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

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

Petar Charalambous,1,2 Louise A. Hurst,1,3 and Solon Thanos1,2

1 2 PURPOSE. Injury to the adult optic nerve, caused mechanically provided by a peripheral nerve graft or culture in vitro, or by diseases, is still not reparable because the retinal ganglion regeneration is possible. Three obstacles stand in the way of cells (RGCs) are not allowed to regrow their axons and die regeneration within the optic nerve: first, the presence of retrogradely, although they possess the intrinsic propensity to inhibitory molecules, such as Nogos,3,4 chondroitin sulfate regenerate axons in experimental conditions. proteoglycans (CSPGs),5–7 and the reactive astrocytes that ETHODS make-up the glial scar; second, lack of appropriate stimulatory M . In vitro propagated embryonic stem cells derived 8–10 from the early chicken neural tube (NTSCs) were used to cues such as neurotrophic factors ; third, loss of retinal ganglion cells (RGCs) through the initial primary assault on the examine whether transplanted NTSCs produce growth-pro- 11,12 moting factors and pave the microenvironment, thus facilitat- optic nerve. ing axonal regeneration within the rat optic nerve. Numerous studies have been conducted to unravel the molecular mechanisms associated with the inhibition of RESULTS. NTSCs survived within the site where the optic nerve growth cone formation and stimulation of regrowth. However, had been cut and continued to be nestin-positive, thus preserv- the mechanisms underlying regrowth of axons from cut axonal ing their undifferentiated cell phenotype. Transplanted NTSCs stumps (de lesio formation of growth cones) are unclear. Based activated the matrix metalloproteases (MMP)-2 and -14 in glial on the appearance of Nogos within the optic nerve,3,13 strat- fibrillary acidic protein (GFAP)-positive optic nerve astrocytes. egies intended to block inhibitory molecules within the distal MMP2 production correlated with immunohistochemically visible optic nerve have been developed.13–18 Alternatively, stimula- degradation of inhibitory chondroitin sulfate proteoglycans tion of axonal regeneration was successful when the intrareti- (CSPGs). In addition, NTSCs produced a panoply of neurite-pro- nal compartments of the RGCs were treated with various moting factors including oncomodulin, ciliary neurotrophic fac- ␤ ␥ neurotrophic factors, including induction of inflammatory factor, brain-derived neurotrophic factor and crystallins and . Cut tors8,10,17–23 and noninflammatory activities such as lenticular axons intermingled with NTSCs and passed through the zone of factors acting directly on RGCs.24–26 All strategies of treatment injury to enter the distal optic nerve over long distances, arriving have to take into account that after transection of the optic at the thalamus and midbrain. nerve, there is delayed death by apoptosis of the RGCs11,27 CONCLUSIONS. This study showed evidence that paving of the accompanied by abortive sprouting.28 To this end, approaches distal optic nerve microenvironment with proteolytically ac- to the support of regeneration of the optic nerve should fulfill tive MMPs and providing stem-cell–derived growth factors is a the criteria of reducing death of the RGCs, of stimulating RGCs suitable method for facilitating regenerative repair of the optic to regrow their axons, and of remodeling the optic nerve nerve. Understanding the molecular mechanisms of this repair microenvironment for successful elongation of axons. has fundamental implications for development of NTSC-based One promising strategy that may fulfill the criteria set for subsidiary therapy after neural injuries. (Invest Ophthalmol Vis successful regeneration is the use of neural stem cells (NSCs)29–34 Sci. 2008;49:3513–3524) DOI:10.1167/iovs.07-1473 or neural progenitor cells (NPCs), which activate a matrix metalloproteinase (MMP)–dependent proteolytic mechanism35 and dult retinal ganglion cells (RGCs) fail to extend axons support intraretinal neurite growth via proteolysis of CSPGs.6,36 Awithin the interior of the optic nerve after injury. This Given the capability of NTSCs to produce multiple factors with failure of regeneration is commonly attributed to inhibitory potential activity on axonal growth, we developed a model of factors associated with myelin components and/or the glial NTSC propagation in culture and transplanted these cells at the scar, which is composed of cells and extracellular matrix. site where the optic nerve was cut. The rationale of using chicken However, under the correct permissive cues, such as those 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 From the 1Department of Experimental Ophthalmology, School of showed that xenografted NTSCs (1) are tolerated within the Medicine, University Eye Hospital Mu¨nster, Mu¨nster, Germany; the injured optic nerve, (2) induce the production of MMPs which 2Interdisciplinary Centre of Clinical Research (IZKF), Mu¨nster, Ger- regulate CSGPs, and (3) produce multiple growth-promoting fac- many; and the 3Biomedical Research Centre, Sheffield Hallam Univer- tors such as oncomodulin, ciliary neurotrophic factor (CNTF), sity, Sheffield, United Kingdom. brain-derived neurotrophic factor (BDNF), and crystallins, which Supported by Deutsche Forschungsgemeinschaft (DFG) Grant Th may contribute in an orchestrated way of stimulating lengthy 386/10-3 (ST) and the IZKF, Mu¨nster. axonal regeneration. Indeed, we observed an enhanced regener- Submitted for publication November 16, 2007; revised March 14 ation of RGC axons. and April 2, 2008; accepted May 30, 2008. Disclosure: P. Charalambous, None; L.A. Hurst, None; S. Tha- nos, None MATERIALS AND METHODS The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertise- Preparation and Culture of Chick Embryo Neural ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. Stem Cells Corresponding author: Solon Thanos, Department of Experimental Ophthalmology, University Eye Hospital Mu¨nster, School of Medicine, Fertilized White Leghorn chicken eggs provided by a local supplier Domagkstrasse 15, 48149 Mu¨nster, Germany; [email protected]. were incubated in a 42% humidified incubator at 37°C for approxi-

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mately 45 to 49 hours. This period allowed the embryos to reach Immunocytochemistry approximately stage 10 of embryogenesis according to the Hamburger Primary antibodies included rabbit anti-BDNF (1:500; Chemicon, Te- and Hamilton scale (HH10; 1951; for review see Ref. 37; Fig. 1A). mecula, CA), mouse anti-CNTF (1:100; Chemicon Inc.), rabbit anti- Neural tube–derived NTSCs were obtained by puncturing the tapered calbindin (1:1000; Chemicon), rabbit anti-calcineurin (1:500; Chemi- and windowed end of the egg, removing 5 mL of egg albumen, and con), mouse anti-calretinin (1:1000; Chemicon), rabbit anti-crystallin-␤ microinjecting sterilized Indian ink under the embryo so as to discern (1:400, BSR Laboratory Department of Biochemistry, Hyderabad, In- the orientation of the neural tube and the position of it rostral edge. dia), rabbit anti-crystallin-␥ (1:100; Samuel Ziegler, National Eye Insti- With a pair of tweezers, the chorioallantoic membrane was torn away, tute, Bethesda, MD), mouse anti-CSPG (neurocan, 1:100; Abcam, Cam- and the prospective embryonic head was dissected and transferred bridge, UK), mouse anti-ED1 (1:500; Serotec, Oxford, UK), mouse into the culture medium, which consisted either of chemically defined, anti-GFAP (for astrocytes; at 1:500; Sigma-Aldrich, St. Louis, MO), serum-free microglia-astrocyte medium (Promocell, Heidelberg, Ger- mouse anti-MAP2 (1:500; Sigma-Aldrich), mouse anti-MMP2 (1:200; many) or of serum-containing Dulbecco’s modified Eagle’s medium Chemicon), mouse anti-MMP14 (1:400; Chemicon), mouse anti-neuro- (DMEM, containing high glucose and L-glutamine, but lacking sodium filament 200 kDa (for neurons; 1:400; Sigma-Aldrich), rabbit anti-neu- pyruvate; PAA Laboratories GmbH, Pasching, Austria) supplemented rofilament 70 kDa (1:200; Chemicon), rabbit anti-nestin (for stem cells, with 10% fetal calf serum (FCS, Gold-FCS; PAA Laboratories GmbH), 1:200; Chemicon), and rabbit anti-oncomodulin (1:500 Swant, Bell- containing 1% penicillin and 1% streptomycin (PAA Laboratories inzona, Switzerland), anti rHu-bFGF (C60201, 1:500; Promocell). These GmbH). The cells were dissociated in a solution of phosphate-buffered were visualized with the following secondary antibodies: TRITC-con- saline (PBS, containing 0.3% BSA and 0.1% DNase already dissolved), jugated goat anti-mouse (at 1:300; Sigma), Cy2-conjugated goat anti- methylcellulose (also containing 0.3% BSA and 0.1% DNase already mouse (1:200; Jackson ImmunoResearch Laboratories, Inc.), TRITC- dissolved; Mann-Pharma, Berlin, Germany), and papain (15 U/mL, conjugated goat anti-rabbit (1:400; Sigma-Aldrich), and Cy2-conjugated Sigma-Aldrich, Taufkirchen, Germany), followed by trituration and goat anti-rabbit (1:200; Jackson ImmunoResearch Laboratories Inc.). washing. The final pellet was resuspended in the culture medium, and All antibodies were diluted in PBS. the cells were maintained within the same culture at 37°C with 5% CO2 For immunocytochemical analyses, the cells were fixed with 4% and fed twice per week by replacing half the culture medium. Typi- PFA for 10 minutes. They were subsequently permeabilized by incu- ϫ 4 ϫ 5 cally, 8.75 10 to 2.0 10 cells were seeded in 0.5 to 1.0 mL of bation at Ϫ20°C in methanol for 10 minutes. After three rinses with medium in four wells of a 12-well culture dish (Corning CoStar, Cam- PBS, the cells were incubated in PBS containing 10% FCS (PAA Labo- 5 bridge, MA). Typically, 1.8 ϫ 10 cells were seeded into two wells of ratories GmbH) for blocking before they were stored overnight at 4°C a 12-well culture dish (Corning CoStar). After the cells were cultured, with the appropriate primary antibodies. The following day, the slides viability was assessed with the telomeric repeat amplification protocol were again rinsed three times with PBS before they were incubated (TRAP kit; Roche Medicine, Mannheim, Germany), which assesses with the secondary antibody at room temperature for 1 hour. After 38 telomerase activity. three final rinses in PBS, the slides were embedded in Mowiol with DAPI (1 mg/mL; 4Ј, 6-diamidino-2-phenylindole; Sigma-Aldrich) to stain TRAP Assay and Live/Dead Assay the nuclei. The number of rinses was reduced in later immunocytochemical procedures, to lower cell loss. Control experiments consisted In the TRAP assay, approximately 1 ϫ 105 cells were collected and of incubation of the cells with only the secondary antibody. To quan- washed in PBS. A lysis solution was added and the proteins were tify the data from immunocytochemistry, labeled cells in 12 random- isolated. The telomerase elongated biotin-conjugated primers. After ized fields were counted with the corresponding filter. The numbers of this, PCR was performed, its product denatured and hybridized with cells stained was used to determine the arithmetic mean Ϯ SD. The digoxigenin (DIG)-labeled telomeric sequences, and a peroxidase cou- same 12 fields were then counted with phase-contrast optics to deter- pled anti-DIG antibody added. ELISA was performed to determine the mine the total number of cells. The proportion of cells stained is concentration of the amplified sequence. Briefly, the product was put presented as a percentage of the total. On each glass slide, negative in avidin-coated microplates, TMB (substrate for peroxidase) was controls were stained with the secondary antibody only, and counting was executed as for the experimental samples. 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 Surgical Procedures for ON Regeneration value of the specimen (both arbitrary units for light absorption) greater Experiments were performed with 50 male and female adult rats of the than 0.2, were treated as positive. Sprague-Dawley strain, weighing 200 to 230 g and aged between 8 and The live/dead assay was performed with a kit from Invitrogen- 10 weeks. All experiments were performed in accordance with the Molecular Probes (Eugene, OR). It determined cell viability based on ARVO Statement for The Use of Animals in Ophthalmic and Vision the properties of calcein AM and ethidium homodimer (EthD-1). The Research and were approved by the local Committee for Animal Care. calcein is able to pass through the cell membrane of either living or For microsurgery, the animals received an intraperitoneal injection of dead cells, but only in living cells is it converted by the general action a mixture of 0.2–0.3 mL ketamine sulfate (50–60 mg/kg; Parke-Davis- of esterases, which render it fluorescent (494/517 nm) and imperme- Pfizer, Karlsruhe, Germany) and 0.1 mL xylazine (10–15 mg/kg; Sanofi- able through the cell membrane. On the other hand, EthD-1 permeates Aventis, Frankfurt, Germany) per 200 g body weight. only the membrane of dead cells, wherein it binds to the nucleic acids, Before axotomy of the RGCs, the head of the anesthetized animal which enhances its fluorescence (528/617 nm) approximately 50 was positioned and fixed in a self-constructed headholder. The left times. optic nerve (ON) was surgically exposed in its intraorbital segment, In addition, the live/dead assay with calcein AM was performed which spans 2 to 3 mm beyond the eye cup, taking care to leave the with a kit from Invitrogen-Molecular Probes according to the protocol retinal vascularization intact.10,39 The lacrimal gland was displaced, but of the supplier. Briefly, the cells were incubated with 150 ␮Lofthe left intact, and the superior extraocular muscles were spread to allow reagent containing 2 ␮M calcein AM and 4 ␮M EthD-1 at room tem- access to the ON. An incision was made in the eye-retractor muscle and perature for 30 minutes. Fluorescence was recorded with a fluores- the meninges perpendicular to the axonal orientation, extending over cence microscope (Axiovert; Carl Zeiss Meditec, GmbH, Oberkochen, one third of the dorsal ON. The ON was completely cut with microscis- Germany). Dead red-fluorescing cells were counted in five areas of sors at a distance of 1 to 2 mm behind the eye, leaving the retinal 100,000 ␮m2. The percentage of dying cells was expressed as the vascularization unaffected. The cut ends of the distal and proximal number of red-fluorescing cells divided by the total number of stained nerve stumps were realigned by tightening with two meningeal cells. threads (10.0 silk; Ethicon, Hamburg, Germany). This method ensured

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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 disap- peared, 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 be- came conﬂuent after 2 days and maintained this morphology at 1 (I) and4(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) Cal- cein-determined vitality of the cells when determined weekly up to 8 weeks in culture.

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that no gap existed between the nerve stumps and that the nerve 400ϫ). Because RGC density varies across the retinal eccentricity, we fascicles were topographically realigned. At this point, 10 ␮L of the cell counted cells at each eccentricity (outer, middle, central) from each suspension at a concentration of 0.5 to 1 ϫ 106 cells/mm3 was injected quadrant of the retina, for a total of 12 fields of cells per retina. between the unified nerve stumps with a pulled 50-␮L glass microcap- Results are expressed as the mean Ϯ SD. Statistical analysis was illary adjusted on a syringe (Hamilton, Reno, NV). Slow injection over performed with commercial software (SPSS, Chicago, IL), using the 5 minutes was performed to assure that as many cells as possible nonparametric, unpaired, one-tailed Mann-Whitney test. Significance remained within the cavity. However, despite all preventative mea- was assumed at the 95% confidence level. sures, only part of the fluid and cells remained within the ON cavity, and the remainder drained into the orbital space. Control rats (n ϭ 5) RESULTS underwent the same procedure as the experimental animals, but received a PBS injection into the cut ON instead of the cell suspension. Morphologic Characterization of Cell Phenotypes Sham-operation control animals underwent all steps of surgery, but no nerve transaction and no cell injection (n ϭ 5). After this, the intraor- The chicken embryos reproducibly reached stage HH10 (Fig. bital cavity was overlaid with a sponge (Gelfoam; Pfizer, Zurich, Swit- 1A) after 45 to 49 hours of incubation. Removal of the pro- zerland), to reduce the outflow of cells from the site of injection. The spective head region (Fig. 1B) and collection of approximately entire procedure was performed within 30 to 40 minutes. All micro- 40 heads per vial (Fig. 1C) were sufficient to produce and seed surgery was performed with the aid of a surgical microscope (OPMI 19; NTSCs for the culturing experiments (Fig. 1D). Carl Zeiss Meditec, GmbH) equipped with a camera that enabled the After 24 hours in culture, typical cell cultures were not surgical procedure to be documented photographically in all the ani- confluent (Fig. 1E), and the cells showed either spherical shape mals. The ON head was examined ophthalmoscopically immediately or were differentiated cells, the latter showing either a spindle- after surgery, to ensure that the retinal vasculature was intact. shaped (Fig. 1F) or flattened membranous morphology. The proportion of spindlelike cells was approximately 20% during Tissue Preparation and Immunohistochemistry the first day and decreased to almost 0% after 7 days in culture (Fig. 1G), whereas round cells made up almost 100% of the After survival times ranging 3 days to 2 months, the animals were given cultures by day 7 (Fig. 1H), indicating that the NTSCs may be a lethal dose of anesthetic. The eyes with the nerve segments, with the less differentiated with time in culture. chiasm still attached, were dissected from the muscles and connective When cultured in serum-containing medium the typical tissue and submersion fixed in 4% PFA at 4°C overnight. The speci- cells in the culture dish were of a spherical shape after 48 mens were then embedded in OCT compound (Tissue Tek; Sakura hours in culture (Fig. 1G). This spherical morphology was Finetek, Torrance, CA) and frozen in liquid nitrogen. Longitudinal maintained after 1 (Fig. 1I) to 4 (Fig. 1K) weeks in culture. The frozen sections (12 ␮m) were cut through the ON/chiasm. Sections viability of the cells was assessed with calcein-staining, which were thaw mounted on gelatin-coated glass slides (Superfrost Plus, revealed a high percentage of living cells (Figs. 1L–N). When Fisher Scientific, Pittsburgh, PA) and stored at Ϫ20°C until use for quantified, more than 80% of all cells were viable throughout immunohistochemistry with the antibodies described earlier. the time of observation, which covered 8 weeks in culture (Fig. The number of axons extending beyond the site of the cut and 1P). The doubling time of cultured cells was examined over 5 suture was averaged from 30 longitudinal ON (12-␮m) sections per 1 weeks and was found to take between 5.8 days (Fig. 1O) and and 6 mm from the site of the cut. To calculate the total number of indicated a typical growth of NTSCs in vitro. axons, we used the equation of Leon et al.,21: ⌺ ad ϭ ␲r2x(average Next, markers for undifferentiated cells were analyzed, axons/mm)/t. The number of axons per millimeter was obtained by starting with nestin, a characteristic filament protein. It ap- measuring the ON width at 1 and 6 mm from the site of the cut, where peared that nestin was expressed in the cultured cells (Fig. t represents thickness of the section (10–12 ␮m), and r represents the 2A–C). Quantification revealed that approximately 80% ex- radius of the ON. Statistical analysis was performed with commercial pressed this marker (Fig. 2D), whereas approximately 49.5% software (SPSS ver. 12.0 for Windows; SPSS, Chicago, IL), using a showed positive staining for GFAP, and only less than 2.4% of nonparametric, unpaired, one-tailed Mann-Whitney test. Significance all cells expressed neuron-specific markers such as ␤-III-tubulin was assumed at the 95% level of confidence. and MAP-2 (Fig. 2D). Double staining for GFAP and nesting revealed that approximately 15% of all nestin-positive NTSCs Retrograde Labeling of the Regenerating Pathway expressed GFAP, too (data not shown). When the telomerase The characteristics and number of RGCs contributing to regeneration activity of the cells was assessed in the TRAP assay between the time of seeding and 60 days in culture, the difference ratio was of the retinas were determined by labeling the RGCs in a retrograde Ͼ fashion from the reinnervated superior colliculus (SC) with gold label 0.2 at all times of examination, compared with the ratio in (Fluorogold; Fluorochrome, Englewood, CO) 6 to 8 weeks after ON control cultures (Fig. 2E), indicating that the cells retained the surgery, with analysis 5 to 6 days later. A midline skin incision was features of undifferentiated cells. It appeared from these stud- made over the midbrain area, and a hole was drilled through the bone. ies that most of the NTSCs cultured from the anterior chicken A pulled-glass capillary (tip diameter, 20 ␮m) was filled with 10 ␮Lof neural tube remained largely undifferentiated when cultured in 3% gold label and attached to a syringe (Hamilton). The cortical tissue vitro. To examine whether the NTSCs produce trophic factors over the right SC was aspirated by means of a vacuum pump, to allow that support their own survival, anti-bFGF staining was per- access to the visible SC and injection of the gold label into the formed and showed that bFGF was expressed in the NTSCs superficial layers of the SC. Retrogradely labeled RGCs were examined (Figs. 2F–H), which likely explains why the cells can be cul- in retinal wholemounts. For this, the rats were killed by an overdose of tured without additional supplementation with FGF. anesthetic (7% chloral hydrate); their eyes were enucleated; the cor- Production of Factors with nea, 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 Growth-Promoting Potential washed with PBS for 5 minutes, and the retina was dissected from the We hypothesized that cultured NTSCs are capable of express- remaining tissue and flatmounted on sartorius nitrocellulose filter with ing factors that may be involved in support of axonal growth the photoreceptor layer facing the filter. The retinas were stored in 4% and examined this possibility using immunohistochemistry. PFA in PBS at 4°C until they were used for RGC counting. Cell density When tested with antibodies to crystallins of the ␤ and ␥ was assessed with the aid of a fluorescence microscope equipped with families, they appeared to produce both classes of crystallins a grid of 200 ϫ 275 ␮m (e.g., 49,000 ␮m2 at a final magnification of (Figs. 3A–F). One of these substances, crystallin ␤-b-2 (crybb2)

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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 ␤-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 Ͼ0.2 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.

has recently been identified to be innate to RGCs and supports polygonal cell shapes and typical features of NTSCs. The cells elongation of regenerating ganglion cell axons in culture.40,41 were detached from the culture and washed in PBS, transferred In contrast, the cells were negative to the ␣-crystallins (data not into DMEM and injected into the cut ON of adult rats. After the shown). Of the neurotrophic factors tested, NTSCs were stain- meninges were resutured to reunify the ON edges and a sur- able with antibodies to BDNF (Figs. 3G–I) and to CNTF (Figs. vival period of 1 to several weeks, the rats were euthanatized 3J–L). Both factors have been tested to influence survival of and the ONs were cryosectioned and processed for immuno- axotomized RGCs and axonal regrowth in vivo and in histochemistry. With the use of antibodies to axonin-1, which vitro.8,11,42 Of the calcium-binding proteins tested, approxi- is a specific marker for chicken neurons, transplanted cells mately 30% of the cells stained positive to calbindin, but more could be identified within the ON and mainly within the site of than 60% of the cells expressed calcineurin (Figs. 3M–O) and the ON cut (Figs. 5A, 5B). Concerning regeneration of axons in calretinin (Figs. 3P–R). More than 60% of the NTSCs expressed control ONs without NTSCs, GAP-43-positive axonal stumps oncomodulin (Figs. 3S–U), a member of the calcium-binding were restricted to the proximal ON (Fig. 5C), and no axons proteins that has recently been shown to be a macrophage- were seen behind the site of injury (Fig. 5C) or more distally derived protein with high potential for supporting axonal re- (Fig. 5D). Using double staining with antibodies to GAP-43, we 23 generation both in vitro and in vivo. This part of the study could observe ON axons to have grown over considerable showed that cultured NTSCs expressed several different pro- distances beyond the site of the cut and NTSC engraftment. In teins that may become regeneration-supporting factors when particular, when double-staining with nestin antibody and the cells are transplanted into the injured ON. To examine GAP-43-antibody was performed, regenerating axons were whether NTSCs produce similar neurite-promoting factors in seen to pass through the site of the injury (Fig. 5E), which was vivo, we examined the injured and cell-injected ONs (n ϭ 3) populated with NTSCs (Figs. 5E, 5F). Although few labeled with immunoblot analysis and compared them with control NTSCs were visualized within each cryostat section (Figs. 5E, ϭ specimens (n 3) lacking NTSC injection. It appeared that no 5F), the mean number of cells determined from the total ␤ ␥ -or -crystallin and no CNTF were detectable in the control sections of each nerve was 163 Ϯ 112 NTSCs (n ϭ 7), indicat- specimens (Fig. 4). In contrast, all three proteins were highly ing that many of the cells injected were displaced. Both the stained in the ONs after engrafting of the NTSCs (Figs. 4), proximal and distal stumps of the ONs were evenly populated indicating that the cells also induced the production of regen- with axons (Figs. 5G, 5H, respectively). When the axons were eration-promoting factors in vivo. quantified from sections throughout the ON, up to 300 axons were found within the most distal ON, whereas close to the Implantation of Precultured NTSCs injury, it was populated with more than 1000 axons (Fig. 5I). To examine further whether implanted NTSCs support re- Most distal axons were detected close to the chiasm region growth of retinofugal axons, precultured NTSCs were en- which is several millimeters away from the site of the cut. In grafted into an ON cavity formed at the site of the ON cut. For addition to axons interacting with the implanted NSCs, regen- this purpose, cells remained in culture up to 4 weeks after erating axons have to pass along ON astrocytes and extracel- seeding on laminin and formed an almost confluent layer with lular matrix while traversing its interior. Double staining with

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FIGURE 4. Engrafted cells induce neurite growth–promoting factors in vivo. (A) Western blot analysis showing that implantation of NTSCs induces expression of ␤- and ␥-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.

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–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

FIGURE 3. Expression of neurotrophic and calcium-binding factors. Cultured NTSCs expressed ␤-crystallin (A–C), ␥-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 Ϯ SD of three different samples after 2 weeks in culture.

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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 contralat- eral 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 ϳ600 RGCs/mm2 (mean Ϯ SD, n ϭ 3 retinas).

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–C). 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 non- phosphorylated neurofilament NF70 staining which is specific FIGURE 5. (A) Scheme of the eye and ON showing the site of cut and to chicken NTSCs, there was a high degree of colocalization implantation. (B) Engrafted NTSCs remained within the site of ON cut (Figs. 7D–F), indicating that NTSCs express oncomodulin after (green, axonin-1 marker). Some of the cells migrated into the distal transplantation. In contrast, no oncomodulin- or NF70-positive part of the ON and intermingled with GFAP-stained astrocytes (red). cells were seen in the control ONs which either underwent a Blue: DAPI. (C, D) Control ON without NTSCs shows GAP-43-stained sham operation or a cut of the nerve without engraftment. axon stumps within the proximal ON stump, but virtually no penetra- Approximately 10% of all cells within the site of injury were tion through the site of the cut (black arrow between A and C). (E, F) either NF70- or oncomodulin-positive (Fig. 6H) of the cells Engrafted NTSCs (nestin-staining, green) within the injury rim resulted were shown to express both proteins. Of this population, in vigorous repopulation of the distal ON with GAP-43 (red) axons. Some axons were visible passing thought the site of the cut. Blue: which accounts for one fifth of all cells, 70% were double- DAPI. (G) Typical alignment of axons (red) within the distal ON stained and account for approximately 14% of the total popu- segment close to the injury (1 mm). (H) Alignment of axons within the lation. most distant part (6 mm) of the distal ON. (I) Quantification of GAP-43 positive axons in control specimens revealed a decrease in axon Alterations of the ON-Environment of NTSCs stumps within both the proximal and distal ON segments over the 4 in Grafting weeks after injury. When NTSCs were engrafted at the site of injury, a marked number of axons were determined within the nerve 2 weeks Immunohistochemistry to MMP-2, MMP-14, and chondroitin later and had even increased by 4 weeks after surgery. sulfate proteoglycans (CSGPs) was performed to examine po-

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FIGURE 7. Identiﬁcation of engrafted NTSCs (nestin, NF70) in rela- tion 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).

tential mechanisms of NTSC-transplantation on axonal growth. DISCUSSION When MMP-2 was studied, marginal immunostaining was observed in untreated control ONs and in control nerves that Identify of NTSCs in Culture were cut, but did not receive transplants (Figs. 8A–C). Quan- The generation of chicken NTSCs for characterization and tification of MMP-2-positive cells in these controls revealed implantation into the cut rat ON was the purpose of this study, only individual cells that were weakly stained and were not which promises to be a new approach to understanding mech- countable. In contrast, MMP-2 was highly upregulated in cut anisms of ON repair. Chicken neural tube–derived cells with ONs that received NTSCs (Figs. 8D–F). The MMP-2 immuno- stem cell features are defined as heterogeneous cells that are staining colocalized with GFAP (Fig. 8F), indicating that astro- capable of self-renewal and also multipotentiality.43–45 In the cytes accounted for its production. Immunostaining to MMP-14 present study, we sought to determine stem cell phenotypes was virtually absent in control cultures without NTSCs (Figs. by using immunocytochemical staining for nestin (a stem cell 8G–I) and was highly upregulated in ONs that had received marker), MAP2 and ␤-III-tubulin (neuronal markers), and GFAP NTSCs (Figs. 8J–L). MMP-14 also colocalized with astrocytic (an astrocyte marker). The fact that most cells propagated GFAP (Fig. 8L). To examine MMP-2 production within the ON, were nestin-positive and about half of them expressed GFAP we performed Western blot analyses. It appeared that MMP-2 indicates that they remained stem cell typical, with a tendency to share in common GFAP, which points to glial progenitor staining increased when the ON was cut or crushed (Figs. 9A, 46 9C). In control specimens that received NTSCs in the nerve cells that show a potential to promote spinal cord repair. (n ϭ 3) without crush or cut, MMP-2 staining was comparable Therefore, their appearance does not contradict their use for (Fig. 9B). In contrast, a higher MMP-2 staining was observed in engrafting. Almost none of the cells differentiated into neural cells in culture under the culture conditions selected in this cut ONs after NTSCs engraftment (Figs. 9B, 9C). This finding study. In addition to immunocytochemistry, viability and dou- confirmed the immunohistochemistry, which showed that the bling time of the NTSCs indicated that most of the cells re- highest MMP-2 staining occurred in cut and cell engrafted ONs. tained stem cell features. In parallel sections from the same ONs (control and exper- One goal of this study was to maximize cell survival and imental specimens), CSPG was visibly expressed in control proliferation. Fibroblast growth factors or other mitogens that normal and cut ONs without NTSCs (Figs. 8M–O). In contrast, have been found to support proliferation of stem cells derived CSPG staining was drastically reduced in the NTSC-trans- from the mammalian forebrain47 were not added to our cul- planted nerves (Figs. 8P–R). This part of the study indicates tures. Despite the observation that chicken NTSCs behave as a that transplanted NTSCs remodel the CSPG within the ON by cell suspension in the absence of inactivated feeder cells,43,44 inducing MMPs within the astrocytes. The data confirm former they loosely attach to the cell culture dish. Using immunostain- studies within the retina with implanted NTSCs, which induce ing, we found expression of a heterogeneous population of MMPs in Mu¨ller cells and cleave CSPGs, resulting in intraretinal growth factors. The classic neurotrophins BDNF and CNTF are outgrowth of neuronal processes.35 expressed by approximately 70% and 50% of the cells, respec-

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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.

tively. Both factors, as well as other neurotrophins, have been particular interest that at least one of these proteins, onco- shown to support regenerative growth of axons and survival of modulin, has been implicated recently in supporting regener- axotomized RGCs, respectively.8,11,48–51 In addition to these ative axon growth both in dissociated RGCs and in vivo.23 classic neurotrophins, several calcium-binding proteins such as Finally, ␤- and ␥-crystallins are also produced by more than 50% calbindin, calretinin, calcineurin, and oncomodulin were ex- and more than 75% of cells, respectively. At least one member pressed by more that half of the cells, except for calbindin, of the crystallins, crybb2, has been implicated in supporting which was expressed by approximately one third. It is of axonal regeneration in RGCs and neurite-sprouting in primary

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planted 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 ex- plain, 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 scar73 and forms pathways for ingrowing axons. FIGURE 9. Expression of MMP-2 within the ON. (A) Western-blots Within the cut ON metalloproteases degrade the inhibitory showed increasing expression of MMP-2 in cut and crushed ONs 74 compared with the untreated control. (B) In the ONs that received environment and the scar by regenerating axons. Such modulation may occur by changing the NgR/p75 NTR/EGFR axis NTSCs without cut, expression of MMP-2 was comparable with the cut 75 and crushed ONs. In contrast, densitometry of cut and NTSC-treated and may be performed by Schwann cell factors. Likewise, the ONs showed a marked upregulation (C). present study showed degradation of CSPGs and upregulation of MMPs. However, it is not clear whether the MMPs were

41 activated by implanted NTSCs directly or by the regenerating hippocampal neurons in vitro. Conceptually, the cultured axons. Irrespective of how they are induced, astrocyte-derived NTSCs fulfilled the criteria for implantation as a source of MMP2 and -14 seem to play an important role. This confirms producing and perhaps secreting trophic factors to facilitate former studies that neurite outgrowth is enhanced when MPP2 growth of axons within the ON. is added,6 and on the other hand that inhibition of MMP2 with 72 Grafting of Precultured Cells into the Cut Optic GM6001 results in impaired growth of retinal axons. In contrast to this degradation of CSPG, neurocan is regulated in Nerve and Axonal Growth brain injuries and in cytokine-treated astrocytes76 indicating Recent studies have revealed that NTSCs are suitable to be that MMPs are produced by astrocytes. The data are also in transplanted in models of neurodegeneration.52,53 Most of the agreement with the fact that chondroitinase ABC promotes concepts foresee that stem and progenitor cells are capable of recovery of spinal tracts after spinal cord injury,77 whereas recovering lost neuronal function by replacing neurons that embryonic transplants with neurotrophins increase regenera- have degenerated.54–62 The alternative concept is that NTSCs tion after spinal cord trauma.78 or NPCs are capable of rescuing imperiled host neurons by However, regenerative axonal growth is more complex, and their ability to secrete trophic and/or neuroprotective agents no singular mechanism may account for maintenance of the after transplantation.61 As an example, in a recent study, retina- vigorous growth of axons observed. In our study, the NTSCs were and brain-derived NPCs were shown to support growth of positive for several molecules known to stimulate axonal growth neurites within the retina.35 within the ON. These observations confirm the proposed inher- The present study was based on this second concept and ited capacity of these cells to produce and secrete neurotrophic focused on characterization and cultivation of chicken NTSCs factors.61 Of them, BDNF has been shown to support regenera- that can be used for xenotransplantation.62 Xenografted pig tion of retinal axons in vitro,2 whereas CNTF supports survival of olfactory sheath cells promoted axonal regeneration in rat axotomized RGCs.8,11,49,50,79,80 Coexpression of the two factors spinal cord.63 Grafting of human fetal cells into the rat spinal may facilitate better survival and regrowth in the paradigm used in cord restores behavioral deficits,64 whereas mouse embryonic our study. The calcium-binding oncomodulin has been recently stem cells (ESCs) have the ability to survive without immuno- identified as a macrophage-derived growth factor for RGCs and suppression and will migrate and differentiate in RPE-cells peripheral sensory neurons.23 It is likely that NTSC-derived onco- within the host environment.65–67 This includes the ability to modulin operates through a similar mechanism within the ON differentiate into mature neurons, which form synapses with containing engrafted NTSCs. Yet another possibility for the ob- the existing host network.68 Pfeifer et al.69 found that cotrans- served regrowth of axons is that further calcium-binding proteins plantation of neural tube progenitor cells (NTPCs) and fibro- or unidentified neurotrophins are involved. More likely, crystallins blasts results in the loss of the lesion cavity, and the cells of the ␤ and ␥ families are contributors to the regenerative differentiate into glial cells. It was concluded from this study growth. At least one isoform of the ␤-2 crystallins, crybb2, has that the GFAP-positive cells provide a scaffold for neurofila- growth-promoting activity, as shown in cultured RGCs, retinal ment-positive corticospinal axons. Olfactory ensheathing cells explants, and hippocampal neurons in vitro.40,41 The assembly of support sprouting when grafted into the adult ON.70 The growth cones from the proximal stump of the cut ON and their present findings support these data and suggest that trans- movement through the scar and the distal stump is, however, a

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