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Keywords:

  • Oligodendrogenesis;
  • Subventricular zone;
  • Epidermal growth factor;
  • NG2 proteoglycan;
  • Adult neural stem cells;
  • Oligodendrocyte precursors;
  • Lysolecithin

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS/SUMMARY
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

New neurons and oligodendrocytes are continuously produced in the subventricular zone (SVZ) of adult mammalian brains. Under normal conditions, the SVZ primary precursors (type B1 cells) generate type C cells, most of which differentiate into neurons, with a small subpopulation giving rise to oligodendrocytes. Epidermal growth factor (EGF) signaling induces dramatic proliferation and migration of SVZ progenitors, a process that could have therapeutic applications. However, the fate of cells derived from adult neural stem cells after EGF stimulation remains unknown. Here, we specifically labeled SVZ B1 cells and followed their progeny after a 7-day intraventricular infusion of EGF. Cells derived from SVZ B1 cells invaded the parenchyma around the SVZ into the striatum, septum, corpus callosum, and fimbria-fornix. Most of these B1-derived cells gave rise to cells in the oligodendrocyte lineage, including local NG2+ progenitors, and premyelinating and myelinating oligodendrocytes. SVZ B1 cells also gave rise to a population of highly-branched S100β+/glial fibrillary acidic protein (GFAP)+ cells in the striatum and septum, but no neuronal differentiation was observed. Interestingly, when demyelination was induced in the corpus callosum by a local injection of lysolecithin, an increased number of cells derived from SVZ B1 cells and stimulated to migrate and proliferate by EGF infusion differentiated into oligodendrocytes at the lesion site. This work indicates that EGF infusion can greatly expand the number of progenitors derived from the SVZ primary progenitors which migrate and differentiate into oligodendroglial cells. This expanded population could be used for the repair of white matter lesions. STEM CELLS 2009;27:2032–2043


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS/SUMMARY
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

The subventricular zone (SVZ) is the largest source of progenitor cells in the adult mammalian brain. SVZ primary precursors correspond to a subpopulation of astroglial cells (type B1 cells) [1], which give rise to actively proliferating transit amplifying type C cells [2]. In rodents, most C cells generate neuroblasts (Type A cells) that migrate into the olfactory bulb via the rostral migratory stream [3–6]. SVZ progenitors also generate a small number of oligodendrocytes that migrate into the corpus callosum, septum, striatum, and fimbria fornix [7–9].

In the presence of epidermal growth factor (EGF) and/or fibroblast growth factor (FGF), cultured SVZ progenitors self renew, and upon removal of growth factors, they can generate neurons, astrocytes, and oligodendrocytes [10, 11]. In vivo, EGF, FGF2, or transforming growth factor-alpha (TGFα) infusions result in a dramatic enlargement of the SVZ and increased migration of progenitor cells into the surrounding brain parenchyma [12–18]. Growth-factor infusions could therefore be useful in the amplification and mobilization of endogenous progenitor pools for brain repair. Earlier studies suggest that most cells derived from the SVZ after EGF stimulation differentiate into astrocytes [14, 18]. Other studies suggest that in vivo epidermal growth factor receptor (EGFR) signaling promotes SVZ progenitors to differentiate along the oligodendroglial lineage [16, 19], and one study reported that a small number of putative SVZ-derived cells in the striatum expresses neuronal markers [12]. These studies used bromodeoxyuridine (BrdU) or unspecific viral tracing analysis and cannot establish whether the newly generated cells after EGF infusion originated from SVZ primary progenitors or from other progenitors within or around the SVZ. We have previously shown that EGF can induce the proliferation and migration of cells derived from SVZ type B and type C cells [13], but the fate of these cells was not established.

By specifically targeting the SVZ primary precursors, we show here that intraventricular infusion of EGF induces a dramatic expansion of cells derived from SVZ B1 cells. These cells upregulate Olig2 and migrate into the striatum, septum, and white matter tracts, including the corpus callosum and fimbria fornix. Many of these cells differentiate into cells in the oligodendrocyte lineage. In addition, SVZ type-B cells also gave rise to a population of highly branched S100β+/GFAP+ cells and to a population of NG2+ oligodendrocyte progenitor cells (OPCs). Following induction of a demyelinating lesion in the corpus callosum, EGF-responsive cells derived from SVZ B1 cells migrated to the lesion site and differentiated into premyelinating and myelinating oligodendrocytes. Our results indicate that EGF signaling can greatly increase the number of SVZ-derived oligodendrocytes in the adult brain, and demonstrate that these new oligodendrocytes originate from SVZ B1 cells. Therefore, SVZ B1 progenitors can generate, under in vivo growth factor stimulation, a large number of cells that disperse and generate new OPCs and myelin-forming cells.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS/SUMMARY
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Animal Care and Tissue Processing

All animal procedures followed the University of California Committee on Animal Research guidelines. Adult CD-1 (Charles River, Hollister, CA, http://www.criver.com) and GFAP-tva (a kind gift from E. Holland) mice were anesthetized by an intraperitoneal injection of 25-30 μl/g body weight of 2.5% Avertin (2,2,2-tribromoethanol + tert-amyl alcohol, 1:1 w/v). Mice were killed by an overdose of pentobarbital (100 mg per kg of body weight) before transcardial perfusion. For light microscopy (n = 4 per group), mice were perfused with 4% paraformaldehyde (PFA) in 0.1M phosphate buffer (PB), and the brains were postfixed overnight at 4°C in the same fixative. Coronal sections that were 30-μm thick were cut with a vibratome. For electron microscopy (EM), mice were killed by intracardial perfusion with either 2% PFA/0.5% glutaraldehyde for EM-immunocytochemistry or 2% PFA/2.5% glutaraldehyde for conventional EM. For EM-immunocytochemistry (n = 3, per group), brains were postfixed in the same fixative overnight at 4°C, cut coronally at 50 μm, and processed as described below. For conventional EM (n = 3, per group), 200 μm vibratome sections were postfixed in 2% osmium for 2 hours, rinsed, dehydrated, and embedded in Araldite (Durcupan, Fluka BioChemika, Ronkonkoma, NY, http://www.sigmaaldrich.com). Sections (1.5 μm thick) were stained with 1% toluidine blue. To identify individual cell types, ultrathin (0.05 μm) sections were stained with lead citrate and examined using a FEI Tecnai Spirit electron microscope (FEI Company, Hillsboro, OR, http://www.fei.com). For freshly-dissociated cell staining, animals (n = 2 per group) were decapitated immediately after pump infusions and their brains immersed in ice-cold pipes buffer. Ipsilateral dorsal SVZ was dissected (1 mm length x 0.3 mm wide tissue piece from lateral wall of ventricle). SVZ was minced and incubated in 0.25% trypsin-EDTA solution at 37°C for 10 minutes. Then trypsin was removed, fresh F-12 medium added, and tissue triturated with a fire-polished pipette. The resulting cell suspension was placed, dried, and fixed with 3% of PFA onto glass slides. Staining was performed as described below.

Immunocytochemistry

After blocking in 0.1M PBS containing 10% of normal goat serum for 1 hour at room temperature, sections were incubated overnight at 4°C in primary antibodies diluted in blocking solution; 0.1% Triton-X was included for intracellular antigens. The following primary antibodies were used: mouse monoclonal to β-tubulin (1:500) (Covance, Berkeley, CA, http://www.covance.com) and CNPase (1:250, Chemicon, Temecula, CA, http://www.chemicon.com), GFAP (1:500, Chemicon), nestin (1:500, Chemicon), poly-sialated neural cell adhesion molecule (PSA-NCAM) (1:1,000, AbCys, France, http://www.abcysonline.com); rabbit polyclonal antibodies against S100β (1:500, Dako, Denmark, http://www.dakocytomation.com), Olig2 (1:5,000; a kind gift from D. Rowitch), PDGFRα (1:50, Santa Cruz Immuno, Santa Cruz, CA, http://www.scbt.com), NG2 (1:250; Chemicon), green fluorescent protein (GFP) (1:1,000, Abcam, U.K.), and degraded myelin basic protein (dMBP) (1:50, Chemicon). Polyclonal antibodies: guinea pig antidoublecortin (1:1,000, Chemicon), rat antimyelin basic protein (MBP) (1:250; Chemicon), and chicken anti-GFP (1:200; Aves Labs, Tigard, OR, http://www.aveslab.com).

For fluorescent immunocytochemistry (ICC) analysis, (n = 4, per group), tissue sections were rinsed with 0.1 M PBS, incubated with the appropriate AlexaFluor conjugated secondary antibodies (all 1:500, molecular probes) in blocking solution for 1 hour at room temperature, and washed in PBS. For pre-embedding Olig2, GFP, and nestin ICC, sections were washed in PB, incubated in blocking solution with the appropriate biotinylated secondary antibody (1:400, Jackson Immunoresearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com) for 2 hours at room temperature, incubated in avidin-biotin peroxidase complex (ABC) peroxidase kit (Vector) for 1 hour, and revealed with 0.03% diaminobenzidine and 0.01% H2O2. Controls in which primary antibodies were omitted resulted in no detectable staining. For imaging, a Zeiss LSM 510 laser scanning confocal microscope (Carl Zeiss, Jena, Germany, http://www.zeiss.com) was used and optical 0.5-μm serial sections were obtained to colocalize fluorescent signals.

For pre-embedding EM-ICC (n = 3 per group), sections were washed in PB and incubated in blocking solution for 2 hours at room temperature. Sections were then incubated in primary antibody: Olig2 (1:500), GFP (1:500), or Nestin (1:250) for 36 hours at 4°C, rinsed and incubated in biotinylated antirabbit IgG (1:400) overnight at 4°C, incubated in ABC peroxidase kit (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com) for 2 hours, and revealed with 0.03% diaminobenzidine (DAB) and 0.01% H2O2. Since DAB precipitates may be difficult to see under the EM, DAB+ cells are first identified and photographed by light microscopy on semithin 1-μm sections. Then these semithin sections are detached from the glass slides, flat-re-embedded, and ultrathin sectioned to performed EM analysis.

For GFP pre-embedding immunogold (n = 3 per group), 50-μm vibratome sections in 0.1 M PBS + 25% saccharose were subjected to three 1-minute freeze/thaw cycles and sequentially incubated before araldite embedding in: 0.3% bovine serum albumin (BSA)/0.1 M PBS for 1 hour at room temperature; chicken anti-GFP antibody (1:200; Aves Labs) in 0.3% BSA/0.1M PBS for 60 hours; 0.1M PBS, blocked in 0.5% BSA/0.1% fish gelatin/0.1 MPBS for 1 hour at room temperature; colloidal gold-conjugated antichicken IgG (0.8 nm particle diameter) diluted 1:50 in 0.1 M PBS/BSA-gelatin during 24-hours at room temperature; and treated with silver enhancement solution.

Retroviral Injections

Two days before pump infusions, avian leukosis retroviruses expressing the reporter gene green fluorescent protein (RCAS-GFP) (100 nl; titer 3.07 × 107) were stereotaxically injected into adult GFAP-tva mice. Coordinates relative to Bregma and the surface of the brain (anterior, posterior, mediolateral, and dorsoventral, respectively) were 1, 1, 2.3; 0.5, 1.1, 1.7; 0, 1.4, 1.6 mm for the SVZ injections. To label non-SVZ astrocytes, we used: 0, 2.5, 2.5 and 0.5, 2.2, 2.5 mm for striatal injections (n = 3 per group); 1, 1.8, 1.25 mm for CC injections (n = 2 per group); 0, 2.8, 0.8 and 0.5, 2.5, 0.5 mm for cortical injections (n = 4 per group); and 0.5, 2.5, 0.1 for brain surface infusions (n = 2 per group).

Epidermal Growth Factor Infusions

EGF (Upstate Biotechnology, Billerica, MA, http://www.millipore. com) (400 ng/day) in vehicle (BSA/0.9% saline) or vehicle alone was infused for 7 days with a miniosmotic pump (Alzet, Cupertino, CA, http://www.alzet.com) (1,007D flow rate 0.5 ml/hour). For infusions into the lateral ventricle, cannulas were implanted at 0 mm relative to Bregma, 1.1 mm lateral and 2.3 mm deep. For brain surface infusions, the dura mater was opened and the cannula placed at 0, 1.1, 0.5 mm (anterior, lateral, depth). Cannulas were removed on the 7th day and mice were killed at different time points as indicated.

Demyelinating Lesion

Demyelination was induced by injecting lysolecithin into the corpus callosum as described previously [7, 8, 20]. RCAS-GFP was injected into the adult SVZ of GFAP-tva mice. Two days later, 0.5 μl of 1% lysolecithin (LPC; Sigma-Aldrich, St. Louis, MO, http://www.sigmaaldrich.com) in 0.9% sodium chloride was injected ipsilaterally into the corpus callosum at 1.1, 1, 1.5 mm (anterior, lateral, depth relative to bregma). EGF or vehicle was then infused onto the cortical surface, as described above. All animals (n = 3 per group) were killed 30 days after EGF or vehicle pump removal.

Quantification

To quantify the number of Olig2+ cells, eight 30-μm sections that were spaced 200-μm apart were randomly selected (n = 4 per group for 21 and 45 days after infusion; n = 3 per group for 90 and 180 days after infusion). The Olig2+ cells within 500 μm from the ependymal layer around the ventricle on the injected side were counted. A similar procedure was performed to quantify the number of DAPI+ cells and used to calculate the cell density per mm2. Double-labeled cells were counted in ten 30-μm sections, spaced 120-μm apart (n = 3-4, per group). Only cells in which DAPI+ nuclei and overlapping marker expression were observed were counted as double labeled. A similar procedure was used to determine the percentage of colabeling in freshly-dissociated cells, where 200 randomly-selected cells per animal were counted and the percentage of colabeled cells was calculated. Quantifications were made under an Olympus fluorescent microscope (AX70) (Olympus America, Inc., Center Valley, PA, http://www.olympusmicro.com) using a 63X water-immersion objective. For demyelination experiments (n = 3, per group), CNPase+/GFP+ cells in corpus callosum in thirty 30-μm sections encompassing 500 μm rostrally and 500 μm caudally from the lysolecithin injection site were counted. To determine the demyelination volume (n = 3, per group), the whole brain was sectioned and sequential 15-μm coronal slices were collected every 60 μm. The demyelination lesion was calculated from the area of dMBP expression in the corpus callosum in serial sections using an image analyzer (Leica QWIN500I; Leica, Wetzlar, Germany, http://www.leica.com). Lesion area measurements were repeated three times for each brain. All data are expressed as means ± SD. For comparisons of means between groups, Student's t test was used; p < .05 was considered significant. In all cases, quantifications were performed by a researcher blinded to group assignment.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS/SUMMARY
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Epidermal Growth Factor Generates a Population of Olig2+Nestin+ Invasive Cells

EGF signaling induces an expansion of migrating progenitor cells around the SVZ [12–14]. To characterize these migrating cells, we infused EGF or vehicle into the lateral ventricles for 7 days, killed the animals at the end of this treatment, and performed ICCs with neuronal and glial markers. Immediately after 7-day EGF infusions, there were dramatic expansions of infiltrating cells around the SVZ. These infiltrating cells were negative to GFAP, vimentin, doublecortin, PSA-NCAM, and S100β (data not shown). In contrast, there was a 9.6-fold increase in the number of Olig2+ cells around the EGF-infused ventricles (1,690 ± 558 cells per section; n = 4 animals, 8 sections each) as compared with the control-vehicle group (175 ± 19 cells per section; n = 4, 8 sections each, p < .01) (Fig. 1A–1C). Here, we will refer to these cells as EGF-induced progenitors (EIPs). These infiltrating cells were not observed in saline-infused control animals (Figs. 1A, 2A, 2B), but many reactive astrocytes were observed near the needle track, as in the EGF-infused group. We quantified the proportion of proliferating Olig2+ cells by injecting 50 mg/kg i.p. BrdU 1 hour before kill. After a 7-day EGF infusion, 3.9% of the Olig2+ cells were BrdU+ (8 of 201 cells; n = 4) in saline-treated animals. In contrast, in the EGF-infused group 12.1% of the Olig2+ cells were BrdU-labeled (110 of 908 cells; n = 4). The cellular infiltration around the ventricle resulted in an approximately sevenfold increase in the density of cells (DAPI-stained nuclei) around the lateral ventricle (500 μm from the ependymal layer): The control group had 2.97 × 103 cells/mm2 versus 2.08 × 104 cells/mm2 in the EGF-infused group (n = 4 animals per group).

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Figure 1. Epidermal growth factor (EGF) effects after a 7-day intraventricular infusion. Olig2 periventricular expression in the vehicle (A) and (B) EGF group. Olig2+ cells are aligned along blood vessels (BV; arrow heads) and myelin tracts of the striatum (Str; arrows). (C): EGF increases 9.6-fold the Olig2+ expression (Student's t test; p < .05). The inserted drawing depicts the area used for quantifications. Nestin periventricular expression in (D) vehicle- and (E) EGF-infused animals. Nestin expression was also found along blood vessels (arrow heads), and myelin tracts (arrows). (F): Olig2/nestin coexpression in the periventricular region (main square). Inset: Olig2+ cell showing a nestin+ leading process (open arrowhead). (G): Periventricular cell freshly dissociated and immunostained. Scale bars in (A,B,D,E) = 200 μm; (F) and inset = 20 μm. Abbreviations: BV, blood vessels; Sep: septum; Str, striatum; V, ventricle.

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Figure 2. Ultrastructural characterization of epidermal growth factor (EGF)-induced progenitors. No type-A and type-C cells are found in the EGF-infused group (B) compared with the control group (A). (C): Parenchymal astrocytes (white arrow heads) and EGF-induced progenitors (EIPs) (black arrow heads). EGF-induced progenitors infiltrate myelin tracts (MT) and blood vessels (BV). These cells have abundant ribosomes (r) (D), desmosome-like contacts (blue asterisks), endocytic vesicles (E) and mitoses (F). (G–H): Olig2 pre-embedding immunocytochemistry (ICC); DAB brown precipitates show the Olig2 expression. (I,J): Diaminobenzidine precipitates detected by electron microscopy (yellow asterisks). The electron microscopy section was obtained from the semithin section shown in the inset. Scale bars (A,B,G–I) and inset = 10 μm; (C) = 5 μm; (D) = 0.5 μm; (E,F) = 1 μm; (J) = 2 μm. Abbreviations: BV, blood vessels; EGF, epidermal growth factor; MT, myelin tracts; n, nucleus; r, ribosomes; V, ventricle.

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In accordance with previous studies [12, 14], after EGF infusions, we observed a dramatic expansion of nestin-expressing cells around the lateral ventricle. The distribution of nestin+ cells after 7-day EGF infusion was similar to that of Olig2+ cells (Fig. 1D–1E). To determine if these nestin+ cells expressed Olig2, we performed double ICC and confocal analysis. Most, if not all, Olig2+ cells around the lateral ventricle appeared to coexpress nestin (Fig. 1F). In order to confirm this observation, we used acutely dissociated cells from the periventricular region (Fig. 1G). Most (92 ± 3%) of Olig2+ cells coexpressed nestin (1,337 of 1,407 cells; n = 3). We also stained for NG2 and PDGFRα, which are expressed by OPCs, migrating and early differentiating oligodendrocytes [21, 22], but not by neuronal progeny [23, 24]. The number of NG2+ and PGDRFα+ cells was greatly increased around the ventricle of EGF-infused animals compared to controls (supporting information Fig. 1A–1D). These observations indicate that invading cells upon EGF infusion express markers of early neural progenitors or OPCs (nestin, Olig2, NG2 and PDGFRα) [25, 26].

To further define the EIP population, we next studied their ultrastructure. Remarkably, in the SVZ of EGF-infused animals, type B cells were observed, but type-C or -A cells were difficult to identify. Instead, we found large numbers of cells in the SVZ and striatum with a unique set of ultrastructural characteristics (Fig. 2A–2F): irregular cytoplasmic profiles, characteristic electrondense cytosolic inclusions, and large nuclear invaginations. Their nuclei were voluminous and irregular, with heterogeneous chromatin and prominent nucleoli. A number of dictiosomes, abundant ribosomes, profuse rough endoplasm reticuli, and mitochondria were also observed, but no obvious polarization of organelles was found. Electrondense intercellular junctions, intercellular spaces, endocytic vesicles, and mitoses were also frequently found among these invading cells (Fig. 2D, 2F). These cells were usually elongated and had a migratory morphology. Pre-embedding ICC analysis showed that these cells (with distinctive ultrastructural characteristics) expressed Olig2 in their nuclei (Fig. 2G–2J), confirming that they corresponded to the population of infiltrating cells characterized as described above by light microscopy. The EIPs were tightly associated to blood vessels in the vicinity of the SVZ, in the corpus callosum, striatum, and septum (Fig. 2B, 2C, 2G–2J). However, EIPs were not in direct contact with the endothelial cells, but separated from these cells by thin astrocytic processes (supporting information Fig. 1). These results indicate that EIPs cells have unique ultrastructural characteristics different from those of normal SVZ progenitors and express nestin and Olig2.

EGF-Induced Progenitors Are Derived from Subventricular Zone B1 Cells and Differentiate into Oligodendrocyte Precursors

To investigate whether the Olig2+ EIPs were derived from the SVZ B1 cells, we used GFAP-tva mice [27] and the avian leukosis retrovirus expressing the reporter gene green fluorescent protein (RCAS-GFP). In these mice, this vector selectively infects dividing GFAP+ SVZ astrocytes cells in the SVZ, allowing permanent labeling of their progeny [2, 13]. RCAS-GFP was injected into the SVZ of GFAP-tva mice. Two days later, EGF or vehicle was infused into the lateral ventricle for 7 days. Immediately after the 7-day EGF infusions, the animals were killed and many GFP+ cells were observed around the ventricle (Fig. 3A, 3B). These GFP+ cells had identical ultrastructure as the EIPs as described above (Fig. 3C). Since the RCAS-GFP labeling revealed the processes and shape of the EIPs in more detail, we performed three-dimensional Z-stack confocal reconstructions of 36 individual cells. Two general morphologies were observed (Fig. 3D). First, we observed simple, unipolar or bipolar cells. These cells were found preferentially close to the lateral ventricle and were frequently associated to blood vessels or axonal tracts. Their location and morphology suggest that these cells correspond to actively migrating EGF-responsive cells. These cells had a dominant leading process with numerous thin ramifications. In about half of the cells, a smaller trailing process was observed. And second, we observed complex or multipolar cells with three or more primary processes and highly ramified branches. These cells were found throughout the region of invasion. To establish whether the simple and complex cells also had different ultrastructural characteristics, we performed three-dimensional reconstructions by EM (n = 12 cells) (Fig. 3E). Interestingly, the EIPs found near the lateral ventricle had fewer cellular processes, ribosomes, and mitochondria. These cells were also smaller (5-10 μm) than the cells found in the striatum or septum (15-20 μm), which contained abundant electrondense cytosolic inclusions, ribosomes, rough-endoplasm reticuli, and large nuclear invaginations. This more complex morphology suggests that these cells are not migratory; however, we do not know how dynamic the processes on these cells are and whether complex cells could revert to a migratory phenotype.

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Figure 3. Subventricular zone (SVZ) astrocytic stem cells give rise to epidermal growth factor-induced progenitors. Green fluorescent protein (GFP)-expressing cells after vehicle (A) and EGF (B) infusions. A number of GFP+ cells derived from the SVZ astrocytes were observed around the lateral ventricle (V) and infiltrated the brain parenchyma of EGF-infused animals. (C): Axonal tracts showing two GFP-labeled cells analyzed by electron microscopy. Upper inset shows the semithin section from where the ultrathin section was obtained. Lower inset: Higher magnification of the area indicated in (C); arrowheads indicate DAB precipitates. (D): Z-stack reconstructions of GFP+ cells. (E): Schematic three-dimensional reconstructions by electron microscopy. The numbers at the left indicate the distance between each section. Time line and mouse brain schematic drawing show the cannula's spatial position and the injection site to label SVZ astrocytes. Scale bar in B = 100 μm; C = 2.5 μm, inset = 1 μm; E = 5 μm. Abbreviations: BV, blood vessel; CC, corpus callosum; EGF, epidermal growth factor; GFP, green fluorescent protein; Str, striatum; Sep, septum; V, ventricle.

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To establish the phenotype of the RCAS-GFP-labeled EIPs, we performed double ICC and confocal analysis. This analysis confirms that most of the GFP+ cells coexpressed Olig2 (Fig. 4A). Interestingly, a high percentage of them also coexpressed NG2 or PDGFRα (Fig. 4B, 4C). To further verify the double-labeling between GFP and Olig2, NG2, or PDGFRα, and to determine the percentage of double-labeled cells, we dissociated the cells isolated from periventricular regions immediately after the 7-day EGF infusion: 97 ± 1% (205 of 211 cells; n = 2) of GFP cells coexpressed Olig2; 84 ± 17% (273 of 323 cells; n = 2) coexpressed PDGFRα; and 87 ± 14% (201 of 227 cells; n = 2) coexpressed NG2. No PSA-NCAM+ or doublecortin+ cells were observed in the SVZ of the EGF-infused animals (data not shown). These results strongly suggest that the EIPs derived from the type B SVZ cells express markers related to oligodendrocyte precursors.

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Figure 4. Epidermal growth factor-express markers related to oligodendrocyte lineage. Brain sections were costained with anti-GFP and anti-Olig2 (A–A″), anti-PDGRFα (B–B″), and anti-NG2 (C–C″) antibodies. High magnification cell details are shown at bottom left square. Independent experiments and immunolabelings were performed on freshly-dissociated cells (insets: a–a″, b–b″, c–c″). Schematic brain drawing depicts the dissected area (1 mm x 0.3 mm) where fresh-dissociated cells were obtained. Nuclear staining was performed with DAPI (blue). Scale bars = 20 μm. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; EGF, epidermal growth factor; PDGFRα, platelet-derived growth factor receptor alpha; RCAS-GFP, avian leukosis retrovirus expressing the reporter gene green fluorescent protein; V, ventricle.

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To analyze whether non-SVZ dividing astrocytes from other brain regions may give rise to Olig2+ EIPs, we infused EGF into the lateral ventricle and injected RCAS-GFP in four different locations: cortex (n = 4), corpus callosum (n = 2), striatum (n = 3), and brain surface (n = 2) (supporting information Fig. 2). After 7-day EGF infusions, only a small population of GFAP+ astrocytes was found labeled close to the injection site at cortex, corpus callosum, and striatum (supporting information Fig. 2), but no GFP+ cells were observed to be infiltrating the parenchyma as seen when the RCAS virus is targeted to the SVZ. No GFP+ cells were observed when the virus was injected onto the brain surface. These results confirm that the SVZ astrocytes (SVZ B1 cells) are the most important source of the Olig2+NG2+PDGFRα+ cells that appear in the parenchyma after EGF infusion.pt?>

Epidermal Growth Factor-Induced Progenitors Differentiate Along the Oligodendrocyte Lineage

We next investigated the long-term fate of the EGF-invaders in the brain. In order to label the cell progenitors derived from the SVZ astrocytes, we used GFAP-tva mice labeled with RCAS-GFP as described above. After 7-day intraventricular infusions of EGF, the micro-osmotic pumps were removed and animals were allowed to survive for 21 days (n = 5). At this time, many more labeled cells were found in the corpus callosum, septum, striatum, cortex, and fimbria fornix compared with controls (Fig. 5A, 5B). The cells found in the cortex were not only distributed along the needle track, but also found as far as 800 to 1,000 μm from the SVZ or the needle track. We analyzed the cells found in the corpus callosum in detail and found that they had round or elongated cell somas. Clusters of three or four elongated GFP+ cells were frequently observed to align with callosal fiber tracts (Fig. 5C, 5D). To reveal the morphology of these cells in more detail, we performed serial confocal analysis of 10 to 15 optical sections (0.75-1 μm thick) in 68 cells. About half of these cells had morphological characteristics of premyelinating and myelinating oligodendrocytes. In addition, transitional forms between the migrating morphology and the premyelinating oligodendrocytes were also observed [7, 28, 29] (Fig. 5E–5G). Premyelinating oligodendrocytes were characterized by their multiple radial processes and thin bushy branches (Fig. 5E), whereas myelinating oligodendrocytes had the typical elongated sheaths aligned with fiber tracts (Fig. 5F). Oligodendrocytes were also observed in some myelinated tracts in the striatum, cortex, and septum (data not shown). We also killed animals at 45 (n = 4), 90 (n = 3), and 180 (n = 3) days after removal of EGF infusion. Similar morphologies were observed at these longer survival times, suggesting that SVZ cells invade the brain in response to EGF and differentiate into mature oligodendrocytes. Oligodendrocytes in the corpus callosum and fimbria were also observed in saline controls (n = 4, for 21 days and 45 days; n = 3, for 90 and 180 days after pump withdrawal), but their numbers were small compared with the EGF-infused animals. In the saline-infused animals, GFP+ oligodendrocytes were very rare in the septum and striatum, and were not observed in the cortex (supporting information Fig. 3).

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Figure 5. Epidermal growth factor (EGF)-induced progenitors derived from subventricular zone astrocytes give rise to oligodendrocytes. Schematic timeline shows the experimental design. GFP+ cells found in the brain parenchyma 21 days after infusion of vehicle (A) or EGF (B). In the corpus callosum (CC) many cells had characteristic of premyelinating and myelinating oligodendrocytes (C) that frequently formed cellular “cords” (D). Similar findings were observed in all regions at every time point. (E–G): Confocal Z-stack three-dimensional reconstructions of the GFP+ cells found in the corpus callosum. (H–N): Markers expressed by RCAS-GFP-labeled cells found in the corpus callosum. These cells have a close relationship with neurofilaments (NF) (N). Scale bars in (A,B) = 100 μm; (B,C,G–K) = 20 μm; n = 5 μm. Abbreviations: BV, blood vessel; EGF, epidermal growth factor; GFP, green fluorescent protein; MBP, myelin basic protein; Sep, septum; Str, striatum, V, ventricle.

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To further establish the identity of cells derived from EIPs, we counterstained sections with different oligodendrocyte lineage markers. The precursor markers included Olig2 [30, 31], NG2 [21, 22], and PDGFRα [32]. The premyelinating oligodendrocytes markers included CNPase [33] and β-IV tubulin [34], and the myelinating oligodendrocytes included myelin basic protein (MBP) [29]. We also stained sections for S100β, which is typically associated with parenchymal astrocytes [35], but has been recently associated with oligodendrocyte precursors [36, 37]. Most of the GFP+ cells in the corpus callosum expressed oligodendrocyte markers (Fig. 5H–5N). For quantification purposes (Table 1), we selected markers that delineate cell bodies and/or nuclei, such as CNPase, Olig2, S100β, and NG2. We found a 200 to 400% increase in the number of GFP-labeled cells that express these markers at 21, 45, 90, and 180 days after EGF infusion compared with controls (Table 1). These findings indicate that the EGF-expanded cells, which are derived from SVZ B1 cells, invade the corpus callosum and fimbria, then largely differentiate into oligodendrocyte lineage. The increased number of S100β+GFP+ cells in corpus callosum after the EGF infusion was comparable to that observed with other oligodendrocyte markers. Interestingly, within the striatum and septum at 21, 45, 90, and 180 days after EGF infusion, SVZ B1 cells gave rise not only to NG2+ cells, but also to a subpopulation of highly-branched S100β+/GFAP+/NG2-cells (supporting information Fig. 3). The branched morphology and low expression of GFAP [38] may indicate that the SVZ-expanded population under EGF has the ability to differentiate into parenchymal astrocytes. However, these unique cells also express S100β, which could indicate that they are part of the oligodendrocyte lineage (see discussion) [36, 37].

Table 1. Expression of oligodendrocyte lineage markers in the corpus callosum after epidural growth factor removala
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A previous study suggests that a small subpopulation of SVZ progenitors, which migrate into the striatum after EGF infusion, differentiate into neurons [12]. Interestingly, at all ages after EGF infusions, short chains of 4 to 7 GFP+ cells that stained positively for doublecortin and PSA-NCAM were observed from 20 to 50 μm deep from the SVZ in the striatum and septum (supporting information Fig. 4). These chains were never observed in control animals. In order to test whether these cells differentiating into mature neurons, we stained sections with the marker NeuN at 21, 45, 90, and 180 days (3 to 5 animals per age group) after EGF withdrawal. We found no evidence of GFP+ cells differentiating into neurons in the striatum, septum, or cortex, even at longer survival times. Cells in these chains may die or rejoin the network of pathways for chain migration in the SVZ. These observations indicate that after withdrawal of EGF, EIPs differentiate into NG2+ OPCs, mature oligodendrocytes, and a population of S100β+/GFAP+ cells.

Epidermal Growth Factor-Expanded Oligodendrocytes Derived from SVZ B1 Cells Promote Remyelination

We next investigated whether cells derived from SVZ type B cells can contribute to increase the number of oligodendrocytes into demyelinating lesions after EGF infusion. SVZ astrocytes were labeled by injecting RCAS-GFP into the SVZ of adult GFAP-tva mice. Two days later, lysolecithin or vehicle was ipsilaterally injected into the corpus callosum. To avoid an additional injury in the corpus callosum, the cannula for EGF or vehicle infusion was placed onto the brain surface. We found that EGF infusion onto the brain surface expands the number of Olig+ cells that invade surrounding brain parenchyma as seen with intraventricular infusions (supporting information Fig. 5). The cells that leave the SVZ expressed Olig2 and had the same morphological characteristics of the EIPs expanded by intraventricular infusions following EGF-infusion onto the brain surface. Animals were killed at 14 and 28 days post lysolecithin injection (DPL). At 14 DPL, we found a 2.1-fold increase in the number of GFP+/CNPase+ cells in the corpus callosum of EGF-infused animals (181 ± 36, n = 3) compared with the control-vehicle group (84 ± 19, n = 3; p < .05). At 28 DPL, we found a 2.6-fold increase in the number of GFP+/CNPase+ cells in the corpus callosum of EGF-infused brains (251 ± 36 cells, n = 3) versus the control-vehicle group (94 ± 35 cells, n = 3; p < .01) (Fig. 6A, 6B, 6D). These GFP+ cells in the lesion site contacting neurofilaments had the morphology and expressed oligodendrocyte markers (CNPase, MBP, S100β) (Fig. 6C–6G). To quantify the effect of EGF on remyelination, we performed immunohistochemistry for degraded-myelin basic protein (dMBP) at 14 and 28 DPL and calculated the volume of the demyelinating lesion in the corpus callosum (n = 3 per group) (Fig. 6H–6J). In both cases, EGF-treated animals had significantly smaller lesion volumes (at 14 DPL = 0.55 ± 0.04 mm3, and at 28 DPL = 0.23 ± 0.05 mm3) compared with controls (at 14 DPL = 0.33 ± 0.08 mm3, and at 28 DPL = 0.4 ± 0.04 mm3; p < .05, Student's t test). These findings indicate that EGF increases the number of oligodendrocytes derived from a subpopulation of SVZ B1 cells that contribute to remyelination after a demyelinating lesion to the corpus callosum.

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Figure 6. Epidermal growth factor (EGF)-induced progenitors derived from SVZ B cells promote remyelination. Schematic timeline shows the experimental design. GFP+ oligodendrocytes found in the demyelinated area (dotted line) of the EGF group (B) and the control group (A) 14 days post lysolecithin injection (DPL). (C): Confocal Z-stack reconstruction of two cells found in the demyelinated area of an EGF-infused animal. GFP+ cells expressed CNPase (D), myelin basic protein (F), or S100β (G). Ultrastructure of GFP+ cells suggests they correspond to oligodendrocytes (E). The number of CNPase+GFP+ cells in the EGF-infused animals was higher than the control-vehicle group at 14 and 28 DPL (D) (p < .05; Student's t test). There is dMBP expression at 14 DPL in a control (H) and EGF-infused (I) animal. Volume of dMBP+ lesion was reduced by the effect of EGF infusion (J). Bars (A–B) = 50 μm; (D) = 20 μm; (E–F) = 10 μm; (G–H) = 200 μm. Abbreviations: EGF, epidermal growth factor; GFP, green fluorescent protein; MBP, myelin basic protein; NF, neurofilaments; V, ventricle; m, myelinated axon.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS/SUMMARY
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

In this study we show that most of the cells derived from SVZ B1 cells after EGF infusion leave the SVZ and migrate along axonal tracts and blood vessels, express Olig2 and other OPC markers, and differentiate into nonmyelinating and myelinating oligodendrocytes. EGF induces proliferation and migration of the SVZ progenitors in embryonic, early postnatal, and adult brain [12, 14, 17, 39–41]. Consistently, our present results indicate that the EGF infusion into adult brain induces expansion of highly-migratory SVZ progenitors. Interestingly, the majority of these EGF-expanded progenitors express Olig2. This is in sharp contrast to the normal conditions in which only a small subpopulation of SVZ B1 and C cells express Olig2 [7, 25, 42]. Olig2 is essential for the proliferation and differentiation of oligodendrocyte precursors [31, 43–46]. Olig2-expressing progenitors also give rise to motor neurons [44, 47, 48], astrocytes, and ependymal cells [26, 49]. Although Olig2 expression has been reported in OPCs and mature oligodendrocytes [50, 51], it is not possible based on the expression of Olig2 alone to conclude that EIPs are committed to the oligodendroglial lineage.

Interestingly, Olig2 overexpression has also been reported in glioma-like growths induced by PDGFα [52] and in multiple types of brain tumors [50, 53]. Similar to PDGFα, Olig2 is required for neurosphere formation and tumor growth [54]. Interestingly, unlike PDGFα, EGF infusion upregulated Olig2 expression, but did not generate glioma-like masses next to the SVZ. Instead, EGF produced highly-motile Olig2+ cells that infiltrate the brain parenchyma along blood vessels and myelinated and nonmyelinated axonal tracts, which is a behavior comparable to that of invading brain tumor cells [50, 55, 56]. Aberrant EGF signaling has been implicated in glioma progression [55, 56]. Long-lasting constitutive EGF signaling has also been found to lead to diffuse hyperplasia of early glial progenitors in the white matter without differentiation into mature oligodendrocytes [57]. Other studies have reported polyp-like formation after a long-term EGF infusion [14] or transplantation of EGF-treated neural progenitors [58]. However, none of our animals had tumors, including some that survived for 180 days after termination of EGF infusions. Although EGFR mutations confer enhanced tumorigenic behavior, the evidence indicates that EGF signaling alone is not enough to produce tumors [55, 59]. Our data indicate that cell proliferation and infiltration cease soon after termination of EGF infusion, and most of the cells differentiate into cells in the oligodendroglial lineage.

Whereas previous studies have followed the differentiation of EGF-expanded populations from the SVZ [12, 14, 18], none have traced the differentiation of cells derived from the primary SVZ progenitors. Furthermore, since EGF may induce proliferation of SVZ progenitors and other precursors outside of the SVZ, it was important to establish the fate of cells derived from the SVZ B1 cells. Toward this end, we used GFAP-tva mice in which GFAP-expressing SVZ cells can be specifically infected with RCAS retrovirus carrying a GFP reporter gene [2, 13, 27]. In all of our experiments, RCAS labeling was done before EGF-pump implantation to avoid a potential EGF-induced expression of tva receptors in other cells. We found that the EGF-expanded cells that invade the brain parenchyma were derived from the SVZ B1 cells. Interestingly, in addition to Olig2, these EGF-expanded population also expressed PDGFRα and NG2 [60], which are markers associated with oligodendrocyte lineage [24, 61–63].

Most of the GFP-labeled cells (from 21 to 180 days) in the corpus callosum and fimbria not only had the morphology, but also expressed markers consistent for cells in the oligodendrocyte lineage. In addition to oligodendrocytes, we also found that the EIPs gave rise to a population of S100β+ cells. These cells expressed low levels of GFAP and had a “bushy” morphology similar to that of protoplasmic astrocytes. This is consistent with previous work suggesting that intracerebral infusion of EGF results in the generation of astrocytes [12, 14]. Since most, if not all, of the EGF-responsive cells expressed Olig2, these astrocyte-like cells were likely derived from Olig2-expressing cells. This is consistent with recent work suggesting that Olig2-expressing precursors can generate astrocytes in addition to oligodendrocytes [26]. However, S100β, which is typically identified as a marker of astrocytes [35], has recently been found in the oligodendroglial lineage [36, 37]. Therefore, the S100β+ “astrocytes” we observed in gray matter brain regions may also correspond to cells in the OPC lineage. The precise nature of GFAP+/S100β+ cell population remains unresolved. Previous studies reported that a small subpopulation of EGF-expanded SVZ cells differentiate into neurons [12]. We did not observe neuronal differentiation in the striatum, septum, and cortex at any of the studied survival times.

Under normal conditions, the production of oligodendrocytes from SVZ B1 stem cells is low compared with neuronal generation [7]. SVZ oligodendrocyte production increases in response to demyelination [7–9, 64], yet cell recruitment into the lesion is not extensive. These results show that EGF infusion can significantly increase the number of oligodendrocytes derived from B1 SVZ cells, which contribute to myelin repair. This is consistent with recent work showing that EGFR signaling significantly increases the number of oligodendrocytes derived from the SVZ and stimulates migration and remyelination [16]. Developmental studies indicate that changes in EGFR expression onto cell membrane can influence cell fate, and its overexpression pushes cells into glial lineage at the expense of neuron formation [39, 41, 65]. However, further studies are necessary to establish whether asymmetric distribution of EGFR during mitosis is responsible for glial fate inductions in adult brains, as reported in developmental studies [66].

In the adult brain, mature oligodendrocytes are not only produced from the SVZ [7–9], but also from local NG2-expressing OPCs [21, 67]. We found that after EGF stimulation, NG2+ cells in the white and gray matter derived from a subpopulation of SVZ B1 cells. NG2+ cells also referred as OPCs, polidendrocytes [61], NG2-expressing glia [63], and synantocytes [68], are likely functionally diverse. Subsets of NG2-expressing cells express α-amino-3-hydroxy-5-methyl isoxazole propionic acid (AMPA) receptors and participate in glutamatergic synaptic signaling in the hippocampus [69], corpus callosum [70] and cortex [71]. We do not know if the population of NG2-expressing cells that are derived by EGF infusions from SVZ type B cells is homogeneous or if different subpopulations of NG2-expressing glia can be generated by this method. Interestingly, synantocytes or polidendrocytes have been implicated in brain scar formation and response to demyelination [28, 72, 73]. Recent evidence has also shown that erythropoietin/EGF-amplified cells from the SVZ may also contribute to functional cortical repair [74]. This suggests that, in addition to their classic function as local OPCs, the EGF-derived NG2+ populations could play important roles in brain repair.

Remyelination has been observed following neural stem cell transplantation into experimental demyelination models [75, 76]. However, one limitation of this approach is delayed rejection to grafted precursors [77, 78]. Recruitment of new OPCs by stimulation of endogenous precursors circumvents immune rejection [79]. The present work shows that EGF stimulates the endogenous SVZ progenitors to differentiate into myelin-forming cells. Interestingly, a local proliferative response has been described in the human SVZ of patients suffering from multiple sclerosis [80], suggesting that local periventricular oligodendrocyte progenitors may exist in the adult human brain.

CONCLUSIONS/SUMMARY

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS/SUMMARY
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Taken together, our results indicate that EGF infusion in the adult mouse brain significantly increases the number of progenitor cells derived from SVZ stem cells. EGF stimulates these progenitors to migrate long distances in the adult brain [13, 16, 17] (present results), which results in massive infiltration into regions that usually receive few new cells from the SVZ (the corpus callosum, fimbria-fornix, striatum, septum, and cortex). Most of the amplified cells differentiate along the oligodendrocyte lineage and contribute to the repair of demyelinating lesions. Effective precursor infiltration and differentiation of OPCs into myelinating oligodendrocytes are considered crucial steps for the development of new cell repair strategies for demyelinating diseases [81]. Our data indicate that the lineage of cells derived from stem cells in the largest germinal layer of the adult brain can be manipulated by infusion of growth factors to significantly increase the formation of oligodendrocytes.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS/SUMMARY
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

This work was supported by NIH grant HD 32116, the Goldhirsh Foundation, and the John G. Bowes research fund. O.G-P. was supported by a FRABA grant (No. 554/08). A.A-B holds the Heather and Melanie Muss Endowed Chair. J.M.G-V was supported by CIBERNED and Red de Terapia Celular. We would like to thank David Rowitch for providing Olig2 antibody, Magdalena Gotz for the RCAS-GFP DNA construct, Eric Holland for the GFAP-tva mice, and Sonia Luquin for technical support. We are also grateful to Thuhien Nguyen, Cynthia Yaschine, and Kaisorn Chaichana for editorial comments on the manuscript.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS/SUMMARY
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS/SUMMARY
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Additional supporting information available online.

FilenameFormatSizeDescription
STEM_00119_sm_suppfig1.tif6168KSupporting Information Figure 1. NG2 and PDGFRα expression after the 7-day EGF infusions. Upon the growth factor infusions a higher expression of NG2 (A - B) and PDGFRα (C - D) is found around of the lateral ventricles and dorsal SVZ as compared to saline-infused controls. The EM picture shows an EIP (see text) process close to a blood vessel (BV), but do not contact the endotelium (E). Inset shows a higher magnification of the squared area. Ribosomes (r), mitochondria (m), 1 = endothelial cell cytoplasm, 2 = basal lamina, 3 = astrocyte process and 4 = EIP process. V: Ventricle. Scale bar in A – D = 20 μm; E = 2 μm.
STEM_00119_sm_suppfig2.tif4208KSupporting Information Figure 2. EGF infusion does not activated parenchymal astrocytes. EGF was infused in the lateral ventricle and RCAS-GFP was injected in different brain regions (see methods). A: The 7-day EGF infusion induces an over-expression of Olig2 around the ventricle, but the progeny of RCAS-labeled astrocytes remains close to the needle track (arrowheads). Similar findings were found in corpus callosum (B) and cortex (C). No GFP+ cells were found when virus was injected onto brain surface. Sep: Septum, Str: Striatum. V: Ventricle. Scale bar = 200 μm.
STEM_00119_sm_suppfig3.tif18273KSupporting Information Figure 3. EIPs' differentiation in the gray matter upon EGF pump removal. The table shows the quantification of several markers in brain parenchyma. In the saline-infused animals a limited number of SVZ-derived are incorporated into brain parenchyma as compared to EGF-infused mice. Asterisks indicate differences statistically significant (P < 0.05; Student “t” test). A - C: GFP+ cell co-expressing GFAP/S100β. D: Pre-embedding immunogold EM of a highly branched GFP+ cell that has a number of intermediate filaments (arrowheads). Insets: Top left, the same cell under the light microscope; top right, semi-thin section of the GFP+ cell; bottom right, intermediate filaments and silver-enhanced precipitates. E: A subset of GFP+ cells showed few ramifications, non-bushy morphology and expressed NG2. Scale bars: C and E = 10 μm; D = 2 μm; inset = 0.5 μm.
STEM_00119_sm_suppfig4.tif4912KSupporting Information Figure 4. Ectopic migration chains. Upon EGF pump removal, several ectopic chains of migrating neuroblast are commonly found in the brain parenchyma. Arrows show doublecortin+ (A) and PSA-NCAM+ cell chains (B) in septum (Sep). Nuclei are counterstained with DAPI (blue). CC: Corpus callosum; Str: Striatum; dSVZ: Dorsal subventricular zone. Scale bars = 20 μm.
STEM_00119_sm_suppfig5.tif3455KSupporting Information Figure 5. Olig 2 expression after a vehicle or EGF infusion onto brain surface. As found for intraventricular EGF infusions (figure 1 of this report), many Olig2+ cells are observed around the SVZ in animals infused with EGF on the surface of the brain. This was not observed in mice that received vehicle onto the brain surface. Nuclei are counterstained with DAPI (blue). CC: Corpus callosum; Str: Striatum; V: Ventricle. Scale bar = 20 μm.

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