Brain Advance Access originally published online on August 29, 2007
Brain 2007 130(11):2962-2976; doi:10.1093/brain/awm200
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Expression of FGF-2 in neural progenitor cells enhances their potential for cellular brain repair in the rodent cortex
1Department of Neurosciences, University Medical Center, University of Geneva Medical School, Departments of 2Adult Psychiatry and 3Neurosurgery, University Hospital of Geneva, CH-1211 Geneva 4 and 4School of Life Sciences, Ecole Polytechnique Federale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland
Correspondence to: Jozsef Zoltan Kiss, Department of Neurosciences, University Medical Center (CMU), Rue Michel-Servet 1, 1211 Genève 4, Switzerland E-mail: Jozsef.Kiss{at}medecine.unige.ch
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Summary |
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Strategies to enhance the capacity of grafted stem/progenitors cells to generate multipotential, proliferative and migrating pools of cells in the postnatal brain could be crucial for structural repair after brain damage. We investigated whether the over-expression of basic fibroblast growth factor 2 (FGF-2) in neural progenitor cells (NPCs) could provide a robust source of migrating NPCs for tissue repair in the rat cerebral cortex. Using live imaging we provide direct evidence that FGF-2 over-expression significantly enhances the migratory capacity of grafted NPCs in complex 3D structures, such as cortical slices. Furthermore, we show that the migratory as well as proliferative properties of FGF-2 over-expressing NPCs are maintained after in vivo transplantation. Importantly, after transplantation into a neonatal ischaemic cortex, FGF-2 over-expressing NPCs efficiently invade the injured cortex and generate an increased pool of immature neurons available for brain repair. Differentiation of progenitor cells into immature neurons was correlated with a gradual down-regulation of the FGF-2 transgene. These results reveal an important role for FGF-2 in regulating NPCs functions when interacting with the host tissue and offer a potential strategy to generate a robust source of migrating and immature progenitors for repairing a neonatal ischaemic cortex.
Key Words: Brain repair; neonatal ischemia; neural progenitors; transplantation; migration; FGF-2
Abbreviations:
DCX, doublecortin; DIV, days in vitro; GABA, (
-aminobutyric acid; GAD-67, glutamic acid decarboxylase 67; NPCs, neural progenitor cells; SVZ, subventricular zone; TU, transducing units
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Received May 17, 2007. Revised July 20, 2007. Accepted August 2, 2007.
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Introduction |
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The postnatal cortex has a very limited ability to regenerate neural tissue after brain insults. This is due in part to the lack of a resident population of neural progenitor cells (NPCs) responsive to signals derived from the damaged tissue. Compensatory cortical neurogenesis has been reported after induced apoptotic degeneration (Magavi et al., 2000
) or after stroke (Jiang et al., 2001; Jin et al., 2003), but the number of newly generated neurons, if present at all (Arvidsson et al., 2002), remains insufficient to restore a normal cortical function (Bjorklund and Lindvall, 2000). To overcome this limitation, the manipulation of NPCs has developed into a key strategy for brain repair. Attempts have been made to stimulate the postnatal subventricular zone (SVZ) with growth hormones in order to recruit a population of NPCs towards the lesioned cortex. These approaches have been supported by the finding that growth hormones such as the basic fibroblast growth factor (FGF-2) are capable of increasing the proliferation of SVZ progenitors and promote olfactory bulb neurogenesis (Kuhn et al., 1997). Furthermore, chronic infusion of FGF-2 and the epidermal growth factor in the lateral ventricles has been shown to be critical in regenerating new hippocampal neurons after global ischaemia (Nakatomi et al., 2002). However, successful replacement of damaged cortical neurons using NPCs has not been reported. Among the multiple factors limiting the efficiency of NPC transplantation in the postnatal cortex is the fact that grafted NPCs rapidly lose their immature, proliferative and migratory properties. It would therefore be of considerable interest to supply a source population of multipotential, proliferative and migrating NPCs competent to respond to chemoattractant cues secreted by the site of injury. Support for this hypothesis derives from in vitro work showing that FGF-2-stimulated NPCs can respond to a chemoattractant cue such as vascular endothelial growth factor (Zhang et al., 2003).
Autocrine/paracrine signalling of FGF-2 appears to play a key role in sustaining self-renewal of neural progenitor/stem cells in vitro (Maric et al., 2003) and maintaining immature proliferative populations in neurogenic niches in vivo (Zheng et al., 2004). We therefore tested the hypothesis that over-expression of FGF-2 in transplanted NPCs may provide robust sources of migrating NPCs for tissue repair after brain damage.
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All animal experiments were conducted in accordance with Swiss laws, previously approved by the Geneva Cantonal Veterinary Authority.
Isolation, cultures and differentiation of NPCs in vitro
The SVZ from coronal slices of newborn rat brains were dissected, mechanically dissociated and trypsinized. NPCs were purified using a 22% Percoll gradient centrifugation as previously described (Lim et al., 2000; Zhang et al., 2003) and seeded at 4 x 105 cells/dish concentration onto matrigel-coated dishes (1:500). Cells were allowed to expand in neurobasal medium (Invitrogen) supplemented with 20 ng/ml FGF-2 (human recombinant, R&D), 2% B27 supplement (Invitrogen), 2 mM L-glutamine, 1 mM sodium pyruvate, 2 mM N-acetyl-cysteine and 1% penicillin–streptomycin. At DIV3, half of the medium was replaced with fresh medium containing 20 ng/ml of FGF-2 with either the control lentiviral vector or FGF-2 lentiviral vector. For FGF-2 deprivation experiments, the dishes were washed and 2 ml of fresh medium was added with or without FGF-2 (20 ng/ml). To induce differentiation, NPCs were trypsinized at DIV5, seeded at 5 x 104 cells/dish concentration onto matrigel-coated dishes (1:500), allowed to expand during 48 h in the presence of 20 ng/ml FGF-2. Differentiation was induced by removing FGF-2 and adding 20 ng/ml BDNF and 1% fetal calf serum (Invitrogen).
Design and production of lentiviral vectors
The pWPI_SPbFGF lentiviral vector coding for FGF-2 was constructed as follows. A cDNA coding for the 18 kDa form of the human FGF-2 (basic FGF) fused to an immunoglobulin signal peptide facilitating the secretion of FGF-2 (Rinsch et al., 2001) was cloned into the pWPI bicistronic lentiviral vector. pWPI is an HIV-1 derived SIN vector containing the EF1 alpha promoter and an EMCV-IRES-GFP cistron (http://tronolab.epfl.ch/). FGF-2 was cloned in the PmeI site located between the EF1 alpha promoter and the IRES_GFP sequences. Control lentiviral vectors were the following: pFUGW contains the ubiquitin promoter controlling the expression of GFP, pWPXL contains the EF1 alpha promoter controlling the expression of GFP (http://tronolab.epfl.ch/) and RIX-PGK-Tom-W vector was constructed by inserting the tdTomato gene (Shaner et al., 2004) downstream of the hPGK promoter, in place of the GFP gene of the RIX-PGK-GFP-W vector. EF1 alpha, ubiquitin and PGK promoters are ubiquitous and active in neural cells. Details on pWPI plasmid and on the RIX-PGK-Tom-W vector can be obtained at http://www.medecine.unige.ch/~salmon/.
Lentiviral vectors were produced, concentrated and titrated according to standard protocols. Details on procedures can be obtained at http://www.medecine.unige.ch/~salmon/. Control and FGF-2 lentiviral vectors had titers ranging from 108 to 109 transducing units (TU)/ml. Transduction of NPCs was done at DIV3 except at DIV2 for the BrdU deprivation study. NPCs (
50 000–75 000 cells per 35 mm culture dish) were transduced using doses ranging from 5 x 104 to 5 x 105 TU of either control or FGF-2 lentiviral vectors.
FGF-2 secretion in the medium
To measure the secretion of FGF-2 by NPCs, cultures were transduced at DIV3 with the GFP control lentiviral vector and the FGF-2 lentiviral vector (1.5 x 105 TU/ml). At DIV6, cultures were washed with fresh medium containing no FGF-2. At DIV8, the medium was removed and the amount of FGF-2 secreted in the medium was quantified using a standard ELISA detection method (Quantikine, R&D). Cells were trypsinized, counted and the percentage of GFP positive cells was analysed by FACS.
Tissue processing and immunohistochemistry
Cultures and cortical slices were fixed overnight at 4°C with cold 4% paraformaldehyde (PFA) (pH 7.4). Rats were anesthesized by pentobarbital and sacrificed by intra-cardial perfusion of 0.9% saline followed by 4% PFA (pH 7.4). Brains were extracted from the skull and post-fixed in 4% PFA (pH 7.4) at 4°C overnight and cryoprotected with sucrose 30% if cut on a cryostat. For histology processing, 20 µm thick sections were cut on a cryostat or 60 µm thick sections were cut on a Vibratome 1500; sections or slices were washed three times with 0.1 M (PBS); incubated overnight at 4°C with a primary antibody diluted in PBS/0.5% bovine serum albumine (BSA)/0.3% Triton X-100; washed in PBS; incubated with the secondary antibodies against the appropriate species; nuclear counterstained with 33 258 bisbenzimide (Invitrogen) or TO-PRO-3 (Invitrogen). The following primary antibodies were used: monoclonal mouse anti-FGF-2 (1:250; Upstate), monoclonal mouse anti-nestin (1:1000, Chemicon), polyclonal rabbit anti-NCAM (1:1000) (Zhang et al., 2003), polyclonal rabbit anti-NG2 (1:250; Chemicon), polyclonal rabbit anti-GFAP (1:500; Dakopatts), polyclonal goat anti-doublecortin (1:100; Santa Cruz), monoclonal mouse anti-GAD67 (1:1000; Chemicon), polyclonal rabbit anti-GFP (1:1000; Molecular Probes, Invitrogen), monoclonal mouse anti-NeuN (1:250; Chemicon), monoclonal mouse anti-BrdU (1:100; Boehringer-Mannheim), monoclonal rat anti-BrdU (1:100; Oxford Biotech. Ltd.), polyclonal rabbit anti-calretinin (1:1000; Swant, Switzerland), monoclonal mouse anti-calbindin (1:5000; Swant, Switzerland), goat anti-parvalbumin (1:5000; Swant, Switzerland), mouse anti-FGFR1(1:100; Upstate), rabbit anti-FGFR2 (SC-122) (1:100; Santa-Cruz). The following secondary antibodies were used: anti-rabbit Alexa-568 and Alexa-488, anti-mouse Alexa-488 and Alexa-568, anti-goat Alexa-555 and Alexa-647 (Invitrogen). For BrdU labelling, cultures and cortical slices were incubated 30 min in 2 N HCL for DNA denaturation followed by standard incubation.
BrdU incorporation experiments
For culture experiments, NPCs were expanded in FGF-2 (20 ng/ml) during two days. At DIV2, the medium was removed and replaced by (i) medium supplemented with FGF-2 (20 ng/ml) and containing control lentiviral vector (1.5 x 105 TU/ml) (ii) medium containing control lentiviral vector (1.5 x 105 TU/ml) without exogenous FGF-2 (iii) medium containing the FGF-2 lentiviral vector at two different doses (0.75 x 105 TU/ml and 1.5 x 105 TU/ml) without exogenous FGF-2. At DIV6, BrdU (10 µM) was added to the medium during 16 h before PFA fixation. For BrdU incorporation experiments on cortical slices, BrdU (10 mM) was added to the medium during 6 h before PFA fixation either at DIV1 or at DIV2 after NPC deposition. For in vivo experiments, intra-peritoneal BrdU (50 mg/kg) was injected twice daily from P7 to P9 or from P37 to P39.
Cortical slice preparation and in vitro NPC transplantation
The brains of P0 Sprague Dawley pups were dissected; 200 µm coronal sections were cut on a Vibratome in ice-cold Hanks medium and cultured on porous nitrocellulose filters (Millicell-CM); Details for cortical slice cultures and in vitro NPC transplantation can be found in the Supplementary Material. For time-lapse imaging slices were placed in a microscope chamber maintained at 37° and 5% CO2. For ex–in vivo slices, rats were sacrificed at P5 after in vivo transplantation at P3 and slices were cut as described above.
In vivo NPC transplantation
For in vivo transplantation in the intact cortex, Wistar pups at postnatal day 3 (P3) were anesthetized with a mixture of Isofluran (Foren® 100%), O2 30% and air 70%, and maintained in a stereotaxic frame. Hypoxia-ischaemia injury was performed at P3 as described previously (Sizonenko et al., 2003). Details on the number of animals and the survival time points can be found in the Supplementary Material.
Image processing
Epifluorescent time-lapse images were acquired with a digital camera (Retiga EX; Qimaging) linked to a fluorescent microscope (Eclipse TE2000-U; Nikon Corp.) equipped with Nikon Plan Fluor 4x/0.13, 10x/0.30 objectives and Nikon Plan Apo 60x A/1.40/oil objective. Time-lapse images were quantified with the Openlab software (version 3.1.2) and post hoc images were transferred to image J (NIH) software for quantification. Confocal images were acquired with a LSM 510 confocal microscope using a Plan-Neofluar 40x/1.3 oil objective. Unpaired t-test and 
2 test were performed using the software SigmaStat® 3.1. Details on quantification of cells can be obtained in the Supplementary Material.
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Effects of FGF-2 over-expression on NPCs in culture
To study the effects of FGF-2 over-expression in multipotential NPCs, SVZ cells were isolated from newborn rats and expanded as described previously (Zhang et al., 2003
). Cultures were transduced after 3 days in vitro (DIV3) with either the control green fluorescent protein (GFP) lentiviral vector or the FGF-2-GFP lentiviral vector. At DIV6, moderate FGF-2 immunoreactivity was detected in control NPCs whereas a much stronger signal was detected in FGF-2-transduced NPCs, showing that FGF-2 transduction can efficiently increase FGF-2 expression (Fig. 1A). Elisa quantification demonstrated that FGF-2 transduction increased the amount of FGF-2 into the culture media by 10-fold (Fig. 1B). Control and FGF-2-transduced NPCs expressed the FGF receptor 1 (FGFR1) and the FGF receptor 2 (FGFR2) at the mRNA level and protein level (Supplementary Fig. 1), demonstrating that NPCs could bind secreted FGF-2. Quantification of the mRNA levels using real-time PCR revealed that FGFR1 and FGFR2 mRNA levels were not significantly modified after FGF-2 transduction (Supplementary Fig. 1). To investigate if FGF-2 over-expression could modify proliferation, cultures were exposed to the S-phase marker bromodeoxyuridine (BrdU) at DIV6. Proliferation significantly decreased in cultures deprived of FGF-2, but could be restored after FGF-2 transduction in a dose-dependent manner (Fig. 1C and D). Taken together, these experiments demonstrate that the FGF-2 transduction of NPCs increases the secretion of FGF-2 and maintains the proliferation of NPCs in the absence of exogenous FGF-2.
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To study in vitro the effects of FGF-2 over-expression in NPCs undergoing differentiation, we applied a protocol that has been shown to induce neurogenesis (Zhang et al., 2003
). After 6 days of differentiation, we observed that FGF-2-transduced cells had a strikingly different morphology compared to control cells: the vast majority of FGF-2-transduced cells displayed elongated processes strongly immunoreactive for nestin and a cell body faintly GFAP positive, whereas a high proportion of control cells displayed a much larger cell body strongly positive for GFAP and variable levels of nestin immunoreactivity (Fig. 1E, F and H). Furthermore, a significantly higher proportion of control cells expressed the immature neuronal marker doublecortin compared to FGF-2-transduced cells, indicating that FGF-2 transduction maintained the cells in an undifferentiated phenotype (Fig. 1H). Only a small fraction of cells expressed the oligodendrocyte precursor marker NG2 in both conditions (Fig. 1H). FGF-2 immunohistochemistry revealed a robust expression of FGF-2 in the cytoplasm and nucleus of almost all FGF-2-transduced NPCs, whereas FGF-2 immunoreactivity was located only in a fraction of control NPCs (Fig. 1G and H). These experiments indicate that in vitro FGF-2 over-expression can efficiently maintain NPCs in an immature state and prevent them from undergoing differentiation.
FGF-2 over-expression promotes the proliferation and migration of transplanted NPCs on brain slices
To explore the behaviour of control and FGF-2-transduced NPCs in a complex environment such as brain tissue, we deposited small islets of NPCs on the surface of sub-acute cortical slices (Fig. 2A). Twenty-four hours after transplantation, FGF-2 transduction significantly increased the size of transplanted islets compared to controls (Fig. 2D). A combination of both proliferation and migration could explain this increase. To investigate these possibilities, BrdU pulse labelling was performed and revealed a significantly higher proliferation rate in FGF-2-transduced NPCs compared to control cells (Fig. 2B and E). Furthermore, live-imaging of transplanted cells revealed that FGF-2 transduction induced a significant shift in the migration velocity (distance traveled per hour) of FGF-2-transduced NPCs compared to controls (Fig. 2F).
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To explore whether this pool of FGF-2-transduced NPCs displayed enhanced invasive capacities, cortical slices were fixed at DIV2 and analysed with a confocal microscope (Fig. 3). Z-sectioning revealed a significant shift in the depth migration between the two populations indicating that FGF-2 transduction enables NPCs to invade more efficiently cortical slices. These results indicate that FGF-2 lentiviral transduction not only maintains a pool of immature proliferative NPCs but also promotes migratory properties and enhances the invasion of cortical tissue.
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Transplantation of FGF-2-transduced NPCs in vivo
To further assess the effects of FGF-2 over-expression, NPCs were transplanted in the cortex of postnatal day 3 (P3) pups. Two days later, animals were sacrificed and cortical slices were prepared. Comparison of images taken at DIV0 and DIV2 in both conditions revealed that FGF-2-transduced NPCs could efficiently disperse in the surrounding cortical tissue whereas the majority of control NPCs remained near the locus of transplantation (Fig. 4A). Time-lapse recordings done over a period of 12 h revealed that control cells displayed limited migratory capacities. (Fig. 4B, F1, Supplementary movie 1). In contrast, the majority of FGF-2-transduced NPCs displayed a robust migratory activity over the 12 h of time-lapse recordings. (Fig. 4C, F2, Supplementary movie 2). Single cell tracking analysis revealed that NPCs adopted a migratory strategy characterized by the dynamic extension and retraction of several processes allowing cells to switch from a bipolar morphology with a leading process exhibiting lamellipodia activity to a transient multipolar morphology (Fig. 4C, Supplementary movie 2). Time-lapse recordings at DIV1 revealed a significant shift in the migration velocity of control versus FGF-2-transduced NPCs (Fig. 4E). Furthermore, we observed that a small fraction (1.66%) of FGF-2-transduced cells divided en route after an initial phase of migration, whereas this phenomenon was less frequent (0.33%) in the control situation (Fig. 4C).
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To explore the long-term effects of FGF-2 over-expression on the dispersion of grafted NPCs, we transplanted control versus FGF-2-transduced NPCs in the cortex of rat pups and analysed their brains at several survival time points. Two weeks after transplantation, FGF-2-transduced cells were found at increasing distances from the center of the cortical transplantation site compared to control cells (Fig. 5A and B). To know if the local secretion of FGF-2 by FGF-2-transduced NPCs could be sufficient to increase the dispersion of co-transplanted control NPCs, we transplanted a mixture of the same amount of control tomato-labelled NPCs and FGF-2-GFP-transduced NPCs in the cortex of neonatal rats. Analysis of brains 8 days after transplantation revealed that a significant proportion of GFP-FGF-2-transduced cells had dispersed in the surrounding cortex, whereas co-transplanted tomato-labelled cells mainly remained at the locus of transplantation, indicating that FGF-2-transduction did not significantly modify the behaviour of co-transplanted control NPCs (Fig. 5C and D).
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To investigate whether the increased dispersion of FGF-2-transduced NPCs was linked to a change in their proliferation index, BrdU labelling was performed between P6 and P9 and animals were sacrificed at P10. Interestingly, at this time point a significantly higher proportion of FGF-2-transduced NPCs had incorporated BrdU compared to controls (Fig. 5E and G). To explore if the phenotype of transplanted NPCs was modified by FGF-2 transduction, nestin staining was performed 2 weeks after transplantation and revealed that a significant proportion of FGF-2-transduced cells remained nestin positive whereas very few control cells expressed this immature neural marker (Fig. 5F and G). In contrast, staining for NG2, a marker for oligodendrocyte precursors but also endothelial cells, revealed that the majority of control cells expressed NG2 compared to only a small amount of FGF-2-transduced cells. Staining for neuronal markers such as doublecortin and NeuN revealed that only a very limited number of control or FGF-2-transduced NPCs (<0.5%) differentiated into immature or mature neurons after transplantation in the normal cortex. These results indicate that FGF-2-transduction of NPCs increases their cortical invasiveness in vivo and that this phenomenon is associated with the preservation of an immature and proliferative state.
Constitutive FGF-2 over-expression in NPCs raises the concern of potential tumour formation. To evaluate the fate of FGF-2-transduced NPCs at longer survival time points, we sacrificed rats 40 days after transplantation in the intact cortex. At this time point, grafted FGF-2-transduced NPCs were still more dispersed than control cells and no tumour formation was detected. Immunohistochemistry quantification revealed that the majority of GFP positive cells had differentiated into NG2 positive cells (73.3 ± 3.4% mean ± SEM) or GFAP positive astroctyes (5.1 ± 1.0% mean ± SEM). Only a very small fraction of grafted NPCs maintained an immature nestin phenotype (0.7 ± 0.3% mean ± SEM). To confirm that grafted cells had lost their proliferative properties, BrdU injections were performed three days before sacrifice. Only 2.7 ± 0.8% (mean ± SEM) of FGF-2-transduced cells still incorporated BrdU.
To investigate if the transient proliferative and migratory properties of FGF-2-transduced cells could be correlated with a change in the levels of FGF-2 production, we tracked FGF-2 protein expression at different survival time points after transplantation. We observed that at the early survival time points during which FGF-2-transduced NPCs displayed proliferative and migratory properties, the majority of FGF-2-transduced NPCs were strongly immunoreactive for FGF-2, whereas at longer survival time points this percentage progressively decreased (Fig. 5I). Taken together, these results indicate that the proliferative and migratory properties of FGF-2-transduced NPCs as well as their immature phenotype is transient and that the loss of these properties is strongly correlated with a gradual down-regulation of FGF-2 production.
FGF-2 transduction increases the pool of grafted olfactory bulb neurons
To test the behaviour of FGF-2-transduced NPCs in a physiological neurogenic system, we transplanted tomato-labelled control NPCs and FGF-2-transduced NPCs in the P0 anterior subventricular zone (SVZ). Brains analysed at different time points after transplantation revealed that both FGF-2-transduced NPCs and control NPCs were found in the SVZ, along the rostral migratory stream and in the olfactory bulb where they had differentiated into immature neurons expressing doublecortin (Fig. 6A1,A2 and C). Six weeks after transplantation, numerous GFP positive neurons with well-developed processes could be observed in the olfactory bulb. These cells extended typical dendrites, expressed the more mature neuronal marker NeuN and displayed the characteristic morphology of granular interneurons (Fig. 6D). Quantification of the ratio of GFP versus tomato-labelled neurons at 4 weeks in the olfactory bulb revealed that neurons derived from FGF-2-transduced NPCs were 4 times more abundant than control neurons indicating that FGF-2 transduction could efficiently increase the amount of transplanted neurons in the olfactory bulb (Fig. 6B). To further confirm that the loss of an immature phenotype strongly correlates with a gradual down-regulation of the levels of FGF-2, we quantified the amount of GFP positive cells immunoreactive for FGF-2 in the rostral migratory stream and in the olfactory bulb 1 month after transplantation. We found that only a very small fraction of FGF-2-transduced neuroblasts in the rostral migratory stream (1.8 ± 0.6% mean ± SEM) and FGF-2-transduced neurons in the olfactory bulb (4.4 ± 0.7% mean ± SEM) continued to express FGF-2, indicating that the loss of an immature phenotype is strongly correlated with FGF-2 down-regulation.
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Transplantation of FGF-2-transduced NPCs after neonatal ischaemia
To study the behaviour of FGF-2-transduced NPCs in an ischaemic environment, we used an animal model of neonatal ischaemia. In this model, rats at postnatal day 3 undergo right carotid artery coagulation followed by 6% hypoxia for 30 min. This moderate hypoxic-ischaemic injury leads to selective neuronal loss in the infragranular layers of the somatosensory cortex (Sizonenko et al., 2005
). After grafting NPCs at the base of the cortex, close to the ischaemic sites (Fig. 7A), the migration pattern of labelled cells was determined in fixed tissues at different intervals. Two weeks after transplantation, we observed that both control and FGF-2-transduced NPCs, had survived, migrated out of the injection site and dispersed in a region that included the infragranular ischaemic cortex and the margin between the cortex and the corpus callosum (Fig. 7A). When comparing the two groups, we observed that FGF-2 transduction significantly increased the number of FGF-2-transduced cells located in the ischaemic regions compared to control cells. Quantification of the ratio of GFP versus tomato-labelled cells at 2 weeks in the ischaemic cortex revealed that FGF-2-transduced NPCs were 2.4 times more abundant than control NPCs indicating that FGF-2 transduction could efficiently increase the pool of grafted NPCs in the ischaemic cortex (Fig. 7I). At this survival time point, only 31.0 ± 4.4% (mean ± SEM) of FGF-2-transduced NPCs still expressed FGF-2, indicating that FGF-2 down-regulation also occurred in an ischaemic environment and was correlated with neural differentiation. The differentiation process was similar in both the control condition and after FGF-2 transduction (Fig. 7J). A large fraction of grafted cells remained nestin positive (Fig. 6D), while a proportion of them were found expressing the astrocytic marker GFAP or the oligodendrocytic marker NG2 (Fig. 6J). Most importantly 50% of grafted cells expressed the immature neuronal marker doublecortin (DCX) (Fig. 7B,C and J). Quantification revealed that the number of grafted immature doublecortin positive neurons was significantly increased after FGF-2 transduction (447.1 ± 96.6 (mean ± SEM) cells/mm2) compared to the control condition (135.3 ± 19.4 (mean ± SEM) cells/mm2) (P < 0.05, t-test), indicating that the ischaemic environment could promote neurogenesis, in contrast to the non-ischaemic cortex where very few neurons were observed. In the ischaemic lesion, grafted cells could differentiate into neurons expressing the -aminobutyric acid (GABA) synthesizing enzyme glutamic acid decarboxylase 67 (GAD67) (Fig. 7F) and a later survival time points the calcium-binding protein calretinin (Fig. 7G). No FGF-2-transduced cells were immunoreactive for calbindin or parvalbumin. Some FGF-2-transduced cells had acquired a more complex neuronal morphology and expressed the neuronal marker NeuN (Fig. 7H). These results indicate that an ischaemic environment can dramatically change the phenotypic fate of both control and FGF-2-transduced NPCs and that FGF-2 over-expression increases the pool of NPCs available for brain repair.
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The identification of molecular signals that could enhance the capacity of grafted NPCs to invade an injured area in the brain is of key importance for neural cell replacement and structural repair. Here we developed a novel system allowing the over-expression of FGF-2 in NPCs and studied its effects on several cellular functions after transplantation into the early postnatal rat cortex. We provide direct evidence for the first time that FGF-2 over-expression not only enhances the proliferative activity, as has been shown previously, but that it also dramatically enhances the migratory properties of grafted NPCs. When transplanted into a neurogenic region such as the SVZ, FGF-2-expressing NPCs gave rise to a significantly increased pool of new interneurons in the olfactory bulb, without altering their differentiation potential. A striking observation of the present study is that differentiation of progenitor cells into immature neurons was systematically correlated with a down-regulation of the FGF-2 transgene. Finally and most importantly, we show that after transplantation into a neonatal ischaemic cortex, FGF-2 over-expressing NPCs efficiently invade the injured cortex and generate an increased pool of immature neurons available for brain repair. These results reveal an important role for FGF-2 in regulating NPCs functions when interacting with the host tissue and offer a potential strategy to generate a robust source of migrating neural progenitors for repairing a neonatal ischaemic cortex.
Using a lentiviral gene transfer system, we successfully over-expressed FGF-2 in NPCs and showed that this technology could be a reliable and non-toxic tool to genetically engineer primary cultures of NPCs. FACS analysis allowed us to estimate the number of copies that transduced NPCs had incorporated in their genome. According to our calculations, using lentiviral vector doses of 1.5 x 105 TU/ml, 2–3 copies of the FGF-2 lentiviral vector were incorporated in the genome. With this range of copy number, FGF-2 transduction was able to produce a 10-fold increase in the amount of FGF-2 secreted in the culture medium. Our data suggest that in vitro this relatively low amount of secreted FGF-2 had a potent biological effect since it could maintain the proliferation of NPCs in culture at a level equivalent to that measured after the exogenous addition of FGF-2 at a dose of 20 ng/ml. Noteworthy that control NPCs secrete low but detectable amounts of FGF-2 into culture media hence confirming previous reports (Maric et al., 2003) and supporting the concept that autocrine/paracrine axes of FGF signalling might regulate biological properties of progenitor cells.
Previous studies demonstrated that FGF-2 signalling is a potent regulator of mammalian neurogenesis (Ghosh and Greenberg, 1995; Temple and Qian, 1995; Palmer et al., 1999; Ford-Perriss et al., 2001). FGF-2 appears to be a major determinant in maintaining proliferative and undifferentiated populations of NPCs in vitro (Maric et al., 2003) and in neurogenic zones such as the SVZ in vivo (Zheng et al., 2004). It has also been reported to be critical in the reprogramming of primordial germ cells into pluripotent stem cells (Durcova-Hills et al., 2006) and in maintaining pluripotency in human embryonic stem cells (Amit et al., 2000). However, its role in regulating dynamic interactions of grafted NPCs with the recipient host tissue was unknown. This is a critical issue since NPCs rapidly differentiate after grafting into postnatal brain tissue. Using a lentiviral-based approach, we demonstrated that FGF-2 over-expression in grafted NPCs was sufficient to maintain their immature phenotype as well as their proliferative and migratory properties in a tissue context. In striking contrast, grafted control cells rapidly lost their migratory and proliferative properties as well as their immature phenotype while differentiating into glial cells. Co-transplantation experiments with tomato-labelled control NPCs mixed with GFP-labelled FGF-2 over-expressing NPCs, revealed that the presence of FGF-2-transduced cells was not sufficient to confer increased migratory properties to control cells. Thus, cross-talk through paracrine signalling of FGF-2 between NPCs may not be sufficient to maintain an undifferentiated and migratory phenotype and we speculate that an autocrine signalling loop of FGF-2 might underlie the observed biological effects of FGF-2 transduction. The expression of FGF receptor 1 and FGF receptor 2 at the mRNA and protein level in FGF-2-transduced NPCs further support the hypothesis that over-secretion of FGF-2 in the extracellular compartment could maintain the biological properties of FGF-2-transduced NPCs by signalling through specific FGF-2 receptors. In addition to this mechanism, it remains possible that the secreted 18 kDa FGF-2 isoform after binding to its receptors could be internalized and reach the nucleus where it could regulate cellular processes in an intracrine fashion (Sorensen et al., 2006).
We systematically observed that the migratory and proliferative properties of FGF-2-transduced NPCs were transient and were lost at longer survival time points. We never detected tumour formation and the dispersion of grafted cells was limited to a few hundred micrometres from the injection site. Cell fusion events have been reported after transplantation of stem cells in various organs. However, several arguments indicate that in our system grafted NPCs do not appear to fuse to resident cells. Using time-lapse imaging, we were able to directly monitor the behaviour of GFP-labelled NPCs after engraftment in various brain regions. No cell fusion events were detected in time-lapse movies monitoring the migration of NPCs in the cortex, in the rostral migratory stream and in the olfactory bulb. Furthermore, no evidence for multinucleated cells was detected after confocal imaging of hundreds of grafted NPCs in various brain regions. Although we cannot totally exclude the possibility that rare cell fusion events could occur after transplantation, these rare events would not modify the results obtained in this study.
By tracking the level of FGF-2 expression in grafted cells at different survival time points and in different transplantation sites, we found that the loss of the proliferative and migratory properties of FGF-2 over-expressing NPCs was strongly correlated with a spontaneous and gradual down-regulation of FGF-2 production. Furthermore, we found that FGF-2 down-regulation was also strongly correlated with the appearance of grafted cells capable of differentiating into a wide range of glial and neuronal cells displaying normal morphologies and expressing standard markers of differentiation. Several mechanisms could account for the observed FGF-2 down-regulation, such as decreased transcription levels, increased mRNA instability, decreased mRNA translation and increased protein degradation. Interestingly, the majority of FGF-2-transduced NPCs grafted in the anterior SVZ continued to express FGF-2 1 month after transplantation (unpublished data), suggesting that in vivo FGF-2 down-regulation may occur in a region-dependent fashion.
The combination of confocal video time-lapse microscopy and cortical slice preparations allowed us to directly observe the migration of FGF-2-transduced NPCs in a 3D tissue context. Imaging of FGF-2-transduced NPCs in cortical slices revealed an important pool of individually migrating NPCs. This pool of migrating cells was significantly reduced but not absent in the control situation, indicating that FGF-2 transduction increases the fraction of migrating cells but does not induce a mode of migration which is absent in the control situation. These observations are consistent with previous reports showing that FGF signalling stimulates the migration of astrocytes (Holland and Varmus, 1998; Sorensen et al., 2006), myoblasts (Allen et al., 2003), oligodendrocyte progenitors (Simpson and Armstrong, 1999) and germ cells (Takeuchi et al., 2005). Interestingly mitosis could be directly observed online after a phase of migration in a small fraction of FGF-2-transduced NPCs and more rarely with control NPCs. These time-lapse observations indicate that immature NPCs are able to migrate and divide in a complex 3D structure. Post hoc confocal Z-stacks taken throughout the cortical slice demonstrated that FGF-2-transduced NPCs had the ability to migrate inside the cortical tissue, whereas control NPCs mainly remained on the surface of the slice, further demonstrating the enhanced invasive migratory properties of FGF-2-transduced NPCs. The molecular mechanisms of this effect remain to be determined. One of the possibilities is that FGF-2 facilitates migration through stimulating the secretion of matrix degrading enzymes such as MMP 2 and 9 (Tsuboi et al., 1990; Kenagy et al., 1997).
Overall our results indicate that the over-expression of FGF-2 in NPCs prior to transplantation could be of considerable interest to generate a larger pool of proliferative and migrating neural progenitor cells available for brain repair (creation of a ‘launch pad’ in situ). Furthermore, we show that FGF-2-transduced NPCs can be recruited towards sites of brain injury where they generate dense clusters of immature neurons. The addition of a large pool of immature neurons in a damaged neonatal cortex represents a promising strategy to achieve efficient neuronal replacement. Additional studies are needed to determine if this strategy could lead to functional improvements in transplanted rats. Furthermore, the behaviour of FGF-2-transduced NPCs in an adult ischaemic cortex remains to be determined. Finally, it remains a long-term goal to understand the molecular mechanisms that regulate the survival and integration of immature neuronal precursors in a damaged cortex.
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TopSummaryIntroductionMaterials and methodsResultsDiscussionSupplementary materialReferences |
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Supplementary material is available at Brain online.
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*These authors contributed equally to this work.
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We wish to thank S. Chliate and C. Saadi for technical assistance. This work was supported by the Swiss National Foundation grant 31-64030.00, the Eagle Foundation and by the European Community Grant Promemoria No. 512012-2005 to Jozsef Z. Kiss.
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