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Glaucoma  |   November 2014
Neural Stem Cell–Based Intraocular Administration of Ciliary Neurotrophic Factor Attenuates the Loss of Axotomized Ganglion Cells in Adult Mice
Author Affiliations & Notes
  • Kai Flachsbarth
    Department of Ophthalmology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
  • Katharina Kruszewski
    Department of Ophthalmology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
  • Gila Jung
    Department of Ophthalmology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
  • Wanda Jankowiak
    Department of Ophthalmology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
  • Kristoffer Riecken
    Research Department Cell and Gene Therapy, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
  • Lars Wagenfeld
    Department of Ophthalmology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
  • Gisbert Richard
    Department of Ophthalmology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
  • Boris Fehse
    Research Department Cell and Gene Therapy, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
  • Udo Bartsch
    Department of Ophthalmology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
  • Correspondence: Udo Bartsch, Department of Ophthalmology, University Medical Center Hamburg- Eppendorf, Martinistr. 52, 20246 Hamburg, Germany; ubartsch@uke.uni-hamburg.de
Investigative Ophthalmology & Visual Science November 2014, Vol.55, 7029-7039. doi:10.1167/iovs.14-15266
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      Kai Flachsbarth, Katharina Kruszewski, Gila Jung, Wanda Jankowiak, Kristoffer Riecken, Lars Wagenfeld, Gisbert Richard, Boris Fehse, Udo Bartsch; Neural Stem Cell–Based Intraocular Administration of Ciliary Neurotrophic Factor Attenuates the Loss of Axotomized Ganglion Cells in Adult Mice. Invest. Ophthalmol. Vis. Sci. 2014;55(11):7029-7039. doi: 10.1167/iovs.14-15266.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: To analyze the neuroprotective effect of intravitreally grafted neural stem (NS) cells genetically modified to secrete ciliary neurotrophic factor (CNTF) on intraorbitally lesioned retinal ganglion cells (RGCs) in adult mice.

Methods.: Adherently cultivated NS cells were genetically modified to express a secretable variant of mouse CNTF together with the fluorescent reporter protein Venus. Clonal CNTF-secreting NS cell lines were established using fluorescence activated cell sorting, and intravitreally grafted into adult mice 1 day after an intraorbital crush of the optic nerve. Brn-3a-positive RGCs were counted in flat-mounted retinas at different postlesion intervals to evaluate the neuroprotective effect of the CNTF-secreting NS cells on the axotomized RGCs. Anterograde axonal tracing experiments were performed to analyze the regrowth of the injured RGC axons in CNTF-treated retinas.

Results.: Intravitreally grafted NS cells preferentially differentiated into astrocytes that survived in the host eyes, stably expressed CNTF, and significantly attenuated the loss of the axotomized RGCs over a period of at least 4 months, the latest postlesion time point analyzed. Depending on the postlesion interval analyzed, the number of RGCs in eyes with grafted CNTF-secreting NS cells was 2.8-fold to 6.4-fold higher than in eyes with grafted control NS cells. The CNTF-secreting NS cells additionally induced long-distance regrowth of the lesioned RGC axons.

Conclusions.: Genetically modified clonal NS cell lines may serve as a useful tool for preclinical studies aimed at evaluating the therapeutic potential of a sustained cell-based intravitreal administration of neuroprotective factors in mouse models of glaucoma.

Introduction
Glaucoma is among the leading causes of blindness, with an estimated 80 million people being affected worldwide in 2020.1 It is a complex neurodegenerative disorder of the retina that is characterized by a progressive loss of retinal ganglion cells (RGCs) and their axons, resulting in visual field loss and eventually irreversible blindness.2,3 The pathomechanisms leading to the apoptotic death of RGCs in this age-related and multifactorial disease are not fully understood. Clinically, an increased IOP is known to be a major risk factor for the development of glaucomatous optic neuropathy. Other factors that have been implicated in causing progressive degeneration of RGCs include neurotrophic factor deprivation, mitochondrial dysfunction, excitotoxic damage, vascular dysfunction, oxidative stress, inflammation, and glial cell activation.25 Currently, lowering IOP is the only proven treatment for glaucoma. However, in a significant proportion of glaucoma patients, the disease progresses despite successful IOP reduction,6,7 indicating the need for alternative treatments. 
It has been proposed that impaired axonal transport as a result of elevated IOP leads to a scarcity of target-derived neurotrophic factors and subsequent apoptotic degeneration of RGCs.8,9 Stimulation of prosurvival signaling pathways by the supplementation of neurotrophic factors has therefore been extensively explored as a strategy to protect RGCs from degeneration. These studies have identified a number of neurotrophic factors that are capable of delaying the degeneration of RGCs in various animal models of RGC loss.2,1013 For instance, intraocular administration of brain-derived neurotrophic factor (BDNF) or glial cell line–derived neurotrophic factor has been demonstrated to significantly delay the degeneration of RGCs in animal models of optic nerve injury1420 or ocular hypertension.2124 
Ciliary neurotrophic factor (CNTF), a member of the IL-6 family of cytokines,25 is another neurotrophic factor that has been shown to potently rescue RGCs in various pathological conditions.19,2633 In addition to protecting RGCs from degeneration, the cytokine has been demonstrated to promote long-distance regrowth of injured RGC axons in the adult mammalian optic nerve.26,3438 However, single intravitreal injections of recombinant CNTF resulted in only limited neuroprotective and axon growth–promoting effects, due to the short half-life of the protein.39 In comparison, more robust long-term effects were observed after sustained intraocular administration of the cytokine, which, in most studies, was achieved by virus-mediated gene transfer to the retina.2,10,13 
Intraocular transplantations of genetically modified cells represent another strategy to continuously deliver neuroprotective factors to the retina.40,41 Importantly, the use of ex vivo modified cells offers the possibility to adjust the amount of neurotrophic factors administered to the retina before the transplantation. Furthermore, in terms of potential clinical applications, the modified cells can be encapsulated into semipermeable polymer devices that allow diffusion of the neurotrophic factors from the implant to the retina, while protecting the transplanted cells from the immune system of the host and the host retina from potential adverse effects of the grafted cells. Moreover, the encapsulated cell implants can be retrieved from the vitreous in case of complications, adding another important safety aspect to this approach.41-43 In fact, the therapeutic potential of a cell-based intraocular administration of a neurotrophic factor is currently being evaluated in patients with RP or geographic atrophy, using intravitreal implants of an encapsulated RPE cell line genetically modified to secrete CNTF.4448 
In the present study, we used a polycistronic lentiviral vector to generate clonal neural stem cell lines stably expressing a secretable variant of CNTF. The modified cells were grafted into the vitreous cavity of adult wild-type mice 1 day after an intraorbital optic nerve crush to evaluate the effects of a sustained cell-based intraocular administration of the cytokine on axonal regrowth and RGC survival in this animal model of injury-induced RGC loss. 
Materials and Methods
Animals
Neural stem cells were isolated from the cerebral cortex of 14-day-old C57BL/6J mouse embryos. Intraorbital optic nerve lesions and intravitreal neural stem (NS) cell transplantations were performed on adult (i.e., at least 2 months old) C57BL/6J mice. Animals were obtained from the animal facility of the University Medical Center Hamburg-Eppendorf (Hamburg, Germany). All animal experiments were approved by the University and State of Hamburg Animal Care Committees and were in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Lentiviral Vectors, NS Cell Transductions, and Generation of Modified Clonal NS Cell Lines
The cDNA of mouse CNTF was ligated in frame with the Ig κ-chain leader sequence of pSecTag2 B (Life Technologies, Darmstadt, Germany) and cloned into the polycistronic lentiviral vector pCAG-IRES-Venus-2A-ZEO encoding the internal ribosome entry site from the encephalomyocarditis virus, a Venus reporter gene, a P2A sequence of porcine teschovirus-1, and a zeocin resistance gene under regulatory control of the cytomegalovirus enhancer/chicken β-actin promoter, giving rise to pCAG-CNTF-IRES-Venus-2A-ZEO.49 Lentiviral particles were pseudotyped with the envelope G protein of the vesicular stomatitis virus and produced by transient transfection of HEK 293T cells as described elsewhere (http://www.LentiGo-Vectors.de).50 
To generate clonal NS cell lines with high expression levels of transgenes, we again transduced previously established CNTF-secreting NS cell lines (CNTF-NS cells) and NS cell lines for control experiments (control-NS cells)49 with pCAG-CNTF-IRES-Venus-2A-ZEO and pCAG-IRES-Venus-2A-ZEO, respectively. In brief, NS cells were seeded into 24-well plates coated with 0.1% Matrigel (BD Bioscience, Heidelberg, Germany) and cultivated in Dulbecco's modified Eagle's medium/F12 (Life Technologies) supplemented with 2 mM glutamine, 5 mM HEPES, 3 mM sodium bicarbonate, 0.3% glucose (all from Sigma-Aldrich Corp., St. Louis, MO, USA; in the following termed “NS cell medium”), 10 ng/mL epidermal growth factor (EGF) and 10 ng/mL FGF-2 (both from TEBU, Offenbach, Germany), and 1% N2 and 1% B27 (both from Life Technologies). Transduction of cells was performed by spinoculation in the presence of 8 μg/mL hexadimethrine bromide (Polybrene; Sigma-Aldrich). Positive cells were selected by further cultivating the cells under adherent conditions4951 in culture flasks coated with poly-L-ornithine (Sigma-Aldrich) and 0.1% Matrigel in the same medium but additionally containing 200 μg/mL zeocin (Invivogen, San Diego, CA, USA). Single CNTF-NS cells with the highest expression level of the reporter gene were then sorted into 96-well plates by fluorescence-activated cell sorting (FACS; FACSAriaIIIu, BD Bioscience, San Diego, CA, USA) to establish clonal CNTF-NS cell lines with elevated expression levels of the cytokine. CNTF levels in culture supernatants of the CNTF-NS cell clones were compared by Western blot analysis using polyclonal rabbit anti-CNTF antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and horseradish peroxidase–conjugated anti-rabbit secondary antibodies (Jackson Immunoresearch Laboratories, West Grove, PA, USA). The clonal cell lines with the highest expression levels of the cytokine were selected, and CNTF levels in the culture supernatants were estimated by Western blot analyses using recombinant mouse CNTF (Biomol, Hamburg, Germany) as a reference. Densitometric analysis of immunoreactive bands was performed using ImageJ software (http://imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA). 
In Vitro Differentiation of NS Cells and Immunocytochemistry
Differentiation of NS cells into astrocytes was induced by cultivating the cells in NS cell medium containing 1% fetal calf serum (Life Technologies) and 2% B27. Astrocytes were maintained for up to 2 months in culture. Neuronal differentiation of NS cells was induced by maintaining the cells for 3 days in NS cell medium supplemented with 5 ng/mL FGF-2, 1% N2, and 2% B27, followed by an additional cultivation period of 4 days in a 1:1 mixture of NS cell medium and Neurobasal medium (Life Technologies) supplemented with 0.25% N2 and 2% B27. 
For immunocytochemical analyses of CNTF expression, CNTF-NS and control-NS cell cultures were fixed in 4% paraformaldehyde (PA; Carl Roth GmbH, Karlsruhe, Germany) in PBS (pH 7.4), blocked in PBS containing 0.1% BSA and 0.3% Triton X-100 (both from Sigma-Aldrich), and incubated with polyclonal rabbit anti-CNTF antibodies (Santa Cruz Biotechnology, Inc.). Cultures were simultaneously incubated with monoclonal mouse anti-glial fibrillary acidic protein (GFAP) antibodies (Sigma-Aldrich) or monoclonal mouse anti-microtubule associated protein 2 (MAP2) antibodies (Sigma-Aldrich) to identify astrocytes or neurons, respectively. Primary antibodies were detected with anti-rabbit Cy3- and anti-mouse Cy5-conjugated secondary antibodies (Jackson Immunoresearch Laboratories). Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). 
Intraorbital Optic Nerve Lesions and Intravitreal NS Cell Transplantations
Animals were deeply anesthetized by an intraperitoneal injection of Ketanest S (Parke Davis GmbH, Berlin, Germany) and Rompun (Bayer Vital GmbH, Leverkusen, Germany), and the optic nerve was intraorbitally crushed with watchmaker's forceps for 15 seconds at a distance of 0.5 to 1.0 mm from the eye.52,53 Loss of the pupillary light reflex, the presence of well-preserved blood vessels, and lack of retinal bleeding were considered as criteria for a successful nerve crush. In a fraction of animals, RGC axons were anterogradely labeled (see below) to further control the nerve crush. One day after the crush, animals were again deeply anesthetized, and 2 μL vitreous fluid were removed from the eye with a fine glass micropipette that was inserted into the vitreous at the junction between sclera and cornea. Subsequently, 2 μL PBS containing either 7.6 × 105 CNTF-NS cells or control-NS cells were slowly injected into the vitreous cavity.49,54 Some animals received intravitreal injections of 2 μL PBS without cells. Particular care was taken not to damage the lens during the removal of the vitreous fluid or the injection of the cells or the vehicle solution. 
Characterization of Intravitreally Grafted NS Cells
Animals were killed 4 months after transplantation, and eyes were immersion-fixed for 1 hour in PBS containing 4% PA. Lenses with attached donor cells were removed and incubated with rabbit anti-CNTF antibodies, mouse anti-GFAP antibodies, rabbit anti-ß-tubulin III antibodies (Sigma-Aldrich), or rat anti-myelin basic protein (MBP) antibodies (Millipore, Bedford, MA, USA) to evaluate the expression of CNTF and to analyze the differentiation of the grafted NS cells. Primary antibodies were detected with anti-rabbit Cy3-, anti-rat Cy3-, or anti-mouse Cy5-conjugated secondary antibodies (Jackson Immunoresearch Laboratories). Lenses with attached donor cells were stained with DAPI, and confocal z-stacks of the posterior poles of the lenses with attached donor cells were prepared with an Olympus FV 1000 confocal microscope (Olympus, Hamburg, Germany) and processed using FV10-ASW software (Olympus). Confocal z-stacks of flat-mounted retinas that were prepared 4 months after cell transplantation and stained with anti-Brn-3a antibodies (see below) were taken with a Leica TCS SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany), and processed using Imaris software (Bitplane, Zürich, Switzerland). Some eyes with grafted CNTF- or control-NS cells were immersion-fixed in 4% PA, cryoprotected in an ascending series of sucrose, frozen, serially sectioned at a thickness of 25 μm, and stained with DAPI. 
Anterograde Axonal Tracing
To anterogradely label RGC axons, a saturated solution of biotin-N-hydroxysuccinimidester (Sigma-Aldrich) in dimethylformamide (Carl Roth GmbH) was diluted 1:1 with ethanol, and intravitreally injected into the eyes with crushed optic nerves.53,55 After 24 hours, animals were killed and eyes with attached optic nerves were immersion-fixed in 4% PA, cryoprotected, and frozen. Longitudinal sections of optic nerves, 25 μm in thickness, were prepared with a cryostat and incubated with Cy3-conjugated streptavidin (Jackson Immunoresearch Laboratories). The distance between the distal margin of the lesion site and the tip of the longest regrown axon in mice with grafted control-NS or CNTF-NS cells (n = 6 for each experimental group) was determined 1 month after the crush. Analysis of sections was done with an Olympus IX51 fluorescence microscope (Olympus). 
Analysis of RGC Survival
Animals were killed 1, 2, 3, or 4 months after the optic nerve crush, and eyes were fixed for 15 minutes in 4% PA. Retinas were flat-mounted on nitrocellulose membranes (Sartorius AG, Göttingen, Germany), fixed again in 4% PA for 1 hour, blocked in PBS containing 0.1% BSA and 1% Triton X-100, and incubated with polyclonal goat anti-Brn-3a antibodies (Santa Cruz Biotechnology, Inc.) overnight at room temperature. Subsequently, retinas were incubated with Cy3-conjugated secondary antibodies, stained with DAPI, and mounted onto slides. Retinas were number-coded, and five photomicrographs from the center to the periphery of the superior, inferior, nasal, and temporal retinal quadrant were taken, covering a total retinal area of approximately 1.9 mm2. All Brn-3a–positive RGCs visible on these 20 photomicrographs were counted using Adobe Photoshop CS3 software (Adobe Systems, Inc., San Jose, CA, USA), and the number of RGCs per mm2 was calculated. Six eyes with grafted CNTF-NS cells or control-NS cells were analyzed for each postlesion interval (i.e., 1, 2, 3, and 4 months after the optic nerve crush). Retinal ganglion cell densities were additionally determined in eyes with intravitreally injected PBS 1 month after the crush (n = 6), and in normal untreated eyes (n = 6). Statistical analysis of data was performed using the Student's t-test. 
Results
Neural Stem Cells and Lentiviral Vectors
To express CNTF in adherently cultivated NS cells51,56 from the embryonic mouse brain (in the following termed “CNTF-NS cells”), we generated a polycistronic lentiviral vector that is based on the lentiviral “gene ontology” (LeGO) vectors.50,57 The vector encoded a secretable variant of mouse CNTF together with a Venus reporter gene and a zeocin resistance gene under regulatory control of the strong and ubiquitously active cytomegalovirus enhancer/chicken β-actin CAG promoter (pCAG-CNTF-IRES-Venus-2A-ZEO; Fig. 1Aa) to ensure robust transgene expression in undifferentiated NS cells and their differentiated progeny. Neural stem cells for control experiments (in the following termed “control-NS cells”) were transduced with the same vector but lacking the CNTF cDNA (pCAG-IRES-Venus-2A-ZEO; Fig. 1Ab).49 
Figure 1
 
Lentiviral vectors and expression of CNTF and Venus in genetically modified clonal NS cell lines. (A) Neural stem cells were transduced with a polycistronic lentiviral vector encoding a secretable variant of mouse CNTF under regulatory control of the human CMV enhancer/chicken β-actin (CAG) promoter. The vector additionally encoded a Venus reporter gene and a zeocin (ZEO) resistance gene separated from each other by a P2A sequence (2A) of porcine teschovirus-1 (Aa). The same vector but lacking the CNTF cDNA was used to transduce NS cells for control experiments (Ab). (B) Clonally derived CNTF-NS (Ba, Bb) and control-NS cell lines (Bc, Bd) were generated using FACS and immunostained with anti-CNTF antibodies. Whereas all cells in the CNTF-NS (Ba) and control-NS cell lines (Bc) expressed Venus, expression of CNTF was detectable only in the CNTF-NS cell clone (Bb; some cells labeled with arrowheads in Ba and Bb), but not in the control-NS cell clone (Bd). (C) Western blot analyses revealed secretion of CNTF into the supernatant of CNTF-NS cell cultures (CNTF-NS), whereas culture supernatants from control-NS cells (control-NS) lacked detectable levels of the cytokine. Ψ, packaging signal; cPPT, central polypurine tract; IRES, internal ribosome entry site; LoxP, recognition site of Cre recombinase; rmCNTF, recombinant mouse ciliary neurotrophic factor; RRE, rev-responsive element; SIN-LTR, self-inactivating long-terminal repeat; wPRE, Woodchuck hepatitis virus posttranscriptional regulatory element. Scale bar in (Bd) for (BaBd): 25 μm.
Figure 1
 
Lentiviral vectors and expression of CNTF and Venus in genetically modified clonal NS cell lines. (A) Neural stem cells were transduced with a polycistronic lentiviral vector encoding a secretable variant of mouse CNTF under regulatory control of the human CMV enhancer/chicken β-actin (CAG) promoter. The vector additionally encoded a Venus reporter gene and a zeocin (ZEO) resistance gene separated from each other by a P2A sequence (2A) of porcine teschovirus-1 (Aa). The same vector but lacking the CNTF cDNA was used to transduce NS cells for control experiments (Ab). (B) Clonally derived CNTF-NS (Ba, Bb) and control-NS cell lines (Bc, Bd) were generated using FACS and immunostained with anti-CNTF antibodies. Whereas all cells in the CNTF-NS (Ba) and control-NS cell lines (Bc) expressed Venus, expression of CNTF was detectable only in the CNTF-NS cell clone (Bb; some cells labeled with arrowheads in Ba and Bb), but not in the control-NS cell clone (Bd). (C) Western blot analyses revealed secretion of CNTF into the supernatant of CNTF-NS cell cultures (CNTF-NS), whereas culture supernatants from control-NS cells (control-NS) lacked detectable levels of the cytokine. Ψ, packaging signal; cPPT, central polypurine tract; IRES, internal ribosome entry site; LoxP, recognition site of Cre recombinase; rmCNTF, recombinant mouse ciliary neurotrophic factor; RRE, rev-responsive element; SIN-LTR, self-inactivating long-terminal repeat; wPRE, Woodchuck hepatitis virus posttranscriptional regulatory element. Scale bar in (Bd) for (BaBd): 25 μm.
Generation of Clonal NS Cell Lines With High Expression Levels of Transgenes
To further increase expression levels of transgenes in previously established clonal CNTF-NS and control-NS cell lines,49 cells were again transduced with pCAG-CNTF-IRES-Venus-2A-ZEO and pCAG-IRES-Venus-2A-ZEO, respectively. Expansion of the transduced NS cells in the presence of zeocin gave rise to CNTF-NS and control-NS cell bulk cultures that were exclusively composed of Venus-positive cells. Single cells with the highest expression level of Venus in these bulk cultures were isolated using FACS and clonally expanded. After four rounds of transductions and clonal expansions, cell lines were analyzed by fluorescence microscopy, immunocytochemistry, and Western blot. Analyses confirmed elevated expression levels of CNTF and Venus or the reporter gene in the CNTF-NS or control-NS cell lines, respectively, when compared with the original clonal cell lines. The clones with the highest expression levels of transgenes were selected for further experiments. Immunocytochemical analyses revealed that all cells in the CNTF-NS cell line coexpressed Venus and CNTF (Figs. 1Ba, 1Bb). Control-NS cells were also Venus-positive, but lacked detectable levels of CNTF immunoreactivity (Figs. 1Bc, 1Bd). Immunoblot analyses demonstrated secretion of CNTF into the culture supernatant of the CNTF-NS cell lines, whereas supernatants of control-NS cell clones lacked detectable levels of the cytokine (Fig. 1C). Expression of CNTF in the clonal CNTF-NS cell line was detectable by immunocytochemical and immunoblot analyses for more than 30 passages, corresponding to a culture period of approximately 6 months. Furthermore, immunoblot analyses (n = 3) of culture supernatants from this cell line revealed secretion of 87.2 ± 10.1 ng (mean ±SEM) CNTF per 105 cells in 24 hours at passage 15. Culture supernatants from passage 34 contained similar quantities of CNTF, indicating stable expression of the cytokine in this clonal cell line. 
To analyze expression of CNTF in differentiated neural cell types in vitro, we next differentiated the CNTF-NS and control-NS cell lines into neurons or astrocytes (Fig. 2). Whereas all MAP2-positive neurons derived from CNTF-NS and control-NS cells were positive for Venus, expression of CNTF was detectable only in neurons derived from CNTF-NS cells, but not in neurons derived from control-NS cells (Figs. 2A–F). Similarly, GFAP-positive astrocytes from both CNTF-NS and control-NS cells were positive for Venus, whereas expression of CNTF was detectable only in astrocytes derived from CNTF-NS cells, but not in astrocytes derived from control-NS cells (Figs. 2G–L). Of note, expression of CNTF in cultured astrocytes derived from the CNTF-NS cell line remained detectable for at least 2 months (Fig. 2I), the longest cultivation period analyzed. 
Figure 2
 
Expression of CNTF and Venus in differentiated clonal NS cell lines. CNTF-NS (ac, gi) and control-NS cell clones (df, j–l) were differentiated into MAP2-positive neurons (af) or GFAP-positive astrocytes (gl). The reporter gene Venus was expressed in all neurons (a, d) and astrocytes (g, j) derived from the CNTF-NS (a, g) and control-NS cell lines (d, j). CNTF was detectable only in neurons (c) and astrocytes (i) derived from CNTF-NS cells, but not in neurons (f) and astrocytes (l) derived from control-NS cells. Neurons and astrocytes were analyzed for expression of Venus and CNTF 1 week and 2 months after induction of differentiation, respectively. Scale bar in (l) (for al): 50 μm.
Figure 2
 
Expression of CNTF and Venus in differentiated clonal NS cell lines. CNTF-NS (ac, gi) and control-NS cell clones (df, j–l) were differentiated into MAP2-positive neurons (af) or GFAP-positive astrocytes (gl). The reporter gene Venus was expressed in all neurons (a, d) and astrocytes (g, j) derived from the CNTF-NS (a, g) and control-NS cell lines (d, j). CNTF was detectable only in neurons (c) and astrocytes (i) derived from CNTF-NS cells, but not in neurons (f) and astrocytes (l) derived from control-NS cells. Neurons and astrocytes were analyzed for expression of Venus and CNTF 1 week and 2 months after induction of differentiation, respectively. Scale bar in (l) (for al): 50 μm.
Intravitreal Transplantations of CNTF-NS and Control-NS Cells
Intravitreally grafted CNTF-NS (Figs. 3A–D; Supplementary Fig. S1) and control-NS cells (Figs. 3E–H) were identified in the host eyes by their expression of the reporter gene Venus (Figs. 3A, 3E; Supplementary Figs. S1A–S1E). Of note, both cell populations survived in the vitreous cavity of the recipient eyes for at least 4 months, the longest posttransplantation interval investigated. Both, CNTF-NS and control-NS cells had formed layers of Venus-positive cells that were either attached to the posterior pole of the lenses (Fig. 3; Supplementary Fig. S1A) or to the vitreal surface of the retinas (Supplementary Figs. S1B–E). Evidence for the formation of tumors by the grafted cells, or for integration of Venus-positive donor cells into the host retinas (Supplementary Figs. S1B, S1D, S1E) was not observed. However, we found some small-sized retinal folds in locally restricted regions of the flat-mounted host retinas. These retinal folds were observed in a fraction of animals with grafted CNTF-NS cells, but not in animals with grafted control-NS cells. 
Figure 3
 
Neural differentiation and expression of CNTF in intravitreally grafted NS cell lines. Analyses of eyes 4 months after intravitreal transplantations of CNTF-NS (ad) and control-NS cell lines (eh) revealed the presence of Venus-positive donor cells (a, e) that were attached to the posterior poles of the lenses. Virtually all CNTF-NS cells (b) and control-NS cells (f) were differentiated into GFAP-positive astrocytes. Expression of CNTF was detectable in cells derived from CNTF-NS cells (c), but not in cells derived from control-NS cells (g). (d) and (h) are merged images of (a) and (c), and (e) and (g), respectively. Some donor cells coexpressing Venus and CNTF are marked with white arrows in (a), (c) and (d). Scale bar in (h) (for ah): 100 μm.
Figure 3
 
Neural differentiation and expression of CNTF in intravitreally grafted NS cell lines. Analyses of eyes 4 months after intravitreal transplantations of CNTF-NS (ad) and control-NS cell lines (eh) revealed the presence of Venus-positive donor cells (a, e) that were attached to the posterior poles of the lenses. Virtually all CNTF-NS cells (b) and control-NS cells (f) were differentiated into GFAP-positive astrocytes. Expression of CNTF was detectable in cells derived from CNTF-NS cells (c), but not in cells derived from control-NS cells (g). (d) and (h) are merged images of (a) and (c), and (e) and (g), respectively. Some donor cells coexpressing Venus and CNTF are marked with white arrows in (a), (c) and (d). Scale bar in (h) (for ah): 100 μm.
Immunostainings of donor cells that were attached to the posterior poles of the lenses revealed that most of the CNTF-NS and control-NS cells were differentiated into GFAP-positive astrocytes (Figs. 3B, 3F). A few CNTF-NS and control-NS cells were differentiated into ß tubulin-III–positive nerve cells (data not shown). Differentiation of grafted cells into MBP-positive oligodendrocytes was not observed. Importantly, robust expression of CNTF was detectable in astrocytes derived from the grafted CNTF-NS cell clone for at least 4 months after transplantation, the latest posttransplantation time point investigated (Figs. 3C, 3D). Astrocytes derived from the control-NS cell clone, in contrast, lacked detectable expression of the cytokine (Figs. 3G, 3H). 
Intravitreally Grafted CNTF-NS Cells Attenuate the Degeneration of Axotomized RGCs
The numbers of RGCs in untreated animals and animals that had received an intraorbital crush of the optic nerve were determined in flat-mounted retinas that were stained with antibodies to Brn-3a, a reliable marker for RGCs.58 Quantitative analysis of retinas from untreated animals (n = 6) revealed the presence of 3993.8 ± 54.4 (mean ± SEM) RGCs/mm2. In animals that received an intraorbital crush of the optic nerve and intravitreal transplantation of control-NS cells, RGC numbers decreased to 189.8 ± 13.5 RGCs/mm2, 97.0 ± 12.8 RGCs/mm2, 50.2 ± 4.2 RGCs/mm2, and 62.7 ± 5.0 RGCs/mm2 at 1, 2, 3, and 4 months after the lesion, respectively (n = 6 for each postlesion interval; Figs. 4E–H, Fig. 5). In animals with intravitreally grafted CNTF-NS cells, in comparison, we detected 529.0 ± 20.9 RGCs/mm2 1 month after the lesion, 429.8 ± 30.7 RGCs/mm2 2 months after the lesion, 320.5 ± 11.5 RGCs/mm2 3 months after the lesion, and 302.7 ± 4.3 RGCs/mm2 4 months after the lesion (n = 6 for each postlesion interval; Figs. 4A–D, Fig. 5). CNTF-treated retinas thus contained 2.8-, 4.4-, 6.4-, and 4.8-fold more surviving RGCs than control retinas at the 1-, 2-, 3-, and 4-month postlesion interval, respectively. This difference between RGC numbers in CNTF-treated and control retinas was statistically significant at all postlesion time points analyzed (P < 0.001 according to the Student's t-test; Fig. 5). Anterograde axonal tracing experiments performed in a fraction of animals from the different experimental groups at the different postlesion intervals confirmed complete transections of RGC axons in all lesioned nerves analyzed. 
Figure 4
 
Intravitreally grafted CNTF-NS cells attenuate degeneration of axotomized retinal ganglion cells in adult mice. Adult mice received intravitreal injections of a CNTF-NS cell clone (ad) or a control-NS cell clone (eh) 1 day after an intraorbital optic nerve crush. Analysis of flat-mounted retinas 1 (a, e), 2 (b, f), 3 (c, g), and 4 months (d, h) after the lesion revealed the presence of significantly more Brn-3a–positive ganglion cells in eyes with grafted CNTF-NS cells (ad) than in eyes with grafted control-NS cells (eh) at all postlesion intervals. Scale bar in (h) (for ah): 100 μm.
Figure 4
 
Intravitreally grafted CNTF-NS cells attenuate degeneration of axotomized retinal ganglion cells in adult mice. Adult mice received intravitreal injections of a CNTF-NS cell clone (ad) or a control-NS cell clone (eh) 1 day after an intraorbital optic nerve crush. Analysis of flat-mounted retinas 1 (a, e), 2 (b, f), 3 (c, g), and 4 months (d, h) after the lesion revealed the presence of significantly more Brn-3a–positive ganglion cells in eyes with grafted CNTF-NS cells (ad) than in eyes with grafted control-NS cells (eh) at all postlesion intervals. Scale bar in (h) (for ah): 100 μm.
Figure 5
 
Quantitative analysis of the neuroprotective effect of intravitreally grafted CNTF-NS cells on axotomized RGCs. The number of Brn-3a–positive RGCs was determined in eyes with intravitreally grafted CNTF-NS cells (filled bars) or control-NS cells (open bars) 1, 2, 3, and 4 months after an intraorbital optic nerve crush. Note that the CNTF-treated retinas contained significantly more RGCs than the control retinas at all postlesion intervals. Each bar represents the mean number (±SEM) of RGCs per mm2 from six retinas. ***P < 0.001 according to the Student's t-test.
Figure 5
 
Quantitative analysis of the neuroprotective effect of intravitreally grafted CNTF-NS cells on axotomized RGCs. The number of Brn-3a–positive RGCs was determined in eyes with intravitreally grafted CNTF-NS cells (filled bars) or control-NS cells (open bars) 1, 2, 3, and 4 months after an intraorbital optic nerve crush. Note that the CNTF-treated retinas contained significantly more RGCs than the control retinas at all postlesion intervals. Each bar represents the mean number (±SEM) of RGCs per mm2 from six retinas. ***P < 0.001 according to the Student's t-test.
To evaluate whether control-NS cells also exerted neuroprotective effects on axotomized RGCs, we additionally compared the number of RGCs between eyes that had received intravitreal grafts of control-NS cells and eyes that had received intravitreal injections of the vehicle solution only. One month after an optic nerve crush, eyes with grafted control-NS cells contained 189.8 ± 13.5 RGCs/mm2, a value not significantly different from that obtained for retinas that received injections of the vehicle (156.2 ± 23.0 RGCs/mm2; n = 6 for each experimental group). 
Regrowth of Axotomized RGC Axons in CNTF-Treated Retinas
Anterograde axonal tracing experiments were performed to analyze whether the sustained cell-based intraocular administration of CNTF had stimulated regrowth of the injured RGC axons. To estimate the extent of axonal regrowth, we determined the distance between the distal margin of the lesion site and the tip of the longest regrown axon in longitudinally sectioned optic nerves from animals with grafted control-NS cells and grafted CNTF-NS cells (n = 6 for each experimental group) 1 month after an intraorbital nerve crush (Fig. 6). RGC axons in control animals extended for only short distances (459.3 ± 47.0 μm (mean ± SEM); Figs. 6A, 6B, 6E) across the lesion site into the distal optic nerve stump. In CNTF-treated animals, in comparison, axotomized RGC axons regrew for up to 2800 μm across the lesion site into the distal optic nerve stump (2302.7 ± 162.8 μm; Figs. 6C, 6D, 6E). Axons in the distal optic nerve stump of CNTF-treated animals followed an irregular course (Figs. 6C, 6D), indicating that they corresponded to regrown axons and not to axons that had escaped the nerve crush. 
Figure 6
 
Long-distance regrowth of axotomized RGC axons in CNTF-treated retinas. Retinal ganglion cell axons in eyes with intravitreally grafted control-NS cells (a, b) or CNTF-NS cells (c, d) were anterogradely labeled 1 month after an intraorbital optic nerve crush. Retinal ganglion cell axons in control animals extended only a short distance across the lesion site (asterisk in [a]) into the distal optic nerve stump (a), and no regrown axons were present 1 mm distal to the lesion (b). In CNTF-treated retinas, in comparison, numerous axons were grown across the lesion site (asterisk in [c]) into the distal nerve stump, and some were detectable 1 mm distal to the lesion (arrows in [d]). Note the irregular trajectory of the labeled axons (c, d). The length of the longest regrown axon in animals with grafted control-NS cells was 459.3 ± 47.0 μm (mean ± SEM), compared with 2302.7 ± 162.8 μm in animals with grafted CNTF-NS cells (n = 6 for each experimental group; [e]). Arrows in (e) indicate mean values.
Figure 6
 
Long-distance regrowth of axotomized RGC axons in CNTF-treated retinas. Retinal ganglion cell axons in eyes with intravitreally grafted control-NS cells (a, b) or CNTF-NS cells (c, d) were anterogradely labeled 1 month after an intraorbital optic nerve crush. Retinal ganglion cell axons in control animals extended only a short distance across the lesion site (asterisk in [a]) into the distal optic nerve stump (a), and no regrown axons were present 1 mm distal to the lesion (b). In CNTF-treated retinas, in comparison, numerous axons were grown across the lesion site (asterisk in [c]) into the distal nerve stump, and some were detectable 1 mm distal to the lesion (arrows in [d]). Note the irregular trajectory of the labeled axons (c, d). The length of the longest regrown axon in animals with grafted control-NS cells was 459.3 ± 47.0 μm (mean ± SEM), compared with 2302.7 ± 162.8 μm in animals with grafted CNTF-NS cells (n = 6 for each experimental group; [e]). Arrows in (e) indicate mean values.
Discussion
Cell-replacement strategies are among the approaches that are currently being explored to develop therapies for yet untreatable degenerative retinal disorders. Recent studies have indeed demonstrated the feasibility to replace dysfunctional or degenerated photoreceptor cells or RPE cells by cell transplantation.5962 However, for retinal disorders characterized by the loss of RGCs, cell-replacement strategies are complicated by the fact that the transplanted cells not only have to integrate as functional RGCs into the host retinas, but additionally have to grow their axons over long distances to project in a topographically appropriate manner to the visual centers of the brain. Current therapeutic strategies for glaucomatous or nonglaucomatous optic neuropathies are therefore primarily aimed at delaying the loss of endogenous RGCs rather than at replacing degenerated RGCs.2,3,5,10,13 
Neurotrophic factor deprivation due to impaired axonal transport has been suggested to contribute to the loss of RGCs in glaucomatous optic neuropathies, and neurotrophic factor supplementation has therefore been extensively studied as a potential treatment option for glaucoma. Because neurotrophic factors usually have short half-life times and do not ordinarily cross the blood-retina barrier, robust and long-lasting neuroprotective effects likely depend on a sustained intraocular delivery of these factors. A sustained intraocular supply of neurotrophic factors has been achieved by intravitreal implantations of slow-release devices and viral or nonviral gene transfer to retina cells.2,10,11,13 Transplantations of cells that have been genetically modified to overexpress neurotrophic factors represent another strategy to continuously deliver these factors to glaucomatous retinas that has been successfully used in preclinical studies. For instance, intravitreal implants of encapsulated cells engineered to secrete glucagon-like peptide-1 have recently been reported to delay degeneration of RGCs in a rat model of optic nerve crush.63 Moreover, intravitreal injections of mesenchymal stem cells lentivirally modified to secrete BDNF resulted in significant attenuation of RGC loss in a rat model of ocular hypertension.64 Furthermore, simultaneous intraocular administration of neurotrophin-3, FGF-2, and BDNF through intravitreal transplantations of transfected fibroblasts has been shown to synergistically promote survival and axonal regrowth of axotomized RGCs in adult rats.65 
In the present study, we explored the use of neural stem cells from the embryonic mouse brain as cellular vectors to continuously deliver neurotrophic factors to adult mouse retinas with a lesion-induced degeneration of RGCs. When these cells are cultivated under adherent conditions in the presence of EGF and FGF-2, they give rise to cultures consisting of homogeneous populations of symmetrically dividing clonogenic stem cells which, in analogy to embryonic stem cells, have been termed NS cells.51,56 To evaluate the efficacy of an NS cell–based neuroprotective approach in an animal model of optic nerve injury, we took advantage of the potent rescue effects of CNTF on RGCs in various pathological conditions,19,26-33 and expressed the cytokine in NS cells using polycistronic lentiviral vectors. In a recent study, we analyzed the neuroprotective potential of CNTF-NS cells in Pde6brd1 and Pde6brd10 mutant mice, two animal models of retinitis pigmentosa characterized by an early onset and rapid degeneration of photoreceptor cells.49 We found that intravitreal transplantations of CNTF-NS cells resulted in significant attenuation of photoreceptor degeneration in both mouse mutants, in line with reports that have demonstrated protective effects of the cytokine on photoreceptor cells in a variety of animal models of inherited or acquired retinal degeneration.41,66 However, retinas of Pde6brd1 and Pde6brd10 mice were analyzed already 18 and 16 days after transplantation of the CNTF-NS cells, respectively.49 One major interest of the present study was therefore to evaluate the survival, transgene expression, and neuroprotective effects of the CNTF-NS cells on axotomized RGCs in long-term experiments. 
To this aim, we first increased the expression level of CNTF in a previously established clonal CNTF-NS line49 by repeated transductions and subsequent clonal expansions of NS cells with the strongest expression of the reporter gene. After several rounds of transductions and clonal expansions, a clonal cell line with high expression levels of CNTF was selected for all further experiments. When this cell line was differentiated into astrocytes in vitro, the glial cells stably expressed the reporter gene and the cytokine for at least 2 months, the longest cultivation period evaluated. More importantly, grafted NS cells survived for at least 4 months in the vitreous cavity of the recipient eyes. Here, the Venus-positive donor cells formed dense cell layers that were attached to the posterior pole of the lenses or the vitreal surface of the retinas. Integration of donor cells into the host retinas was not observed. In addition, there was no evidence for ongoing proliferation of the grafted cells, in line with our previous findings that the proliferation marker Ki-67 was expressed in only less than 4% and 2% of the donor cells 8 and 16 days after intravitreal transplantations of NS cells into Pde6brd1 and Pde6brd10 mice, respectively.49 Furthermore, we found some small-sized and locally restricted retinal folds in a fraction of eyes with grafted CNTF-NS cells. Retinal folds also have been described in dystrophic and normal retinas of cats that had received repeated intravitreal injections of the human CNTF analogue axokine.67 Formation of retinal folds thus appears to be among the complications associated with the intraocular delivery of CNTF,6771 provided the cytokine is administered in high amounts and over an extended period of time. 
The vast majority of grafted CNTF-NS cells were differentiated into astrocytes that still expressed CNTF 4 months after transplantation. Astrocytes derived from control-NS cells, in contrast, lacked detectable expression levels of the cytokine. Of note, sustained expression of the cytokine in eyes with CNTF-NS cell–derived astrocytes correlated with a significant attenuation of RGC loss, as demonstrated by the presence of 2.8-, 4.4-, 6.4-, and 4.8-fold more surviving RGCs in CNTF-treated retinas than in control retinas at the 1-, 2-, 3-, and 4-month postlesion intervals, respectively. Although protection of axotomized RGCs by the CNTF-NS cell grafts was transient and degeneration of RGCs progressed also in CNTF-treated eyes, we consider it remarkable that the NS cell–based intraocular delivery of CNTF attenuated RGC loss over an extended period. Given that intraorbital optic nerve lesions induce a rapid apoptotic degeneration of RGCs,72 it will be interesting to evaluate the neuroprotective potential of the clonal CNTF-NS cell line in animal models of ocular hypertension that more closely mimic the slowly progressing RGC loss in human glaucoma patients.7375 
In addition to protecting RGCs from degeneration, CNTF has been shown to promote regrowth of injured RGC axons.26,35,37,38 In line with these studies, we observed long-distance regeneration of lesioned RGC axons in eyes with grafted CNTF-NS cells. One month after the crush, some axons in CNTF-treated eyes were regrown for more than 2 mm into the distal nerve stump, whereas axons in eyes with grafted control-NS cells extended for only approximately 0.5 mm into the distal nerve stump. These data further confirm sustained delivery of functionally relevant quantities of the cytokine from the intravitreally located CNTF-secreting donor cells to the adult murine retina. 
Recent transplantation studies have identified a variety of cell types that exert neuroprotective effects on RGCs without prior genetic modification, including Schwann cells, olfactory ensheathing cells, oligodendrocyte precursor cells, and mesenchymal stem cells.40,76 Although the precise mechanisms by which these cell types rescued RGCs from degeneration are largely unknown, the neuroprotective effects have usually been attributed to the secretion of endogenously expressed neurotrophic factors and/or to immune modulatory effects of the grafted cell types. To analyze whether nonmodified NS cells also exert neuroprotective effects on RGCs, we determined the number of RGCs in animals that had received intravitreal injections of control-NS cells or the vehicle only, and found similar numbers of surviving RGCs in both experimental groups 1 month after the nerve crush. 
In summary, we have shown that an intravitreally grafted CNTF-secreting clonal NS cell line survived in the vitreous cavity of the host eyes and significantly attenuated a lesion-induced degeneration of RGCs over a period of 4 months, the longest posttransplantation time period analyzed. Furthermore, we also demonstrated that the grafted NS cells promoted long-distance regrowth of intraorbitally lesioned RGC axons in adult mice. The combined data suggest that genetically modified clonal NS cell lines may represent a useful tool for preclinical studies aimed at evaluating the therapeutic potential of a sustained cell-based intraocular administration of neuroprotective factors in mouse models of optic nerve injury or other retinal disorders characterized by a loss of RGCs. 
Acknowledgments
The authors thank Elke Becker, Sabine Helbing, Stephen Peters, and Stefanie Schlichting for excellent technical assistance. The authors also thank the FACS-Core Unit and the microscopy and imaging facility of the University Medical Center Hamburg-Eppendorf for technical support, and to Ali Derin and Susanne Conrad for animal care. 
Supported by the Stiftung Dr. Liselotte and Dr. Karl-Robert Brauns Foundation (Hamburg) (UB). 
Disclosure: K. Flachsbarth, None; K. Kruszewski, None; G. Jung, None; W. Jankowiak, None; K. Riecken, None; L. Wagenfeld, None; G. Richard, None; B. Fehse, None; U. Bartsch, None 
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Figure 1
 
Lentiviral vectors and expression of CNTF and Venus in genetically modified clonal NS cell lines. (A) Neural stem cells were transduced with a polycistronic lentiviral vector encoding a secretable variant of mouse CNTF under regulatory control of the human CMV enhancer/chicken β-actin (CAG) promoter. The vector additionally encoded a Venus reporter gene and a zeocin (ZEO) resistance gene separated from each other by a P2A sequence (2A) of porcine teschovirus-1 (Aa). The same vector but lacking the CNTF cDNA was used to transduce NS cells for control experiments (Ab). (B) Clonally derived CNTF-NS (Ba, Bb) and control-NS cell lines (Bc, Bd) were generated using FACS and immunostained with anti-CNTF antibodies. Whereas all cells in the CNTF-NS (Ba) and control-NS cell lines (Bc) expressed Venus, expression of CNTF was detectable only in the CNTF-NS cell clone (Bb; some cells labeled with arrowheads in Ba and Bb), but not in the control-NS cell clone (Bd). (C) Western blot analyses revealed secretion of CNTF into the supernatant of CNTF-NS cell cultures (CNTF-NS), whereas culture supernatants from control-NS cells (control-NS) lacked detectable levels of the cytokine. Ψ, packaging signal; cPPT, central polypurine tract; IRES, internal ribosome entry site; LoxP, recognition site of Cre recombinase; rmCNTF, recombinant mouse ciliary neurotrophic factor; RRE, rev-responsive element; SIN-LTR, self-inactivating long-terminal repeat; wPRE, Woodchuck hepatitis virus posttranscriptional regulatory element. Scale bar in (Bd) for (BaBd): 25 μm.
Figure 1
 
Lentiviral vectors and expression of CNTF and Venus in genetically modified clonal NS cell lines. (A) Neural stem cells were transduced with a polycistronic lentiviral vector encoding a secretable variant of mouse CNTF under regulatory control of the human CMV enhancer/chicken β-actin (CAG) promoter. The vector additionally encoded a Venus reporter gene and a zeocin (ZEO) resistance gene separated from each other by a P2A sequence (2A) of porcine teschovirus-1 (Aa). The same vector but lacking the CNTF cDNA was used to transduce NS cells for control experiments (Ab). (B) Clonally derived CNTF-NS (Ba, Bb) and control-NS cell lines (Bc, Bd) were generated using FACS and immunostained with anti-CNTF antibodies. Whereas all cells in the CNTF-NS (Ba) and control-NS cell lines (Bc) expressed Venus, expression of CNTF was detectable only in the CNTF-NS cell clone (Bb; some cells labeled with arrowheads in Ba and Bb), but not in the control-NS cell clone (Bd). (C) Western blot analyses revealed secretion of CNTF into the supernatant of CNTF-NS cell cultures (CNTF-NS), whereas culture supernatants from control-NS cells (control-NS) lacked detectable levels of the cytokine. Ψ, packaging signal; cPPT, central polypurine tract; IRES, internal ribosome entry site; LoxP, recognition site of Cre recombinase; rmCNTF, recombinant mouse ciliary neurotrophic factor; RRE, rev-responsive element; SIN-LTR, self-inactivating long-terminal repeat; wPRE, Woodchuck hepatitis virus posttranscriptional regulatory element. Scale bar in (Bd) for (BaBd): 25 μm.
Figure 2
 
Expression of CNTF and Venus in differentiated clonal NS cell lines. CNTF-NS (ac, gi) and control-NS cell clones (df, j–l) were differentiated into MAP2-positive neurons (af) or GFAP-positive astrocytes (gl). The reporter gene Venus was expressed in all neurons (a, d) and astrocytes (g, j) derived from the CNTF-NS (a, g) and control-NS cell lines (d, j). CNTF was detectable only in neurons (c) and astrocytes (i) derived from CNTF-NS cells, but not in neurons (f) and astrocytes (l) derived from control-NS cells. Neurons and astrocytes were analyzed for expression of Venus and CNTF 1 week and 2 months after induction of differentiation, respectively. Scale bar in (l) (for al): 50 μm.
Figure 2
 
Expression of CNTF and Venus in differentiated clonal NS cell lines. CNTF-NS (ac, gi) and control-NS cell clones (df, j–l) were differentiated into MAP2-positive neurons (af) or GFAP-positive astrocytes (gl). The reporter gene Venus was expressed in all neurons (a, d) and astrocytes (g, j) derived from the CNTF-NS (a, g) and control-NS cell lines (d, j). CNTF was detectable only in neurons (c) and astrocytes (i) derived from CNTF-NS cells, but not in neurons (f) and astrocytes (l) derived from control-NS cells. Neurons and astrocytes were analyzed for expression of Venus and CNTF 1 week and 2 months after induction of differentiation, respectively. Scale bar in (l) (for al): 50 μm.
Figure 3
 
Neural differentiation and expression of CNTF in intravitreally grafted NS cell lines. Analyses of eyes 4 months after intravitreal transplantations of CNTF-NS (ad) and control-NS cell lines (eh) revealed the presence of Venus-positive donor cells (a, e) that were attached to the posterior poles of the lenses. Virtually all CNTF-NS cells (b) and control-NS cells (f) were differentiated into GFAP-positive astrocytes. Expression of CNTF was detectable in cells derived from CNTF-NS cells (c), but not in cells derived from control-NS cells (g). (d) and (h) are merged images of (a) and (c), and (e) and (g), respectively. Some donor cells coexpressing Venus and CNTF are marked with white arrows in (a), (c) and (d). Scale bar in (h) (for ah): 100 μm.
Figure 3
 
Neural differentiation and expression of CNTF in intravitreally grafted NS cell lines. Analyses of eyes 4 months after intravitreal transplantations of CNTF-NS (ad) and control-NS cell lines (eh) revealed the presence of Venus-positive donor cells (a, e) that were attached to the posterior poles of the lenses. Virtually all CNTF-NS cells (b) and control-NS cells (f) were differentiated into GFAP-positive astrocytes. Expression of CNTF was detectable in cells derived from CNTF-NS cells (c), but not in cells derived from control-NS cells (g). (d) and (h) are merged images of (a) and (c), and (e) and (g), respectively. Some donor cells coexpressing Venus and CNTF are marked with white arrows in (a), (c) and (d). Scale bar in (h) (for ah): 100 μm.
Figure 4
 
Intravitreally grafted CNTF-NS cells attenuate degeneration of axotomized retinal ganglion cells in adult mice. Adult mice received intravitreal injections of a CNTF-NS cell clone (ad) or a control-NS cell clone (eh) 1 day after an intraorbital optic nerve crush. Analysis of flat-mounted retinas 1 (a, e), 2 (b, f), 3 (c, g), and 4 months (d, h) after the lesion revealed the presence of significantly more Brn-3a–positive ganglion cells in eyes with grafted CNTF-NS cells (ad) than in eyes with grafted control-NS cells (eh) at all postlesion intervals. Scale bar in (h) (for ah): 100 μm.
Figure 4
 
Intravitreally grafted CNTF-NS cells attenuate degeneration of axotomized retinal ganglion cells in adult mice. Adult mice received intravitreal injections of a CNTF-NS cell clone (ad) or a control-NS cell clone (eh) 1 day after an intraorbital optic nerve crush. Analysis of flat-mounted retinas 1 (a, e), 2 (b, f), 3 (c, g), and 4 months (d, h) after the lesion revealed the presence of significantly more Brn-3a–positive ganglion cells in eyes with grafted CNTF-NS cells (ad) than in eyes with grafted control-NS cells (eh) at all postlesion intervals. Scale bar in (h) (for ah): 100 μm.
Figure 5
 
Quantitative analysis of the neuroprotective effect of intravitreally grafted CNTF-NS cells on axotomized RGCs. The number of Brn-3a–positive RGCs was determined in eyes with intravitreally grafted CNTF-NS cells (filled bars) or control-NS cells (open bars) 1, 2, 3, and 4 months after an intraorbital optic nerve crush. Note that the CNTF-treated retinas contained significantly more RGCs than the control retinas at all postlesion intervals. Each bar represents the mean number (±SEM) of RGCs per mm2 from six retinas. ***P < 0.001 according to the Student's t-test.
Figure 5
 
Quantitative analysis of the neuroprotective effect of intravitreally grafted CNTF-NS cells on axotomized RGCs. The number of Brn-3a–positive RGCs was determined in eyes with intravitreally grafted CNTF-NS cells (filled bars) or control-NS cells (open bars) 1, 2, 3, and 4 months after an intraorbital optic nerve crush. Note that the CNTF-treated retinas contained significantly more RGCs than the control retinas at all postlesion intervals. Each bar represents the mean number (±SEM) of RGCs per mm2 from six retinas. ***P < 0.001 according to the Student's t-test.
Figure 6
 
Long-distance regrowth of axotomized RGC axons in CNTF-treated retinas. Retinal ganglion cell axons in eyes with intravitreally grafted control-NS cells (a, b) or CNTF-NS cells (c, d) were anterogradely labeled 1 month after an intraorbital optic nerve crush. Retinal ganglion cell axons in control animals extended only a short distance across the lesion site (asterisk in [a]) into the distal optic nerve stump (a), and no regrown axons were present 1 mm distal to the lesion (b). In CNTF-treated retinas, in comparison, numerous axons were grown across the lesion site (asterisk in [c]) into the distal nerve stump, and some were detectable 1 mm distal to the lesion (arrows in [d]). Note the irregular trajectory of the labeled axons (c, d). The length of the longest regrown axon in animals with grafted control-NS cells was 459.3 ± 47.0 μm (mean ± SEM), compared with 2302.7 ± 162.8 μm in animals with grafted CNTF-NS cells (n = 6 for each experimental group; [e]). Arrows in (e) indicate mean values.
Figure 6
 
Long-distance regrowth of axotomized RGC axons in CNTF-treated retinas. Retinal ganglion cell axons in eyes with intravitreally grafted control-NS cells (a, b) or CNTF-NS cells (c, d) were anterogradely labeled 1 month after an intraorbital optic nerve crush. Retinal ganglion cell axons in control animals extended only a short distance across the lesion site (asterisk in [a]) into the distal optic nerve stump (a), and no regrown axons were present 1 mm distal to the lesion (b). In CNTF-treated retinas, in comparison, numerous axons were grown across the lesion site (asterisk in [c]) into the distal nerve stump, and some were detectable 1 mm distal to the lesion (arrows in [d]). Note the irregular trajectory of the labeled axons (c, d). The length of the longest regrown axon in animals with grafted control-NS cells was 459.3 ± 47.0 μm (mean ± SEM), compared with 2302.7 ± 162.8 μm in animals with grafted CNTF-NS cells (n = 6 for each experimental group; [e]). Arrows in (e) indicate mean values.
Supplementary Figure S1
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