Oligodendrocyte progenitor cells (OPCs) constitute around 5% of all cells in the adult brain1,2 and generate mature, myelinating oligodendrocytes throughout life3–6. Fate-mapping studies have found region-dependent differences in OPCs’ differentiation, with the majority of cells located in the white matter differentiating into myelinating oligodendrocytes and OPCs located in the gray matter generating fewer mature cells3–6. We asked whether this is a result of intrinsic differences among adult OPCs located in these regions or the distinct environment of the white and gray matter.
To answer this question, we transplanted adult gray or white matter–derived cells from actin-EGFP mice7, which allowed us to detect the grafted cells by GFP expression, independent of their identity and fate. To identify mature oligodendrocytes, we crossed these mice with PLP-DsRed mice, in which DsRed expression is driven by the proteolipid protein (Plp1) promoter8. We isolated cells from the cerebral cortex of adult offspring and determined their identity in vitro by immunocytochemistry 3 h after dissociation (Fig. 1). The vast majority of cells derived from both gray and white matter were oligodendroglia (75–82% expressed Olig2, of which 51% were OPCs and 32% were mature oligodendrocytes; Fig. 1b and Supplementary Fig. 1), whereas only a few were astrocytes (7–10% expressed both GFAP and S100β) or microglia (8–12% expressed CD45; Fig. 1b). Notably, cell composition was hardly affected by selective survival of specific cell populations shortly after transplan-tation (4 d post-transplantation, dpt) into the cerebral cortex (white or gray matter) of adult C57Bl/6 mice. Indeed, the majority of the cells expressed oligodendroglial markers such as NG2, PDGFRα, PLP-DsRed and Olig2, and only a minor fraction belonged to other
cell types (Fig. 1a,c,d). Notably, the composition of white and gray matter–derived cells was comparable.
To examine the differentiation of these cells, we analyzed them at 6 and 11 weeks post-transplantation (wpt) in either their original environment or the respective heterotopic one. To our surprise, both white and gray matter–derived cells showed a higher proportion of PLP-DsRed+ cells at 11 wpt when transplanted into the white matter (~80%; Figs. 1f and 2a,b). Thus, both white and gray matter–derived cells matured at equal rates when exposed to the white matter environment, even though gray matter–derived cells seemed to take longer (Fig. 1f).
In contrast, only white matter–derived cells showed an increase in the proportion of differentiated PLP-DsRed+ cells when trans-planted into the gray matter (Fig. 1g). Accordingly, the proportion of PDGFRα+ OPCs decreased from 24.8 ± 4.2% (n = 4) at 6 wpt to 12.4 ± 7.2% (n = 3) at 11 wpt, supporting the observation of differen-tiating cells in the gray matter. Some cells could also myelinate in the white and gray matter, as shown by staining with paranodal (Caspr) and myelin (MAG) markers, as well as by immunoelectron micros-copy for GFP in the corpus callosum (Fig. 1h,i and Supplementary Figs. 2 and 3).
To determine whether this difference is a result of a local niche effect exerted in the cluster of grafted white matter cells, we diluted these transgenic-derived cells with wild-type (not expressing GFP or DsRed) cells from the gray matter (ratio of 1:2) before transplantation into the gray matter. Notably, at 11 wpt, the differentiation of GFP+ white matter–derived cells into PLP-DsRed+ cells was not significantly different (65.3 ± 7.2%, n = 5) when compared with that of the pure white matter population at the same time point, suggesting that the community effect in the transplants has only a minor role, if any.
The difference in the proportion of transplanted cells differentiat-ing into mature oligodendrocytes may also be the result of selective cell survival, proliferation or differentiation. To examine the prolifera-tion of the transplanted cells, we administered the thymidine analog BrdU to mice for 4 dpt. Notably, both gray and white matter–derived cells proliferated at a similar rate after being transplanted into gray and white matter (Supplementary Fig. 4a). We did not observe a considerable change in the numbers of transplanted cells between 4 dpt and 11 wpt, indicating that selective survival and proliferation do not have a substantial effect on the composition of the transplanted cells. Colocalization of BrdU with DsRed+ cells at later time points (6 wpt) further confirmed that the transplanted cells differentiated into oligodendrocytes (Supplementary Fig. 4b). Taken together, our data show that white matter–derived cells differentiate into mature oligodendrocytes with a higher efficiency than gray matter–derived cells when transplanted into the gray matter.
Transplantation reveals regional differences in oligodendrocyte differentiation in the adult brainFrancesca Viganò1–3, Wiebke Möbius4,5, Magdalena Götz1,2,6 & Leda Dimou1,2
To examine the role of gray and white matter niches for oligodendrocyte differentiation, we used homo- and heterotopic transplantations into the adult mouse cerebral cortex. White matter–derived cells differentiated into mature oligodendrocytes in both niches with equal efficiency, whereas gray matter–derived cells did not. Thus, white matter promotes oligodendrocyte differentiation, and cells from this niche differentiate more easily, even in the less supportive gray matter environment.
1Physiological Genomics, Institute of Physiology, Ludwig-Maximilians University, Munich, Germany. 2Institute for Stem Cell Research, HelmholtzZentrum, Neuherberg, Germany. 3Department of Pharmacological Sciences, University of Milan, Milan, Italy. 4Department of Neurogenetics, Max Planck Institute of Experimental Medicine, Göttingen, Germany. 5Center for Nanoscale Microscopy and Molecular Physiology of the Brain, Göttingen, Germany. 6Munich Cluster for Systems Neurology (SyNergy), Munich, Germany. Correspondence should be addressed to L.D. ([email protected]).
Received 21 March; accepted 26 July; published online 1 September 2013; doi:10.1038/nn.3503
As described above, the white matter envi-ronment promotes differentiation of gray matter–derived cells. However, we noted some morphological differences among GFP+ DsRed+ oligodendrocytes. Although the majority of white matter–derived cells at 11 wpt had a morphology that was typical of myelinating oligodendrocytes, with long and parallel processes, cells from the gray matter appeared to be more immature, with ramified processes, despite expressing DsRed (Fig. 2a,b). Classification of the cells as being round, having ramified processes (OPCs and immature oligodendrocytes) and having parallel processes (mature oligodendro-cytes; Fig. 2c,d) revealed significantly fewer cells with parallel processes amongst gray matter–derived cells (Mann Whitney test, P = 0.0357), suggesting that, even in a supportive environment (such as the white matter), white matter–derived cells differentiated into myelinating oligodendrocytes faster, which in turn indicates that there are intrinsic differences between white and gray matter OPCs.
Taken together, our results demonstrate that cells from the white matter differentiate efficiently into mature, myelinating oligodendro-cytes in both more (white matter) and less (gray matter) supportive environments, whereas gray matter–derived cells do so less efficiently. Our data suggest that there are intrinsic differences between adult OPCs from the white and gray matter, which may be a result of the long residence of the cells in these environments. However, the limited differentiation of gray matter–derived cells could be overcome, to some extent, by transplantation into the supportive white matter environment. Indeed, niche factors, such as extracellular matrix cues and diffusible ligands, as well as the spatial constraints result-ing from a high density of axonal fibers9–11, have been shown to influence oligodendrocyte maturation. Whatever the exact molecular components are, this concept of regional differences in oligodendrocyte maturation is of great importance, as OPCs are con-sidered to be a good source of endogenous precursors for repair in
demyelinating diseases. Moreover, considering that regional differences have also been observed in the limited remyelination ability of OPCs12,13, our results shed light on the efficacy of any therapeutic approach.
MeThodsMethods and any associated references are available in the online version of the paper.
Note: Any Supplementary Information and Source Data files are available in the online version of the paper.
AcknowledgmentSWe thank J. Trotter (Johannes Gutenberg-University) for the NG2-EYFP and F. Kirchhoff (University of Saarland) for the PLP-DsRed mouse lines, K. Karram for advice regarding cell isolation procedures, J. Ninkovic for help with the statistics, and E. Violette and S. Sirko for carefully reading the manuscript. We are also grateful to B. Sadowski for technical help with the electron microscope, as well as the technical assistants in the Physiological Genomics. W.M. is funded by the European Research Council Advanced Investigator Grant AXOGLIA to K.-A. Nave and is supported by the Cluster of Excellence and DFG research Center Nanoscale Microscopy and Molecular Physiology of the Brain. F.V. was, in part, supported by a doctorate fellowship from the University of Milan and an International Brain Research Organization InEurope grant. This work was mainly supported by the SFB 596 and the SFB 870 of the Deutsche Forschungsgemeinschaft, the Helmholtz Association of Mental Aging and the Friedrich Bauer Stiftung.
Figure 1 Transplantation and differentiation of adult OPCs. (a) Confocal images of GFP+ cells from the gray or white matter labeled with Olig2, GFAP/S100β or CD45. Inlays show higher magnifications. (b) Characterization of GFP+ gray or white matter cells 3 h after dissociation (n = 3 experiments). (c) Characterization of GFP+ gray or white matter cells at 4 dpt (n = 3–6 transplantations). (d,e) Confocal images of GFP+ cells from the white matter at 4 dpt (d) and 6 wpt (e). (f,g) Percentage GFP+ cells that also expressed PLP-DsRed at different time points after transplantation into white (f) or gray matter (g) (n = 3–5 mice per time point and area). *P = 0.0357, **P = 0.0079, #P = 0.0159, Mann Whitney test. (h) Confocal stack image showing the colocalization of white matter– derived cells with the paranodal marker Caspr. Inlays show higher magnification images of single confocal planes. (i) Immunoelectron microscopy image showing GFP in the paranodal myelin. All data are presented as mean ± s.e.m. Arrows indicate double-positive cells. Scale bars represent 50 µm (a,d,e,h), 20 µm (inlays) and 200 nm (i).
AUtHoR contRIBUtIonSF.V. performed and designed the experiments, analyzed the data, and wrote the manuscript. W.M. performed and analyzed the immunoelectron microscopy. M.G. helped to design some experiments and wrote the manuscript. L.D. designed and supervised experiments, originally developed the transplantation technique, and wrote the manuscript. F.V., M.G. and L.D. discussed the results and implications and commented on the manuscript at all stages.
comPetIng FInAncIAl InteReStSThe authors declare no competing financial interests.
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1. Dawson, M.R., Polito, A., Levine, J.M. & Reynolds, R. Mol. Cell. Neurosci. 24, 476–488 (2003).
11. Rosenberg, S.S., Kelland, E.E., Tokar, E., De la Torre, A.R. & Chan, J.R. Proc. Natl. Acad. Sci. USA 105, 14662–14667 (2008).
12. Gudi, V. et al. Brain Res. 1283, 127–138 (2009).13. Albert, M., Antel, J., Bruck, W. & Stadelmann, C. Brain Pathol. 17,
129–138 (2007).
Figure 2 Morphology of transplanted cells at 11 wpt. (a–c) Confocal images of white (a) and gray matter–derived (b) cells transplanted into the white matter at 11 wpt. Cells were categorized depending on their morphology, as shown by confocal images of gray matter cells (c). (d) Proportion of gray or white matter–derived cells with different morphologies at 11 wpt (n = 3–5 mice). Data are presented as mean ± s.e.m. Scale bars represent 50 µm.
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oNLINe MeThodsmice. Adult mice (2–3 months old) of the following lines were used: C57Bl/6J, TgN(beta-act-EGFP)7 (referred to in the text as actin-EGFP mice), NG2-EYFP mice14 and TgN(PLP-DsRed1)8 (referred to in the text as PLP-DsRed mice).
cell isolation. Cells were isolated from brains of adult (2–3 months old) actin-EGFP or actin-EGFP; PLP-DsRed mice7,8. A coronal cut at the level of the optic chiasm was performed to exclude the neurogenic subependymal zone. Tissue from the gray and white matter was dissected from the remaining cerebral cortex and collected separately. To dissociate cells, we adapted a previously described protocol15. Briefly, tissue was first dissociated mechanically and then papain (3.75 U ml−1, Sigma-Aldrich), DNaseI (500 U ml−1, Roche) and ovomucoid (0.1 mg ml−1, Sigma-Aldrich) were added. After a sucrose gradient and a final centrifugation step of 8,000g (at 4 °C), isolated cells were resuspended at a density of 90,000 cells per µl in Sato medium without serum or growth factors (DMEM, Life Technologies; 5 µg ml−1 transferrin, 10 µg ml−1 insulin, 100 µg ml−1 putrescin, 200 nM progesterone, 500 pM tri-iodo-thyronine, 220 nM sodium selenite, 520 nM l-thyroxine, 25 µg ml−1 gentamicin, Sigma-Aldrich).
transplantation procedure. Adult wild-type mice (C57Bl/6J, 2–3 month old) were deeply anesthetized as described16. Approximately 1 µl of cell suspension was injected in either the white or gray matter of the somatosensory cortex. Transplantations were performed with a Hamilton syringe (Hamilton, 75 SN 5µl; ga 33 L 30mm) into the white matter at −0.7 mm (anterio-posterior), 1.1 mm (medio-lateral), −1.2 mm (dorso-ventral) relative to the bregma or into the gray matter at −0.7 mm (anterio-posterior), 1.1 mm (medio-lateral), −1 to −0.4 mm (dorso-ventral) relative to the bregma. Experimental mice were killed at 4 d, or 6 or 11 weeks post-transplantation. Surgeries were performed in accord-ance with the guidelines on the use of animals and humans in Neuroscience Research, revised and approved by the Society of Neuroscience and licensed by the State of Upper Bavaria.
BrdU administration in the drinking water. After transplantation, mice received BrdU (Sigma-Aldrich) in the drinking water (1 mg ml−1 BrdU supplemented with 1% sucrose) for 4 d or 2 weeks. Mice were killed immediately or 4 weeks after BrdU administration, respectively.
Immunohistochemistry. Mice were anesthetized and then transcardially per-fused with 4% paraformaldehyde (PFA, wt/vol). Brains were collected, shortly postfixed in 4% PFA and cryoprotected in 30% sucrose (wt/vol). Sections (60 µm) were cut and stained as described3,16 with the following primary anti-bodies: chick antibody to GFP (1:1,000, GFP-1020, Aves Lab), rabbit antibody to RFP (1:500, 600-401-379, Rockland/Biomol), rabbit antibody to NG2 (1:500, AB5320, Millipore), mouse antibody to MAG (1:250, MAB1567, Millipore), rat antibody to PDGFRα (1:250, 558774, BD), rabbit antibody to Olig2 (1:250, AB9610, Millipore), rabbit antibody to or mouse antibody to GFAP (1:500, Z0334, Dako; G3893, Sigma-Aldrich), rabbit antibody to or mouse antibody to S100β (1:500, S2644 or S2532, Sigma-Aldrich), rat antibody to CD45 (1:500, 550539, BD), rat antibody to BrdU (1:250, BZL20630, Biozol), mouse antibody to Caspr (1:100, NeuroMab, clone K65/35). For Olig2 and BrdU labeling, staining of other antigens was performed first, and sections were then fixed with 4% PFA (10 min) before boiling in 0.01 M citrate buffer (pH 6.0) for 20 min and further staining. Fluorochrome-conjugated secondary antibodies were chosen according to the primary antibody: antibody to chick A488 (1:500, A11039, Life Technologies), antibody to rabbit Cy3, Cy5 or DyLight 649 (1:500, 711-165-152, 111-175-144 or 111-495-144, respectively, Dianova), antibody to rat A647 (1:500, A-21247, Life Technologies) or Cy3 (1:500, 112-165-167, Dianova), antibody to mouse Cy3, Cy5 or DyLight 649 (1:500, 115-165-003, 115-176-072 or 115-496-072, respec-tively, Dianova). Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole, 1:10,000, D9564, Sigma-Aldrich). Images were collected with a Zeiss confocal microscope system (LSM710).
Immunoelectron microscopy. Brains at 11 wpt were fixed as described above, and small pieces (1 mm2) of vibratome sections (60 µm) containing corpus callosum, including transplanted cells, were stained with Toluidine blue and infil-trated with 2.3 M sucrose overnight. Vibratome sections were then mounted onto
aluminum pins using small blocks of sucrose-infiltrated 10% gelatine (wt/vol) as support and frozen in liquid nitrogen. Ultrathin cryosections were cut at −110 °C using a cryo-immuno diamond knife (Diatome) and an UC6 ultracryomicrotome (Leica) until the blue tissue slice was completely consumed. Immunolabeling was performed as described17,18 using rabbit antibody to GFP (1:100, A6455, Invitrogen), which was subsequently detected with protein A–gold (10 nm). The colloidal 10-nm gold–Protein A conjugates were obtained from the Cell Microscopy Center, University Medical Center Utrecht. Sections were analyzed with a LEO EM912 Omega (Zeiss) and digital micrographs were obtained with an on-axis 2,048 × 2,048-CCD camera (TRS).
Immunocytochemistry. For the analysis of the cells immediately after dissocia-tion, cells were plated on coated coverslips (poly-l-lysine, 0.1 mg ml−1; laminin, 10 µg ml−1; Sigma-Aldrich) for 3 h and then fixed with 4% PFA for 10 min. Primary antibody staining was performed overnight in blocking solution (10% normal goat serum (vol/vol), Life Technologies; 0.1% Triton X-100 (vol/vol), Sigma-Aldrich) at 4 °C, with antibodies listed above at double the dilution indicated. We also used mouse antibody to NeuN (1:200, MAB377, Millipore), rat antibody to CD31 (1:200, 550274, BD) and rat antibody to RFP (1:200, 5F8, Chromotek). Secondary antibody and nuclei staining was performed as described above. Images were collected with a Zeiss epifluorescent microscope (Axio ImagerM2).
Quantification and statistical analysis. Immunohistochemical analysis was performed on multi-channel, confocal stacks, using Zeiss ZEN 2010 software and the cell counter plug-in for ImageJ. Quantitative analysis was performed by counting marker-positive cells among all GFP+ cells in the site of transplanta-tion. For differentiation analysis (Fig. 1f,g), we used gray matter–derived cells transplanted into the gray matter (4 dpt, n = 5 mice; 6 wpt, n = 3; 11 wpt, n = 3), white matter–derived cells transplanted into the gray matter (4 dpt, n = 4 mice; 6 wpt, n = 4; 11 wpt, n = 5), gray matter–derived cells transplanted into the white matter (4 dpt, n = 3 mice; 6 wpt, n = 5; 11 wpt, n = 3) and white matter–derived cells transplanted into the white matter (4 dpt, n = 5 mice; 6 wpt, n = 4; 11 wpt, n = 5). A total of 6,008 cells were counted. For the morphological analysis (Fig. 2d), we used n = 3 mice for gray matter–derived cells and n = 5 mice for white matter–derived cells transplanted into the white matter; a total of 1,153 cells were counted. For the characterization of transplanted cells at 4 dpt (Fig. 1c), we used stained cells for Olig2, CD45 and GFAP/S100β (gray matter cells: Olig2, n = 3 transplantations; CD45, n = 3; GFAP/S100β, n = 3; white matter cells: Olig2, n = 3; transplantations, CD45, n = 6; GFAP/S100β, n = 4); a total of 1,553 cells were counted. For the proliferation analysis (Supplementary Fig. 4a), we transplanted gray matter–derived cells into gray matter (n = 5 mice) and white matter (n = 5 mice), and white matter–derived cells into gray matter (n = 5 mice) and white matter (n = 6 mice); a total of 1,681 cells were counted. For the niche effect analysis, we used n = 5 mice; a total of 263 cells were counted. For the PDGFRα analysis, we used n = 4 mice at 6 wpt and n = 3 mice at 11 wpt; a total of 429 cells were counted.
Immunocytochemical analysis was performed on multi-channel fields acquired with Zeiss software and the cell counter plug-in for Fiji. For quantita-tive analysis, the number of GFP+ cells that were also immune-positive for a cell type–specific antigen was counted and expressed as a percentage of GFP+ cells (n = 3 independent experiments, a total of 2,903 cells were counted). To quantify the percentage of OPCs or mature oligodendrocytes over Olig2+ cells, we used the transgenic mouse lines NG2-EYFP14 and PLP-DsRed8, respectively. The number of reporter+ cells that were also immunopositive for the Olig2 antigen was counted and expressed as a percentage of Olig2+ cells (n = 3 independent experiments for each reporter mouse, a total of 2,096 cells was counted).
Results are represented as mean ± s.e.m. No statistical methods were used to predetermine sample sizes, but our sample sizes are similar to those generally employed in the field. As our data are not normally distributed, we used the Mann-Whitney test as a non-parametric test. We also tested the significance of our quantifications by one-way ANOVA with Tukey post-test, which is well suited for a small sample number, and confirmed the significance observed with the Mann Whitney test. Data were considered to be significant at P < 0.05. Statistics was performed with GraphPad Prism 4.0 and OriginPro 8.6. All data were collected and processed randomly. For identity of cells at 3 h after plating, two independent people analyzed the data (one person blinded) and the average
