remained in the explants, the interaction between oligodendrocytes and neurites in co-culture were observed clearly and dynamically without immunostaining. © 2009 Elsevier B.V. All rights reserved.
1.
Introduction
In the nervous system, myelinated axons are wrapped tightly by the continuous membrane layers of glial cells, i.e., oligodendrocytes in the central nervous system (CNS) or Schwann cells in the peripheral nervous system (PNS). The in vitro models developed to investigate the growth and myelination of axons, such as dorsal root ganglion (DRG)–Schwann cell co-culture (Bunge and Wood, 1987; Einheber et al., 1993; Paivalainen et al., 2008; Seilheimer and Schachner, 1988), DRG neuron–oligodendrocyte co-culture (Wang et al., 2007), retinal ganglion–Schwann cell co-culture (Bahr et al., 1991) and CNS neuron–oligodendrocyte co-culture (Fex Svenningsen et al.,
2003; Thomson et al., 2006), have revealed the mechanisms that underlie the interaction between neurons and myelinforming cells. To better understand the complex process of axonal growth and myelination during CNS development and spinal cord repair, we established an oligodendrocyte–spinal cord explant co-culture model. The neurites extended radially from the explants, but their somas remained in the explants, which facilitated the observation and measurement of the neurites during co-culture. In addition, the oligodendrocytes in coculture were induced from GFP-oligodendrocyte precursor cells (GFP-OPCs), which could be observed directly by fluorescence microscopy. Thus, in our co-culture model, as without
⁎ Corresponding author. Fax: +86 21 64453296. E-mail address: [email protected] (P.-H. Lu). 0006-8993/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2009.10.060
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the interference of non-GFP somas, the interaction between GFP-oligodendrocytes and neurites could be observed clearly and dynamically without immunostaining.
2.
Results
2.1. Attachment and neurite outgrowth of spinal cord explants
least 95% of the explants were attached to the coverslips and fine neurites growing from the edge of the explants were visible (Fig. 1E). Three days after culture, the neurites from the explants were extended significantly, some even up to 1000 μm (Fig. 1F). The results indicated that, in contrast to poly-L-lysine, laminin and fibronectin, Matrigel not only speeded up the attachment of spinal cord explants, but also promoted neurite outgrowth from the explants.
2.2. Spinal cord explants were plated initially onto poly-L-lysine substratum and cultured with spinal cord explant culture medium, which is commonly used for neurons (Calabrese and Halpain, 2005; Jiang et al., 2006). After 3 days in vitro, most explants remained detached, and the explant was devoid of neurites (Fig. 1A). To improve the attachment, we used laminin and fibronectin to substitute for poly-L-lysine respectively. The results showed that after 3 days culture, on both laminin-coated (Fig. 1B) and fibronectin-coated (Fig. 1C) coverslips, 50–60% of explants could attach to the coverslips. And the neurites from explants could be seen, but in low density and short length. It has been reported that Matrigel encourages the attachment and axonal growth of neurons (Khan et al., 2002; Yu et al., 1994). To further improve the culture environment for spinal cord explants, we tried to coat the coverslips with 2% Matrigel instead of poly-L-lysine. The results showed that 4 h after plating, more than 60% of the explants were attached to the Matrigel-coated coverslips and neurites budding from the explants were observed (Fig. 1D). Twelve hours after plating, at
Co-culture of spinal cord explants with GFP-OPCs
As mature oligodendrocytes express myelin-associated proteins (Nogo, MAG and OMgp) that inhibit axonal outgrowth in neuron (Domeniconi et al., 2005; GrandPre et al., 2002; Wang et al., 2002), it is difficult to co-culture neurons with mature oligodendrocytes in vitro (Ma et al., 2009). In addition, the previous study has revealed that maturation of oligodendrocytes is regulated largely by axonal signals in the development of the CNS (Miller, 2002). Therefore, we co-cultured spinal cord explants with GFP-OPCs rather than mature oligodendrocytes. We believe that during co-culture with neurons, the OPCs gradually differentiated into mature oligodendrocytes, and the GFP expressed in OPCs facilitated the dynamic observation of the interaction between oligodendrocytes and neurites without immunostaining. At first, to make sure that Matrigel also facilitated the growth of GFP-OPCs, we compared the viability of OPCs on different substratums. The results showed that 1 day after seeding, OPCs on poly-L-lysine attached poorly to coverslip, and cell status was inconsistent (Fig. 2A). OPCs on fibronectin could attach well
Fig. 1 – Effects of culture substratums on attachment and neurite outgrowth of spinal cord explants. Phase contrast micrographs showed representative explants under different conditions. (A–C) After 3 days culture, the spinal cord explant attached poorly to the poly-L-lysine-coated coverslip, therefore, the explant was devoid of neurites (A), while on laminin-coated (B) and fibronectin-coated (C) coverslips, 50–60% of explants could attach to the coverslips. The neurites from the explants were in low density, and the length of neurites was restricted to 500 μm. (D) Four hours after plating, more than 60% of the explants were attached to 2% Matrigel-coated coverslip, and outgrowth of neurites from the explants occurred (box). (E) Twelve hours after seeding, at least 95% of explants were attached to the Matrigel-coated coverslip, and many neurites extended straight out of the explants. (F) After 3 days culture on the Matrigel-coated coverslip, the neurites from the spinal cord explants were extended significantly, some of them up to 1000 μm. Scale bar: A–E, 66 μm; F, 100 μm.
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to the coverslip, but cell status was also inconsistent (Fig. 2B). OPCs on laminin (Fig. 2C) and on Matrigel (Fig. 2D) all grew well and were in good status, but obviously, cells on Matrigel showed even better attachment. It indicated that Matrigel was the best substratum for both spinal cord explants and OPCs. Before co-culture with spinal cord explants, the GFP-OPCs in 2% Matrigel were seeded onto the glass coverslips in OPM (details as described in Experimental procedures). On the next day, GFP cells attached to the coverslips displayed typical bipolar or tri-polar morphology of OPCs (Fig. 2D–F). Immunostaining showed that more than 90% of GFP cells expressed A2B5 (Fig. 2G) and PDGFR (Fig. 2H), the markers for OPCs, and
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no RIP+ cells (oligodendrocytes) were observed (Fig. 2I). After 1 day culture in OPM, the GFP-OPCs were ready for co-culture, OPM was replaced with CoM (details as described in Experimental procedures), and the spinal cord explants were plated subsequently into the culture. At first, CoM1 and CoM2 that can support the survival of oligodendrocytes and neurons on Matrigel were used simultaneously. The co-culture results revealed that, although OPCs/oligodendrocytes and neurons all grew well in both media, there were more somas emigrating out of the explants in CoM1 (containing PDGF and bFGF) than in CoM2 (containing T3). After 3 days culture in CoM1, at least 2500 somas were
Fig. 2 – Identification of GFP-OPCs induced from GFP-NPCs before co-culture. (A–D) Phase contrast micrographs of OPCs cultured on different substratum. OPCs cultured on poly-L-lysine showed poor attachment and inconsistent cell status (A); OPCs attached well on fibronectin, but also presented inconsistent cell status (B). OPCs on laminin (C) and on Matrigel (D) were healthy, but cells on Matrigel showed even better attachment. (D–F) Photomicrographs of live OPCs cultured on Matrigel showed that the GFP-OPCs induced from GFP-NSCs displayed bi- or tri-polar morphology, the typical morphology of OPCs. (G–I) Confocal images of immunostaining showed that most GFP-OPCs expressed A2B5 (G) and PDGFR (H), the specific markers for OPCs, but did not express RIP (I), a specific marker for oligodendrocytes. Cells in G–I were counterstained with Hoechst 33342 (blue), a nuclear dye. Scale bar: A–F, 40 μm; G–I, 10 μm.
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observed surrounding each spinal cord explant, whether cultured alone or with GFP-OPCs (Figs. 3A–C), while in CoM2, only a few somas emigrated from each explant (Figs. 3D–F). Further identification of those migrated cells in CoM1 indicated that most of them were O4+ pre-oligodendrocytes and GFAP+ astrocytes (Figs. 3G–I). This was consistent with the previous findings that PDGF and bFGF have a great tendency to promote neural cell migration (Frost et al., 2009) and prolifer-
ation (Noguchi et al., 2006; Reimers et al., 2001). Since too many emigrated somas would have been inconvenient for the observation of oligodendrocyte–neurite interaction and the measurement of their growth parameters, CoM1 was not appropriate for co-culture, thus, CoM2 was adopted as the medium for subsequent co-culture. Three days after co-culture, the neurites from spinal cord explants had grown into GFP-OPCs monolayer in
Fig. 3 – Effects of CoM1 and CoM2 on spinal cord explant cultures, alone or with GFP-OPCs. (A and D) Phase contrast micrographs showed that 3 days after explants were cultured alone in CoM1, there were many somas moving out of the explant (A). In CoM2, only a few somas emigrated from the explant (D). (B, C, E and F) Immunofluorescence micrographs showed that 3 days after co-culture with GFP-OPCs, the neurites (NF, red) from explants were extended and had grown into the GFP-OPCs monolayer (GFP, green) in CoM1 (B and C) and CoM2 (E and F). There were also many somas moving out of the explant (C, Hoechst), when cultured in CoM1. This did not happen in CoM2 (F). (G–I) Identification of migrated cells, there were numerous somas surrounding the explant after cultured in CoM1 (G). Immunostaining assay showed the cells around the explant were O4 positive (H) and GFAP positive (I), which indicated that the cells migrated from explant were pre-oligodendrocytes and astrocytes. * Represents the location of spinal cord explant. (J–L) High-magnification micrographs revealed that 3 days after co-culture in CoM2, the GFP-OPCs displayed more branches (J) than original OPCs, and attached closely to the neurite (NF, red) outgrowth from the explant (K and L). Scale bar: A–F, 100 μm; G–I, 25 μm; J–L, 15 μm.
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scattered fibers (Figs. 3E and F), and the growing neurites made direct contact with the surface of the OPCs (Fig. 3L). Meanwhile, the GFP-OPCs in co-culture had differentiated into pre-oligodendrocytes that displayed more branches than the original OPCs (Figs. 3J and L). Our co-culture model showed that OPCs/pre-oligodendrocytes and neurites could co-exist, even though they were from different individuals.
2.3. Myelination of neurites growing from spinal cord explants in co-culture Neurites from spinal cord explants in co-culture elongated rapidly during the first 3 days. From the fourth day onwards,
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the growth rate of the main neurites slowed down, but the branches of the neurites were growing denser. Eight days after co-culture, some GFP cells exhibited multipolar morphology, which indicated that some GFPOPCs had differentiated into more mature oligodendrocytes. Combined phase contrast and fluorescence microscopy showed two-dimensional interaction between GFP cells and neurites in co-culture. It was demonstrated clearly that the processes of some GFP-oligodendrocytes had touched and elongated along the neurites (Figs. 4A–C). Moreover, the fine branch networks of GFP-oligodendrocytes also attached tightly to the neurites or their branches (Figs. 4D–F). The green sheaths formed by GFP-oligodendrocytes were observed clearly. In general, the processes of one mature oligodendro-
Fig. 4 – Myelination of neurites by GFP-oligodendrocytes in co-culture. (A–C) Live-cell images showed that 8 days after co-culture, a GFP-expressing oligodendrocyte contacted the neurites by its processes that were growing along with the neurites (C, arrow). (D–F) High-magnification images of the box in picture C showed that the fine branch networks of the GFP-oligodendrocyte were also attached closely to the neurites (F, arrows). (G–I) Confocal images showed that after 8 days co-culture, the GFP-oligodendrocytes made close contact with the neurites (arrows), which expressed MBP (G and I,) and RIP (H), the specific markers of mature oligodendrocytes. (J–M) Confocal images showed that after 15 days co-culture, the myelinated neurites were observed clearly. The neurites were labeled with NF (L) and myelin sheaths were co-labeled with GFP (J) and MBP (K) (arrows). (N and O) The presence of compact myelin in 15-day co-cultures was confirmed by electron microscopy. Scale bar: A–C, 15 μm; D–F, 5 μm; G, 20 μm; H and I, 10 μm; J–M, 5 μm; N, 1 μm; O, 40 nm.
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cyte made simultaneous contact with several segments of neurites or their branches. Immunostaining indicated that these GFP-oligodendrocytes expressed RIP and MBP, the markers of more mature oligodendrocytes, and about 32.5% of the GFP cells associated with neurites expressed MBP (Figs. 4G–I). Fifteen days after co-culture, immunostaining showed that the myelinated neurites could be observed clearly (Figs. 4J–M). The three-dimensional sheaths after 15 days co-culture were further identified by electron microscopy. The electron microscopic images showed that the neurites were surrounded by compact myelin sheaths (Figs. 4N and O).
3.
Discussion
3.1. Attachment and neurite outgrowth of spinal cord explants depend on culture substratum Initially, we used conventional poly-L-lysine substratum for co-culture. However, on poly-L-lysine-coated coverslips, spinal cord explants cultured in spinal cord explant culture medium (commonly used for culture of neurons) could not attach well to the coverslips (even extended waiting time to 5– 7 days, or reduced the amount of medium to 100–300 μl per well), and no neurites grew out from the explants. This was completely different from dissociated neurons that were cultured on the same substratum with the same medium, which did adhere to the coverslips and displayed extended neurites. A previous study has suggested that neurite outgrowth from the explants is promoted by an astrocyte bed layer, since during normal development, axonal growth and myelination are guided by an astrocytic scaffold (Thomson et al., 2006). We did not adopt astrocytes to support the growth of neurites from the explants, because the astrocyte bed layers were inconvenient for the direct observation of the interaction between oligodendrocytes and neurites during co-culture. Thus, we focused on improvement of the culture substratum, and plated the spinal cord explants on fibronectin, laminin or Matrigel. We found that Matrigel was the best substratum for explant attachment and neurite outgrowth. For the convenience of observation by conventional microscopy, we diluted Matrigel to a concentration of 2% to form a monolayer culture substratum, which did not impair the attachment and neurite outgrowth of spinal cord explants.
3.2. Migration of somas from spinal cord explants regulated by culture medium In order to determine an appropriate medium for our coculture system, two different media (CoM1 and CoM2) were tested. CoM1 was a combination of two culture media, one for neurons and the other for OPCs, and CoM2 was also a combination of two culture media, one for neurons and the other facilitating the differentiation of OPCs into mature oligodendrocytes. CoM1 and CoM2 both supported the survival of spinal cord explants and GFP-OPCs, promoted neurite outgrowth from explants, and induced differentiation of OPCs into oligodendrocytes. However, many somas moved
out of the explants when they were cultured in CoM1. Further identification of these migrated cells indicated that most of them were astrocytes and pre-oligodendrocytes. Since CoM1 contained PDGF and bFGF, while CoM2 did not, we considered that stimulation by PDGF and bFGF might be the major reason for the proliferation and migration of the glial cells from the explants, which was consistent with the previous study (de Castro and Bribian, 2005). Since too many emigrated somas would have interfered with the observation of the interaction between oligodendrocytes and neurites, CoM1 was not considered as an appropriate CoM for our model. CoM2 contained T3, which is a strong candidate for the extracellular signals that are required for oligodendrocyte precursor cells to stop dividing and differentiate into mature oligodendrocytes (Barres et al., 1994; Raff, 2006). CoM2 prevented the proliferation and migration of glial cells within explants; therefore, it was an ideal medium for our co-culture model.
3.3. Survival and viability of OPCs/oligodendrocytes play an important role in co-culture The survival and viability of GFP-OPCs/oligodendrocytes in coculture exert a great influence on the success of the OPC– spinal cord explant co-culture model. Accordingly, several approaches were used to improve the activity of GFP-OPC/ oligodendrocytes in our co-culture model. Firstly, Matrigel helped the GFP-OPCs attach well to the coverslips, and better attachment of OPCs ensured their viability during culture. Secondly, the right seeding order for GFP-OPCs and spinal cord explants may increase the activity of OPCs. Our results indicated that when GFP-OPCs were plated after the spinal cord explants, some OPCs were floating in the medium, had difficulty in attaching to the coverslips, and eventually died. Moreover, the surviving OPCs under such conditions were inclined to aggregate around the explants or neurites (Fig. 5A), and most of them would miss the chance to get in touch with neurites. If the GFP-OPCs were seeded before the spinal cord explants, the OPCs attached very well to the coverslips, and did not impede the attachment of spinal cord explants in the next day (Fig. 5B). Thirdly, GFP-OPCs should be preincubated in OPM for 1 or 2 days before co-cultured with spinal cord explants, because OPM contains PDGF and bFGF, which are important growth factors for the early generation of oligodendrocyte precursors (McKinnon et al., 1990; Robinson and Miller, 1996), whereas the selected co-culture medium lacks both of them. Lastly, co-culture medium should be renewed more frequently at later stage of co-culture, because in this stage, the neurites from the explants were long and extensive, and the GFP-oligodendrocytes were more mature, exhibiting multipolar morphology with dense processes, thus, they needed more nutrients to meet the requirement of their blooming growth and myelination.
3.4. Application of oligodendrocyte–spinal cord explant co-culture model Most of the previous co-culture models that contained oligodendrocytes or Schwann cells were co-cultured with
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Fig. 5 – The effect of seeding order on co-culture for 3 days. (A) GFP-OPCs were inclined to aggregate as they were seeded 1 day after spinal cord explants plating. (B) GFP-OPCs maintained dispersed as they were plated 1 day before spinal cord explants plating. Scale bar: 100 μm.
DRG or dissociated DRG neurons (Bunge and Wood, 1987; Einheber et al., 1993; Paivalainen et al., 2008; Seilheimer and Schachner, 1988; Wang et al., 2007). Although these models have been successfully utilized to study axon–myelin interaction, there are some limitations to these models in the study of axon–myelin interaction in the CNS, because DRG neurons are the components of the peripheral nervous system (PNS) and have different cellular and molecular characters from CNS neurons (Chan et al., 2004). In our coculture model, the oligodendrocytes and spinal cord explants were all derived from the spinal cord, which may better mimic the interaction between oligodendrocytes and neurons in the CNS, especially in the spinal cord. Thus, our model is very useful for the study of neural regeneration and myelination in spinal cord injury and demyelinating diseases of the spinal cord. Of course, there have been a few co-culture models using CNS neurons, but the observations on myelination in these models are not ideal enough. For example, in a previously reported neuron–oligodendrocyte co-culture model, the neurons were dissociated from CNS, and the myelination of the neurons was very robust (Fex Svenningsen et al., 2003; Thomson et al., 2006), however, the reticular distribution of the neurons in co-culture made it difficult to measure their growth parameters. Thomson et al. (2006) cultured the spinal cord explants on a bed layer of astrocytes, under such conditions, the extended neurites were myelinated by the oligodendrocytes migrated out of the explants, which made it easy to observe neurite–glial interaction, track individual myelinated neurites and determine their growth parameters. However, in this model, the neurite–glia interaction could only be observed after immunostaining, and also, the astrocyte bed layer on coverslip would cause some trouble in live observation. Considering these factors, we adopted Matrigel instead of astrocytes for the culture of spinal cord explants, and used exogenous GFP-OPCs as the source of the myelin-forming cells. Interestingly, with the combination of Matrigel and different culture media, the migration of neuronal somas and glia cells from the spinal cord explants were inhibited, thus the neurites grew out of the explant could be observed
clearly. Furthermore, since the oligodendrocytes in coculture were derived from GFP transgenic rats, with standard light microscopy and fluorescence microscopy, we could observe dynamically the growth of neurites, the interaction between oligodendrocytes and neurites, and the process of myelination, without immunostaining. The use of GFP-oligodendrocytes has shown clearly that the neurites and oligodendrocytes in co-culture can recognize and associate with each other, and form myelin structures. As the oligodendrocytes in our co-culture system were exogenous, in future study, we can try to genetically modify OPCs before co-culture and assess various factors involved in axonal growth and myelination, which would help with the study of the mechanisms and therapeutics of spinal cord injury and demyelinating diseases, such as multiple sclerosis.
4.
Experimental procedures
4.1.
Culture media
4.1.1.
Spinal cord explant culture medium
The medium consisted of Neurobasal medium (Invitrogen, USA), 2% B-27 Serum-Free Supplement (B27, Invitrogen) and 2 mM glutamine (Invitrogen).
4.1.2. GFP-neural precursor cell (GFP-NPC) culture medium (NPM) The medium consisted of Dulbecco's Modified Eagle's Medium/F12 (D/F12; Invitrogen), 1% N-2 Supplement (N2; Invitrogen), 1% B27, 2 mM glutamine, 3 μg/ml heparin (Sigma–Aldrich, USA), 20 ng/ml basic fibroblast growth factor (bFGF; Invitrogen) and 20 ng/ml epidermal growth factor (EGF; Sigma–Aldrich).
4.1.3. GFP-oligodendrocyte precursor cell (GFP-OPC) culture medium (OPM) The medium used to encourage the induction of OPCs from NPCs, consisted of D/F12, 1% N2, 1% B27, 2 mM glutamine, 3 μg/ml heparin, 0.1% bovine serum albumin, (BSA; PAA,
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USA), 10 ng/ml biotin (Sigma–Aldrich), 10 ng/ml bFGF and 10 ng/ml platelet-derived growth factor (PDGF; R&D, USA).
4.1.4.
Co-culture medium (CoM)
CoM1 consisted of 1:1 D/F12 and Neurobasal, 1% N2, 2% B27, 2 mM glutamine, 3 μg/ml heparin, 0.1% BSA, 10 ng/ml biotin, 10 ng/ml bFGF and 10 ng/ml PDGF. CoM2 consisted of 1:1 D/F12 and Neurobasal, 1% N2, 2% B27, 2 mM glutamine, 0.1% BSA, 10 ng/ml biotin and 30 ng/ml 3,3′, 5-triiodo-L-thyronine (T3; Sigma–Aldrich).
4.2.
Cell or tissue culture
4.2.1.
Culture of GFP-OPCs
GFP-OPCs were induced from GFP-NPCs which derived from the spinal cord of GFP transgenic E16 rats (The GFP transgenic SD rats were provided kindly by Dr. Okabe, University of Osaka, Japan). Induction was carried out as described previously (Fu et al., 2007). Freshly dissociated NPCs from the spinal cord were seeded at 1 × 105 cells/ml in NPM and cultured for 1 or 2 days to generate small neurospheres. The NPM was gradually replaced with OPM every other day for 3–4 times until the OPCs migrated out of the neurospheres and attached to the bottom of the flask. After removal of the necrotic spheres and fragmented cells, the OPCs were cultured for an additional 5–7 days until visible oligospheres (50–200 cells/sphere) were formed. The cultures were incubated with Accutase (PAA), 2 ml per T25 flask (Corning, USA) for 8–10 min at 37°C and washed with 8 ml HBSS. The cell suspension was triturated gently by a fire-polished Pasteur pipette to dissociate the oligospheres, centrifuged at 800– 1000 rpm for 8 min at room temperature (RT), and then the cells were resuspended with OPM. Finally, 2 × 104/100 μl GFPOPCs were seeded onto glass coverslips coated with poly-Llysine (200 μg/ml; Sigma–Aldrich), fibronectin (5 μg/ml; Invitrogen), laminin (10 μg/ml; Invitrogen) or 2% Matrigel (BD Biosciences, USA), or onto plastic coverslips (plastic coverslips, EMS, USA; the cultures on plastic coverslips were especially for electron microscopy) coated with 2% Matrigel. These cultures were placed in 24-well plates (Corning). After 1 h incubation, an extra 400 μl OPM was added to each well, and the cells were ready for co-culture after further overnight incubation. One day later, GFP-OPCs were ready for immunostaining or co-culture.
4.2.2.
Preparation and plating of spinal cord explants
Embryonic Sprague–Dawley (SD) rats at 14 days of gestation (E14) were removed from their pregnant mothers and transferred to a 100-mm Falcon culture dish (Becton Dickinson Labware, USA) that contained Hanks' balanced salt solution (HBSS; Invitrogen). Their vertebral canals were opened and spinal cords were removed rapidly into another 100-mm dish that contained HBSS, using sterile fine-tipped forceps and micro-spring scissors. After the meninges and blood vessels were removed carefully, the embryonic spinal cords were cut into small pieces (no more than 0.5 mm in length) by scissors, which would be used as spinal cord explants. To test different substrates, spinal cord explants were plated onto glass coverslips coated with poly-L-lysine (200 μg/ml),
fibronectin (5 μg/ml), laminin (10 μg/ml) or 2% Matrigel respectively. For co-culture, spinal cord explants were plated onto GFP-OPCs, after OPM was replaced with CoM. To assess the percentages of the attachment of plated explants, a total of 24 wells (one explant per well) from at least three independent experiments were accounted for each group. The above cultures were all maintained in a humidified incubator at 37 °C and 5% CO2. All animal care, surgical procedures, and post-operative euthanasia were performed in accordance with the National Institutes of Health Guide for the care and use of laboratory animals (NIH Publications No. 80-23; revised 1996) and were approved by the Animal Care Committee of the Use of Laboratory Animals at Shanghai Jiaotong University School of Medicine.
4.3.
Immunocytochemistry
Before immunostaining, coverslips were fixed with 4% paraformaldehyde (PFA) for 10 min at RT, washed three times for 5 min each in 0.01 M PBS, penetrated by 0.3% Triton X-100 [except A2B5 and PDGF receptor (PDGFR) staining], blocked with 10% normal goat serum for 1 h at RT, and incubated with primary antibodies: mouse A2B5 IgM hybridoma supernatant, 1:1; mouse RIP IgG hybridoma supernatant, 1:1 (generous gifts from Dr. Xiao-ming Xu, Indiana University); rabbit anti-rat PDGFR polyclonal antibody, 1:100 (Neomarkers, Fremont, CA, USA); mouse antiMBP monoclonal antibody, 1:20 (Chemicon, CA, USA) rabbit anti-neurofilament (NF) polyclonal antibody, 1:80 (Sigma– Aldrich). Incubation was overnight at 4 °C, except for A2B5, which was incubated for 1 h at RT. After three washes (10 min each) in PBS, the cultures were incubated with the secondary antibodies: rhodamine-conjugated goat antimouse IgM, 1:200 (Sigma–Aldrich); Alexa 546 goat antimouse IgG, 1:1000 (Molecular probes, Invitrogen); Alexa 546 goat anti-rabbit IgG, 1:1000 (Molecular probes, Invitrogen); Alexa 633 goat anti-rabbit IgG, 1:1000 (Molecular probes, Invitrogen). Incubation was for 30 min at RT, followed by rinsing five times (5 min each) in PBS, and mounting with Gel/Mount aqueous mounting media (Biomeda Corp., CA, USA) that contained Hoechst 33342 (Sigma–Aldrich) to stain cell nuclei.
4.4.
Co-culture preparation for electron microscopy
Preparation for electron microscopy was as described previously (Zhang et al., 2009). Co-cultures on plastic coverslips were transferred to 2% glutaraldehyde and 3% sucrose in 0.1 M phosphate buffer (pH 7.4) for 30 min, followed by 2% osmium tetroxide in 0.1 M PBS for 1 h. Cultures were then dehydrated in graded ethanol, and embedded with EM-812. Ultrathin sections (70–90 nm) were cut in an orientation that cut axons transversely.
4.5.
Microscopy
Phase-contrast images of the live cultures and some immunofluorescence images were taken with an Olympus
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IX70 microscope equipped with a digital camera. Confocal images of immunofluorescence were captured with a Zeiss LSM 510 META laser scanning microscope. Electron microscopy was performed with a Philips CM-120 transmission electron microscope (Eindhoven, The Netherlands), at the EM Image Facility of Shanghai Jiaotong University School of Medicine.
4.6.
Cell counting
The number of immunofluorescent cells was counted by the software of Image Pro-plus 5.0. The purity of GFP-OPCs was expressed as percentage of A2B5- or PDGFR-positive cells relative to the total number of GFP-positive cells. The cell number of migrated cells around spinal cord explant when cultured alone was exactly the number of Hoechst-labeled cells. The cell number of migrated cells around spinal cord explant when co-cultured with GFP-OPCs was equal to the total number of Hoechst-labeled cells minus the number of GFP-positive cells. The proportion of myelinating GFP-oligodendrocytes was expressed as the percentage of the cells associated with neurites and co-expressed GFP and MBP relative to the total GFP-cells associated with neurites.
Acknowledgments This work was funded by the 973 project (2003CB515302), Shanghai Educational Committee Technology Foundation (06BZ006) and the Young Teachers' Science and Technology Ability Upgrading Project of Institutes of Medical Sciences, Shanghai Jiaotong University School of Medicine.
REFERENCES
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Einheber, S., Milner, T.A., Giancotti, F., Salzer, J.L., 1993. Axonal regulation of Schwann cell integrin expression suggests a role for alpha 6 beta 4 in myelination. J. Cell Biol. 123, 1223–1236. Fex Svenningsen, A., Shan, W.S., Colman, D.R., Pedraza, L., 2003. Rapid method for culturing embryonic neuron–glial cell cocultures. J. Neurosci. Res. 72, 565–573. Frost, E.E., Zhou, Z., Krasnesky, K., Armstrong, R.C., 2009. Initiation of oligodendrocyte progenitor cell migration by a PDGF-A activated extracellular regulated kinase (ERK) signaling pathway. Neurochem. Res 34, 169–181. Fu, S.L., Hu, J.G., Li, Y., Wang, Y.X., Jin, J.Q., Xui, X.M., Lu, P.H., 2007. A simplified method for generating oligodendrocyte progenitor cells from neural precursor cells isolated from the E16 rat spinal cord. Acta Neurobiol. Exp. (Wars) 67, 367–377. GrandPre, T., Li, S., Strittmatter, S.M., 2002. Nogo-66 receptor antagonist peptide promotes axonal regeneration. Nature 417, 547–551. Jiang, X.Y., Fu, S.L., Nie, B.M., Li, Y., Lin, L., Yin, L., Wang, Y.X., Lu, P.H., Xu, X.M., 2006. Methods for isolating highly-enriched embryonic spinal cord neurons: a comparison between enzymatic and mechanical dissociations. J. Neurosci. Methods 158, 13–18. Khan, Z., Ferrari, G., Kasper, M., Tonge, D.A., Steiner, J.P., Hamilton, G.S., Gordon-Weeks, P.R., 2002. The non-immunosuppressive immunophilin ligand GPI-1046 potently stimulates regenerating axon growth from adult mouse dorsal root ganglia cultured in Matrigel. Neuroscience 114, 601–609. Ma, Z., Cao, Q., Zhang, L., Hu, J., Howard, R.M., Lu, P., Whittemore, S.R., Xu, X.M., 2009. Oligodendrocyte precursor cells differentially expressing Nogo-A but not MAG are more permissive to neurite outgrowth than mature oligodendrocytes. Exp. Neurol. 217, 184–196. McKinnon, R.D., Matsui, T., Dubois-Dalcq, M., Aaronson, S.A., 1990. FGF modulates the PDGF-driven pathway of oligodendrocyte development. Neuron 5, 603–614. Miller, R.H., 2002. Regulation of oligodendrocyte development in the vertebrate CNS. Prog. Neurobiol. 67, 451–467. Noguchi, Y., Okamoto, A., Kasama, T., Imajoh-Ohmi, S., Karatsu, T., Nogawa, H., 2006. Lysophosphatidic acid cooperates with EGF in inducing branching morphogenesis of embryonic mouse salivary epithelium. Dev. Dyn. 235, 403–410. Paivalainen, S., Nissinen, M., Honkanen, H., Lahti, O., Kangas, S.M., Peltonen, J., Peltonen, S., Heape, A.M., 2008. Myelination in mouse dorsal root ganglion/Schwann cell cocultures. Mol. Cell Neurosci. 37, 568–578. Raff, M., 2006. The mystery of intracellular developmental programmes and timers. Biochem. Soc. Trans. 34, 663–670. Reimers, D., Lopez-Toledano, M.A., Mason, I., Cuevas, P., Redondo, C., Herranz, A.S., Lobo, M.V., Bazan, E., 2001. Developmental expression of fibroblast growth factor (FGF) receptors in neural stem cell progeny. Modulation of neuronal and glial lineages by basic FGF treatment. Neurol. Res. 23, 612–621. Robinson, S., Miller, R., 1996. Environmental enhancement of growth factor-mediated oligodendrocyte precursor proliferation. Mol. Cell Neurosci. 8, 38–52. Seilheimer, B., Schachner, M., 1988. Studies of adhesion molecules mediating interactions between cells of peripheral nervous system indicate a major role for L1 in mediating sensory neuron growth on Schwann cells in culture. J. Cell Biol. 107, 341–351. Thomson, C.E., Hunter, A.M., Griffiths, I.R., Edgar, J.M., McCulloch, M.C., 2006. Murine spinal cord explants: a model for evaluating axonal growth and myelination in vitro. J. Neurosci. Res. 84, 1703–1715.
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Research Report
Oligodendrocyte–spinal cord explant co-culture: An in vitro model for the study of myelination Zhifang Chen, Zhengwen Ma, Yanxia Wang, Ying Li, Hezuo Lü, Saili Fu, Qin Hang, Pei-Hua Lu⁎ Department of Neurobiology, Shanghai Jiaotong University School of Medicine, 280 South Chongqing Road, Shanghai 200025, P.R. China
A R T I C LE I N FO
AB S T R A C T
Article history:
The in vitro models developed to investigate the growth and myelination of axons, such as
Accepted 24 October 2009
dorsal root ganglion (DRG)–Schwann cell co-culture, DRG–oligodendrocyte co-culture and
Available online 30 October 2009
central nervous system (CNS) neuron–oligodendrocyte co-culture, have provided an effective way to reveal the mechanisms that underlie the interaction between neurons
Keywords:
and myelin-forming cells. In order to better understand the complex process of myelination
Spinal cord explant
during CNS development and spinal cord repair, we established a rat spinal cord neuron–
Neurite
oligodendrocyte co-culture model. In this co-culture system, the spinal cord explants were
Oligodendrocyte
used as the source of neurons, and the oligodendrocytes were induced from GFP-
Myelin
oligodendrocyte precursor cells (GFP-OPCs). The results showed that the GFP-
GFP
oligodendrocytes that differentiated from GFP-OPCs in co-culture attached to the neurites
Co-culture
growing out from the spinal cord explants and formed myelin structures. As the oligodendrocytes expressed GFP, and the neuron somas remained in the explants, the interaction between oligodendrocytes and neurites in co-culture were observed clearly and dynamically without immunostaining. © 2009 Elsevier B.V. All rights reserved.
1.
Introduction
In the nervous system, myelinated axons are wrapped tightly by the continuous membrane layers of glial cells, i.e., oligodendrocytes in the central nervous system (CNS) or Schwann cells in the peripheral nervous system (PNS). The in vitro models developed to investigate the growth and myelination of axons, such as dorsal root ganglion (DRG)–Schwann cell co-culture (Bunge and Wood, 1987; Einheber et al., 1993; Paivalainen et al., 2008; Seilheimer and Schachner, 1988), DRG neuron–oligodendrocyte co-culture (Wang et al., 2007), retinal ganglion–Schwann cell co-culture (Bahr et al., 1991) and CNS neuron–oligodendrocyte co-culture (Fex Svenningsen et al.,
2003; Thomson et al., 2006), have revealed the mechanisms that underlie the interaction between neurons and myelinforming cells. To better understand the complex process of axonal growth and myelination during CNS development and spinal cord repair, we established an oligodendrocyte–spinal cord explant co-culture model. The neurites extended radially from the explants, but their somas remained in the explants, which facilitated the observation and measurement of the neurites during co-culture. In addition, the oligodendrocytes in coculture were induced from GFP-oligodendrocyte precursor cells (GFP-OPCs), which could be observed directly by fluorescence microscopy. Thus, in our co-culture model, as without
⁎ Corresponding author. Fax: +86 21 64453296. E-mail address: [email protected] (P.-H. Lu). 0006-8993/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2009.10.060
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the interference of non-GFP somas, the interaction between GFP-oligodendrocytes and neurites could be observed clearly and dynamically without immunostaining.
2.
Results
2.1. Attachment and neurite outgrowth of spinal cord explants
least 95% of the explants were attached to the coverslips and fine neurites growing from the edge of the explants were visible (Fig. 1E). Three days after culture, the neurites from the explants were extended significantly, some even up to 1000 μm (Fig. 1F). The results indicated that, in contrast to poly-L-lysine, laminin and fibronectin, Matrigel not only speeded up the attachment of spinal cord explants, but also promoted neurite outgrowth from the explants.
2.2. Spinal cord explants were plated initially onto poly-L-lysine substratum and cultured with spinal cord explant culture medium, which is commonly used for neurons (Calabrese and Halpain, 2005; Jiang et al., 2006). After 3 days in vitro, most explants remained detached, and the explant was devoid of neurites (Fig. 1A). To improve the attachment, we used laminin and fibronectin to substitute for poly-L-lysine respectively. The results showed that after 3 days culture, on both laminin-coated (Fig. 1B) and fibronectin-coated (Fig. 1C) coverslips, 50–60% of explants could attach to the coverslips. And the neurites from explants could be seen, but in low density and short length. It has been reported that Matrigel encourages the attachment and axonal growth of neurons (Khan et al., 2002; Yu et al., 1994). To further improve the culture environment for spinal cord explants, we tried to coat the coverslips with 2% Matrigel instead of poly-L-lysine. The results showed that 4 h after plating, more than 60% of the explants were attached to the Matrigel-coated coverslips and neurites budding from the explants were observed (Fig. 1D). Twelve hours after plating, at
Co-culture of spinal cord explants with GFP-OPCs
As mature oligodendrocytes express myelin-associated proteins (Nogo, MAG and OMgp) that inhibit axonal outgrowth in neuron (Domeniconi et al., 2005; GrandPre et al., 2002; Wang et al., 2002), it is difficult to co-culture neurons with mature oligodendrocytes in vitro (Ma et al., 2009). In addition, the previous study has revealed that maturation of oligodendrocytes is regulated largely by axonal signals in the development of the CNS (Miller, 2002). Therefore, we co-cultured spinal cord explants with GFP-OPCs rather than mature oligodendrocytes. We believe that during co-culture with neurons, the OPCs gradually differentiated into mature oligodendrocytes, and the GFP expressed in OPCs facilitated the dynamic observation of the interaction between oligodendrocytes and neurites without immunostaining. At first, to make sure that Matrigel also facilitated the growth of GFP-OPCs, we compared the viability of OPCs on different substratums. The results showed that 1 day after seeding, OPCs on poly-L-lysine attached poorly to coverslip, and cell status was inconsistent (Fig. 2A). OPCs on fibronectin could attach well
Fig. 1 – Effects of culture substratums on attachment and neurite outgrowth of spinal cord explants. Phase contrast micrographs showed representative explants under different conditions. (A–C) After 3 days culture, the spinal cord explant attached poorly to the poly-L-lysine-coated coverslip, therefore, the explant was devoid of neurites (A), while on laminin-coated (B) and fibronectin-coated (C) coverslips, 50–60% of explants could attach to the coverslips. The neurites from the explants were in low density, and the length of neurites was restricted to 500 μm. (D) Four hours after plating, more than 60% of the explants were attached to 2% Matrigel-coated coverslip, and outgrowth of neurites from the explants occurred (box). (E) Twelve hours after seeding, at least 95% of explants were attached to the Matrigel-coated coverslip, and many neurites extended straight out of the explants. (F) After 3 days culture on the Matrigel-coated coverslip, the neurites from the spinal cord explants were extended significantly, some of them up to 1000 μm. Scale bar: A–E, 66 μm; F, 100 μm.
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to the coverslip, but cell status was also inconsistent (Fig. 2B). OPCs on laminin (Fig. 2C) and on Matrigel (Fig. 2D) all grew well and were in good status, but obviously, cells on Matrigel showed even better attachment. It indicated that Matrigel was the best substratum for both spinal cord explants and OPCs. Before co-culture with spinal cord explants, the GFP-OPCs in 2% Matrigel were seeded onto the glass coverslips in OPM (details as described in Experimental procedures). On the next day, GFP cells attached to the coverslips displayed typical bipolar or tri-polar morphology of OPCs (Fig. 2D–F). Immunostaining showed that more than 90% of GFP cells expressed A2B5 (Fig. 2G) and PDGFR (Fig. 2H), the markers for OPCs, and
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no RIP+ cells (oligodendrocytes) were observed (Fig. 2I). After 1 day culture in OPM, the GFP-OPCs were ready for co-culture, OPM was replaced with CoM (details as described in Experimental procedures), and the spinal cord explants were plated subsequently into the culture. At first, CoM1 and CoM2 that can support the survival of oligodendrocytes and neurons on Matrigel were used simultaneously. The co-culture results revealed that, although OPCs/oligodendrocytes and neurons all grew well in both media, there were more somas emigrating out of the explants in CoM1 (containing PDGF and bFGF) than in CoM2 (containing T3). After 3 days culture in CoM1, at least 2500 somas were
Fig. 2 – Identification of GFP-OPCs induced from GFP-NPCs before co-culture. (A–D) Phase contrast micrographs of OPCs cultured on different substratum. OPCs cultured on poly-L-lysine showed poor attachment and inconsistent cell status (A); OPCs attached well on fibronectin, but also presented inconsistent cell status (B). OPCs on laminin (C) and on Matrigel (D) were healthy, but cells on Matrigel showed even better attachment. (D–F) Photomicrographs of live OPCs cultured on Matrigel showed that the GFP-OPCs induced from GFP-NSCs displayed bi- or tri-polar morphology, the typical morphology of OPCs. (G–I) Confocal images of immunostaining showed that most GFP-OPCs expressed A2B5 (G) and PDGFR (H), the specific markers for OPCs, but did not express RIP (I), a specific marker for oligodendrocytes. Cells in G–I were counterstained with Hoechst 33342 (blue), a nuclear dye. Scale bar: A–F, 40 μm; G–I, 10 μm.
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observed surrounding each spinal cord explant, whether cultured alone or with GFP-OPCs (Figs. 3A–C), while in CoM2, only a few somas emigrated from each explant (Figs. 3D–F). Further identification of those migrated cells in CoM1 indicated that most of them were O4+ pre-oligodendrocytes and GFAP+ astrocytes (Figs. 3G–I). This was consistent with the previous findings that PDGF and bFGF have a great tendency to promote neural cell migration (Frost et al., 2009) and prolifer-
ation (Noguchi et al., 2006; Reimers et al., 2001). Since too many emigrated somas would have been inconvenient for the observation of oligodendrocyte–neurite interaction and the measurement of their growth parameters, CoM1 was not appropriate for co-culture, thus, CoM2 was adopted as the medium for subsequent co-culture. Three days after co-culture, the neurites from spinal cord explants had grown into GFP-OPCs monolayer in
Fig. 3 – Effects of CoM1 and CoM2 on spinal cord explant cultures, alone or with GFP-OPCs. (A and D) Phase contrast micrographs showed that 3 days after explants were cultured alone in CoM1, there were many somas moving out of the explant (A). In CoM2, only a few somas emigrated from the explant (D). (B, C, E and F) Immunofluorescence micrographs showed that 3 days after co-culture with GFP-OPCs, the neurites (NF, red) from explants were extended and had grown into the GFP-OPCs monolayer (GFP, green) in CoM1 (B and C) and CoM2 (E and F). There were also many somas moving out of the explant (C, Hoechst), when cultured in CoM1. This did not happen in CoM2 (F). (G–I) Identification of migrated cells, there were numerous somas surrounding the explant after cultured in CoM1 (G). Immunostaining assay showed the cells around the explant were O4 positive (H) and GFAP positive (I), which indicated that the cells migrated from explant were pre-oligodendrocytes and astrocytes. * Represents the location of spinal cord explant. (J–L) High-magnification micrographs revealed that 3 days after co-culture in CoM2, the GFP-OPCs displayed more branches (J) than original OPCs, and attached closely to the neurite (NF, red) outgrowth from the explant (K and L). Scale bar: A–F, 100 μm; G–I, 25 μm; J–L, 15 μm.
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scattered fibers (Figs. 3E and F), and the growing neurites made direct contact with the surface of the OPCs (Fig. 3L). Meanwhile, the GFP-OPCs in co-culture had differentiated into pre-oligodendrocytes that displayed more branches than the original OPCs (Figs. 3J and L). Our co-culture model showed that OPCs/pre-oligodendrocytes and neurites could co-exist, even though they were from different individuals.
2.3. Myelination of neurites growing from spinal cord explants in co-culture Neurites from spinal cord explants in co-culture elongated rapidly during the first 3 days. From the fourth day onwards,
13
the growth rate of the main neurites slowed down, but the branches of the neurites were growing denser. Eight days after co-culture, some GFP cells exhibited multipolar morphology, which indicated that some GFPOPCs had differentiated into more mature oligodendrocytes. Combined phase contrast and fluorescence microscopy showed two-dimensional interaction between GFP cells and neurites in co-culture. It was demonstrated clearly that the processes of some GFP-oligodendrocytes had touched and elongated along the neurites (Figs. 4A–C). Moreover, the fine branch networks of GFP-oligodendrocytes also attached tightly to the neurites or their branches (Figs. 4D–F). The green sheaths formed by GFP-oligodendrocytes were observed clearly. In general, the processes of one mature oligodendro-
Fig. 4 – Myelination of neurites by GFP-oligodendrocytes in co-culture. (A–C) Live-cell images showed that 8 days after co-culture, a GFP-expressing oligodendrocyte contacted the neurites by its processes that were growing along with the neurites (C, arrow). (D–F) High-magnification images of the box in picture C showed that the fine branch networks of the GFP-oligodendrocyte were also attached closely to the neurites (F, arrows). (G–I) Confocal images showed that after 8 days co-culture, the GFP-oligodendrocytes made close contact with the neurites (arrows), which expressed MBP (G and I,) and RIP (H), the specific markers of mature oligodendrocytes. (J–M) Confocal images showed that after 15 days co-culture, the myelinated neurites were observed clearly. The neurites were labeled with NF (L) and myelin sheaths were co-labeled with GFP (J) and MBP (K) (arrows). (N and O) The presence of compact myelin in 15-day co-cultures was confirmed by electron microscopy. Scale bar: A–C, 15 μm; D–F, 5 μm; G, 20 μm; H and I, 10 μm; J–M, 5 μm; N, 1 μm; O, 40 nm.
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cyte made simultaneous contact with several segments of neurites or their branches. Immunostaining indicated that these GFP-oligodendrocytes expressed RIP and MBP, the markers of more mature oligodendrocytes, and about 32.5% of the GFP cells associated with neurites expressed MBP (Figs. 4G–I). Fifteen days after co-culture, immunostaining showed that the myelinated neurites could be observed clearly (Figs. 4J–M). The three-dimensional sheaths after 15 days co-culture were further identified by electron microscopy. The electron microscopic images showed that the neurites were surrounded by compact myelin sheaths (Figs. 4N and O).
3.
Discussion
3.1. Attachment and neurite outgrowth of spinal cord explants depend on culture substratum Initially, we used conventional poly-L-lysine substratum for co-culture. However, on poly-L-lysine-coated coverslips, spinal cord explants cultured in spinal cord explant culture medium (commonly used for culture of neurons) could not attach well to the coverslips (even extended waiting time to 5– 7 days, or reduced the amount of medium to 100–300 μl per well), and no neurites grew out from the explants. This was completely different from dissociated neurons that were cultured on the same substratum with the same medium, which did adhere to the coverslips and displayed extended neurites. A previous study has suggested that neurite outgrowth from the explants is promoted by an astrocyte bed layer, since during normal development, axonal growth and myelination are guided by an astrocytic scaffold (Thomson et al., 2006). We did not adopt astrocytes to support the growth of neurites from the explants, because the astrocyte bed layers were inconvenient for the direct observation of the interaction between oligodendrocytes and neurites during co-culture. Thus, we focused on improvement of the culture substratum, and plated the spinal cord explants on fibronectin, laminin or Matrigel. We found that Matrigel was the best substratum for explant attachment and neurite outgrowth. For the convenience of observation by conventional microscopy, we diluted Matrigel to a concentration of 2% to form a monolayer culture substratum, which did not impair the attachment and neurite outgrowth of spinal cord explants.
3.2. Migration of somas from spinal cord explants regulated by culture medium In order to determine an appropriate medium for our coculture system, two different media (CoM1 and CoM2) were tested. CoM1 was a combination of two culture media, one for neurons and the other for OPCs, and CoM2 was also a combination of two culture media, one for neurons and the other facilitating the differentiation of OPCs into mature oligodendrocytes. CoM1 and CoM2 both supported the survival of spinal cord explants and GFP-OPCs, promoted neurite outgrowth from explants, and induced differentiation of OPCs into oligodendrocytes. However, many somas moved
out of the explants when they were cultured in CoM1. Further identification of these migrated cells indicated that most of them were astrocytes and pre-oligodendrocytes. Since CoM1 contained PDGF and bFGF, while CoM2 did not, we considered that stimulation by PDGF and bFGF might be the major reason for the proliferation and migration of the glial cells from the explants, which was consistent with the previous study (de Castro and Bribian, 2005). Since too many emigrated somas would have interfered with the observation of the interaction between oligodendrocytes and neurites, CoM1 was not considered as an appropriate CoM for our model. CoM2 contained T3, which is a strong candidate for the extracellular signals that are required for oligodendrocyte precursor cells to stop dividing and differentiate into mature oligodendrocytes (Barres et al., 1994; Raff, 2006). CoM2 prevented the proliferation and migration of glial cells within explants; therefore, it was an ideal medium for our co-culture model.
3.3. Survival and viability of OPCs/oligodendrocytes play an important role in co-culture The survival and viability of GFP-OPCs/oligodendrocytes in coculture exert a great influence on the success of the OPC– spinal cord explant co-culture model. Accordingly, several approaches were used to improve the activity of GFP-OPC/ oligodendrocytes in our co-culture model. Firstly, Matrigel helped the GFP-OPCs attach well to the coverslips, and better attachment of OPCs ensured their viability during culture. Secondly, the right seeding order for GFP-OPCs and spinal cord explants may increase the activity of OPCs. Our results indicated that when GFP-OPCs were plated after the spinal cord explants, some OPCs were floating in the medium, had difficulty in attaching to the coverslips, and eventually died. Moreover, the surviving OPCs under such conditions were inclined to aggregate around the explants or neurites (Fig. 5A), and most of them would miss the chance to get in touch with neurites. If the GFP-OPCs were seeded before the spinal cord explants, the OPCs attached very well to the coverslips, and did not impede the attachment of spinal cord explants in the next day (Fig. 5B). Thirdly, GFP-OPCs should be preincubated in OPM for 1 or 2 days before co-cultured with spinal cord explants, because OPM contains PDGF and bFGF, which are important growth factors for the early generation of oligodendrocyte precursors (McKinnon et al., 1990; Robinson and Miller, 1996), whereas the selected co-culture medium lacks both of them. Lastly, co-culture medium should be renewed more frequently at later stage of co-culture, because in this stage, the neurites from the explants were long and extensive, and the GFP-oligodendrocytes were more mature, exhibiting multipolar morphology with dense processes, thus, they needed more nutrients to meet the requirement of their blooming growth and myelination.
3.4. Application of oligodendrocyte–spinal cord explant co-culture model Most of the previous co-culture models that contained oligodendrocytes or Schwann cells were co-cultured with
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Fig. 5 – The effect of seeding order on co-culture for 3 days. (A) GFP-OPCs were inclined to aggregate as they were seeded 1 day after spinal cord explants plating. (B) GFP-OPCs maintained dispersed as they were plated 1 day before spinal cord explants plating. Scale bar: 100 μm.
DRG or dissociated DRG neurons (Bunge and Wood, 1987; Einheber et al., 1993; Paivalainen et al., 2008; Seilheimer and Schachner, 1988; Wang et al., 2007). Although these models have been successfully utilized to study axon–myelin interaction, there are some limitations to these models in the study of axon–myelin interaction in the CNS, because DRG neurons are the components of the peripheral nervous system (PNS) and have different cellular and molecular characters from CNS neurons (Chan et al., 2004). In our coculture model, the oligodendrocytes and spinal cord explants were all derived from the spinal cord, which may better mimic the interaction between oligodendrocytes and neurons in the CNS, especially in the spinal cord. Thus, our model is very useful for the study of neural regeneration and myelination in spinal cord injury and demyelinating diseases of the spinal cord. Of course, there have been a few co-culture models using CNS neurons, but the observations on myelination in these models are not ideal enough. For example, in a previously reported neuron–oligodendrocyte co-culture model, the neurons were dissociated from CNS, and the myelination of the neurons was very robust (Fex Svenningsen et al., 2003; Thomson et al., 2006), however, the reticular distribution of the neurons in co-culture made it difficult to measure their growth parameters. Thomson et al. (2006) cultured the spinal cord explants on a bed layer of astrocytes, under such conditions, the extended neurites were myelinated by the oligodendrocytes migrated out of the explants, which made it easy to observe neurite–glial interaction, track individual myelinated neurites and determine their growth parameters. However, in this model, the neurite–glia interaction could only be observed after immunostaining, and also, the astrocyte bed layer on coverslip would cause some trouble in live observation. Considering these factors, we adopted Matrigel instead of astrocytes for the culture of spinal cord explants, and used exogenous GFP-OPCs as the source of the myelin-forming cells. Interestingly, with the combination of Matrigel and different culture media, the migration of neuronal somas and glia cells from the spinal cord explants were inhibited, thus the neurites grew out of the explant could be observed
clearly. Furthermore, since the oligodendrocytes in coculture were derived from GFP transgenic rats, with standard light microscopy and fluorescence microscopy, we could observe dynamically the growth of neurites, the interaction between oligodendrocytes and neurites, and the process of myelination, without immunostaining. The use of GFP-oligodendrocytes has shown clearly that the neurites and oligodendrocytes in co-culture can recognize and associate with each other, and form myelin structures. As the oligodendrocytes in our co-culture system were exogenous, in future study, we can try to genetically modify OPCs before co-culture and assess various factors involved in axonal growth and myelination, which would help with the study of the mechanisms and therapeutics of spinal cord injury and demyelinating diseases, such as multiple sclerosis.
4.
Experimental procedures
4.1.
Culture media
4.1.1.
Spinal cord explant culture medium
The medium consisted of Neurobasal medium (Invitrogen, USA), 2% B-27 Serum-Free Supplement (B27, Invitrogen) and 2 mM glutamine (Invitrogen).
4.1.2. GFP-neural precursor cell (GFP-NPC) culture medium (NPM) The medium consisted of Dulbecco's Modified Eagle's Medium/F12 (D/F12; Invitrogen), 1% N-2 Supplement (N2; Invitrogen), 1% B27, 2 mM glutamine, 3 μg/ml heparin (Sigma–Aldrich, USA), 20 ng/ml basic fibroblast growth factor (bFGF; Invitrogen) and 20 ng/ml epidermal growth factor (EGF; Sigma–Aldrich).
4.1.3. GFP-oligodendrocyte precursor cell (GFP-OPC) culture medium (OPM) The medium used to encourage the induction of OPCs from NPCs, consisted of D/F12, 1% N2, 1% B27, 2 mM glutamine, 3 μg/ml heparin, 0.1% bovine serum albumin, (BSA; PAA,
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USA), 10 ng/ml biotin (Sigma–Aldrich), 10 ng/ml bFGF and 10 ng/ml platelet-derived growth factor (PDGF; R&D, USA).
4.1.4.
Co-culture medium (CoM)
CoM1 consisted of 1:1 D/F12 and Neurobasal, 1% N2, 2% B27, 2 mM glutamine, 3 μg/ml heparin, 0.1% BSA, 10 ng/ml biotin, 10 ng/ml bFGF and 10 ng/ml PDGF. CoM2 consisted of 1:1 D/F12 and Neurobasal, 1% N2, 2% B27, 2 mM glutamine, 0.1% BSA, 10 ng/ml biotin and 30 ng/ml 3,3′, 5-triiodo-L-thyronine (T3; Sigma–Aldrich).
4.2.
Cell or tissue culture
4.2.1.
Culture of GFP-OPCs
GFP-OPCs were induced from GFP-NPCs which derived from the spinal cord of GFP transgenic E16 rats (The GFP transgenic SD rats were provided kindly by Dr. Okabe, University of Osaka, Japan). Induction was carried out as described previously (Fu et al., 2007). Freshly dissociated NPCs from the spinal cord were seeded at 1 × 105 cells/ml in NPM and cultured for 1 or 2 days to generate small neurospheres. The NPM was gradually replaced with OPM every other day for 3–4 times until the OPCs migrated out of the neurospheres and attached to the bottom of the flask. After removal of the necrotic spheres and fragmented cells, the OPCs were cultured for an additional 5–7 days until visible oligospheres (50–200 cells/sphere) were formed. The cultures were incubated with Accutase (PAA), 2 ml per T25 flask (Corning, USA) for 8–10 min at 37°C and washed with 8 ml HBSS. The cell suspension was triturated gently by a fire-polished Pasteur pipette to dissociate the oligospheres, centrifuged at 800– 1000 rpm for 8 min at room temperature (RT), and then the cells were resuspended with OPM. Finally, 2 × 104/100 μl GFPOPCs were seeded onto glass coverslips coated with poly-Llysine (200 μg/ml; Sigma–Aldrich), fibronectin (5 μg/ml; Invitrogen), laminin (10 μg/ml; Invitrogen) or 2% Matrigel (BD Biosciences, USA), or onto plastic coverslips (plastic coverslips, EMS, USA; the cultures on plastic coverslips were especially for electron microscopy) coated with 2% Matrigel. These cultures were placed in 24-well plates (Corning). After 1 h incubation, an extra 400 μl OPM was added to each well, and the cells were ready for co-culture after further overnight incubation. One day later, GFP-OPCs were ready for immunostaining or co-culture.
4.2.2.
Preparation and plating of spinal cord explants
Embryonic Sprague–Dawley (SD) rats at 14 days of gestation (E14) were removed from their pregnant mothers and transferred to a 100-mm Falcon culture dish (Becton Dickinson Labware, USA) that contained Hanks' balanced salt solution (HBSS; Invitrogen). Their vertebral canals were opened and spinal cords were removed rapidly into another 100-mm dish that contained HBSS, using sterile fine-tipped forceps and micro-spring scissors. After the meninges and blood vessels were removed carefully, the embryonic spinal cords were cut into small pieces (no more than 0.5 mm in length) by scissors, which would be used as spinal cord explants. To test different substrates, spinal cord explants were plated onto glass coverslips coated with poly-L-lysine (200 μg/ml),
fibronectin (5 μg/ml), laminin (10 μg/ml) or 2% Matrigel respectively. For co-culture, spinal cord explants were plated onto GFP-OPCs, after OPM was replaced with CoM. To assess the percentages of the attachment of plated explants, a total of 24 wells (one explant per well) from at least three independent experiments were accounted for each group. The above cultures were all maintained in a humidified incubator at 37 °C and 5% CO2. All animal care, surgical procedures, and post-operative euthanasia were performed in accordance with the National Institutes of Health Guide for the care and use of laboratory animals (NIH Publications No. 80-23; revised 1996) and were approved by the Animal Care Committee of the Use of Laboratory Animals at Shanghai Jiaotong University School of Medicine.
4.3.
Immunocytochemistry
Before immunostaining, coverslips were fixed with 4% paraformaldehyde (PFA) for 10 min at RT, washed three times for 5 min each in 0.01 M PBS, penetrated by 0.3% Triton X-100 [except A2B5 and PDGF receptor (PDGFR) staining], blocked with 10% normal goat serum for 1 h at RT, and incubated with primary antibodies: mouse A2B5 IgM hybridoma supernatant, 1:1; mouse RIP IgG hybridoma supernatant, 1:1 (generous gifts from Dr. Xiao-ming Xu, Indiana University); rabbit anti-rat PDGFR polyclonal antibody, 1:100 (Neomarkers, Fremont, CA, USA); mouse antiMBP monoclonal antibody, 1:20 (Chemicon, CA, USA) rabbit anti-neurofilament (NF) polyclonal antibody, 1:80 (Sigma– Aldrich). Incubation was overnight at 4 °C, except for A2B5, which was incubated for 1 h at RT. After three washes (10 min each) in PBS, the cultures were incubated with the secondary antibodies: rhodamine-conjugated goat antimouse IgM, 1:200 (Sigma–Aldrich); Alexa 546 goat antimouse IgG, 1:1000 (Molecular probes, Invitrogen); Alexa 546 goat anti-rabbit IgG, 1:1000 (Molecular probes, Invitrogen); Alexa 633 goat anti-rabbit IgG, 1:1000 (Molecular probes, Invitrogen). Incubation was for 30 min at RT, followed by rinsing five times (5 min each) in PBS, and mounting with Gel/Mount aqueous mounting media (Biomeda Corp., CA, USA) that contained Hoechst 33342 (Sigma–Aldrich) to stain cell nuclei.
4.4.
Co-culture preparation for electron microscopy
Preparation for electron microscopy was as described previously (Zhang et al., 2009). Co-cultures on plastic coverslips were transferred to 2% glutaraldehyde and 3% sucrose in 0.1 M phosphate buffer (pH 7.4) for 30 min, followed by 2% osmium tetroxide in 0.1 M PBS for 1 h. Cultures were then dehydrated in graded ethanol, and embedded with EM-812. Ultrathin sections (70–90 nm) were cut in an orientation that cut axons transversely.
4.5.
Microscopy
Phase-contrast images of the live cultures and some immunofluorescence images were taken with an Olympus
BR A IN RE S EA RCH 1 3 09 ( 20 1 0 ) 9 –1 8
IX70 microscope equipped with a digital camera. Confocal images of immunofluorescence were captured with a Zeiss LSM 510 META laser scanning microscope. Electron microscopy was performed with a Philips CM-120 transmission electron microscope (Eindhoven, The Netherlands), at the EM Image Facility of Shanghai Jiaotong University School of Medicine.
4.6.
Cell counting
The number of immunofluorescent cells was counted by the software of Image Pro-plus 5.0. The purity of GFP-OPCs was expressed as percentage of A2B5- or PDGFR-positive cells relative to the total number of GFP-positive cells. The cell number of migrated cells around spinal cord explant when cultured alone was exactly the number of Hoechst-labeled cells. The cell number of migrated cells around spinal cord explant when co-cultured with GFP-OPCs was equal to the total number of Hoechst-labeled cells minus the number of GFP-positive cells. The proportion of myelinating GFP-oligodendrocytes was expressed as the percentage of the cells associated with neurites and co-expressed GFP and MBP relative to the total GFP-cells associated with neurites.
Acknowledgments This work was funded by the 973 project (2003CB515302), Shanghai Educational Committee Technology Foundation (06BZ006) and the Young Teachers' Science and Technology Ability Upgrading Project of Institutes of Medical Sciences, Shanghai Jiaotong University School of Medicine.
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