Engrafted Chicken Neural Tube–Derived Stem Cells Support the Innate Propensity for Axonal Regeneration within the Rat Optic Nerve

Abstract

purpose. Injury to the adult optic nerve, caused mechanically or by diseases, is still not reparable because the retinal ganglion cells (RGCs) are not allowed to regrow their axons and die retrogradely, although they possess the intrinsic propensity to regenerate axons in experimental conditions.

methods. In vitro propagated embryonic stem cells derived from the early chicken neural tube (NTSCs) were used to examine whether transplanted NTSCs produce growth-promoting factors and pave the microenvironment, thus facilitating axonal regeneration within the rat optic nerve.

results. NTSCs survived within the site where the optic nerve had been cut and continued to be nestin-positive, thus preserving their undifferentiated cell phenotype. Transplanted NTSCs activated the matrix metalloproteases (MMP)-2 and -14 in glial fibrillary acidic protein (GFAP)-positive optic nerve astrocytes. MMP2 production correlated with immunohistochemically visible degradation of inhibitory chondroitin sulfate proteoglycans (CSPGs). In addition, NTSCs produced a panoply of neurite-promoting factors including oncomodulin, ciliary neurotrophic factor, brain-derived neurotrophic factor and crystallins β and γ. Cut axons intermingled with NTSCs and passed through the zone of injury to enter the distal optic nerve over long distances, arriving at the thalamus and midbrain.

conclusions. This study showed evidence that paving of the distal optic nerve microenvironment with proteolytically active MMPs and providing stem-cell–derived growth factors is a suitable method for facilitating regenerative repair of the optic nerve. Understanding the molecular mechanisms of this repair has fundamental implications for development of NTSC-based subsidiary therapy after neural injuries.

Adult retinal ganglion cells (RGCs) fail to extend axons within the interior of the optic nerve after injury. This failure of regeneration is commonly attributed to inhibitory factors associated with myelin components and/or the glial scar, which is composed of cells and extracellular matrix. However, under the correct permissive cues, such as those provided by a peripheral nerve graft ¹ or culture in vitro, ² regeneration is possible. Three obstacles stand in the way of regeneration within the optic nerve: first, the presence of inhibitory molecules, such as Nogos, ^{3 4} chondroitin sulfate proteoglycans (CSPGs), ^{5 6 7} and the reactive astrocytes that make-up the glial scar; second, lack of appropriate stimulatory cues such as neurotrophic factors ^{8 9 10} ; third, loss of retinal ganglion cells (RGCs) through the initial primary assault on the optic nerve. ^{11 12}  

Numerous studies have been conducted to unravel the molecular mechanisms associated with the inhibition of growth cone formation and stimulation of regrowth. However, the mechanisms underlying regrowth of axons from cut axonal stumps (de lesio formation of growth cones) are unclear. Based on the appearance of Nogos within the optic nerve, ^{3 13} strategies intended to block inhibitory molecules within the distal optic nerve have been developed. ^{13 14 15 16 17 18} Alternatively, stimulation of axonal regeneration was successful when the intraretinal compartments of the RGCs were treated with various neurotrophic factors, including induction of inflammatory factors ^{8 10 17 18 19 20 21 22 23} and noninflammatory activities such as lenticular factors acting directly on RGCs. ^{24 25 26} All strategies of treatment have to take into account that after transection of the optic nerve, there is delayed death by apoptosis of the RGCs ^{11 27} accompanied by abortive sprouting. ²⁸ To this end, approaches to the support of regeneration of the optic nerve should fulfill the criteria of reducing death of the RGCs, of stimulating RGCs to regrow their axons, and of remodeling the optic nerve microenvironment for successful elongation of axons. 

One promising strategy that may fulfill the criteria set for successful regeneration is the use of neural stem cells (NSCs) ^{29 30 31 32 33 34} or neural progenitor cells (NPCs), which activate a matrix metalloproteinase (MMP)–dependent proteolytic mechanism ³⁵ and support intraretinal neurite growth via proteolysis of CSPGs. ^{6 36} Given the capability of NTSCs to produce multiple factors with potential activity on axonal growth, we developed a model of NTSC propagation in culture and transplanted these cells at the site where the optic nerve was cut. The rationale of using chicken cells was their relative abundance within the neural tube, the accessibility of chicken embryos at early stages of development, and the availability of ethically harmless stem cells. Our results showed that xenografted NTSCs (1) are tolerated within the injured optic nerve, (2) induce the production of MMPs which regulate CSGPs, and (3) produce multiple growth-promoting factors such as oncomodulin, ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), and crystallins, which may contribute in an orchestrated way of stimulating lengthy axonal regeneration. Indeed, we observed an enhanced regeneration of RGC axons. 

Materials and Methods

Preparation and Culture of Chick Embryo Neural Stem Cells

Fertilized White Leghorn chicken eggs provided by a local supplier were incubated in a 42% humidified incubator at 37°C for approximately 45 to 49 hours. This period allowed the embryos to reach approximately stage 10 of embryogenesis according to the Hamburger and Hamilton scale (HH10; 1951; for review see Ref. ³⁷ ; Fig. 1A ). Neural tube–derived NTSCs were obtained by puncturing the tapered and windowed end of the egg, removing 5 mL of egg albumen, and microinjecting sterilized Indian ink under the embryo so as to discern the orientation of the neural tube and the position of it rostral edge. With a pair of tweezers, the chorioallantoic membrane was torn away, and the prospective embryonic head was dissected and transferred into the culture medium, which consisted either of chemically defined, serum-free microglia-astrocyte medium (Promocell, Heidelberg, Germany) or of serum-containing Dulbecco’s modified Eagle’s medium (DMEM, containing high glucose and l-glutamine, but lacking sodium pyruvate; PAA Laboratories GmbH, Pasching, Austria) supplemented with 10% fetal calf serum (FCS, Gold-FCS; PAA Laboratories GmbH), containing 1% penicillin and 1% streptomycin (PAA Laboratories GmbH). The cells were dissociated in a solution of phosphate-buffered saline (PBS, containing 0.3% BSA and 0.1% DNase already dissolved), methylcellulose (also containing 0.3% BSA and 0.1% DNase already dissolved; Mann-Pharma, Berlin, Germany), and papain (15 U/mL, Sigma-Aldrich, Taufkirchen, Germany), followed by trituration and washing. The final pellet was resuspended in the culture medium, and the cells were maintained within the same culture at 37°C with 5% CO₂ and fed twice per week by replacing half the culture medium. Typically, 8.75 × 10⁴ to 2.0 × 10⁵ cells were seeded in 0.5 to 1.0 mL of medium in four wells of a 12-well culture dish (Corning CoStar, Cambridge, MA). Typically, 1.8 × 10⁵ cells were seeded into two wells of a 12-well culture dish (Corning CoStar). After the cells were cultured, viability was assessed with the telomeric repeat amplification protocol (TRAP kit; Roche Medicine, Mannheim, Germany), which assesses telomerase activity. ³⁸  

TRAP Assay and Live/Dead Assay

In the TRAP assay, approximately 1 × 10⁵ cells were collected and washed in PBS. A lysis solution was added and the proteins were isolated. The telomerase elongated biotin-conjugated primers. After this, PCR was performed, its product denatured and hybridized with digoxigenin (DIG)-labeled telomeric sequences, and a peroxidase coupled anti-DIG antibody added. ELISA was performed to determine the concentration of the amplified sequence. Briefly, the product was put in avidin-coated microplates, TMB (substrate for peroxidase) was added, and the photometric measurement was performed after H₂SO₄ was added to stop the reaction. According to the description of the manufacturer, all differences between the negative control and the value of the specimen (both arbitrary units for light absorption) greater than 0.2, were treated as positive. 

The live/dead assay was performed with a kit from Invitrogen-Molecular Probes (Eugene, OR). It determined cell viability based on the properties of calcein AM and ethidium homodimer (EthD-1). The calcein is able to pass through the cell membrane of either living or dead cells, but only in living cells is it converted by the general action of esterases, which render it fluorescent (494/517 nm) and impermeable through the cell membrane. On the other hand, EthD-1 permeates only the membrane of dead cells, wherein it binds to the nucleic acids, which enhances its fluorescence (528/617 nm) approximately 50 times. 

In addition, the live/dead assay with calcein AM was performed with a kit from Invitrogen-Molecular Probes according to the protocol of the supplier. Briefly, the cells were incubated with 150 μL of the reagent containing 2 μM calcein AM and 4 μM EthD-1 at room temperature for 30 minutes. Fluorescence was recorded with a fluorescence microscope (Axiovert; Carl Zeiss Meditec, GmbH, Oberkochen, Germany). Dead red-fluorescing cells were counted in five areas of 100,000 μm². The percentage of dying cells was expressed as the number of red-fluorescing cells divided by the total number of stained cells. 

Immunocytochemistry

Primary antibodies included rabbit anti-BDNF (1:500; Chemicon, Temecula, CA), mouse anti-CNTF (1:100; Chemicon Inc.), rabbit anti-calbindin (1:1000; Chemicon), rabbit anti-calcineurin (1:500; Chemicon), mouse anti-calretinin (1:1000; Chemicon), rabbit anti-crystallin-β (1:400, BSR Laboratory Department of Biochemistry, Hyderabad, India), rabbit anti-crystallin-γ (1:100; Samuel Ziegler, National Eye Institute, Bethesda, MD), mouse anti-CSPG (neurocan, 1:100; Abcam, Cambridge, UK), mouse anti-ED1 (1:500; Serotec, Oxford, UK), mouse anti-GFAP (for astrocytes; at 1:500; Sigma-Aldrich, St. Louis, MO), mouse anti-MAP2 (1:500; Sigma-Aldrich), mouse anti-MMP2 (1:200; Chemicon), mouse anti-MMP14 (1:400; Chemicon), mouse anti-neurofilament 200 kDa (for neurons; 1:400; Sigma-Aldrich), rabbit anti-neurofilament 70 kDa (1:200; Chemicon), rabbit anti-nestin (for stem cells, 1:200; Chemicon), and rabbit anti-oncomodulin (1:500 Swant, Bellinzona, Switzerland), anti rHu-bFGF (C60201, 1:500; Promocell). These were visualized with the following secondary antibodies: TRITC-conjugated goat anti-mouse (at 1:300; Sigma), Cy2-conjugated goat anti-mouse (1:200; Jackson ImmunoResearch Laboratories, Inc.), TRITC-conjugated goat anti-rabbit (1:400; Sigma-Aldrich), and Cy2-conjugated goat anti-rabbit (1:200; Jackson ImmunoResearch Laboratories Inc.). All antibodies were diluted in PBS. 

For immunocytochemical analyses, the cells were fixed with 4% PFA for 10 minutes. They were subsequently permeabilized by incubation at −20°C in methanol for 10 minutes. After three rinses with PBS, the cells were incubated in PBS containing 10% FCS (PAA Laboratories GmbH) for blocking before they were stored overnight at 4°C with the appropriate primary antibodies. The following day, the slides were again rinsed three times with PBS before they were incubated with the secondary antibody at room temperature for 1 hour. After three final rinses in PBS, the slides were embedded in Mowiol with DAPI (1 mg/mL; 4′, 6-diamidino-2-phenylindole; Sigma-Aldrich) to stain the nuclei. The number of rinses was reduced in later immunocytochemical procedures, to lower cell loss. Control experiments consisted of incubation of the cells with only the secondary antibody. To quantify the data from immunocytochemistry, labeled cells in 12 randomized fields were counted with the corresponding filter. The numbers of cells stained was used to determine the arithmetic mean ± SD. The same 12 fields were then counted with phase-contrast optics to determine the total number of cells. The proportion of cells stained is presented as a percentage of the total. On each glass slide, negative controls were stained with the secondary antibody only, and counting was executed as for the experimental samples. 

Surgical Procedures for ON Regeneration

Experiments were performed with 50 male and female adult rats of the Sprague-Dawley strain, weighing 200 to 230 g and aged between 8 and 10 weeks. All experiments were performed in accordance with the ARVO Statement for The Use of Animals in Ophthalmic and Vision Research and were approved by the local Committee for Animal Care. For microsurgery, the animals received an intraperitoneal injection of a mixture of 0.2–0.3 mL ketamine sulfate (50–60 mg/kg; Parke-Davis- Pfizer, Karlsruhe, Germany) and 0.1 mL xylazine (10–15 mg/kg; Sanofi-Aventis, Frankfurt, Germany) per 200 g body weight. 

Before axotomy of the RGCs, the head of the anesthetized animal was positioned and fixed in a self-constructed headholder. The left optic nerve (ON) was surgically exposed in its intraorbital segment, which spans 2 to 3 mm beyond the eye cup, taking care to leave the retinal vascularization intact. ^{10 39} The lacrimal gland was displaced, but left intact, and the superior extraocular muscles were spread to allow access to the ON. An incision was made in the eye-retractor muscle and the meninges perpendicular to the axonal orientation, extending over one third of the dorsal ON. The ON was completely cut with microscissors at a distance of 1 to 2 mm behind the eye, leaving the retinal vascularization unaffected. The cut ends of the distal and proximal nerve stumps were realigned by tightening with two meningeal threads (10.0 silk; Ethicon, Hamburg, Germany). This method ensured that no gap existed between the nerve stumps and that the nerve fascicles were topographically realigned. At this point, 10 μL of the cell suspension at a concentration of 0.5 to 1 × 10⁶ cells/mm³ was injected between the unified nerve stumps with a pulled 50-μL glass microcapillary adjusted on a syringe (Hamilton, Reno, NV). Slow injection over 5 minutes was performed to assure that as many cells as possible remained within the cavity. However, despite all preventative measures, only part of the fluid and cells remained within the ON cavity, and the remainder drained into the orbital space. Control rats (n = 5) underwent the same procedure as the experimental animals, but received a PBS injection into the cut ON instead of the cell suspension. Sham-operation control animals underwent all steps of surgery, but no nerve transaction and no cell injection (n = 5). After this, the intraorbital cavity was overlaid with a sponge (Gelfoam; Pfizer, Zurich, Switzerland), to reduce the outflow of cells from the site of injection. The entire procedure was performed within 30 to 40 minutes. All microsurgery was performed with the aid of a surgical microscope (OPMI 19; Carl Zeiss Meditec, GmbH) equipped with a camera that enabled the surgical procedure to be documented photographically in all the animals. The ON head was examined ophthalmoscopically immediately after surgery, to ensure that the retinal vasculature was intact. 

Tissue Preparation and Immunohistochemistry

After survival times ranging 3 days to 2 months, the animals were given a lethal dose of anesthetic. The eyes with the nerve segments, with the chiasm still attached, were dissected from the muscles and connective tissue and submersion fixed in 4% PFA at 4°C overnight. The specimens were then embedded in OCT compound (Tissue Tek; Sakura Finetek, Torrance, CA) and frozen in liquid nitrogen. Longitudinal frozen sections (12 μm) were cut through the ON/chiasm. Sections were thaw mounted on gelatin-coated glass slides (Superfrost Plus, Fisher Scientific, Pittsburgh, PA) and stored at −20°C until use for immunohistochemistry with the antibodies described earlier. 

The number of axons extending beyond the site of the cut and suture was averaged from 30 longitudinal ON (12-μm) sections per 1 and 6 mm from the site of the cut. To calculate the total number of axons, we used the equation of Leon et al., ²¹ : Σ ad = πr ² x (average axons/mm)/t. The number of axons per millimeter was obtained by measuring the ON width at 1 and 6 mm from the site of the cut, where t represents thickness of the section (10–12 μm), and r represents the radius of the ON. Statistical analysis was performed with commercial software (SPSS ver. 12.0 for Windows; SPSS, Chicago, IL), using a nonparametric, unpaired, one-tailed Mann-Whitney test. Significance was assumed at the 95% level of confidence. 

Retrograde Labeling of the Regenerating Pathway

The characteristics and number of RGCs contributing to regeneration of the retinas were determined by labeling the RGCs in a retrograde fashion from the reinnervated superior colliculus (SC) with gold label (Fluorogold; Fluorochrome, Englewood, CO) 6 to 8 weeks after ON surgery, with analysis 5 to 6 days later. A midline skin incision was made over the midbrain area, and a hole was drilled through the bone. A pulled-glass capillary (tip diameter, 20 μm) was filled with 10 μL of 3% gold label and attached to a syringe (Hamilton). The cortical tissue over the right SC was aspirated by means of a vacuum pump, to allow access to the visible SC and injection of the gold label into the superficial layers of the SC. Retrogradely labeled RGCs were examined in retinal wholemounts. For this, the rats were killed by an overdose of anesthetic (7% chloral hydrate); their eyes were enucleated; the cornea, lens, and ciliary body removed; and the remaining eye cups immersion fixed in 4% PFA in PBS for 1 hour. Then, the eye cup was washed with PBS for 5 minutes, and the retina was dissected from the remaining tissue and flatmounted on sartorius nitrocellulose filter with the photoreceptor layer facing the filter. The retinas were stored in 4% PFA in PBS at 4°C until they were used for RGC counting. Cell density was assessed with the aid of a fluorescence microscope equipped with a grid of 200 × 275 μm (e.g., 49,000 μm² at a final magnification of 400×). Because RGC density varies across the retinal eccentricity, we counted cells at each eccentricity (outer, middle, central) from each quadrant of the retina, for a total of 12 fields of cells per retina. 

Results are expressed as the mean ± SD. Statistical analysis was performed with commercial software (SPSS, Chicago, IL), using the nonparametric, unpaired, one-tailed Mann-Whitney test. Significance was assumed at the 95% confidence level. 

Results

Morphologic Characterization of Cell Phenotypes

The chicken embryos reproducibly reached stage HH10 (Fig. 1A)after 45 to 49 hours of incubation. Removal of the prospective head region (Fig. 1B)and collection of approximately 40 heads per vial (Fig. 1C)were sufficient to produce and seed NTSCs for the culturing experiments (Fig. 1D) . 

After 24 hours in culture, typical cell cultures were not confluent (Fig. 1E) , and the cells showed either spherical shape or were differentiated cells, the latter showing either a spindle-shaped (Fig. 1F)or flattened membranous morphology. The proportion of spindlelike cells was approximately 20% during the first day and decreased to almost 0% after 7 days in culture (Fig. 1G) , whereas round cells made up almost 100% of the cultures by day 7 (Fig. 1H) , indicating that the NTSCs may be less differentiated with time in culture. 

When cultured in serum-containing medium the typical cells in the culture dish were of a spherical shape after 48 hours in culture (Fig. 1G) . This spherical morphology was maintained after 1 (Fig. 1I)to 4 (Fig. 1K)weeks in culture. The viability of the cells was assessed with calcein-staining, which revealed a high percentage of living cells (Figs. 1L 1M 1N) . When quantified, more than 80% of all cells were viable throughout the time of observation, which covered 8 weeks in culture (Fig. 1P) . The doubling time of cultured cells was examined over 5 weeks and was found to take between 5.8 days (Fig. 1O)and indicated a typical growth of NTSCs in vitro. 

Next, markers for undifferentiated cells were analyzed, starting with nestin, a characteristic filament protein. It appeared that nestin was expressed in the cultured cells (Fig. 2A 2B 2C) . Quantification revealed that approximately 80% expressed this marker (Fig. 2D) , whereas approximately 49.5% showed positive staining for GFAP, and only less than 2.4% of all cells expressed neuron-specific markers such as β-III-tubulin and MAP-2 (Fig. 2D) . Double staining for GFAP and nesting revealed that approximately 15% of all nestin-positive NTSCs expressed GFAP, too (data not shown). When the telomerase activity of the cells was assessed in the TRAP assay between the time of seeding and 60 days in culture, the difference ratio was >0.2 at all times of examination, compared with the ratio in control cultures (Fig. 2E) , indicating that the cells retained the features of undifferentiated cells. It appeared from these studies that most of the NTSCs cultured from the anterior chicken neural tube remained largely undifferentiated when cultured in vitro. To examine whether the NTSCs produce trophic factors that support their own survival, anti-bFGF staining was performed and showed that bFGF was expressed in the NTSCs (Figs. 2F 2G 2H) , which likely explains why the cells can be cultured without additional supplementation with FGF. 

Production of Factors with Growth-Promoting Potential

We hypothesized that cultured NTSCs are capable of expressing factors that may be involved in support of axonal growth and examined this possibility using immunohistochemistry. When tested with antibodies to crystallins of the β and γ families, they appeared to produce both classes of crystallins (Figs. 3A 3B 3C 3D 3E 3F) . One of these substances, crystallin β-b-2 (crybb2) has recently been identified to be innate to RGCs and supports elongation of regenerating ganglion cell axons in culture. ^{40 41} In contrast, the cells were negative to the α-crystallins (data not shown). Of the neurotrophic factors tested, NTSCs were stainable with antibodies to BDNF (Figs. 3G 3H 3I)and to CNTF (Figs. 3J 3K 3L) . Both factors have been tested to influence survival of axotomized RGCs and axonal regrowth in vivo and in vitro. ^{8 11 42} Of the calcium-binding proteins tested, approximately 30% of the cells stained positive to calbindin, but more than 60% of the cells expressed calcineurin (Figs. 3M 3N 3O)and calretinin (Figs. 3P 3Q 3R) . More than 60% of the NTSCs expressed oncomodulin (Figs. 3S 3T 3U) , a member of the calcium-binding proteins that has recently been shown to be a macrophage-derived protein with high potential for supporting axonal regeneration both in vitro and in vivo. ²³ This part of the study showed that cultured NTSCs expressed several different proteins that may become regeneration-supporting factors when the cells are transplanted into the injured ON. To examine whether NTSCs produce similar neurite-promoting factors in vivo, we examined the injured and cell-injected ONs (n = 3) with immunoblot analysis and compared them with control specimens (n = 3) lacking NTSC injection. It appeared that no β- or γ-crystallin and no CNTF were detectable in the control specimens (Fig. 4) . In contrast, all three proteins were highly stained in the ONs after engrafting of the NTSCs (Figs. 4) , indicating that the cells also induced the production of regeneration-promoting factors in vivo. 

Implantation of Precultured NTSCs

To examine further whether implanted NTSCs support regrowth of retinofugal axons, precultured NTSCs were engrafted into an ON cavity formed at the site of the ON cut. For this purpose, cells remained in culture up to 4 weeks after seeding on laminin and formed an almost confluent layer with polygonal cell shapes and typical features of NTSCs. The cells were detached from the culture and washed in PBS, transferred into DMEM and injected into the cut ON of adult rats. After the meninges were resutured to reunify the ON edges and a survival period of 1 to several weeks, the rats were euthanatized and the ONs were cryosectioned and processed for immunohistochemistry. With the use of antibodies to axonin-1, which is a specific marker for chicken neurons, transplanted cells could be identified within the ON and mainly within the site of the ON cut (Figs. 5A 5B) . Concerning regeneration of axons in control ONs without NTSCs, GAP-43-positive axonal stumps were restricted to the proximal ON (Fig. 5C) , and no axons were seen behind the site of injury (Fig. 5C)or more distally (Fig. 5D) . Using double staining with antibodies to GAP-43, we could observe ON axons to have grown over considerable distances beyond the site of the cut and NTSC engraftment. In particular, when double-staining with nestin antibody and GAP-43-antibody was performed, regenerating axons were seen to pass through the site of the injury (Fig. 5E) , which was populated with NTSCs (Figs. 5E 5F) . Although few labeled NTSCs were visualized within each cryostat section (Figs. 5E 5F) , the mean number of cells determined from the total sections of each nerve was 163 ± 112 NTSCs (n = 7), indicating that many of the cells injected were displaced. Both the proximal and distal stumps of the ONs were evenly populated with axons (Figs. 5G 5H , respectively). When the axons were quantified from sections throughout the ON, up to 300 axons were found within the most distal ON, whereas close to the injury, it was populated with more than 1000 axons (Fig. 5I) . Most distal axons were detected close to the chiasm region which is several millimeters away from the site of the cut. In addition to axons interacting with the implanted NSCs, regenerating axons have to pass along ON astrocytes and extracellular matrix while traversing its interior. Double staining with GFAP and GAP-43 revealed a close relationship of axons and astrocytes throughout the ON (data not shown). 

After passing through the distal ON and tract, regenerating axons were expected to arrive at thalamic and midbrain visual centers approximately 6 weeks after injury and NSC implantation. Retrograde staining of the retina from the SC 2 and 4 weeks after surgery did not show any labeled RGCs within the retina, confirming a previous study. ³⁹ Six weeks after injury, only individual RGCs were retrogradely stained from the SC (Figs. 6A 6B) , with a density of less than 10 cells/mm² retina (Fig. 6F) . When the cells were retrogradely stained 8 weeks after surgery, they appeared scattered throughout the retinal eccentricity with a cell density of approximately 600 cells/mm² (Figs. 6C 6D 6E) . The results of the study show that once permitted and stimulated to grow, retinal axons arrived at the midbrain approximately 6 weeks later. 

Persistence and Identification of NTSCs within the ON

In addition to interacting with axons, astrocytes, and ECM molecules, NTSCs encounter interneural macrophages that clear away injury-induced debris. A localization study with ED-1 antibody to detect macrophages and nestin-antibody to detect NTSCs indicated that the cells intermingled within the site of implantation (Figs. 7A 7B 7C) . There was no double staining, with approximately 60% of cells showing NTSC features; approximately 35% were macrophages (Fig. 7G) . When OX-42 staining was performed to distinguish ON microglia from macrophages, no double-stained cells were observed with nestin or ED-1 (data not shown). Approximately 5% of the cells remained unlabeled with either antibody. 

When oncomodulin staining was combined with the nonphosphorylated neurofilament NF70 staining which is specific to chicken NTSCs, there was a high degree of colocalization (Figs. 7D 7E 7F) , indicating that NTSCs express oncomodulin after transplantation. In contrast, no oncomodulin- or NF70-positive cells were seen in the control ONs which either underwent a sham operation or a cut of the nerve without engraftment. Approximately 10% of all cells within the site of injury were either NF70- or oncomodulin-positive (Fig. 6H)of the cells were shown to express both proteins. Of this population, which accounts for one fifth of all cells, 70% were double-stained and account for approximately 14% of the total population. 

Alterations of the ON-Environment of NTSCs in Grafting

Immunohistochemistry to MMP-2, MMP-14, and chondroitin sulfate proteoglycans (CSGPs) was performed to examine potential mechanisms of NTSC-transplantation on axonal growth. When MMP-2 was studied, marginal immunostaining was observed in untreated control ONs and in control nerves that were cut, but did not receive transplants (Figs. 8A 8B 8C) . Quantification of MMP-2-positive cells in these controls revealed only individual cells that were weakly stained and were not countable. In contrast, MMP-2 was highly upregulated in cut ONs that received NTSCs (Figs. 8D 8E 8F) . The MMP-2 immunostaining colocalized with GFAP (Fig. 8F) , indicating that astrocytes accounted for its production. Immunostaining to MMP-14 was virtually absent in control cultures without NTSCs (Figs. 8G 8H 8I)and was highly upregulated in ONs that had received NTSCs (Figs. 8J 8K 8L) . MMP-14 also colocalized with astrocytic GFAP (Fig. 8L) . To examine MMP-2 production within the ON, we performed Western blot analyses. It appeared that MMP-2 staining increased when the ON was cut or crushed (Figs. 9A 9C) . In control specimens that received NTSCs in the nerve (n = 3) without crush or cut, MMP-2 staining was comparable (Fig. 9B) . In contrast, a higher MMP-2 staining was observed in cut ONs after NTSCs engraftment (Figs. 9B 9C) . This finding confirmed the immunohistochemistry, which showed that the highest MMP-2 staining occurred in cut and cell engrafted ONs. 

In parallel sections from the same ONs (control and experimental specimens), CSPG was visibly expressed in control normal and cut ONs without NTSCs (Figs. 8M 8N 8O) . In contrast, CSPG staining was drastically reduced in the NTSC-transplanted nerves (Figs. 8P 8Q 8R) . This part of the study indicates that transplanted NTSCs remodel the CSPG within the ON by inducing MMPs within the astrocytes. The data confirm former studies within the retina with implanted NTSCs, which induce MMPs in Müller cells and cleave CSPGs, resulting in intraretinal outgrowth of neuronal processes. ³⁵  

Discussion

Identify of NTSCs in Culture

The generation of chicken NTSCs for characterization and implantation into the cut rat ON was the purpose of this study, which promises to be a new approach to understanding mechanisms of ON repair. Chicken neural tube–derived cells with stem cell features are defined as heterogeneous cells that are capable of self-renewal and also multipotentiality. ^{43 44 45} In the present study, we sought to determine stem cell phenotypes by using immunocytochemical staining for nestin (a stem cell marker), MAP2 and β-III-tubulin (neuronal markers), and GFAP (an astrocyte marker). The fact that most cells propagated were nestin-positive and about half of them expressed GFAP indicates that they remained stem cell typical, with a tendency to share in common GFAP, which points to glial progenitor cells that show a potential to promote spinal cord repair. ⁴⁶ Therefore, their appearance does not contradict their use for engrafting. Almost none of the cells differentiated into neural cells in culture under the culture conditions selected in this study. In addition to immunocytochemistry, viability and doubling time of the NTSCs indicated that most of the cells retained stem cell features. 

One goal of this study was to maximize cell survival and proliferation. Fibroblast growth factors or other mitogens that have been found to support proliferation of stem cells derived from the mammalian forebrain ⁴⁷ were not added to our cultures. Despite the observation that chicken NTSCs behave as a cell suspension in the absence of inactivated feeder cells, ^{43 44} they loosely attach to the cell culture dish. Using immunostaining, we found expression of a heterogeneous population of growth factors. The classic neurotrophins BDNF and CNTF are expressed by approximately 70% and 50% of the cells, respectively. Both factors, as well as other neurotrophins, have been shown to support regenerative growth of axons and survival of axotomized RGCs, respectively. ^{8 11 48 49 50 51} In addition to these classic neurotrophins, several calcium-binding proteins such as calbindin, calretinin, calcineurin, and oncomodulin were expressed by more that half of the cells, except for calbindin, which was expressed by approximately one third. It is of particular interest that at least one of these proteins, oncomodulin, has been implicated recently in supporting regenerative axon growth both in dissociated RGCs and in vivo. ²³ Finally, β- and γ-crystallins are also produced by more than 50% and more than 75% of cells, respectively. At least one member of the crystallins, crybb2, has been implicated in supporting axonal regeneration in RGCs and neurite-sprouting in primary hippocampal neurons in vitro. ⁴¹ Conceptually, the cultured NTSCs fulfilled the criteria for implantation as a source of producing and perhaps secreting trophic factors to facilitate growth of axons within the ON. 

Grafting of Precultured Cells into the Cut Optic Nerve and Axonal Growth

Recent studies have revealed that NTSCs are suitable to be transplanted in models of neurodegeneration. ^{52 53} Most of the concepts foresee that stem and progenitor cells are capable of recovering lost neuronal function by replacing neurons that have degenerated. ^{54 55 56 57 58 59 60 61 62} The alternative concept is that NTSCs or NPCs are capable of rescuing imperiled host neurons by their ability to secrete trophic and/or neuroprotective agents after transplantation. ⁶¹ As an example, in a recent study, retina- and brain-derived NPCs were shown to support growth of neurites within the retina. ³⁵  

The present study was based on this second concept and focused on characterization and cultivation of chicken NTSCs that can be used for xenotransplantation. ⁶² Xenografted pig olfactory sheath cells promoted axonal regeneration in rat spinal cord. ⁶³ Grafting of human fetal cells into the rat spinal cord restores behavioral deficits, ⁶⁴ whereas mouse embryonic stem cells (ESCs) have the ability to survive without immunosuppression and will migrate and differentiate in RPE-cells within the host environment. ^{65 66 67} This includes the ability to differentiate into mature neurons, which form synapses with the existing host network. ⁶⁸ Pfeifer et al. ⁶⁹ found that cotransplantation of neural tube progenitor cells (NTPCs) and fibroblasts results in the loss of the lesion cavity, and the cells differentiate into glial cells. It was concluded from this study that the GFAP-positive cells provide a scaffold for neurofilament-positive corticospinal axons. Olfactory ensheathing cells support sprouting when grafted into the adult ON. ⁷⁰ The present findings support these data and suggest that transplanted chicken NTSCs possess the ability to survive and support axonal regeneration. This potential was documented by immunohistochemical detection of axons that were seen to transcend the region of surgery and elongate within the ON distal to the site of injury. The fact that the parent cell bodies within the retina can be retrogradely labeled with gold tracer from the midbrain 6 to 8 weeks after implantation suggests that their axons have arrived there by this time after surgery. 

Which Factors Permit and Facilitate Regrowth of Axons?

The upregulation of MMP2 and -14 observed within the grafted ONs may be one of the mechanisms responsible for degradation of CSPGs and thus rendering the microenvironment a permissive one. The local production of MMPs does not explain, however, the growth of axons in the more distal regions within the optic tract and thalamus. Once stimulated to grow through the site of injury, axons may modulate the noninjured environment by growth cone–induced MMPs, although this has to be shown experimentally. However, it is unlikely that implanted NTSCs account for these distant effects. MMP2 plays a crucial role during development and is involved in cell migration, neurite outgrowth, and synaptic plasticity. ^{71 72} Within the injured spinal cord, MMP2 shows strong activity within the inhibitory scar ⁷³ and forms pathways for ingrowing axons. Within the cut ON metalloproteases degrade the inhibitory environment and the scar by regenerating axons. ⁷⁴ Such modulation may occur by changing the NgR/p75 NTR/EGFR axis and may be performed by Schwann cell factors. ⁷⁵ Likewise, the present study showed degradation of CSPGs and upregulation of MMPs. However, it is not clear whether the MMPs were activated by implanted NTSCs directly or by the regenerating axons. Irrespective of how they are induced, astrocyte-derived MMP2 and -14 seem to play an important role. This confirms former studies that neurite outgrowth is enhanced when MPP2 is added, ⁶ and on the other hand that inhibition of MMP2 with GM6001 results in impaired growth of retinal axons. ⁷² In contrast to this degradation of CSPG, neurocan is regulated in brain injuries and in cytokine-treated astrocytes ⁷⁶ indicating that MMPs are produced by astrocytes. The data are also in agreement with the fact that chondroitinase ABC promotes recovery of spinal tracts after spinal cord injury, ⁷⁷ whereas embryonic transplants with neurotrophins increase regeneration after spinal cord trauma. ⁷⁸  

However, regenerative axonal growth is more complex, and no singular mechanism may account for maintenance of the vigorous growth of axons observed. In our study, the NTSCs were positive for several molecules known to stimulate axonal growth within the ON. These observations confirm the proposed inherited capacity of these cells to produce and secrete neurotrophic factors. ⁶¹ Of them, BDNF has been shown to support regeneration of retinal axons in vitro, ² whereas CNTF supports survival of axotomized RGCs. ^{8 11 49 50 79 80} Coexpression of the two factors may facilitate better survival and regrowth in the paradigm used in our study. The calcium-binding oncomodulin has been recently identified as a macrophage-derived growth factor for RGCs and peripheral sensory neurons. ²³ It is likely that NTSC-derived oncomodulin operates through a similar mechanism within the ON containing engrafted NTSCs. Yet another possibility for the observed regrowth of axons is that further calcium-binding proteins or unidentified neurotrophins are involved. More likely, crystallins of the β and γ families are contributors to the regenerative growth. At least one isoform of the β-2 crystallins, crybb2, has growth-promoting activity, as shown in cultured RGCs, retinal explants, and hippocampal neurons in vitro. ^{40 41} The assembly of growth cones from the proximal stump of the cut ON and their movement through the scar and the distal stump is, however, a complex choreographed event that is likely to involve more factors than described. 

 Supported by Deutsche Forschungsgemeinschaft (DFG) Grant Th 386/10-3 (ST) and the IZKF, Münster.

 Submitted for publication November 16, 2007; revised March 14 and April 2, 2008; accepted May 30, 2008.

 Disclosure: P. Charalambous, None; L. A. Hurst, None; S. Thanos, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement ” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Isolation and dissociation of neural tube–derived cells. (A, B) The rostral neural tube (e.g., prospective brain) region of chicken embryos was microdissected and collected in a vial containing papain (C). After dissociation, the cells were cultured in 12-well plates (D). Initially, the cells showed various morphologies (E). At later stages in culture, the spindle cells disappeared, and round cells of uniform morphology were predominant (F). (G, H) Counts revealed approximately 20% of spindle cells at 1 day in culture and virtually none after 7 days. (I–K) The spherical cells became confluent after 2 days and maintained this morphology at 1 (I) and 4 (J, K) weeks in culture. (L–N) Calcein-staining (L) (after 8 weeks in subconfluent culture) revealed that more than 80% of the cells were stained, and only approximately 20% were dead (M). In the overlay (N), the ratio of dead and alive cells is visible. (O) The mean doubling time of cultured cells was 5.8 days over 5 weeks in vitro, and therefore typical of undifferentiated NSCs. (P) Calcein-determined vitality of the cells when determined weekly up to 8 weeks in culture.

Figure 2.

 

Embryonic NTSC markers. (A–C) immunocytochemistry with nestin-antibody (A) revealed that most of the cells seen in phase-contrast (B) were stainable (C). (D) Quantification of different markers revealed that only 2% were β-III-tubulin and 2.4% MAP-2 positive; 49.5% expressed GFAP, and 80% were nestin-positive. (E) The TRAP-assay showed that the ratio between the positive and negative control was >0.2 throughout the time of culture up to 60 days. (F–H) Staining with antibodies to bFGF revealed that NTSCs were positive and indicated that the cells produced bFGF for their own survival.

Figure 3.

 

Expression of neurotrophic and calcium-binding factors. Cultured NTSCs expressed β-crystallin (A–C), γ-crystallin (D–F), BDNF (G–I), CNTF (J–L), calcineurin (M–O), calretinin (P–R), and oncomodulin (S–U). (V) Quantification of the cells producing either of the proteins as a percentage of the total cells counted. Mean ± SD of three different samples after 2 weeks in culture.

Figure 4.

 

Engrafted cells induce neurite growth–promoting factors in vivo. (A) Western blot analysis showing that implantation of NTSCs induces expression of β- and γ-crystallin and CNTF within the cut ON when compared with control ONs with no cell implantation. (B) Densitometric quantification of the bands shows a clear upregulation of factor production. Actin was used as the control.

Figure 5.

 

(A) Scheme of the eye and ON showing the site of cut and implantation. (B) Engrafted NTSCs remained within the site of ON cut (green, axonin-1 marker). Some of the cells migrated into the distal part of the ON and intermingled with GFAP-stained astrocytes (red). Blue: DAPI. (C, D) Control ON without NTSCs shows GAP-43-stained axon stumps within the proximal ON stump, but virtually no penetration through the site of the cut (black arrow between A and C). (E, F) Engrafted NTSCs (nestin-staining, green) within the injury rim resulted in vigorous repopulation of the distal ON with GAP-43 (red) axons. Some axons were visible passing thought the site of the cut. Blue: DAPI. (G) Typical alignment of axons (red) within the distal ON segment close to the injury (1 mm). (H) Alignment of axons within the most distant part (6 mm) of the distal ON. (I) Quantification of GAP-43 positive axons in control specimens revealed a decrease in axon stumps within both the proximal and distal ON segments over the 4 weeks after injury. When NTSCs were engrafted at the site of injury, a marked number of axons were determined within the nerve 2 weeks later and had even increased by 4 weeks after surgery.

Figure 6.

 

Assessment of RGCs by retrograde staining from the brain. (A, B) Retrogradely filled RGCs within the flatmounted retina 6 weeks after surgery and 1 week after injection of gold label into the contralateral SC (C, D). Eight weeks after surgery and 1 week after dye injection into the SC, numerous retrogradely filled RGCs were seen throughout the retina. (E) At higher magnification, different sizes of ganglion cells bodies were discernible. (F) Counts of retrogradely labeled RGCs at 6 and 8 weeks showed a marked increase to ∼600 RGCs/mm² (mean ± SD, n = 3 retinas).

Figure 7.

 

Identification of engrafted NTSCs (nestin, NF70) in relation to immigrating macrophages (ED-1). (A–C) Localization of ED-1-positive macrophages (green) and nestin-positive NTSCs (red) revealed an intermingling of the cells at the site of injury (G). (D–F) Localization of oncomodulin and NF70 showed a high degree of double staining and confirmed that oncomodulin was mainly expressed by NTSCs (H).

Figure 8.

 

Longitudinal sections though the ON stained for MMPs. (A–C) Control ONs lacking NTSCs showed marginal staining for MMP-2 (A), intense staining with GFAP (B), and colocalization of both antigens (C). Blue: DAPI staining of astrocytic palisades. (D–F) Implantation of NTSCs resulted in enhanced staining of MMP-2 (D) and strong colocalization with GFAP-positive astrocytes (E, F). (G–I) Staining for MMP-14 in control samples lacking NTSCs showed marginal expression around the astrocytes. (J–L) When the NTSCs were implanted, an upregulation and colocalization with GFAP-positive astrocytes was observed (L). (M–O) CSPG expression within control ONs lacking NTSCs. Staining for CSPG (M) showed periastrocytic localization of the antigen (N, O). In contrast, the staining substantially decreased (P) in ONs engrafted with NTSCs (P–R). Whenever visible, CSPGs were in close proximity to GFAP-positive astrocytes.

Figure 9.

 

Expression of MMP-2 within the ON. (A) Western-blots showed increasing expression of MMP-2 in cut and crushed ONs compared with the untreated control. (B) In the ONs that received NTSCs without cut, expression of MMP-2 was comparable with the cut and crushed ONs. In contrast, densitometry of cut and NTSC-treated ONs showed a marked upregulation (C).

The authors thank Mechthild Langkamp-Flock and Mechthild Wissing for technical assistance, Peter Sonderegger for providing the antiserum to axonin-1, Uwe Schröer for critical discussions during preparation of the work, and Magdalena Reis for typing the manuscript.