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THE STEM CELL NICHE

Increased Neurogenesis and Astrogenesis from Neural Progenitor Cells Grafted in the Hippocampus of GFAP^–/–Vim^–/– Mice

Center for Brain Repair and Rehabilitation, Department of Clinical Neuroscience and Rehabilitation, Institute of Neuroscience and Physiology, Sahlgrenska Academy at Göteborg University, Göteborg, Sweden

Key Words. Glial fibrillary acidic protein • Vimentin • Intermediate filaments • Astrocytes • Reactive gliosis

Correspondence: Milos Pekny, M.D., Ph.D., Center for Brain Repair and Rehabilitation, Department of Clinical Neuroscience and Rehabilitation, Institute of Neuroscience and Physiology, Sahlgrenska Academy, Göteborg University, Medicinaregatan 9A, SE-413 90 Göteborg, Sweden. Telephone: 46-31-786-32-69; Fax: 46-31-416108; e-mail: milos.pekny{at}medkem.gu.se

Received February 12, 2007; accepted for publication June 27, 2007.
First published online in STEM CELLS EXPRESS   July 12, 2007.

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

 
After neurotrauma, ischemia, or neurodegenerative disease, astrocytes upregulate their expression of the intermediate filament proteins glial fibrillary acidic protein (GFAP), vimentin (Vim), and nestin. This response, reactive gliosis, is attenuated in GFAP^–/–Vim^–/– mice, resulting in the promotion of synaptic regeneration after neurotrauma and improved integration of retinal grafts. Here we assessed whether GFAP^–/–Vim^–/– astrocytes affect the differentiation of neural progenitor cells. In coculture with GFAP^–/–Vim^–/– astrocytes, neural progenitor cells increased neurogenesis by 65% and astrogenesis by 124%. At 35 days after transplantation of neural progenitor cells into the hippocampus, adult GFAP^–/–Vim^–/– mice had more transplant-derived neurons and astrocytes than wild-type controls, as well as increased branching of neurite-like processes on transplanted cells. Wnt3 immunoreactivity was readily detected in hippocampal astrocytes in wild-type but not in GFAP^–/–Vim^–/– mice. These findings suggest that GFAP^–/–Vim^–/– astrocytes allow more neural progenitor cell-derived neurons and astrocytes to survive weeks after transplantation. Thus, reactive gliosis may adversely affect the integration of transplanted neural progenitor cells in the brain.

Disclosure of potential conflicts of interest is found at the end of this article.

	INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

 
In the adult central nervous system (CNS), diverse injuries, such as neurotrauma, ischemic damage, and administration of neural grafts, result in astrocyte activation and reactive gliosis. Reactive gliosis was proposed to constitute an environment hostile for CNS regeneration [1–6], but the molecular mechanism remains incompletely understood.

Upregulation of glial fibrillary acidic protein (GFAP), vimentin (Vim), and nestin and increased abundance of intermediate filaments are hallmarks of reactive astrocytes [1, 2]. In mice, reactive gliosis can be attenuated by genetic ablation of GFAP and vimentin [7], two intermediate filament proteins that are components of the astrocyte cytoskeleton. The attenuation of reactive gliosis in GFAP^–/–Vim^–/– mice slows healing after neurotrauma [7]. However, at later stages, these mice display better synaptic recovery [8] and improved axonal regeneration after spinal cord injury [9] or after severing of the optic nerve [10]. Introduction of neural transplants into the CNS also leads to a marked activation of astrocytes [11–13]. We have shown that transplanted retinal cells from postnatal mice up to 30 days of age integrate more robustly in retinas of GFAP^–/–Vim^–/– mice than in those of wild-type mice [14].

These results suggest that GFAP^–/–Vim^–/– astrocytes influence the differentiation of neural progenitor cells and that attenuation of reactive gliosis favors the differentiation and survival of neural progenitor cells in the brain. Two regions of the mammalian brain showing neurogenesis, even in the adult, are the dentate gyrus of the hippocampus and the subventricular zone of the lateral ventricles [15–17]. Hippocampal neural progenitor cells can be isolated, propagated, and differentiated into neurons and glial cells and are a potential source of cells for transplantation [11, 18, 19]. However, proper neuronal differentiation, integration, and survival of these cells in adult hosts are major hurdles that must be overcome before transplantation of such cells can be turned into a successful therapeutic paradigm [20, 21].

To evaluate the effect of attenuated reactive gliosis on the efficacy of graft integration, we assessed neurogenesis, astrogenesis, and oligogenesis in vitro. We also compared the number of neurons and astrocytes that differentiated from grafted rat hippocampal neural progenitor cells and survived 35 days after transplantation into the hippocampus of GFAP^–/–Vim^–/– and wild-type mice. Signals provided by the microenvironment surrounding the neural progenitor cells regulate their survival, proliferation, and fate commitment, and astrocytes are often a source of such signals [22–24]. It has been shown that Wnt3 is a powerful regulator of neurogenesis from adult hippocampal progenitor cells, both in vitro and in vivo [23]. We therefore explored the expression of this factor in GFAP^–/–Vim^–/– and wild-type mice and in cocultures of rat hippocampal neural progenitor cells and GFAP^–/–Vim^–/– and wild-type astrocytes.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

 
Mice
The GFAP^–/–Vim^–/– and wild-type mice used for transplantation and in vitro coculture experiments were of a mixed genetic background (C57Bl/6, 129Sv, 129Ola) [25, 26]. For transplantation experiments, we used 12 GFAP^–/–Vim^–/– and 13 wild-type female mice, 4.5–5 months old. To avoid host-versus-graft rejection, these mice were null mutants for Rag-1 [27]. The null mutations were confirmed as described [25–27]. The mice were housed in standard cages in a barrier animal facility and had free access to food and water. All experimental protocols were approved by the Ethics Committee of Göteborg University.

Neural Progenitor Cell Cultures
Neural progenitor cells derived from adult rat hippocampus and expressing green fluorescent protein (GFP) [28] were grown on laminin/poly-L-ornithine-coated Petri dishes in Ham's F-12 high-glucose medium (Irvine Scientific, Santa Ana, CA, http://www.irvinesci.com) with 1% N2 supplement (Gibco, Paisley, U.K., http://www.invitrogen.com), 2 mM L-glutamine (Gibco), 20 ng/ml recombinant human basic fibroblast growth factor (BD Biosciences, Erembodegem, Belgium, http://www.bdbiosciences.com), penicillin/streptomycin (100 U/0.1 mg/ml; Gibco), and Fungizone (0.25 µg/ml; Bristol-Myers Squibb, Princeton, NJ, http://www.bms.com). The cells were cultured at 37°C in 5% CO₂.

Assessment of Neural Progenitor Cell Differentiation In Vitro
Primary cultures of astrocytes from 1-day-old GFAP^–/–Vim^–/– and wild-type mice were prepared as described [29]. At confluence, primary cultures were passaged (1:2) onto coverslips and cultured in normal serum-containing medium. Confluent cultures were washed twice with serum-free medium (Dulbecco's modified Eagle's medium/Nut Mix F12 [1:1; Gibco], 2 mM L-glutamine, 1% N2 supplement, 1% penicillin/streptomycin [100 U/0.1 mg/ml; Gibco], and 0.25 µg/ml Fungizone [Bristol-Myers Squibb]) and cultured in serum-free medium or medium supplemented with 1% fetal calf serum (Gibco).

The astrocyte cultures were seeded with GFP-expressing neural progenitor cells [24] at approximately 2.0 x 10³ cells per cm². At 6 days, lineage selection was assayed. Briefly, cells were fixed (4% paraformaldehyde in phosphate-buffered saline [PBS], 4°C, 10 minutes), nonspecific binding was blocked, and cells were immunocytochemically stained (1 hour, room temperature) with mouse anti-microtubule-associated protein 2 subunits a and b (anti-Map2ab) antibodies (1:100; Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), rabbit anti-GFAP antibodies (1:500; DAKO, Glostrup, Denmark, http://www.dako.com), and mouse anti-Rip antibodies (1:50; Developmental Studies Hybridoma Bank, Iowa City, IA, http://www.uiowa.edu/dshbwww) in PBS containing 3% donkey serum (Jackson Immunoresearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com) and 0.05% saponin (Sigma-Aldrich). After three washes in PBS, cells were incubated for 1 hour at room temperature with Alexa Fluor 555-conjugated donkey anti-mouse or anti-rabbit secondary antibodies (1:2,000; Molecular Probes, Eugene, OR, http://www.probes.invitrogen.com) and the nuclear dye bisbenzimide from a stock at 5 µg/ml (Hoechst 33258, 1:80; Sigma-Aldrich).

Differentiation was assessed by counting GFP^pos, GFP^posMap2ab^pos, GFP^posRip^pos, and GFP^posGFAP^pos cells in at least 12 randomly selected, nonoverlapping fields using a Nikon Eclipse 80i microscope (Nikon, Preston, U.K., http://www.nikon.com). All differentiation experiments were done in triplicate (n = 4), and 200–500 cells were counted in each field. Cell counts were performed on blinded samples.

For the analysis of Wnt3 expression, confluent astrocyte cultures and cocultures with neural progenitor cells (2.0 x 10³ cells per cm²) in 1% or 10% of fetal calf serum were fixed and immunocytochemically stained with goat anti-Wnt3 antibodies (1:50; Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com) and rabbit anti-GFAP antibodies (1:500; DAKO) in PBS containing 3% donkey serum and 0.1% Triton X-100 (Merck, Darmstadt, Germany, http://www.merck.com). After three washes in PBS, cells were incubated for 1 hour at room temperature with Alexa Fluor 488-conjugated donkey anti-mouse and Alexa Fluor 555-conjugated donkey anti-goat antibodies (1:2,000; Molecular Probes). The percentage of astrocytes displaying the filamentous pattern of Wnt3 immunoreactivity was assessed by investigating 200 wild-type and 200 GFAP^–/–Vim^–/– astrocytes. All images were processed in Photoshop (version 8.0; Adobe Systems Inc., San Jose, CA, http://www.adobe.com) and ImageJ (version 1.30, National Institutes of Health, Bethesda, MD, http://www.nih.gov).

Neural Progenitor Cell Transplantation
Neural progenitor cells were grown to confluence in the presence of 5 µM 5-bromo-2'-deoxyuridine (BrdU) (Sigma-Aldrich) for 48 hours, trypsinized, washed, resuspended (60,000 cells per microliter) in PBS with 0.15% glucose and 30 ng/ml of basic fibroblast growth factor, and kept on ice until grafting. The mice were anesthetized with avertin and placed in a stereotactic frame, and 1.0 µl of cell suspension was slowly injected (2 minutes) unilaterally into the hippocampus (mediolateral, –3.0 mm; anteriorposterior, –3.5 mm; dorsoventral, –2.7 mm) with a 5-µl Hamilton syringe, which was left in place for an additional 2 minutes to prevent reflux of the cell suspension. The syringe was slowly raised 0.5 mm, 0.2 µl was injected, and the syringe was left in place for 1 minute. After 7 hours on ice, viability of the remaining cell suspension was 90% as assessed by trypan blue exclusion.

Grafted cells were identified with rat anti-BrdU antibodies (1:100; Nordic BioSite, Täby, Sweden, http://www.biosite.se) followed by goat anti-rat Alexa Fluor 488 antibodies (1:500; Molecular Probes). To visualize astrocytes, we used mouse anti-GFAP antibodies (1:100; Sigma-Aldrich) and rabbit antibodies against the endothelin B receptor (ET_BR) (1:100; Alomone Labs, Jerusalem, http://www.alomone.com), followed by Alexa Fluor 488 or 568 goat anti-mouse or Alexa Fluor 488 donkey anti-mouse and Alexa Fluor 568 goat anti-rabbit antibodies (1:500; Molecular Probes), respectively. To assess the number of immature astrocytes, we used rabbit anti-S100 antibodies (1:200; DAKO), followed by biotinylated donkey anti-rabbit antibodies (1:200; Jackson Immunoresearch Laboratories) and streptavidin conjugated with Cy3 (1:100; Sigma-Aldrich) or mouse anti-glutamine synthase (GS) antibodies (1:100; Chemicon, Temecula, CA, http://www.chemicon.com) followed by biotinylated rabbit anti-mouse antibodies (1:400; DAKO) and streptavidin conjugated with Cy3 (1:100; Sigma-Aldrich). To visualize oligodendrocytes, we used mouse anti-Rip antibodies (1:20; Developmental Studies Hybridoma Bank), followed by biotinylated donkey anti-mouse antibodies (1:200; Jackson Immunoresearch Laboratories) and streptavidin conjugated with Alexa 594 (1:1,000; Molecular Probes). To visualize astrocyte and oligodendrocyte progenitors, we used goat anti-Olig2 antibodies (1:200; R&D Systems Inc., Minneapolis, http://www.rndsystems.com) followed by biotinylated donkey anti-goat antibodies (1:200; Jackson Immunoresearch Laboratories) and streptavidin conjugated with Cy3 (1:200; Sigma-Aldrich). To assess neuronal differentiation, we used biotinylated mouse anti-NeuN antibodies (1:100; Chemicon) followed by streptavidin conjugated with Cy3 (1:100; Sigma-Aldrich). To study Wnt3 immunoreactivity, we used goat anti-Wnt3 antibodies (1:50; Santa Cruz Biotechnology) followed by biotinylated donkey anti-goat antibodies (1:200; Jackson Immunoresearch Laboratories) and streptavidin conjugated with Cy3 (1:300; Sigma-Aldrich). We used TO-PRO-3 (1:1,000; Molecular Probes) to counterstain cell nuclei.

Antibodies were diluted in a buffer containing 1% bovine serum albumin and 0.01% Tween 20 in PBS and mounted in Mowiol (Clariant GmbH, Frankfurt, Germany, http://www.clariant.com). The sections were incubated with primary antibodies overnight at 4°C and with secondary antibodies for 4 hours at room temperature.

To study the survival and differentiation of transplanted neural progenitor cells, BrdU^pos transplanted neural progenitor cells were counted in the subgranular zone, granular cell layer, and Ammon's horn of the hippocampus. In each mouse, seven 35-µm horizontal sections through the hippocampus at 140-µm intervals were examined with a Nikon Eclipse 80i epifluorescence microscope (Nikon). On the same sections, BrdU^pos cells were counted in a 70,000-µm² arc 250 µm away from the needle track and in a 120,000-µm² arc 500 µm away from the needle track. For each mouse, 25 BrdU^pos cells in the subgranular zone and 50 BrdU^pos cells in the granular cell layer were examined for S100 and GS positivity (astroglial markers) and NeuN positivity (neuronal marker) by laser-scanning confocal microscopy (Leica TCS, Heidelberg, Germany, http://www.leica.com). BrdU^pos cells in the granular cell layer and the subgranular zone in one and two sections per mouse were examined for Rip and Olig2 positivity, respectively, by laser-scanning confocal microscopy. All images were processed in Photoshop (version 8.0; Adobe Systems) and ImageJ (version 1.30; NIH).

GFP^pos cells in the molecular layer with processes exceeding three cell-body lengths were counted in seven 35-µm-thick horizontal hippocampal sections at 140-µm intervals; the longest process in each cell was measured, and its primary branches were counted (Fig. 5D). We used laser-scanning confocal microscopy to produce images at 1-µm intervals throughout the sections.

SDS-Polyacrylamide Gel Electrophoresis and Western Blot Analysis
Western blot was used to analyze Wnt3 protein expression in vivo and in vitro. Hippocampi from five wild-type and four GFAP^–/–Vim^–/– mice, 18 months of age, were homogenized in 500 µl of lysis buffer (50 mM dithiothreitol, 25 mM Tris HCl, 35 mM Tris base, 0.5% lithium dodecyl sulfate, 2.5% glycerol, 12.5 mM ethylenediaminetetraacetic acid [EDTA] and proteinase inhibitor [Complete Mini cocktail tablet; Roche Diagnostics, Basel, Switzerland, http://www.roche-applied-science.com]) using a motor-driven grinder pestle. The samples were sonicated for 1 minute, agitated for 2 hours, and heated to approximately 80°C for 3 minutes. The crude protein extract was centrifuged at 13,000 rpm for 15 minutes. Primary astrocyte cultures from five wild-type and four GFAP^–/–Vim^–/– mice, 1 day old, and cocultures from these cells and neural progenitors were lysed in lysis buffer and sonicated for 1 minute. For all samples, total protein concentration was determined with the Bradford method using Coomassie Plus (Pierce, Rockford, IL, http://www.piercenet.com) and measuring absorbance at 595 nm.

The sample volume (15–25 µl) loaded on the NuPAGE Novex Bis-Tris 10% acrylamide gel (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) was adjusted to gain an equal loading of 30 µg of total protein. One-dimensional gel electrophoresis was performed using 3-(N-morpholino)propanesulfonic acid (MOPS) SDS running buffer (Invitrogen) at a constant voltage of 200 V. For quantitative Western blot analysis, protein samples were transferred from gels to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, http://www.bio-rad.com). The membranes were blocked, washed, and incubated with goat anti-Wnt3 antibodies (1:200; Santa Cruz Biotechnology) or goat anti-β-actin antibodies (1:500; Abcam, Cambridge, U.K., http://www.abcam.com) followed by several washes and incubation with iodine-125-conjugated donkey anti-goat antibodies (1:1,000; Sigma-Aldrich). After exposure to photographic film, the relative intensities of Wnt3 and β-actin protein bands were analyzed using ImageJ (NIH).

Statistical Analysis
Values are presented as mean ± SEM. Differences were evaluated by two-tailed t test; p < .05 was considered significant.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

 
GFAP^–/–Vim^–/– Astrocytes Enhance the Differentiation of Neural Progenitor Cells into Neurons and Astrocytes
First, we assessed the differentiation of rat hippocampal neural progenitor cells [18] cocultured for 6 days with primary astrocytes from wild-type or GFAP^–/–Vim^–/– mice (Fig. 1). In cultures maintained with medium containing 1% fetal calf serum, the fraction of GFP^posMap2ab^pos neurons was 65% higher in the presence of GFAP^–/–Vim^–/– than wild-type astrocytes (11.16% ± 1.58% vs. 6.76% ± 0.70%; p < .05), and the fraction of GFP^posGFAP^pos astrocytes was 124% higher (10.70% ± 0.91% vs. 4.78% ± 0.99%; p < .01); no difference was found in the proportion of GFP^posRip^pos oligodendrocytes. In cultures maintained in serum-free medium, however, GFAP^–/–Vim^–/– astrocytes had no effect on neuronal and astrocytic lineage selection.

View larger version (38K): [in this window] [in a new window]   Figure 1. Differentiation of neural progenitor cells into neurons (A, D), oligodendrocytes (B, E), and astrocytes (C, F) during coculture with primary gv or wt astrocytes in serum-free medium or in medium with 1% FCS. *, p < .05; **, p < .01. Scale bar = 20 µm. Abbreviations: FCS, fetal calf serum; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein; gv, GFAP–/–Vim–/–; Map2ab, microtubule-associated protein 2 subunits a and b; wt, wild-type.

View larger version (42K): [in this window] [in a new window]   Figure 2. Grafting of neural progenitor cells in the hippocampal region triggered reactive gliosis. (A): Astrocytes were strongly GFAPpos in the vicinity of BrdU-labeled neural progenitor cells in the dentate gyrus of wt mice 35 days after transplantation. (B): Neural progenitor cells in the dentate gyrus of gv mice; no GFAP immunoreactivity was detected. Dashed line (A, B) indicates the granular cell layer. (C, D): Higher-magnification image shows GFPpos transplanted cells in wt (C) and gv mice (D) surrounded in wt mice by reactive GFAPpos astrocytes. ETBR immunoreactivity marks reactive astrocytes in the grafted and the contralateral dentate gyrus of wt mice (E, F) but not gv mice (G, H). Scale bars = 100 µm (A, B, E–H) and 10 µm (C, D). Abbreviations: BrdU, 5-bromo-2'-deoxyuridine; contra, contralateral; ETBR, endothelin B receptor; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein; gv, GFAP–/–Vim–/–; ipsi, ipsilateral; wt, wild-type.

View larger version (27K): [in this window] [in a new window]   Figure 3. Survival and migration of neural progenitor cells in wt and gv mice 35 days after transplantation into the hippocampus. (A): Survival of graft-derived cells in the sgz, gcl, and ah of the dentate gyrus. (B): Migration of grafted cells assessed by counting BrdUpos cells 250 and 500 µm away from the transplantation site (the areas indicated in [C]). *, p < .05. Scale bar = 100 µm. Abbreviations: ah, Ammon's horn; BrdU, 5-bromo-2'-deoxyuridine; gcl, granular cell layer; gv, GFAP–/–Vim–/–; sgz, subgranular zone; wt, wild-type.

GFAP^–/–Vim^–/– Mice Have More Neural Progenitor Cells 35 Days After Grafting
To study the survival and migration of grafted neural progenitors, we assessed the distribution of BrdU^pos cells in the hippocampus. Survival of transplanted cells in the subgranular zone, granular cell layer, and Ammon's horn did not differ significantly between GFAP^–/–Vim^–/– and wild-type mice (p = .081, p = .129, and p = .074, respectively) (Fig. 3A). At 250 and 500 µm from the injection site (Fig. 3C), however, transplanted neural progenitor cells were somewhat more numerous in GFAP^–/–Vim^–/– mice (799 ± 31 vs. 685 ± 42; p < .05) (Fig. 3B).

GFAP^–/–Vim^–/– Mice Have More Graft-Derived S100^pos Astrocytes
To assess the differentiation of grafted neural progenitors into astroglial cells, we used antibodies against S100, a marker of immature and mature astrocytes [32, 33], and GS, a marker of mature astrocytes [34]. At 35 days after grafting, the number of BrdU^posS100^pos cells was 91% higher in the granular cell layer of GFAP^–/–Vim^–/– than wild-type mice (91.2 ± 13 vs. 47.8 ± 12; p < .05); no difference was found in the subgranular zone (Fig. 4A). In both regions combined, there were 83% more BrdU^posS100^pos astrocytes in GFAP^–/–Vim^–/– mice (118 ± 16 vs. 64.8 ± 15; p < .05).

View larger version (32K): [in this window] [in a new window]   Figure 4. Differentiation of grafted neural progenitor cells in the sgz and gcl of the dentate gyrus of the hippocampus 35 days after transplantation. Shown are the number (A) and percentage (B) of grafted cells that were positive for S100 (BrdUposS100pos), indicating immature astroglial cells and mature astrocytes, in wt and gv mice. (C): Orthogonal sections showing BrdUposS100pos cells. Shown are the number (D) and percentage (E) of grafted cells that were positive for GS (BrdUposGSpos), indicating mature astrocytes, in wt and gv mice. (F): Orthogonal sections showing BrdUposGSpos cells. Shown are the number (G) and percentage (H) of grafted cells that were positive for Olig2 (BrdUposOlig2pos), indicating astrocyte progenitors and oligodendrocyte progenitors, in wt and gv mice. (I): Orthogonal sections showing BrdUposOlig2pos cells. *, p < .05. Scale bars = 10 µm. Abbreviations: BrdU, 5-bromo-2'-deoxyuridine; gcl, granular cell layer; GS, glutamine synthase; gv, GFAP–/–Vim–/–; sgz, subgranular zone; wt, wild-type.

The number and percentage of graft-derived GS^pos mature astrocytes in the granular cell layer and subgranular zone were comparable between groups (Fig. 4D, 4E). However, GFAP^–/–Vim^–/– mice tended to have more of these cells in the subgranular zone (4.1 ± 0.9 vs. 1.9 ± 0.7; p = .06) (Fig. 4D).

The differentiation of grafted neural progenitors into Olig2^pos cells (astrocyte progenitors and oligodendrocyte progenitors [35, 36]) was assessed by using antibodies against Olig2. Thirty-five days after grafting, the number of BrdU^posOlig2^pos cells in the granular cell layer was 78% higher in GFAP^–/–Vim^–/– mice than in wild-type mice (14.9 ± 2.5 vs. 8.3 ± 0.8; p < .05), whereas no difference was observed in the subgranular zone (Fig. 4G). Combined data from both regions displayed a 69% increase in the number of BrdU^posOlig2^pos cells in GFAP^–/–Vim^–/– mice (21.1 ± 3.2 vs. 12.5 ± 1.0; p < .05). The percentage of graft-derived BrdU^posOlig2^pos cells was 84% higher in the granular cell layer of GFAP^–/–Vim^–/– mice compared with wild-type mice (11.0 ± 1.7 vs. 6.0 ± 0.8; p < .05); no difference was observed in the subgranular zone (Fig. 4H). When both regions were combined, 74% more BrdU^posOlig2^pos cells were observed in GFAP^–/–Vim^–/– mice (22.5 ± 3.12 vs. 12.9 ± 1.5; p < .05). Consistent with the quantification of oligodendrocyte differentiation in vitro, no difference in the percentage of Rip^pos oligodendrocytes was observed between wild-type and GFAP^–/–Vim^–/– mice (2.1% ± 0.9% and 3.7% ± 0.9%, respectively; p = .23).

GFAP^–/–Vim^–/– Mice Have More Graft-Derived Neurons
Next, we assessed the differentiation of grafted neural progenitor cells into mature neurons by counting BrdU^posNeuN^pos cells 35 days after grafting (Fig. 5). In the granular cell layer, BrdU^posNeuN^pos cells were 45% more numerous in GFAP^–/–Vim^–/– mice (208 ± 15 vs. 144 ± 26; p < .05), but no difference was seen in the subgranular zone (21.0 ± 2.8 in GFAP^–/–Vim^–/– vs. 13.7 ± 3.2 in wild-type; p = .113). In the two areas combined, mature neurons were 51% more numerous in GFAP^–/–Vim^–/– mice (229 ± 17 vs. 151.7 ± 28; p < .05). There was no difference in the percentage of transplant-derived neurons in the granular cell layer (44.2% ± 2.6% in GFAP^–/–Vim^–/– vs. 37.1% ± 3.6% in wild-type; p = .15).

View larger version (54K): [in this window] [in a new window]   Figure 5. Neuronal differentiation of grafted neural progenitor cells in the sgz and gcl of the dentate gyrus of the hippocampus 35 days after transplantation. Shown are the number (A) and percentage (B) of grafted cells that were positive for the neuronal marker NeuN (BrdUposNeuNpos) in wt and gv mice. (C): Orthogonal sections showing BrdUposNeuNpos cells. Shown are transplanted GFPpos cells with neurite-like cellular processes and their branching in wt and gv mice. (D, E): At 35 days after transplantation, the number of GFPpos cells in the molecular layer of the hippocampus whose longest neurite-like cellular process exceeded three body lengths and had one or more primary branches was twice as high in gv mice (D) than in wt mice (E). (F): The distribution of such cells with different number of primary branches in wt and gv mice. *, p < .05. Scale bars = 10 µm. Abbreviations: BrdU, 5-bromo-2'-deoxyuridine; gcl, granular cell layer; GFP, green fluorescent protein; gv, GFAP–/–Vim–/–; sgz, subgranular zone; wt, wild-type.

Wnt3 Immunoreactivity Is Detected in Hippocampal Astrocytes of Wild-Type but Not GFAP^–/–Vim^–/– Mice
Using immunohistochemistry and Western blot analysis, we assessed the expression and distribution of Wnt3 protein, an important regulator of adult hippocampal neurogenesis [23], in the hippocampus of nongrafted wild-type and GFAP^–/–Vim^–/– mice. In wild-type hippocampus, there was a distinct Wnt3 immunoreactivity associated with astrocytes and blood vessels (Fig. 6A, 6C, 6E). In contrast, Wnt3 immunoreactivity was absent from hippocampal astrocytes of GFAP^–/–Vim^–/– mice, whereas Wnt3 immunoreactivity associated with blood vessels was retained (Fig. 6B, 6D, 6F). Western blot analysis of Wnt3 showed comparable amounts of the Wnt3 protein in the hippocampus of wild-type and GFAP^–/–Vim^–/– mice (Fig. 6G, 6H).

View larger version (34K): [in this window] [in a new window]   Figure 6. Wnt3 immunoreactivity was detected in astrocytes in the dentate gyrus of the hippocampus of wt (A, C) but not gv mice (B, D). (C): Higher magnification images show that astrocytes were Wnt3-positive only in wt mice, whereas Wnt3 immunoreactivity was associated with blood vessels in both wt and gv mice (C, D). Astrocytes positive for GS showed immunoreactivity for Wnt3 and GFAP in wt mice (E) but not in gv mice (F). Western blot analysis showed comparable levels of Wnt3 in the hippocampus of wt and gv mice (G, H). Equal protein loading was confirmed by immunoblotting for β-actin (G, I). Scale bars = 100 µm (A, B) and 25 µm (C–F). Abbreviations: GFAP, glial fibrillary acidic protein; GS, glutamine synthase; gv, GFAP–/–Vim–/–; TOPRO, TO-PRO-3; wt, wild-type.

View larger version (71K): [in this window] [in a new window]   Figure 7. In primary cultures of wt astrocytes, Wnt3 immunoreactivity had a filamentous appearance and showed partial colocalization with bundles of GFAP-positive intermediate filaments both in 1% (A–C) and 10% (G–I) fetal calf serum. In contrast, intermediate filament-free gv astrocytes exhibited a diffuse nonfilamentous pattern of Wnt3 immunoreactivity (D–F). Scale bars = 20 µm. Abbreviations: GFAP, glial fibrillary acidic protein; gv, GFAP–/–Vim–/–; wt, wild-type.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

In this study, when cocultured with neural progenitor cells, GFAP^–/–Vim^–/– astrocytes enhanced neurogenesis by 65% and astrogenesis by 124%. In GFAP^–/–Vim^–/– mice, neurons derived from neural progenitors showed improved survival and enhanced branching of neurite-like cellular processes 35 days after transplantation into the hippocampus. These findings suggest that the survival and differentiation of transplanted neural progenitor cells can be improved by modifying the astroglial environment of the host. In the presence of 1% serum to mimic conditions leading to astrocyte activation and reactive gliosis in vivo, the number of astrocytes derived from neural progenitor cells increased in cocultures with wild-type or GFAP^–/–Vim^–/– astrocytes, as reported before [28]. However, such astrocytes were more than twice as abundant in cocultures with GFAP^–/–Vim^–/– astrocytes, and neurogenesis was increased in cocultures with GFAP^–/–Vim^–/– in the presence of serum. These findings suggest that nonreactive astrocytes (i.e., in the absence of serum) lacking intermediate filaments have the same effect on neurogenesis and astrogenesis as wild-type astrocytes; however, when activated by serum factors, they increase the rate of neurogenesis and astrogenesis, either by increased permissiveness or instruction of neural progenitor cell differentiation.

Previously, we reported that the retinas of GFAP^–/–Vim^–/– mice are more permissive for the integration of retinal grafts [14], as shown by a sixfold increase in the number of grafted cells that migrated, differentiated into mature neurons, and integrated into the ganglion cell layer. Consistent with those results, the number of graft-derived neurons in the neurogenic subgranular zone and granular cell layer of the hippocampus was 50% higher in GFAP^–/–Vim^–/– mice than in wild-type mice. Moreover, at 35 days after grafting, the molecular layer in GFAP^–/–Vim^–/– mice had twice as many graft-derived cells with long and branched neurite-like processes, implying that they were mature neurons. Thus, reactive gliosis may negatively influence the fate of neural progenitor cells, both in culture and after grafting, and attenuation of reactive gliosis may promote neurogenesis from transplanted cells irrespective of whether the transplants are immature retinal cells [14] or homogenous populations of neural progenitor cells (this study).

In the granular cell layer, the number of graft-derived S100^pos immature and mature astrocytes was 90% higher in GFAP^–/–Vim^–/– mice than wild-type mice. In both groups, a similarly small fraction of these cells was GS^pos, a marker of mature astrocytes, but GFAP^–/–Vim^–/– mice tended to have more GS^pos cells in the subgranular zone (p = .06). This suggests that attenuation of reactive gliosis increases astrogenesis, but few S100^pos astrocytes complete their differentiation. Consistent with this scenario and with the paucity of transplant-derived GFAP^pos cells in the dentate gyrus [11, 39], graft-derived GFAP^pos cells were virtually absent in both wild-type and GFAP^–/–Vim^–/– mice (not shown). In the granular cell layer, the number of graft-derived Olig2^pos cells (astrocyte progenitors and oligodendrocyte progenitors) was 78% higher in GFAP^–/–Vim^–/– mice than in wild-type mice. Similar to the low number of fully mature graft-derived astrocytes in the hosts, the number of Rip^pos oligodendrocytes was very low but comparable in GFAP^–/–Vim^–/– and wild-type hosts.

We found that a proportion of wild-type astrocytes in primary cultures or cocultures with neural progenitor cells exhibited a filamentous pattern of Wnt3 immunoreactivity, which seemed, at least to some extent, to colocalize with bundles of intermediate filaments. Astrocytes in the hippocampus of wild-type mice were clearly Wnt3-positive, but the Wnt3 immunostaining was undetectable in astrocytes in the hippocampus of GFAP^–/–Vim^–/– mice, suggesting that Wnt3 associates with intermediate filaments also in vivo. It is possible that the association of Wnt3 with cytoskeletal components in astrocytes is important for the intracellular distribution of Wnt3 and might affect its secretion and consequently its effect on neural progenitor cells. Hippocampal neural progenitor cells were shown to express receptor and signaling components for Wnt proteins, and increased levels of Wnt3 were demonstrated to increase neurogenesis from hippocampal neural progenitor cells both in vitro and in vivo [23]. Thus, the differential distribution of Wnt3 in wild-type and GFAP^–/–Vim^–/– astrocytes could contribute to the increased neurogenesis from neural progenitor cells observed in GFAP^–/–Vim^–/– cocultures or when grafted into GFAP^–/–Vim^–/– hosts compared with wild-type.

We conclude that inhibition of reactive astrocytes facilitates the generation of mature neurons and astrocytes from hippocampal neural progenitor cells in vitro and in vivo. The survival and differentiation of transplanted neural progenitor cells might be improved by modifying the astroglial environment of the host.

	DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

Top Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

 
The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

Top Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

 
We dedicate this article to our great colleague Professor Peter Eriksson, a pioneer in the field of adult human neural stem cells, who died on August 2, 2007, at only 48 years old. We thank Dr. Fred Gage for providing us with neural progenitor cells, Dr. Marcela Pekna for comments on the manuscript, Dr. Susann Teneberg for iodine conjugation, and the Swegene Center for Cellular Imaging at Göteborg University. This work was supported by grants from the Swedish Medical Research Council (Projects 11548 and 5174), Swedish Stroke Foundation, Torsten and Ragnar Söderberg Foundations, the region of Västra Götaland (RUN), Frimurare Foundation, the Swedish Society for Medicine, W. and M. Lundgren Foundation, Tornspiran Foundation, John and Brit Wennerström's Foundation for Neurological Research, Foundation Edit Jacobson's Donation Fund, Trygg-Hansa, Hjärnfonden, and ALF Göteborg.

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