Neural Stem/Progenitor Cells Transplanted to the Hypoglossal Nucleus Integrates with the Host CNS in Adult Rats and Promotes Motor Neuron Survival

Abstract

Transplantation of neural stem cells and the mobilization of endogenous neuronal precursors in the adult brain have been proposed as therapeutic strategies for central nervous system disorders and injuries. The aim of the present study was to investigate the possible survival and integration of grafted neural progenitor cells (NPCs) from the subventricular zone (SVZ) in a hypoglossal nerve avulsion model with substantial neuronal loss. Adult neural progenitor cells (NPCs) from the subventricular zone (SVZ) were cultured from inbred transgenic eGFP Lewis rats and transplanted to the hypoglossal nucleus of inbred Lewis rat from the same family but that were not carrying the eGFP strain after avulsion of the hypoglossal nerve. Grafted cells survived in the host more than 3 months and differentiated into neurons [βIII tubulin (Tuj-1 staining)] with fine axon- and dendrite-like processes as well as astrocytes (GFAP) and oligodendrocytes (O4) with typical morphology. Staining for synaptic structures (synaptophysin and bassoon) indicated integration of differentiated cells from the graft with the host CNS. Furthermore, transplantation of NPCs increased the number of surviving motoneurons in the hypoglossal nucleus after nerve avulsion that, if untreated, result in substantial neuronal death. The NPCs used in this study expressed VEGF in vitro as well as in vivo following transplantation that may mediate the rescue effect of the axotomized motoneurons.

Keywords

Brain stem Neural progenitor cells Nerve injury Motor neurons Transplantation Integration

Introduction

The adult mammalian central nervous system (CNS) has for long been considered to lack regenerative capacity after birth. However, Reynolds and Weiss in 1992 (36) showed that neural stem cells taken from adult brain can be propagated in vitro. These cells have the capacity to generate the major cell types of the central nervous system (CNS): neurons, astrocytes, and oligodendrocytes (36). Neuronal stem cells are generated in two areas of the adult rodent brain, the subventricular zone (SVZ) of the lateral ventricle wall, where cells proliferate and migrate via the rostral migratory stream to the olfactory bulb differentiating into interneurons (3,18, 28 –30). In the dentate gyrus of the hippocampus, newly generated neuronal cells are added to the granule cell layer; they differentiate into mature neurons and extend axonal projections to the CA3 area (5,6,10). Activation of the neural progenitor cells (NPCs) including proliferation, migration, and differentiation has been found in disease and injury models (9,13,22,23,33,34).

In the present study, we have used a hypoglossal nerve injury model (32) to investigate if transplanted adult SVZ NPCs could survive, differentiate, and integrate with host CNS tissue in the hypoglossal nucleus. In this study we hypothesized that survival of the graft, differentiation, and integration would be optimized in a model where the major histocompatibility complex (MHC) of the donor and the receiver were compatible. For this we used a strain of inbred Lewis rats that carried the enhanced green fluorescent protein (eGFP) gene and transplanted NPCs to their wild-type siblings. A potential effect of transplantation on survival of axotomized motoneurons in the hypoglossal nucleus was also addressed.

Materials and Methods

All animal experiments were approved by the local ethical committee on animal research and animal care was in accordance with institutional guidelines.

Animal Breeding and Genotyping

Animals were housed in an environmentally controlled room temperature at 21 ± 1°C with a 12:12-h light/dark cycle, with food and water administered ad libitum. A total of 52 male Lewis rats were used for cell harvesting in the study. Genotyping was performed by following the genotyping protocol from RRRC-Missouri (http://www.nrrrc.missouri.edu). A REDExtract-N-AMP Tissue PCR Kit from sigma (cat# XNAT 100 reactions) was used to extract the DNA from the ear tip biopsy from each animal. Animals that were homozygous for the eGFP transgene were selected as donors, and eGFP noncarrier siblings were selected as the receivers of the transplant.

Cell Culture

Subventricular zone (SVZ) adult stem/progenitor cells were obtained from Lewis Rats (RRRC Missouri University, Columbia, MO (http://www.nrrrc.missouri.edu), carrying an insertion of the enhanced green fluorescent protein (eGFP transgene), under the ubiquitin C promoter on chromosome 5. The rats were 7–8 weeks old at day of cell isolation. Cells were isolated in accordance to a protocol described by Johansson et al. (22). For transplantation, the cells were propagated for two passages, in Dulbecco's modified Eagle's medium/F-12 (DMEM/F12) containing B27 supplement, penicillin (100 U/ml), streptomycin (100 μg/ml), epidermal growth factor (EGF, 20 ng/ml), and basic fibroblast growth factor (bFGF, 10 ng/ml). After the second passage, neurospheres were dissociated and cells were resuspended in a solution that contained 100,000 cells/5 μl in DMEM/F12 medium enriched with EGF (20 ng/ml) and bFGF (10 ng/ml).

For the in vitro differentiation analysis, spheres were split after two passages into single cell suspensions, seeded on poly-d-lysine-coated plates, and cultured in medium lacking mitogens but supplemented with 1% fetal calf serum (FCS). The cultures were differentiated for 7 days.

Animal Surgery

Offspring from the same strain as described above were genotyped and wild-type males were used as recipients in the transplantation experiments. Animals were divided into four groups: I) uninjured animals, II) injury only (i.e., no injections into the brain stem), III) injury and injection of cell medium only, and IV) injury and cell transplantation. Adult male wild-type Lewis rats, weight 300–450 g, were anesthetized using intraperitoneal injection of medetomidine (Domitor® Vet. 1 mg/ml, OrionPharma, Espoo, Finland) 0.5 mg/kg, and ketamine (Ketalar® 50 mg/ml, Pfizer, Sollentuna, Sweden) 75mg/kg, and the hypoglossal nerve on the right side was avulsed at the level where it passes the carotid artery (groups II–IV).

Transplantation of eGFP NPCs Into Hypoglossal Nucleus

Nine days after the avulsion injury the animals were reanesthetized and placed in a stereotaxic frame (Kopf®, David Kopf Instruments, Tujunga, CA) (groups III–IV). In these animals the brain stem was exposed and the obex identified. In group IV cells were injected through a glass micropipette (Clark Electromedical Instruments, Reading, England) pulled to an aperture of 90 μm, adapted, and sealed onto a Hamilton syringe (Hamilton Company, Bonaduz, Switzerland) connected to a Univentor 802 syringe pump (Univentor, Zejtun, Malta). At 0.3 mm caudal and 0.1 mm lateral (right) to the obex 100,000 cells in 5 μl culture medium D-MEM/F12 enriched with mitogens as previously described (see Cell Culture) were injected into the right hypoglossal nucleus.

Cells were injected as single cells and the volume was administered at 2 μl/min. The glass micropipette was left in place for 5 min and then withdrawn. Animals in group III received 5 μl DMEM/F12 enriched with mitogens in the same proportion but with no cells. All animals with brain stem injections received injection of cyclosporin 10 mg/kg (Sandimmun® 50 mg/ml, Novartis, Täby, Sweden) subcutaneously once daily starting 1 day prior to transplantation. After 4 weeks the injections were given three times/week. Survival times were 2, 7, and 28 days and 3 months (n = 8 in each group and survival time). At sacrifice animals were deeply anesthetized as above and sacrificed by transcardial perfusion of body temperature (37°C) saline followed by cold (4°C) 4% paraformaldehyde in PBS.

The brain stems were removed and postfixed for 120 min in cold (4°C) 4% paraformaldehyde in PBS followed by rinse in PBS and cryoprotected for 48 h in 15% sucrose (w/v) in PBS. The hypoglossal nucleus was serially cryosectioned (14 μm) using a Leica CM3000 (Leica Microsystems, Kista, Sweden) cryostat and mounted on SuperFrost® Plus microscope slides (Menzel-Gläser, Braunschweig, Germany).

Immunohistochemistry

The sections were washed in PBS for 30 min and then incubated for 60 min in PBS containing 1% bovine serum albumin, 0.3% Triton-X, and 0.1% sodium azide to prevent nonspecific binding. All of the primary antibodies used were diluted in this solution. Sections were incubated with one (two for double staining) of the following antibodies: polyclonal rabbit GFP (Chemicon/Millipore Sweden AB, Solna, Sweden) (dilution 1:500) for transplanted transgene expressing cells; polyclonal rabbit (sex determining region Y)-box 2 (Sox2; Chemicon/Millipore) (1:200) and monoclonal mouse Nestin (Chemicon/Millipore) (1:100) for stem/progenitor cells; polyclonal rabbit glial fibrillary acidic protein (GFAP; DAKO Sweden AB, Stockholm, Sweden) (1:1000) and monoclonal mouse GFAP (Chemicon/Millipore) (1: 1000) for astrocytes; monoclonal mouse O4 (Chemicon/Millipore) (1:50) for oligodendrocytes; monoclonal mouse tubulin β-III isoform (TUJ1; Chemicon/Millipore) (1:100) and monoclonal mouse NeuN (Chemicon/Millipore) (1:100) for neurons; mouse monoclonal choline acetyltransferase (ChAT; Abcam, Cambridge, UK) (1:100) for motoneurons; polyclonal rabbit synaptophysin (Abcam, Cambridge, UK) (1:100) and rabbit antibasson (Abcam, Cambridge, UK) (1:100) for synaptic structures; rabbit anti-VEGF (R&D Systems, UK) (1:50) for vascular endothelial growth factor and rabbit polyclonal BDNF (Chemicon/Millipore) (1:1000) for brain derived neurotrophic factor. All sections were incubated with the primary antibody for 24 h at +4°C, rinsed in PBS, and subsequently incubated with species-specific secondary antibodies conjugated with cyanine 3 (Cy3; goat anti-mouse, donkey anti-mouse, goat ant-rabbit, or donkey ant-rabbit) (Jackson ImmunoResearch Europe Ltd., Suffolk, UK) (1:1000), Alexa 594 (donkey anti-mouse) (Invitrogen/Molecular Probes, Eugene, OR, USA) (1:500), Alexa 633 (goat anti-mouse) (Invitrogen/Molecular Probes) (1:500), and/or Cy5 (goat anti-rabbit) (Jackson ImmunoResearch) (1:200).

Sections were also counterstained with the nuclear marker 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen, Invitrogen Ltd, Paisley, UK) (1:2000) or TO-PRO-3 (Invitrogen) (1:10000). After another step of washing in PBS sections were mounted in Mowiol (Calbiochem, Merck Chemicals Ltd, Nottingham, UK). For negative controls the primary antibody was omitted.

For the avidin-biotin complex (ABC) technique sections were incubated with biotinylated secondary antibodies (Vector Laboratories, Vector Laboratories Ltd, Peterborough, England) (1:200) for 1 h at room temperature, rinsed in PBS, and incubated with ABC (Vectastain® ABC Kit, Vector Laboratories) for 1 h. After another step of rinsing in PBS followed by Tris-hydrogen chloride buffer (0.1 M, pH 7.45) immunoreactivity was revealed by incubation in 3,3′-diaminobenzidine (DAB) by using the DAB Substrate Kit for Peroxidase (Vector Laboratories) for 2–10 min. Sections were rinsed in Tris and dehydrated through a series of graded ethanols to xylene and mounted in a nonaqueous DPX medium.

Confocal Microscopy

Immunolabeling experiments were analyzed by confocal laser-scanning microscopy with a Leica TCS SP II system (Leica Microsystems, Heidelberg, Germany). Argon-krypton and helium-neon lasers were used to excite the fluorochromes at 488 nm (Alexa 488), 543 nm (Cy3), 594 nm (Alexa 594), and 633nm (Cy5, TO-PRO). Fluorescence was detected with 490–520, 560–630, 610–650, and 640–750 band-pass filters, respectively. The images were taken using a 20x [numeric aperture (N/A) 0.7] and 63x (N/A 1.40) objective. Crossover fluorescence was negligible. Each optical section (1 μm) was averaged four times. Pictures were the projection of 25 successive optical sections into one image.

Cell Counting and Statistical Analysis

To carry out a semiquantitative determination of the number of motoneuron survival in the hypoglossal nucleus, we used 8–10 sections from each brain stem of the hypoglossal nucleus at the same level for each experimental and control group. Sections were stained with cresyl violet. Neuronal profiles that had a clear nucleus and nucleolus were included in sections at every 168 μm throughout the hypoglossal nucleus using a wide-field microscope (Leica DM 400B, Leica) with a 40&x objective. The method of Abercrombie was used to correct for volume changes of individual neuronal profiles (1,31). These measurements were followed by a statistical analysis by using unpaired two-tailed Student's t-test, for multiple group comparison one-way analysis of variance followed by Tukey's post test was implied (GraphPad Prism® 5.0, GraphPad Software Inc., La Jolla, CA, USA). Data are presented as mean ± SD, and *p < 0.05 and ***p < 0.001 was defined as statistical significance.

Results

In Vitro Characterization of Cells

The cells cultured from the SVZ were categorized with regards to stemness and differentiation potential. At transplantation more than 90% of the cells expressed Sox 2 (Fig. 1A). After differentiation for 7 days in vitro, the cells also possessed the ability to differentiate into neurons (2–4%), oligodendrocytes (36–38%), and astrocytes (59–61%) (Fig. 1B–D). No difference was seen between cells from the eGFP-transgenic Lewis animals compared to cells from wild-type animals.

Figure 1.

Neural progenitor cell (NPC) culture and differentiation in vitro. (A) The green fluorescent protein (GFP)-positive cells (green) had round-shaped cell bodies; immunocytochemistry on these cells revealed efficient expression of the progenitor cell marker (sex determining region Y)-box 2 (Sox2). More than 90% of the grafted cells were Sox2 positive (red). (B) A number of NPCs (2–4%) had the capacity to differentiate into neurons (Tuj-1, red). (C) The majority of NPCs (59–61%) differentiated into astrocytes [glial fibrillary acidic protein (GFAP), red] and (D) oligodendrocytes (36–38%, O4, red). (E) Anatomical site of transplantation. AP, area postrema; CC, central canal; HN, hypoglossal nucleus; NTS, nucleus tractus solitaries. Scale bars: 40 μm (A) and 20 μm (B–D).

Survival, Differentiation, and Integration of the Graft

Viability of cells before transplantation was assessed with trypan blue, which showed that more than 90% of the cells were viable before transplantation. After grafting, transplanted cells were easily detectable based on their eGFP expression. Single immunolabeling with an antibody against GFP proteins confirmed that these cells were GFP positive, which further supported that these cells were eGFP NPC transplanted cells. The majority of transplanted cells were located in the right hypoglossal nucleus ipsilateral to nerve injury (Fig. 2A). At 2 and 7 days after transplantation the cells appeared without any morphological differentiation (Fig. 2A). The cells were small and round with no immunoreactivity (ir) for neurons, astrocytes, or oligodendrocytes.

Figure 2.

Survival, morphology and differentiation of the grafted cells. (A) The transplanted cells after 7 days into the hypoglossal nucleus; the cells appeared without any morphological differentiation. (B) Higher magnification shows the transplant after 3 months; the cells displayed a more differentiated appearance. (C) Double-immunolabeling with GFAP (red) and (D) O4 (red) indicated that NPCs differentiated towards astrocytes and oligodendrocytes, respectively. (E, F) Double-labeling with Tuj-1 (antibody marker for tubulin β-III; red) and NeuN (blue) revealed that a small proportion of the grafted NPCs differentiated into Tuj-1- and NeuN-positive cells in the hypoglossal nucleus, showing grafted cells with a neuronal appearance. (G) Synaptophysin labeling (red) was detected along fiber extensions of grafted neuronal-like cells. (H) Bassoon immunoreactivity was detected on the cell bodies of NPC grafted cells, indicating active sites of synapse formation. Scale bars: 40 μm (A, C, D, E), 10 μm (B, F), 20 μm (G, H).

At 3 months we estimated the number of surviving cells in the graft. The number was highly variable because cells sometimes tended to retract in the trajectory canal. Approximately 10% of grafted cells were present. Differentiation of transplanted cells was assessed using markers for neurons and glia. After 3 months numerous cells in each section displayed a neuronal-like appearance with long extensions such as for axons or dendrites (Fig. 2B–H).

Grafted cells were commonly found to express GFAP, suggesting differentiation towards the astrocyte lineage (Fig. 2C). Cells coexpressing O4 and eGFP-ir could be detected in the graft (Fig. 2D). These cells mostly maintained a rounded immature appearance, but could occasionally produce a spreading, plane morphology; however, this was rare.

Numerous cells displayed eGFP and were colabeled with Tuj-1 (Fig. 2E). The nuclei of cells with neuronal shape expressed NeuN-IR (Fig. 2F). For cells displaying neuronal differentiation there was no evidence for presence of ChAT-ir (marker for motoneurons) in eGFP transplanted cells, nor could we detect GFP-ir in the motoneurons in light stable analysis (ABC-technique, data not shown).

To determine whether the eGFP transplanted cells were integrated into the hypoglossal nucleus, double-labeling experiments were performed with synaptophysin and bassoon as markers for presynaptic active zone formation. Synaptophysin-ir was detected around the transplanted eGFP-NPCs and along the dendrite-like extensions (Fig. 2G). Bassoon-ir was observed around eGFP-NPC transplanted cell bodies; the immunoreactivity was distributed in punctuates mainly on the cell soma (Fig. 2H).

Increased Motoneuron Survival After Transplantation

In order to examine whether the grafted cells could influence the number of surviving motor neurons, the remaining numbers of neurons were assessed (n = 8 in each group). Avulsion injury caused a progressive death of motoneurons with the time course. Without transplantation 42.6 ± 9.1% and 23.9 ± 5.1% of motor neurons survived at 28 days and 3 months, respectively, after nerve avulsion.

After NPC transplantation the number of surviving motoneurons increased significantly to 55.4 ± 12.7% and 45.8 ± 13.3% at 28 days and 3 months, respectively, compared to control animals as well as to sham-injected animals (Fig. 3A–E). On the grafted side significantly more cells had maintained the normal size, whereas degenerating neurons were of similar size as in the avulsed controls.

Figure 3.

Effect of the transplanted NPCs on the survival of host motoneurons at 28 days and 3 months after hypoglossal nerve avulsion. (A–D) Transverse sections of the right hypoglossal nucleus. (A) Intact control side. (B) Hypoglossal nucleus at 3 months after avulsion with massive loss of motoneurons. (C) Injection of the culture medium without NPCs. (D) NPCs grafted into the hypoglossal nucleus; many motoneurons remain surviving. Note more frequently occurring motoneuronal profiles in comparison to (B) and (C). (E) Numbers of motoneurons in the different groups of animals at 28 days postavulsion as well as 3 months postavulsion. The motoneuronal death was progressive; however, the number of surviving motoneurons was significantly higher in avulsed animals receiving NPCs than what was found in controls. Scale bars: 40 μm (A–D). Data are presented as mean ± SD. *p < 0.05; ***p < 0.001.

Finally, in order to comprehend how the grafted cells affect the motoneuronal survival, staining for BDNF and VEGF was carried out. VEGF-ir cells were observed in the transplantation cells in vitro (Fig. 4A) as well as in the grafted cells after 3 months (Fig. 4B), while there was no evidence for BDNF immunoreactivity (data not shown).

Figure 4.

Confocal images showing vascular endothelial growth factor (VEGF) immunostaining in NPCs. (A) NPCs in culture showed VEGF-ir expression in vitro (before grafting; red). (B) Many NPCs were VEGF positive (VEGF, green) at 3 months postavulsion and grafting. Scale bar: 20 μm (A, B).

Discussion

Since recognition of foreign MHC antigens is believed to be a major determinant in the rejection of neural grafts (15,16,39,49) we used a strain of inbred Lewis rats that carried the eGFP transgene and transplanted to their noncarrier siblings. As the animals were not totally genetically homologous, we still applied a protocol for immunosuppression. We selected the hypoglossal nerve injury model because it is a model where a robust loss of neurons is achieved by the avulsion, without a direct mechanical trauma to the brain stem (19). Comparable progressive neuronal loss has also been shown in ventral root avulsion injuries in the spinal cord where the cell loss is even more pronounced (20,25,51). In this model, cell loss is relatively simple to assess because the intact side is present for control in each section in the immediate vicinity to the axotomized side.

In vitro, the NPCs expressed Sox2 and differentiated into neurons, astrocytes, and oligodendrocytes. We focused our study on the differentiation and integration of the grafted cells into the hypoglossal nucleus after nerve injury. The grafted eGFP NPCs showed morphological maturation and differentiation into all CNS cell types (neurons, astrocytes, and oligodendrocytes) 3 months after transplantation. NPCs have been grafted in various models of diseases and injuries, validating their potential for the treatment of a broad range of CNS diseases (12,35,38). It is generally believed that generation of new neurons occurs mostly in the SVZ and subgranular layer of the hippocampal dentate gyrus. The adult environment may lack the stimulus to direct the NPC differentiation into a neuronal fate (17). Therefore, replacement of neurons by endogenous cells after CNS injury is for the most part restricted and functionally insignificant (37). The difficulties in recruiting and expanding endogenous NPCs have directed focus and attention to transplantation strategies that are capable of generating neurons. The advantage of using defined NPC populations to generate specific classes of neuronal cells has previously been demonstrated (26).

In this study graft-derived neurons also formed a connection and integration with the preexisting network identified by the presence of terminal-like structures, expressing synaptophysin and bassoon immunoreactivity, at the surface of eGFP transplanted cells. Synaptophysin is localized at the synaptic vesicle membrane and forms complexes with synaptobrevin (7) also at the synaptic vesicle membrane; this complex serves to store synaptobrevin that can be recruited during periods of high synaptic activity. Bassoon is a cytoskeletal protein, often found at nascent synapses, and plays a role in the formation of the active zone (47). It has been demonstrated that both synaptophysin and bassoon are expressed and involved in the synaptic fusion and endocytosis in the active zone (14,47). Since synaptophysin and bassoon represent areas of synaptic contact between neurons this suggest integration of the graft. These results are consistent with previous studies that reported that NPCs after engraftment into the neonatal and adult brain and spinal cord integrate with the host circuitry (24,26,48).

The number of motoneurons in the animals with grafted cells was significantly higher compared with the untreated animals at 3 months after injury. We reasoned that either the transplanted cells differentiated into motoneuronal profiles or the grafts increased motoneuron survival. Because we did not detect any ChAT-ir, a marker for motoneurons, in eGFP transplanted cells and none of the surviving motoneurons were GFP positive even when an antibody against GFP was applied, we conclude that grafted cells prevented the long-term loss of motoneurons.

VEGF is known to have such neurotrophic effects in the CNS and has also been shown to be able to increase axonal outgrowth, block neuronal apoptosis, and promote neurogenesis (11,21,40). Moreover, it has been shown that treatment with VEGF is efficient in many in vitro and in vivo models of neurodegeneration (4,46). It has been reported that NPCs are able to express and secrete neurotrophic factors that promote motoneuron survival in diseases and injuries (8,27,42,50). Since the transplanted NPCs expressed VEGF in both in vitro and in vivo, our results are in accordance with these earlier studies and suggest that the NPCs transplanted into the hypoglossal nucleus increase motoneuron survival by means of neuroprotection.

The timing of transplantation (9 days) was chosen since the microglial response, which peaks in the first week after injury (2,43,44), may constitute a hostile environment for the graft. Also, it has been demonstrated that subacute transplantation post-avulsion injury significantly increases the neuronal differentiation of transplanted NPCs compared to acute transplantation or transplantation at 6 weeks after injury (41). Furthermore, long-term integration of human neural stem cells (NSCs) after transplantation to the spinal cord has been shown to be successful at 9 days after injury (45).

In summary, this study demonstrated a beneficial effect of SVZ NPCs transplanted into the lesion area. The grafts displayed morphological maturation into all three mature CNS cells types (neurons, oligodendrocytes, and astrocytes), integration with the host, and promotion of motoneuron survival in the hypoglossal nucleus. Therefore, transplantation of the adult SVZ NPCs to the hypoglossal nucleus after nerve injury as an experimental model shows promising results for treatment of lesions associated with levels of high motoneuron cell death.

Footnotes

Acknowledgments

We thank Mrs. Britt Meijer for excellent technical assistance. This work was supported by Torsten och Ragnar Soderberg Foundation, the Swedish Medical Research Council, and Karolinska Institutet. The authors declare no conflict of interest.

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