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Central migration of neuronal tissue and embryonic stem cells following transplantation along the adult auditory nerve
Brain Research 1026 (2004) 68 – 73 www.elsevier.com/locate/brainres
Research report
Central migration of neuronal tissue and embryonic stem cells following transplantation along the adult auditory nerve Zhengqing Hua,*, Mats Ulfendahla,*, N. Petri Oliviusa,b a
Center for Hearing and Communication Research, and Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden b Department of Otorhinolaryngology, Karolinska University Hospital Solna, Stockholm, Sweden Accepted 10 August 2004 Available online 13 September 2004
Abstract The regeneration of the auditory nerve remains a challenge in restoring hearing. An interesting approach would be to use a cell replacement therapy with the potential to establish connections from the inner ear to the central auditory system. This hypothesis was tested by xenografted (mouse to rat) implantation of embryonic dorsal root ganglion (DRG) neurons and embryonic stem (ES) cells along the auditory nerve in the adult host. DRG neurons were obtained at embryonic day 13–14 in transgenic animals expressing enhanced green fluorescence protein (EGFP). For embryonic stem cells, a tau-GFP ES cell line was used as a donor. The fibers of the auditory nerve in the adult rat were transected through the modiolus at the first cochlear turn, and the biological implants were transplanted into the transection. The transplanted DRG neurons and ES cells survived for a postoperative survival time ranging from 3 to 9 weeks, verified by EGFP/GFP fluorescence, and neurofilament or TUJ1 immunostaining. At 9 weeks following implantation, the implanted DRG neurons were found to have migrated along the auditory nerve in the internal meatus. At the same postoperative time, the ES cells had migrated into the brain stem close to the ventral cochlear nucleus. The results demonstrate not only the survival and migration of xenografted DRG neurons and stem cells along the adult auditory nerve but also the feasibility of a cell replacement therapy in the degenerated auditory system. D 2004 Elsevier B.V. All rights reserved. Theme: Development and regeneration Topic: Sensory systems Keywords: Auditory nerve fiber; Dorsal root ganglion; Hearing loss; Migration; Spiral ganglion; Stem cell
1. Introduction Insults to the sensory epithelium, the organ of Corti, in the mammalian cochlea result in a series of pathological changes in the auditory system. The primary damage is often selective to the sensory epithelium but, as in other afferent systems, degeneration of the spiral ganglion neurons (SGNs) and auditory nerve fibers (ANFs) occurs secondarily to the loss of cochlear sensory epithelium [12,21]. Moreover, in some clinical cases, i.e. following acoustic neuroma surgery, the ANFs have to be completely * Corresponding authors. Tel.: +46 8 51776307; fax: +46 8 301876. E-mail addresses: [email protected] (Z. Hu); [email protected] (M. Ulfendahl). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.08.013
removed. It is thus of interest to explore novel approaches to restore auditory function. One strategy would be to replace degenerated or absent SGNs and ANFs using a cell replacement therapy. Combined with a cochlear prosthesis, the sound can be transduced into electrical signals and then stimulate the implanted cells directly, thus bypass the degenerated sensory epithelium, SGNs and ANFs. Several possible tissues are candidates as donors for a cell replacement strategy in the auditory system. In addition to SGNs other types of neuronal tissues could be feasible. Recent observations have shown that embryonic dorsal root ganglion (DRG) neurons not only survive in the inner ear [9,15,16] but also migrate into the spiral ganglion region and along its peripheral dendrites projecting to the organ of Corti [9]. Stem cells are another interesting candidate for a cell
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replacement therapy. Stem cells have the capacity for selfrenewal and differentiation into diverse cell types. Embryonic stem (ES) cells are pluripotent and thus able to differentiate into a variety of cell types, i.e. ectoderm, mesoderm and endoderm [3,8]. It has been documented that neural stem cell can generate olfactory bulb neurons and after an injury, their proliferation rate increases and their progeny migrates to the site of the lesion, where they differentiate into astrocytes [10]. Further, stem cells have been tested in the treatment of nervous system disease, i.e. Parkinson’s disease, Huntington’s disease, Alzheimer’s disease and spiral cord injury [1,2,23,24,26]. Following transplantation into mature retina, the neuronal differentiation and morphological integration of adult neural progenitor cells were also identified [18,27,28]. We have recently reported that at 4 weeks following transplantation into the mature inner ear, ES cells survived and migrated along the ANFs leading from the organ of Corti where they were seen to differentiate into neurons (Hu et al., submitted). Furthermore, the implantation of DRGs and stem cells along the vestibulocochlear nerve illustrates the survival of implanted cells up to 4 weeks (Regala et al., submitted). In order for a cell replacement therapy to be of potential clinical interest, both a considerable survival time and the migration of implanted cells towards functionally important regions are essential. In this study, we explore the possibilities of embryonic DRG neurons and tau-tagged mouse ES cells to survive for a longer time, e.g. 9 weeks along the adult rat ANFs and to migrate centrally into the brain stem close to the cochlear nucleus.
2. Materials and methods All animal procedures were approved by the regional ethical committee (approval nos. 283a–d/02, 464/03, and 58/03). Implantation was made into a total of 40 Sprague– Dawley rats (body weight 250–280 g), 20 rats transplanted with DRGs and 20 animals transplanted with tau-green fluorescent protein (tau-GFP) ES cells. Animals transplanted with DRGs and tau-GFP ES cells were further divided into the following groups (c.f. Table 1): 3-week survival (n=6), 6-week survival (n=6) and 9-week survival (n=8). 2.1. Preparation of DRG tissue and tau-GFP stem cells The dissection of donor DRG neurons has been described before [9]. Briefly, the DRG tissues were dissected out from pregnant mice (E13–14) belonging to a line with an enhanced green fluorescent protein (EGFP) cDNA under the control of a chicken beta-actin promoter and cytomegalovirus enhancer (strain C57BL/6-TgN (ACTbEGFP) 1Osb from The Jackson Laboratory). Under aseptic conditions and deep anesthesia (Ketalar; 4 mg/100 g; and Rompun; 1 mg/100 g; i.m.) the abdomen and uterus
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Table 1 Survival and migration of DRGs and ES cells along the auditory nerve DRG neurons
ES cells
3 6 9 3 6 9 weeks weeks weeks weeks weeks weeks Number of animals receiving implantation 6 surgery showing surviving implant 5/6 showing the migration of implant at the basal cochlear turn 4/6 within the internal meatus 1/6 within the brain stem 0/6
6
8
6
6
8
2/6
2/8
4/6
2/6
2/8
1/6 1/6 0/6
2/8 2/8 0/8
2/6 2/6 0/6
1/6 2/6 1/6
2/8 2/8 2/8
The survival of dorsal root ganglion neurons and tau-GFP embryonic stem cells following transplantation along the auditory never fibers in the adult rat. The implanted cells were also found to have migrated centrally towards the brain stem, in close vicinity of the cochlear nucleus.
of the pregnant mice were exposed. The embryos were excised and transferred to tissue culture medium (DMEM, Gibco) where the DRGs were dissected out for later transplantation. The tau-GFP ES cell line (E14Tg2aSc4tp6.3, from the Department of Biomedical Science and the Center for Developmental Biology, University of Edinburgh, Scotland) [17] were cultured using DMEM (Gibco-BRL) supplemented with 15% fetal calf serum (HyClone), 1000 U/ml of mouse recombinant leukemia inhibitory factor (LIF, Invitrogen), 0.1 mM 2-mercaptoethanol (Sigma), 0.1 mM nonessential amino acids, 2 mM glutamine, 100 U/ml of penicillin and 100 mg/ml streptomycin (all obtained from Gibco-BRL). The culture solution was put to a 10 cm culture dish and the dish was kept in an incubator at 37 8C, 5% CO2 and humidity environment. Several days later, the ES cells were harvested and cell density was adjusted to around 104/Al with fresh medium. The cells were kept at 4 8C until transplantation. 2.2. DRGs and tau-GFP ES cells transplantation Transplantation of DRGs and stem cells was made as described previously [9]. Briefly, using a postauricular approach with aseptic conditions, the left bulla was exposed and opened to provide access to the cochlea (under deep anesthesia, same drugs as mouse with a body weightadjusted dosage). A small hole was made at the base of the cochlea to expose the modiolus. The modiolus was opened at the basal cochlear turn and the ANFs were transected completely. The DRGs or the ES cells were transplanted at the transection site along the ANFs. For the ES cell transplantation 5 Al culture medium with tau-GFP ES cells (104/Al) was administered using a microsyringe (Exmire microsyringe, Ito, Fuji, Japan). A small piece of fascia was placed over the cochleostomy and the skin incision was approximated with sutures. In order to reduce the risk of postoperative immunological rejection and infection, the transplanted host animals received daily injections of
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cyclosporin (Novartis Sverige, 0.56 mg/100 g body weight) and doxycycline (Nordic Drugs, 0.24 mg/100 g body weight) intraperitoneally until the day of sacrifice. 2.3. Histology At sacrifice, the animals were anaesthetized with an overdose of pentobarbital (i.p.) and transcardially perfused with 0.9% saline at 37 8C followed by 4% paraformaldehyde and 14% picric acid in 0.1 M phosphate buffer (pH 7.4, 4 8C). The auditory nerve together with the brain stem and a part of the cochlea was dissected out and kept in the fixative for 2 h before being transferred to 0.1 M phosphate buffer, in which they were stored until further processing. The specimens were incubated in 30% sucrose overnight, embedded in tissue freezing medium (Leica Instruments), and sectioned on a Leica CM3050S cryostat at a thickness of 14 Am. Every second section was collected from the entire specimen (auditory nerve together with the brain stem and a part of the cochlea). The sections were analyzed using a Zeiss fluorescent microscope with a digital camera (Nikon Coolpix 5000 or Spot RT, Diagnostic Instrument) or confocal microscopy (Zeiss LSM 510). 2.4. Immunohistochemistry Neurofilament antibody was applied to label the DRG neurons along the auditory nerve transplanted with DRG tissues. Following preincubation in 1% goat serum (Santa Cruz Biotechnology; diluted in 0.1 M phosphate buffer saline) for 30 min at room temperature, the sections were incubated with neurofilament monoclonal antibody (NF-L, 1:100, Santa Cruz Biotechnology) overnight at 4 8C. After rinsing, a goat anti-mouse IgG1-Texas red (1:200, Santa Cruz Biotechnology) was applied for 2 h at room temperature. To identify the neuronal differentiation of the ES cells in the animals transplanted with tau-GFP ES cells, TUJ1 antibody, a neuronal marker, was used (primary antibody, TUJ1 antibody, 1:500, Berkeley Antibody; secondary antibody, cy3-conjugated affiniPure F(abV)2 fragment goat anti-mouse IgG, 1:400, Jackson Immunoresearch Laboratories). To identify glial cell differentiation of ES cells, an antibody against the glial fibrillary acidic protein (GFAP) was used (primary antibody, polyclonal rabbit anti-GFAP antibody, 1:250, DAKO; secondary antibody, amca affinity purified goat anti-rabbit IgG (H+L), 1:400, Vector Laboratories). In order to enhance the fluorescence of GFP-expressing cells, an anti-GFP antibody (polyclonal rabbit anti-GFP antibody, 1:500, Abcam) and appropriate secondary antibody (fluorescein (FITC)-conjugated affiniPure donkey anti-rabbit IgG (H+L), 1:500, Jackson Immunoresearch Laboratories) were used in the study. The sections were observed using the same equipment as above with the appropriate fluorescence filter.
2.5. Cell counting Cell counting was performed in the animals with surviving ES cells. The GFP fluorescence was used to detect the surviving implanted cells and the TUJ1 antibody positive staining was applied to identify the neuronal differentiation of ES cells. The sections from the whole specimen (auditory nerve together with the brain stem and a part of the cochlea) were used.
3. Results 3.1. Survival and migration of DRGs along ANFs Implanted DRG neurons were identified (by detection of EGFP fluorescence) in histological sections of ANFs connected with the brain stem for up to 9 weeks following implantation (Fig. 1A,D,E). The morphology of the surviving DRG neurons appeared normal and revealed no signs of degeneration. Neurite outgrowth from the surviving DRG neurons was observed by EGFP fluorescence (Fig. 1A) and neurofilament antibody (Fig. 1B), suggesting the neurons to be in good metabolic state. The number of host animals with surviving DRG implant was decreased with the postoperative time (Table 1). At 3 weeks after implantation, surviving DRG implants were found in five of six (83%) of the host animals, whereas at 6 weeks postoperatively two of six (33%) animals showed surviving DRG neurons along the ANFs. At 9 weeks following implantation surviving DRGs were observed in two of eight (25%) of the host animals. At 3 weeks following implantation, the surviving DRG neurons were mainly located along the ANFs in the modiolus at the first cochlear turn. At 6 and 9 weeks following implantation, a central migration of DRG neurons was observed (Fig. 1D,E). At 9 weeks following transplantation, DRG neurons were found along the ANFs at the internal meatus (Fig. 1E; Table 1). 3.2. Survival and migration of tau-GFP ES cells In rats transplanted with tau-GFP ES cells, implanted cells were easily detected by the GFP fluorescence (Fig. 2A,B,C) up to 9 weeks following the transplantation. At 3 weeks, surviving ES cells were observed in four of six animals (67%), at 6 weeks in two of six (33%) and at 9 weeks postoperatively in two of eight (25%). At 3 weeks survival, the stem cells were located along the ANFs at the basal cochlear turn and the internal meatus (Fig. 2A; Table 1). The implanted cells were observed to migrate further centrally at 6 weeks after the implantation (Fig. 2B). At 9 weeks postoperatively, the ES cells were observed within the brain stem, close to the ventral cochlear nucleus in two host rats (Fig. 2C). The tau-GFP ES cells were found not only labeled with the glial cell marker
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Fig. 1. Embryonic DRG neurons survived and migrated centrally following implantation along the auditory nerve of adult rat. At 3 weeks following transplantation the EGFP-positive DRG neurons were located along the auditory nerve fibers (ANFs) at the basal cochlear turn (in the modiolus) (A). The surviving DRG neurons were further labeled with a neuronal marker, neurofilament antibodies (B), which was merged in panel C. DRG neurons were observed to have migrated centrally along the auditory nerve fibers at the internal meatus at 6 and 9 weeks following transplantation (D, E, respectively). BS: brain stem. Scale bar: 50 Am shown in panels C, D and E.
Fig. 2. Tau-GFP ES cells survived and migrated following transplantation along the adult rat auditory nerve fibers (ANFs). The ES cells were found to have migrated at the internal meatus (A, confocal image) at 3 weeks following implantation. At later stages, the ES cells migrated centrally towards the brain stem (6 weeks survival, B). At 9 weeks following implantation the ES cells were found within the brain stem and towards the ventral cochlear nucleus (C). The implanted ES cells were also double-labeled with a neuronal marker, TUJ1 antibody (D). The arrows show the internal meatus of the auditory nerve fibers and the arrowheads show the margin of the brain stem. Scale bar: 100 Am shown in panels A and B; 200 Am in panel C; and 20 Am in panel D.
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(GFAP antibody) but also labeled with the neuronal marker, TUJ1 antibody (Fig. 2D), suggesting that the implanted stem cells differentiated into a neuronal cell fate in the auditory system. Cell counting revealed a relatively low survival rate (1.1– 1.5%) of the transplanted cells. However, around 7%, 14% and 15% of the surviving cells were observed to be labeled with TUJ1 antibody at 3 weeks, 6 weeks and 9 weeks following transplantation, respectively. At 9 weeks postoperatively, nearly half (46%) of the surviving cells were found within the brain stem.
4. Discussion In the present study, DRG neurons and ES cells survived for up to 9 weeks following transplantation along the auditory nerve of adult mammalians. The DRG neurons were found to migrate from the first cochlear turn to the internal meatus of the ANFs while ES cells were observed to migrate further centrally to the brain stem and close to the ventral cochlear nucleus. Previous studies have shown that it is possible to restore spinal central sensory input from the periphery via transplantation of neuronal tissue. These studies have shown that allogenic and xenogenic grafts of DRG neurons can survive and reinnervate denervated host targets in a variety of locations in the gangliectomized rat [19]. Xenografts of human fetal DRGs have been shown to extend axons through the peripheral nervous system–central nervous system (CNS–PNS) boundary, to migrate into the central nervous system and to form functional connections [11,13]. Fetal DRGs have also been shown to survive for 4 weeks following transplantation into the inner ear [9,15,16] and along the auditory nerve (Regala et al., submitted) as well as to form neurite projections towards the SGNs in the cochlea (Hu et al., in press). In the present study, embryonic DRGs and ES cells were found to survive along the auditory nerve for 9 weeks following the implantation. The results suggest that both differentiated embryonic neuronal tissues and pluripotent ES cells may be candidates for a cell replacement therapy in the auditory system. The survival rate of the implanted cells is quite poor in the present study (1.1– 1.5%). But interestingly, around 7–15% of the surviving cells were labeled with the neuronal marker, TUJ1 antibody. Clearly, in order to have a future clinical application, much more efforts are needed to enhance the survival of implant cells. An essential issue in a cell replacement strategy for the auditory system is that the implant, apart from the long time survival and high survival rate, has a potential to migrate centrally towards the auditory neurons and even form neuronal connections further centrally within the auditory system. In the present study, the first step was taken by illustrating that DRG neurons and ES cells can
migrate centrally to the internal meatus and into the brain stem. This may suggest a possibility for a biological implant to replace degenerated or absent SGNs and ANFs. We speculate that site-specific cues may play an important role in cell migration. We have previously reported that implanted DRG neurons migrated to the spiral ganglion region and along its peripheral dendritic projections [9], a migration possibly directed by the neurotrophic factors released by the SGNs [6,20]. Neural progenitors implanted into the brain showed migration along the rostral migratory pathway to differentiate into neurons in the olfactory bulb [5,25]. A recent study shows that region-specific cues are important in the neuronal differentiation and migration of naive ES cells [7]. It appears that certain factors could bguideQ foreign cell migration. In this study, the DRG neurons and ES cells could have been bdirectedQ by factors released from the auditory neurons (e.g. the cochlear nucleus) in the brain stem, thus illustrating a site-specific migration. We also found that the surviving DRG neurons and ES cells in some animals failed to migrate centrally towards brain stem in the study. The reason is still obscure but we speculate the CNS–PNS boundary (located in the auditory nerve, [4]) of the auditory system may be one of the important factors preventing central migration [11,14,22]. In the present study tau-GFP ES cells were also used. Tau is one of the microtubules which is important for maintenance of the cytoskeleton in nervous tissues. A potential benefit of tau-GFP ES cells is that it may have effect on the cytoskeleton maintenance of the implanted cells. A further potential benefit of tau-GFP over the untagged donor is that the tagged donor is anchored to the cytoskeleton, thereby reducing the risk of the extracellular diffusion of GFP protein during the processes of transplantation and histochemical tissue preparations, which would reduce the labeling specificity and increase background activity. Therefore the tau-GFP fluorescence is easier to detect as compared to the untagged donors [17]. In summary, the present findings demonstrate that xenografted embryonic DRG neuronal implants and tauGFP ES cells survive for up to 9 weeks following transplantation along the adult ANFs. The implanted neurons and stem cells also migrate centrally towards the cochlear nucleus in the brain stem. This illustrates not only the survival of differentiated neuronal tissues and pluripotent ES cells in the adult auditory system but also the ability of these implanted cells to migrate centrally to functionally appropriate locations. The present findings may help to establish whether biologically active neuronal tissue and pluripotent ES cells could be used to restore the degenerated adult auditory system. However, in order to restore hearing, the establishment of potential synaptic contacts between the implanted cells and the host auditory neurons needs to be demonstrated. This will be an exiting topic for future research.
Z. Hu et al. / Brain Research 1026 (2004) 68–73
Acknowledgements This work was supported by Petrus and Augusta Hedlund’s Foundation, The Swedish Research Council, The Swedish Association of Hard of Hearing People, Ollie and Elof Ericsson’s Foundation for Medical Research and the Foundation Tysta Skolan. Dr. Z. Hu was supported by the European Commission Quality of Life Programme (#QLG3-CT-2000-01343), the Swedish Institute, and the Karolinska Institutet.
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Research report
Central migration of neuronal tissue and embryonic stem cells following transplantation along the adult auditory nerve Zhengqing Hua,*, Mats Ulfendahla,*, N. Petri Oliviusa,b a
Center for Hearing and Communication Research, and Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden b Department of Otorhinolaryngology, Karolinska University Hospital Solna, Stockholm, Sweden Accepted 10 August 2004 Available online 13 September 2004
Abstract The regeneration of the auditory nerve remains a challenge in restoring hearing. An interesting approach would be to use a cell replacement therapy with the potential to establish connections from the inner ear to the central auditory system. This hypothesis was tested by xenografted (mouse to rat) implantation of embryonic dorsal root ganglion (DRG) neurons and embryonic stem (ES) cells along the auditory nerve in the adult host. DRG neurons were obtained at embryonic day 13–14 in transgenic animals expressing enhanced green fluorescence protein (EGFP). For embryonic stem cells, a tau-GFP ES cell line was used as a donor. The fibers of the auditory nerve in the adult rat were transected through the modiolus at the first cochlear turn, and the biological implants were transplanted into the transection. The transplanted DRG neurons and ES cells survived for a postoperative survival time ranging from 3 to 9 weeks, verified by EGFP/GFP fluorescence, and neurofilament or TUJ1 immunostaining. At 9 weeks following implantation, the implanted DRG neurons were found to have migrated along the auditory nerve in the internal meatus. At the same postoperative time, the ES cells had migrated into the brain stem close to the ventral cochlear nucleus. The results demonstrate not only the survival and migration of xenografted DRG neurons and stem cells along the adult auditory nerve but also the feasibility of a cell replacement therapy in the degenerated auditory system. D 2004 Elsevier B.V. All rights reserved. Theme: Development and regeneration Topic: Sensory systems Keywords: Auditory nerve fiber; Dorsal root ganglion; Hearing loss; Migration; Spiral ganglion; Stem cell
1. Introduction Insults to the sensory epithelium, the organ of Corti, in the mammalian cochlea result in a series of pathological changes in the auditory system. The primary damage is often selective to the sensory epithelium but, as in other afferent systems, degeneration of the spiral ganglion neurons (SGNs) and auditory nerve fibers (ANFs) occurs secondarily to the loss of cochlear sensory epithelium [12,21]. Moreover, in some clinical cases, i.e. following acoustic neuroma surgery, the ANFs have to be completely * Corresponding authors. Tel.: +46 8 51776307; fax: +46 8 301876. E-mail addresses: [email protected] (Z. Hu); [email protected] (M. Ulfendahl). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.08.013
removed. It is thus of interest to explore novel approaches to restore auditory function. One strategy would be to replace degenerated or absent SGNs and ANFs using a cell replacement therapy. Combined with a cochlear prosthesis, the sound can be transduced into electrical signals and then stimulate the implanted cells directly, thus bypass the degenerated sensory epithelium, SGNs and ANFs. Several possible tissues are candidates as donors for a cell replacement strategy in the auditory system. In addition to SGNs other types of neuronal tissues could be feasible. Recent observations have shown that embryonic dorsal root ganglion (DRG) neurons not only survive in the inner ear [9,15,16] but also migrate into the spiral ganglion region and along its peripheral dendrites projecting to the organ of Corti [9]. Stem cells are another interesting candidate for a cell
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replacement therapy. Stem cells have the capacity for selfrenewal and differentiation into diverse cell types. Embryonic stem (ES) cells are pluripotent and thus able to differentiate into a variety of cell types, i.e. ectoderm, mesoderm and endoderm [3,8]. It has been documented that neural stem cell can generate olfactory bulb neurons and after an injury, their proliferation rate increases and their progeny migrates to the site of the lesion, where they differentiate into astrocytes [10]. Further, stem cells have been tested in the treatment of nervous system disease, i.e. Parkinson’s disease, Huntington’s disease, Alzheimer’s disease and spiral cord injury [1,2,23,24,26]. Following transplantation into mature retina, the neuronal differentiation and morphological integration of adult neural progenitor cells were also identified [18,27,28]. We have recently reported that at 4 weeks following transplantation into the mature inner ear, ES cells survived and migrated along the ANFs leading from the organ of Corti where they were seen to differentiate into neurons (Hu et al., submitted). Furthermore, the implantation of DRGs and stem cells along the vestibulocochlear nerve illustrates the survival of implanted cells up to 4 weeks (Regala et al., submitted). In order for a cell replacement therapy to be of potential clinical interest, both a considerable survival time and the migration of implanted cells towards functionally important regions are essential. In this study, we explore the possibilities of embryonic DRG neurons and tau-tagged mouse ES cells to survive for a longer time, e.g. 9 weeks along the adult rat ANFs and to migrate centrally into the brain stem close to the cochlear nucleus.
2. Materials and methods All animal procedures were approved by the regional ethical committee (approval nos. 283a–d/02, 464/03, and 58/03). Implantation was made into a total of 40 Sprague– Dawley rats (body weight 250–280 g), 20 rats transplanted with DRGs and 20 animals transplanted with tau-green fluorescent protein (tau-GFP) ES cells. Animals transplanted with DRGs and tau-GFP ES cells were further divided into the following groups (c.f. Table 1): 3-week survival (n=6), 6-week survival (n=6) and 9-week survival (n=8). 2.1. Preparation of DRG tissue and tau-GFP stem cells The dissection of donor DRG neurons has been described before [9]. Briefly, the DRG tissues were dissected out from pregnant mice (E13–14) belonging to a line with an enhanced green fluorescent protein (EGFP) cDNA under the control of a chicken beta-actin promoter and cytomegalovirus enhancer (strain C57BL/6-TgN (ACTbEGFP) 1Osb from The Jackson Laboratory). Under aseptic conditions and deep anesthesia (Ketalar; 4 mg/100 g; and Rompun; 1 mg/100 g; i.m.) the abdomen and uterus
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Table 1 Survival and migration of DRGs and ES cells along the auditory nerve DRG neurons
ES cells
3 6 9 3 6 9 weeks weeks weeks weeks weeks weeks Number of animals receiving implantation 6 surgery showing surviving implant 5/6 showing the migration of implant at the basal cochlear turn 4/6 within the internal meatus 1/6 within the brain stem 0/6
6
8
6
6
8
2/6
2/8
4/6
2/6
2/8
1/6 1/6 0/6
2/8 2/8 0/8
2/6 2/6 0/6
1/6 2/6 1/6
2/8 2/8 2/8
The survival of dorsal root ganglion neurons and tau-GFP embryonic stem cells following transplantation along the auditory never fibers in the adult rat. The implanted cells were also found to have migrated centrally towards the brain stem, in close vicinity of the cochlear nucleus.
of the pregnant mice were exposed. The embryos were excised and transferred to tissue culture medium (DMEM, Gibco) where the DRGs were dissected out for later transplantation. The tau-GFP ES cell line (E14Tg2aSc4tp6.3, from the Department of Biomedical Science and the Center for Developmental Biology, University of Edinburgh, Scotland) [17] were cultured using DMEM (Gibco-BRL) supplemented with 15% fetal calf serum (HyClone), 1000 U/ml of mouse recombinant leukemia inhibitory factor (LIF, Invitrogen), 0.1 mM 2-mercaptoethanol (Sigma), 0.1 mM nonessential amino acids, 2 mM glutamine, 100 U/ml of penicillin and 100 mg/ml streptomycin (all obtained from Gibco-BRL). The culture solution was put to a 10 cm culture dish and the dish was kept in an incubator at 37 8C, 5% CO2 and humidity environment. Several days later, the ES cells were harvested and cell density was adjusted to around 104/Al with fresh medium. The cells were kept at 4 8C until transplantation. 2.2. DRGs and tau-GFP ES cells transplantation Transplantation of DRGs and stem cells was made as described previously [9]. Briefly, using a postauricular approach with aseptic conditions, the left bulla was exposed and opened to provide access to the cochlea (under deep anesthesia, same drugs as mouse with a body weightadjusted dosage). A small hole was made at the base of the cochlea to expose the modiolus. The modiolus was opened at the basal cochlear turn and the ANFs were transected completely. The DRGs or the ES cells were transplanted at the transection site along the ANFs. For the ES cell transplantation 5 Al culture medium with tau-GFP ES cells (104/Al) was administered using a microsyringe (Exmire microsyringe, Ito, Fuji, Japan). A small piece of fascia was placed over the cochleostomy and the skin incision was approximated with sutures. In order to reduce the risk of postoperative immunological rejection and infection, the transplanted host animals received daily injections of
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cyclosporin (Novartis Sverige, 0.56 mg/100 g body weight) and doxycycline (Nordic Drugs, 0.24 mg/100 g body weight) intraperitoneally until the day of sacrifice. 2.3. Histology At sacrifice, the animals were anaesthetized with an overdose of pentobarbital (i.p.) and transcardially perfused with 0.9% saline at 37 8C followed by 4% paraformaldehyde and 14% picric acid in 0.1 M phosphate buffer (pH 7.4, 4 8C). The auditory nerve together with the brain stem and a part of the cochlea was dissected out and kept in the fixative for 2 h before being transferred to 0.1 M phosphate buffer, in which they were stored until further processing. The specimens were incubated in 30% sucrose overnight, embedded in tissue freezing medium (Leica Instruments), and sectioned on a Leica CM3050S cryostat at a thickness of 14 Am. Every second section was collected from the entire specimen (auditory nerve together with the brain stem and a part of the cochlea). The sections were analyzed using a Zeiss fluorescent microscope with a digital camera (Nikon Coolpix 5000 or Spot RT, Diagnostic Instrument) or confocal microscopy (Zeiss LSM 510). 2.4. Immunohistochemistry Neurofilament antibody was applied to label the DRG neurons along the auditory nerve transplanted with DRG tissues. Following preincubation in 1% goat serum (Santa Cruz Biotechnology; diluted in 0.1 M phosphate buffer saline) for 30 min at room temperature, the sections were incubated with neurofilament monoclonal antibody (NF-L, 1:100, Santa Cruz Biotechnology) overnight at 4 8C. After rinsing, a goat anti-mouse IgG1-Texas red (1:200, Santa Cruz Biotechnology) was applied for 2 h at room temperature. To identify the neuronal differentiation of the ES cells in the animals transplanted with tau-GFP ES cells, TUJ1 antibody, a neuronal marker, was used (primary antibody, TUJ1 antibody, 1:500, Berkeley Antibody; secondary antibody, cy3-conjugated affiniPure F(abV)2 fragment goat anti-mouse IgG, 1:400, Jackson Immunoresearch Laboratories). To identify glial cell differentiation of ES cells, an antibody against the glial fibrillary acidic protein (GFAP) was used (primary antibody, polyclonal rabbit anti-GFAP antibody, 1:250, DAKO; secondary antibody, amca affinity purified goat anti-rabbit IgG (H+L), 1:400, Vector Laboratories). In order to enhance the fluorescence of GFP-expressing cells, an anti-GFP antibody (polyclonal rabbit anti-GFP antibody, 1:500, Abcam) and appropriate secondary antibody (fluorescein (FITC)-conjugated affiniPure donkey anti-rabbit IgG (H+L), 1:500, Jackson Immunoresearch Laboratories) were used in the study. The sections were observed using the same equipment as above with the appropriate fluorescence filter.
2.5. Cell counting Cell counting was performed in the animals with surviving ES cells. The GFP fluorescence was used to detect the surviving implanted cells and the TUJ1 antibody positive staining was applied to identify the neuronal differentiation of ES cells. The sections from the whole specimen (auditory nerve together with the brain stem and a part of the cochlea) were used.
3. Results 3.1. Survival and migration of DRGs along ANFs Implanted DRG neurons were identified (by detection of EGFP fluorescence) in histological sections of ANFs connected with the brain stem for up to 9 weeks following implantation (Fig. 1A,D,E). The morphology of the surviving DRG neurons appeared normal and revealed no signs of degeneration. Neurite outgrowth from the surviving DRG neurons was observed by EGFP fluorescence (Fig. 1A) and neurofilament antibody (Fig. 1B), suggesting the neurons to be in good metabolic state. The number of host animals with surviving DRG implant was decreased with the postoperative time (Table 1). At 3 weeks after implantation, surviving DRG implants were found in five of six (83%) of the host animals, whereas at 6 weeks postoperatively two of six (33%) animals showed surviving DRG neurons along the ANFs. At 9 weeks following implantation surviving DRGs were observed in two of eight (25%) of the host animals. At 3 weeks following implantation, the surviving DRG neurons were mainly located along the ANFs in the modiolus at the first cochlear turn. At 6 and 9 weeks following implantation, a central migration of DRG neurons was observed (Fig. 1D,E). At 9 weeks following transplantation, DRG neurons were found along the ANFs at the internal meatus (Fig. 1E; Table 1). 3.2. Survival and migration of tau-GFP ES cells In rats transplanted with tau-GFP ES cells, implanted cells were easily detected by the GFP fluorescence (Fig. 2A,B,C) up to 9 weeks following the transplantation. At 3 weeks, surviving ES cells were observed in four of six animals (67%), at 6 weeks in two of six (33%) and at 9 weeks postoperatively in two of eight (25%). At 3 weeks survival, the stem cells were located along the ANFs at the basal cochlear turn and the internal meatus (Fig. 2A; Table 1). The implanted cells were observed to migrate further centrally at 6 weeks after the implantation (Fig. 2B). At 9 weeks postoperatively, the ES cells were observed within the brain stem, close to the ventral cochlear nucleus in two host rats (Fig. 2C). The tau-GFP ES cells were found not only labeled with the glial cell marker
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Fig. 1. Embryonic DRG neurons survived and migrated centrally following implantation along the auditory nerve of adult rat. At 3 weeks following transplantation the EGFP-positive DRG neurons were located along the auditory nerve fibers (ANFs) at the basal cochlear turn (in the modiolus) (A). The surviving DRG neurons were further labeled with a neuronal marker, neurofilament antibodies (B), which was merged in panel C. DRG neurons were observed to have migrated centrally along the auditory nerve fibers at the internal meatus at 6 and 9 weeks following transplantation (D, E, respectively). BS: brain stem. Scale bar: 50 Am shown in panels C, D and E.
Fig. 2. Tau-GFP ES cells survived and migrated following transplantation along the adult rat auditory nerve fibers (ANFs). The ES cells were found to have migrated at the internal meatus (A, confocal image) at 3 weeks following implantation. At later stages, the ES cells migrated centrally towards the brain stem (6 weeks survival, B). At 9 weeks following implantation the ES cells were found within the brain stem and towards the ventral cochlear nucleus (C). The implanted ES cells were also double-labeled with a neuronal marker, TUJ1 antibody (D). The arrows show the internal meatus of the auditory nerve fibers and the arrowheads show the margin of the brain stem. Scale bar: 100 Am shown in panels A and B; 200 Am in panel C; and 20 Am in panel D.
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(GFAP antibody) but also labeled with the neuronal marker, TUJ1 antibody (Fig. 2D), suggesting that the implanted stem cells differentiated into a neuronal cell fate in the auditory system. Cell counting revealed a relatively low survival rate (1.1– 1.5%) of the transplanted cells. However, around 7%, 14% and 15% of the surviving cells were observed to be labeled with TUJ1 antibody at 3 weeks, 6 weeks and 9 weeks following transplantation, respectively. At 9 weeks postoperatively, nearly half (46%) of the surviving cells were found within the brain stem.
4. Discussion In the present study, DRG neurons and ES cells survived for up to 9 weeks following transplantation along the auditory nerve of adult mammalians. The DRG neurons were found to migrate from the first cochlear turn to the internal meatus of the ANFs while ES cells were observed to migrate further centrally to the brain stem and close to the ventral cochlear nucleus. Previous studies have shown that it is possible to restore spinal central sensory input from the periphery via transplantation of neuronal tissue. These studies have shown that allogenic and xenogenic grafts of DRG neurons can survive and reinnervate denervated host targets in a variety of locations in the gangliectomized rat [19]. Xenografts of human fetal DRGs have been shown to extend axons through the peripheral nervous system–central nervous system (CNS–PNS) boundary, to migrate into the central nervous system and to form functional connections [11,13]. Fetal DRGs have also been shown to survive for 4 weeks following transplantation into the inner ear [9,15,16] and along the auditory nerve (Regala et al., submitted) as well as to form neurite projections towards the SGNs in the cochlea (Hu et al., in press). In the present study, embryonic DRGs and ES cells were found to survive along the auditory nerve for 9 weeks following the implantation. The results suggest that both differentiated embryonic neuronal tissues and pluripotent ES cells may be candidates for a cell replacement therapy in the auditory system. The survival rate of the implanted cells is quite poor in the present study (1.1– 1.5%). But interestingly, around 7–15% of the surviving cells were labeled with the neuronal marker, TUJ1 antibody. Clearly, in order to have a future clinical application, much more efforts are needed to enhance the survival of implant cells. An essential issue in a cell replacement strategy for the auditory system is that the implant, apart from the long time survival and high survival rate, has a potential to migrate centrally towards the auditory neurons and even form neuronal connections further centrally within the auditory system. In the present study, the first step was taken by illustrating that DRG neurons and ES cells can
migrate centrally to the internal meatus and into the brain stem. This may suggest a possibility for a biological implant to replace degenerated or absent SGNs and ANFs. We speculate that site-specific cues may play an important role in cell migration. We have previously reported that implanted DRG neurons migrated to the spiral ganglion region and along its peripheral dendritic projections [9], a migration possibly directed by the neurotrophic factors released by the SGNs [6,20]. Neural progenitors implanted into the brain showed migration along the rostral migratory pathway to differentiate into neurons in the olfactory bulb [5,25]. A recent study shows that region-specific cues are important in the neuronal differentiation and migration of naive ES cells [7]. It appears that certain factors could bguideQ foreign cell migration. In this study, the DRG neurons and ES cells could have been bdirectedQ by factors released from the auditory neurons (e.g. the cochlear nucleus) in the brain stem, thus illustrating a site-specific migration. We also found that the surviving DRG neurons and ES cells in some animals failed to migrate centrally towards brain stem in the study. The reason is still obscure but we speculate the CNS–PNS boundary (located in the auditory nerve, [4]) of the auditory system may be one of the important factors preventing central migration [11,14,22]. In the present study tau-GFP ES cells were also used. Tau is one of the microtubules which is important for maintenance of the cytoskeleton in nervous tissues. A potential benefit of tau-GFP ES cells is that it may have effect on the cytoskeleton maintenance of the implanted cells. A further potential benefit of tau-GFP over the untagged donor is that the tagged donor is anchored to the cytoskeleton, thereby reducing the risk of the extracellular diffusion of GFP protein during the processes of transplantation and histochemical tissue preparations, which would reduce the labeling specificity and increase background activity. Therefore the tau-GFP fluorescence is easier to detect as compared to the untagged donors [17]. In summary, the present findings demonstrate that xenografted embryonic DRG neuronal implants and tauGFP ES cells survive for up to 9 weeks following transplantation along the adult ANFs. The implanted neurons and stem cells also migrate centrally towards the cochlear nucleus in the brain stem. This illustrates not only the survival of differentiated neuronal tissues and pluripotent ES cells in the adult auditory system but also the ability of these implanted cells to migrate centrally to functionally appropriate locations. The present findings may help to establish whether biologically active neuronal tissue and pluripotent ES cells could be used to restore the degenerated adult auditory system. However, in order to restore hearing, the establishment of potential synaptic contacts between the implanted cells and the host auditory neurons needs to be demonstrated. This will be an exiting topic for future research.
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Acknowledgements This work was supported by Petrus and Augusta Hedlund’s Foundation, The Swedish Research Council, The Swedish Association of Hard of Hearing People, Ollie and Elof Ericsson’s Foundation for Medical Research and the Foundation Tysta Skolan. Dr. Z. Hu was supported by the European Commission Quality of Life Programme (#QLG3-CT-2000-01343), the Swedish Institute, and the Karolinska Institutet.
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