- Email: [email protected]
A novel cell transplantation protocol and its application to an ALS mouse model
Experimental Neurology 213 (2008) 431–438
Contents lists available at ScienceDirect
Experimental Neurology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y e x n r
A novel cell transplantation protocol and its application to an ALS mouse model Eri Morita a,b,1, Yasuhiro Watanabe a,⁎,1, Miho Ishimoto a,1, Toshiya Nakano a, Michio Kitayama a, Kenichi Yasui a, Yasuyo Fukada a, Koji Doi a, Asanka Karunaratne c, Wayne G Murrell c, Ratneswary Sutharsan c, Alan Mackay-Sim c, Yoshio Hata d, Kenji Nakashima a a
Department of Neurology, Institute of Neurological Sciences, Faculty of Medicine, Tottori University, 36-1 Nishico-cho, Yonago 683-8504, Japan Division of Neurobiology, School of Life Science, Tottori University Graduate School of Medical Science, Yonago 683-8504, Japan National Centre for Adult Stem Cell Research, Eskitis Institute for Cell and Molecular Therapies, Griffith University, Brisbane, Queensland 4111, Australia d Department of Integrative Bioscience, Tottori University Graduate School of Medical Science, Yonago 683-8504, Japan b c
a r t i c l e
i n f o
Article history: Received 13 February 2008 Revised 18 June 2008 Accepted 8 July 2008 Available online 22 July 2008 Keywords: Amyotrophic lateral sclerosis Transplantation Intrathecal Olfactory ensheathing cell Mesenchymal stem cell
a b s t r a c t Amyotrophic lateral sclerosis (ALS) is a lethal neurodegenerative disease, which selectively affects motor neurons throughout the central nervous system. The extensive distribution of motor neurons is an obstacle to applying cell transplantation therapy for the treatment of ALS. To overcome this problem, we developed a cell transplantation method via the fourth cerebral ventricle in mice. We used mouse olfactory ensheathing cells (OECs) and rat mesenchymal stem cells (MSCs) as donor cells. OECs are reported to promote regeneration and remyelination in the spinal cord, while MSCs have a capability to differentiate into several types of specific cells including neural cells. Furthermore both types of cells can be relatively easily obtained by biopsy in human. Initially, we confirmed the safety of the operative procedure and broad distribution of grafted cells in the spinal cord using wild-type mice. After transplantation, OECs distributed widely and survived as long as 100 days after transplantation, with a time-dependent depletion of cell number. In ALS model mice, OEC transplantation revealed no adverse effects but no significant differences in clinical evaluation were found between OEC-treated and non-transplanted animals. After MSC transplantation into the ALS model mice, females, but not males, showed a statistically longer disease duration than the non-transplanted controls. We conclude that intrathecal transplantation could be a promising way to deliver donor cells to the central nervous system. Further experiments to elucidate relevant conditions for optimal outcomes are required. © 2008 Elsevier Inc. All rights reserved.
Introduction Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder that clinically is characterized by skeletal muscle wasting and paralysis. Without respiratory assistance, death usually occurs less than 5 years after the clinical onset (Cleveland and Rothstein, 2001). ALS exhibits progressive loss of both upper and lower motor neurons in the cerebral cortex, brain stem and spinal cord. The cause of ALS is still elusive and no effective treatment exists so far. Approximately 90% of the disease is sporadic and about 10% is familial in form, called FALS. Twenty percent of FALS cases are associated with mutations of the Cu/Zn superoxide dismutase (SOD1) gene (Rosen et al., 1993; Cudkowicz et al., 1997). In recent years, ever-greater hopes have been placed in regenerative medicine using in-vitro-expanded cells to cure a damaged or degenerated central nervous system (CNS). As motor neurons distribute throughout the entire CNS, replenishment using stem cells by ⁎ Corresponding author. Fax: +81 859 38 6759. E-mail address: [email protected] (Y. Watanabe). 1 These authors contributed equally to this work. 0014-4886/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2008.07.011
focal injection would be impractical for the treatment of ALS. To make the transplanted cells distribute diversely, we developed a transplantation method for mice that allows even diffusion of donor cells throughout the CNS. Then we applied this protocol to cell transplant treatment in a mouse model of ALS. We chose olfactory ensheathing cells (OECs) and mesenchymal stem cells (MSCs) as suitable donor cells based on the following evidence. OECs, a specific glial cell type residing in the nose, assists axonal extension of olfactory sensory neurons from the peripheral nervous system to CNS (Doucette, 1995). The OEC promotes regeneration and remyelination of injured spinal pathways and enhances motor recovery in experimental animal models with spinal cord injury (Ramon-Cueto and Nieto-Sampedro, 1994; Li et al., 1997; RamonCueto et al., 1998). Indeed, OECs from patients with spinal cord lesion were grafted autologously as a clinical trial in Australia (Feron et al., 2005). On the other hand, MSCs can differentiate into various kinds of cells, including cardiomyocytes, neurons, and glia (Woodbury et al., 2000). Human MSC transplantation was reported to increase voluntary activity pre-symptomatically after transplantation into ALS model mice (Habisch et al., 2007). Importantly, both OECs and MSCs can be obtained by biopsy in human (Bianco et al., 2004; Mazzini et al., 2006),
432
E. Morita et al. / Experimental Neurology 213 (2008) 431–438
thereby making autologous transplantation feasible. Using these cells we attempted to alleviate clinical signs of motor neuron degeneration in a mouse model of ALS.
Table 1 Number of mice in different groups
Materials and methods
WT mice (n = 20)
Olfactory ensheathing cells (OECs)
Transgenic L126delTT (n = 29) Transgenic L126delTT (n = 34)
Nasal olfactory mucosae were collected from 5-week-old “green” mice: C57BL/6CR background, ubiquitously expressing green fluorescent protein (GFP) (Okabe et al., 1997). The tissues were incubated for 40 min at 37 °C in Dispase II (Roche, Mannheim, Germany) to isolate the lamina propria (LP) from the olfactory epithelium. The LP was then transferred into 0.25% collagenase type IA (Sigma, USA) in Dulbecco's modified Eagle medium/Ham's F12 (DMEM/F12) and incubated for 30 min at 37 °C. After centrifugation, the cell pellet was resuspended in the DMEM/F12 with 10% fetal bovine serum (FBS) and plated on poly-L-lysine (PLL)-coated dishes. At 80–90% confluence the cells were harvested and passaged with DMEM/F12 containing 1% insulin-transferrin-selenium supplement (ITS-X, Gibco, USA) and 50 ng/ml neurotrophin 3 (NT3, PeproTech EC Ltd, UK) onto a PLL-coated dish. Cells were maintained in this medium and passaged 5 to 6 times until OECs were purified to more than 95% (Bianco et al., 2004). Mesenchymal stem cells (MSCs) We isolated MSCs from GFP rats, because the technique to isolate and expand MSCs from mice is difficult especially from GFP transgenic mice (Peister et al., 2004). Rat MSCs were obtained from tibial and femoral bones of a 5-week-old LEW-GFP rat (Hakamata et al., 2001). Both ends of the bones were cut, and the bone marrow was extruded by using 21G needle with α-MEM (NK System, Japan) supplemented with 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin. After centrifugation the cell pellet was suspended in the medium. The cells were seeded on 75 cm2 flasks. Twenty-four hours after the seeding medium was refreshed to remove floating cells. At confluence the cells were passaged and re-plated. This cycle was repeated 3 times, after which the cells were preserved at −80 °C with cell banker (Uji field, Japan) until use (Tohill et al., 2004). As an additional marker, because the GFP fluorescence of rat MSCs proved to be too weak to identify transplanted cells in the host spinal cord, we also labeled them with dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) (Molecular Probes, USA). MSCs were incubated in the culture medium diluting 2 μg/ml DiI for 5 min at 37 °C, and then for an additional 15 min at 4 °C. After labeling, MSCs were washed twice with PBS and then transplanted. To provide immune suppression because MSCs were derived from rat, FK506 (3 mg/kg/day, kindly provided from Fujisawa Co, Japan) was administered orally to MSC-transplanted and sham-operated animals daily from 1 week before the operation for the duration of the study. Animals All the studies were carried out in accordance with the Guidelines for Animal Experimentation at the Faculty of Medicine, Tottori University. As the animal model of ALS, we used transgenic SOD1Leu126delTT mice with a mutated human SOD1 gene. This gene has a 2-bp deletion at codon 126 (Leu126delTT) found in a human FALS family (Nakashima et al., 1995). The mice begin to show clinical symptoms at around 20 to 21 weeks of age. After the disease duration of 1–2 weeks, mice die at 22–23 weeks (Watanabe et al., 2005). In these mice, there are no differences between genders with regard to ages of onset and death, and disease duration (Watanabe et al., 2005). The effects of transplantation on clinical outcomes were assessed by forming two groups of transgenic mice: littermates were dis-
OEC No operation OEC Sham-operated MSC Sham-operated
10-day survival
30-day survival
Endpoint
4 (4) 0 5 (5) 2 (2) 2 (2) 2 (2)
4 0 5 2 2 2
6 (6) 6 (6) 8 (4) 7 (4) 13 (8) 13 (7)
(4) (5) (2) (2) (2)
Clinical analysis was performed only on the endpoint groups. The number of animals used for histo-pathological analysis is shown in parentheses.
tributed equally into cell-transplanted and sham-operated mice, with the groups balanced with regard to date of birth, body weight, and gender. Littermate mice excess to these groups were divided into groups for histo-pathological analysis 10 or 30 days after the transplantation. The groups and numbers of mice used are listed in Table 1. Stereotaxic operation Cell transplantation was performed on wild-type (WT) C57BL/6CR mice and transgenic SOD1-Leu126delTT mice using sterotaxic coordinates (Bai et al., 2003). A hole 0.5 mm in diameter was made in the skull at the site 6 mm caudal to the lambda suture on the midline. A 30-G needle (Dentsply, Sankin, Japan) was then inserted into the fourth ventricle (3.75 mm depth from cerebellar surface) and 10 μl (3 to 4 × 105 cells) of suspension (OECs or MSCs) was injected slowly over 10 min. As sham controls, animals were injected with 10 μl phosphate-buffered saline (PBS) without cells. Prior to cell injection experiments, 5 μl pontamine sky blue (Sigma, USA) was injected using the same procedure, to confirm the accuracy of the operation. Clinical evaluation Clinical evaluation was performed every week from 1 week before the operation. This evaluation included body weight, hind limb extension score, foot print analysis, ages of onset and endpoint, and disease duration. A hind limb extension score was recorded as 0, 0.5, 1.0, 1.5 and 2.0 after evaluation of the reflex of lower limb according to published procedures (Garbuzova-Davis et al., 2003). Foot print analysis provided an indication of whether stride and gait were altered by the treatment. A composite stride-length measure was calculated in the following manner. The rear feet were dipped in ink and the animals were allowed to walk freely on paper. The distance between adjacent footprints was measured, starting with the first right–left pair. Then the distance from this left footprint to the next right footprint was measured. This was repeated for six adjacent consecutive footprints and the average distance between adjacent footprints was calculated, referred to here as “stride length”. Age of disease onset was determined when tremor or subtle weakness was identified during suspension by the tail and/or during voluntary walking. Endpoint was arbitrarily determined when mice were not able to get up from a lying position, or the eyes were highly infected (Jaarsma et al., 2001). These signs of impending death or actual death provided the experimental endpoint for survival and histological analysis. Preparation of tissue sections Wild-type C57BL/6CR mice transplanted with OECs, mice were histologically evaluated at 10, 30 and 100 days (an arbitrary endpoint based on the approximate age of the transgenic mice) after the transplantation (n = 4, 4, and 6 males at each time point, respectively)
E. Morita et al. / Experimental Neurology 213 (2008) 431–438
(Table 1). Wild-type C57BL/6CR mice without any operative procedure were also histologically evaluated for inflammation markers (male 3, female 3). Transgenic SOD1-Leu126delTT mice, transplanted with OECs or MSCs, were histologically evaluated at 10 days, 30 days and at the experimental endpoint. In the OEC-transplanted group, the following numbers of animals were analysed: 10 days, 3 male, 2 female; 30 days, 4 male, 1 female; endpoint, 4 male. In the (OEC) control group, the numbers were: 10 days, 2 female; 30 days, 1 male, 1 female; endpoint 3 male, 1 female. In the MSC-transplanted group the numbers were: 10 days, 2 female; 30 days, 2 female; endpoint, 4 male, 4 female. In the (MSC) control group the numbers were: 10 days, 2 female; 30 days, 1 male, 1 female; endpoint (4 male, 3 female). Animals were deeply anesthetized by intraperitoneal injection of pentobarbital sodium and transcardially perfused with normal saline solution, followed by 4% paraformaldehyde in 0.1 M phosphate-buffered solution. A lumbar spinal cord segment was immersed in the same fixative and cryoprotected in a series of sucrose solutions; 10%, 15% and 20% sucrose in PBS at 4 °C for 2 days. After tissue embedded in Tissue-Tec (Sakura, Japan) and frozen in liquid nitrogen cooled isopentane. Ten micrometer-thick sections were cut transversely using a cryostat. Histopathological and immunohistochemical staining Selected sections were stained with hematoxylin and eosin and with immunostaining for GF P, glial fibrillary acidic protein (GFAP), or Iba1. For immunohistochemical study, the sections were permeabilized with 0.2% Triton X-100 (Wako, Japan) and 4% paraformaldehyde, and blocked with 1 mg/ml bovine serum albumin (BSA, Sigma) and 1% normal goat serum (Funakoshi, Japan) in PBS. Primary antibodies used and their final dilutions were as follows: mouse anti-GFP monoclonal
433
antibody (1:250, MAB3580, Chemicon, USA), rabbit polyclonal antibody against GFP (1:250, MBL, Japan), rabbit polyclonal antibody against GFAP (prediluted, Dako, USA), and rabbit anti Iba1 (1:100, Wako, Japan). The secondary antibody was goat anti-mouse antibody IgG fluorescein isothiocyanate (FITC) (1:100, Santa Cruz Biotechnology, CA, USA), goat anti-rabbit antibody IgG FITC (1:100, Santa Cruz Biotechnology, CA, USA), and goat anti-rabbit antibody IgG Texas Red (TR) (1:100, Santa Cruz Biotechnology, CA, USA). Specimens were incubated with primary antibodies overnight at 4 °C, followed by incubation with secondary antibodies for an hour at room temperature. Immunoperoxidase staining The cryostat sections were first permeabilized with 0.2% Triton X-100 in PBS for 30 min, washed in PBS three times, and incubated 3% H2O2 for an hour at 37 °C. After washing in PBS three times, sections were blocked in 1 mg/ml BSA and 1% normal goat serum in PBS. The sections were then incubated with mouse anti-GFP antibody (1:250) and mouse anti-NeuN antibody (1:500, MAB377, Chemicon, USA) overnight at 4 °C. After washing in PBS three times, sections were incubated labeled polymer-horse radish peroxidase (HRP) anti-mouse (DAKO, USA) overnight at 4 °C. Subsequently, washed in PBS three times, the sections were reacted with 3,3′-diaminobenzidine tetrahydrochloride (DAB) for 15 min, followed by incubation with the same reagent containing 3% H2O2 for 5 min to visualized the antibody-bound HRP. Motor neuron quantification Serial cross-sections of the lumber spinal cords were stained with anti-NeuN antibodies. NeuN positive cells in the ventral gray matter
Fig. 1. Stereotaxic injection into the fourth ventricle in wild-type C57BL/6 mice. (A) Blue dye injected into the fourth ventricle diffuses throughout the surface of the spinal cord and cerebral base. (B) A small hemorrhage was observed resulting from the injection. The arrow indicates the injected region. The arrowhead indicates the fourth ventricle. (C) The spinal cord surface 30 days after GFP-OEC transplantation shown without fixation. (D–F) There were no differences between the OEC-transplanted and non-transplanted mice with regard to long-term motor function after the transplantation but there were minor differences in body weight (n = 6 each). The arrow indicates when the transplant was performed. ⁎P b 0.05. Scale bar = 1 mm in B; 500 μm in C.
434
E. Morita et al. / Experimental Neurology 213 (2008) 431–438
were counted in every fifth section and the results of at least 5 sections were averaged (Pardo et al., 2006). Statistical analysis The quantitative data were expressed as mean ± standard error of mean (SEM). Statistical analysis was performed with repeated measures ANO VA using Statview (Abacus Concept, USA). The statistical analysis of onset age and endpoint was performed with the Kaplan Meier method of SPSS 11.5J for Windows (SPSS inc, USA).
We then evaluated the perioperative and long-term safety of transplantation of OECs into the fourth ventricle in wild-type C57BL/ 6CR mice. Clinical assessments were carried out weekly for 9 weeks from 1 week before the transplantation until the mice were 200 days old. Similar assessments were made of sham-transplanted mice. There were no differences between OEC-transplanted (n = 6) and nontransplanted mice (n = 6) for all parameters (Figs. 1D–F). Examination of the spinal cords by fluorescent microscopy at 10, 30 and 100 days post-transplantation revealed OECs on the surface of the spinal cord for at least 100 days after transplantation while decreasing in a time-dependent manner (Fig. 1C).
Results OEC transplantation Transplantation procedure In an initial study, the brain and spinal cord were observed 3 h after injecting with pontamine sky blue via the fourth ventricle. The dye had diffused throughout the spinal cord and base of brain, with the surfaces of cerebellum and cerebrum free from dye at this time point (Fig. 1A). Microscopic observation revealed the presence of a small hemorrhagic scar in the cerebellum from the injection (Fig. 1B).
OECs derived from GFP mice were transplanted into transgenic SOD1-Leu126delTT animals (n = 8) at 13 weeks of age. Then we compared OEC-transplanted with control (PBS injected) groups (n = 7) by weight, hind limb extension score, stride length, ages of onset and endpoint, and disease duration. On the basis of clinical parameters there were no differences in any of these measures (Figs. 2A–E). Average ages of onset were 139.5 ± 4.5 days and 137.1 ± 6.4 days, and endpoints were 151.1 ± 6.0 days (OEC) and 156.1 ± 6.8 days (control).
Fig. 2. Effect of OEC transplantation in transgenic SOD1-Leu126delTT mice. (A–E) There were no significant differences between transplanted and sham-operated transgenic mice in body weight (A), hind limb extension (B), stride length (C). Kaplan Meier incidence (D) and survival (E) plots show no differences between the two groups — transplantation of OECs did not prolong the survival period of transgenic SOD1-Leu126delTT mice (OEC; n = 8, PBS; n = 7). (F) The surface of the spinal cord showing GFP-OECs 10 days after transplantation shown without fixation. (G) Section of the spinal cord of 30 days after GFP-OEC transplantation. No OECs were observed to enter the spinal cord parenchyma. (H) There was no difference between the OEC-transplanted and non-transplanted mice in the number of motor neurons in the ventral gray matter (n = 4 in each group). Scale bar= 0.33 mm in F; 100 μm in G.
E. Morita et al. / Experimental Neurology 213 (2008) 431–438
OECs reportedly have some beneficial characteristics for transplantation such as the ability to penetrate even to the glial scar area (Oudega., 2007) but our observations revealed that OECs did not invade into spinal cord parenchyma (Fig. 2G) remaining on the surface of the spinal cord (Fig. 2F). The number of motor neurons remaining at endpoint was similar between OEC-transplanted animals (17.1 ± 4.1/ hemi section; n = 4) and sham-operated animals (17.7 ± 2.2/hemi section; n = 4) (Fig. 2H). MSC transplantation MSCs were transplanted into transgenic SOD1-Leu126delTT mice (n = 13). As a control, PBS only was injected into transgenic SOD1Leu126delTT mice (n = 13). Because OEC transplantation at 13 weeks did not show any beneficial effect in transgenic SOD1-Leu126delTT animals, MSC transplantation in transgenic SOD1-Leu126delTT mice was performed at 14 weeks. Compared to controls the MSCtransplanted group did not show any difference on body weight,
435
extension score, and stride length (Figs. 3A–C). There were beneficial trends in ages of onset and endpoint, and disease duration in MSCtransplanted mice compared to control mice, however these did not reach statistical significance (Figs. 3D and E). Intriguingly, when we compared MSC-treated female mice with control female mice (n = 7 each), there was a statistically significant difference for disease duration between the two groups (MSC; 21.6 ± 5.4 days, control; 10.0 ± 2.2 days) (Fig. 3F). We observed that MSCs labeled with DiI stayed on the surface of the spinal cord of 10 and 30 days after transplantation (Figs. 4A and B), moreover some MSCs invaded into spinal cord parenchyma (Figs. 4C and D). In the brain, especially in the Purkinje cell layer in the cerebellum, we were able to observe survival and invasion of MSCs or their progeny (Figs. 4E and F). Using anti-GFP antibody staining, the GFP-positive MSC-derived cells still existed in sections of spinal cord at endpoint (Figs. 4G and H). The number of motor neurons remaining at endpoint was greater in the MSC group compared to the control group but this was not statistically significant (18.5 ± 3.8/hemi section,
Fig. 3. Effect of MSC transplantation in transgenic SOD1-Leu126delTT mice. (A–E) There were no significant differences between transplanted and sham-operated transgenic mice in body weight (A), hind limb extension (B), stride length (C). Kaplan Meier incidence (D) and survival (E) plots show no differences between the two groups — transplantation of MSCs did not prolong the survival period of transgenic SOD1-Leu126delTT mice (n = 13 in each group). (F) When sex is taken into account, disease duration of females treated with MSCs was significantly longer than control females (male; n = 6, female; n = 7 in each treatment group). ⁎P = 0.025. (G) There was no difference between the MSC-transplanted and nontransplanted mice in the number of motor neurons in the ventral gray matter (MSC; n = 8, PBS; n = 7).
436
E. Morita et al. / Experimental Neurology 213 (2008) 431–438
Fig. 4. Histopathological evaluations of the spinal cord and the brain after MSC transplantation. Spinal cord and brain after sham injections with phosphate-buffered saline (PBS: A, C, E, and G) or MSCs (MSC: B, D, F, and H). (B) 10 days after transplantation, many MSCs labeled with DiI survived on the surface of the spinal cord, shown here without fixation, and remained 30 days later (data not shown). (D) Section of the spinal cord 30 days after MSC transplantation. MSCs (DiI labeled, red) entered into spinal cord parenchyma. Astrocytes labeled with anti-GFAP antibody (green). (F) MSC also invaded into parenchyma of the brain, including into the Purkinje cell layer of the cerebellum: DiI labeled MSC (red), GFAP-labeled astrocytes (green). (H) MSCs were detected in sections of lumber spinal cord after fixation by anti-GFP antibody staining (brown). Scale bar = 500 μm in A and B; 100 μm in C–H.
n = 8, and 15.9 ± 3.9/hemi section, n = 7, respectively; Fig. 3G). There were no discernable differences in the numbers of remaining motor neurons in males compared to females. In order to assess the effect of MSC transplantation on suppressing inflammation, we employed immunohistochemical analyses for reactive astrogliosis (using antibodies to glial proteins, GFAP) and for activated microglia (using an antibody to Iba1). There was noticeable immunoreactivity for GFAP and Iba1 in MSC-transplanted animals at 10 days, 30 days and at experimental endpoint (Fig. 5). There was also immunostaining for both proteins in sham-operated control animal injections with PBS alone (Figs. 5C and H). In contrast, non-transgenic, unoperated, wild-type mice showed less immunoreactivity using the same antibodies and similar processing. These data were not
quantified but they suggest that the SOD1-Leu126delTT mice have increased “baseline” inflammation compared to wild-type controls which was not affected by MSC transplantation. Discussion In this study, we demonstrated that intrathecal cell transplantation via the fourth ventricle is feasible and safe in mouse. After transplantation in a mouse model of ALS (Watanabe et al., 2005), OECs showed no beneficial effect whereas MSCs prolonged the survival of female transgenic SOD1-Leu126delTT animals. Previously, a variety of cell transplantation experiments have been employed to treat a mouse model of ALS (the G93A SOD1 transgenic
E. Morita et al. / Experimental Neurology 213 (2008) 431–438
437
Fig. 5. Reactive astrogliosis and microglia activation after MSC transplantation. Spinal cord sections from MSC-transplanted, transgenic SOD1-Leu126delTT mice (A–C, F–H) and PBS injected, transgenic SOD1-Leu126delTT mice (D and I) 10 days, 30 days after transplantation and at experimental endpoint were immunoreactive for GFAP (A–D) and Iba1 (F–I). Less immunoreactivity was observed in non-transgenic mice (WT) of a similar age to the transgenic mice at experimental endpoint (E and J). Scale bar = 100 μm in A–J.
mouse). There is little consensus, however, on the effects of cell transplantation. Some experiments showed beneficial effects on animal clinical parameters (Chen and Ende, 2000; Ende et al., 2000; Willing et al., 2001; Garbuzova-Davis et al., 2002; 2003; Corti et al., 2004), while others showed no beneficial effects (Hemendinger et al., 2005; Habisch et al., 2007). It is difficult to generalize among these results because several parameters vary, namely, the cell type transplanted, the age at transplantation and the administration route. The donor cells included human umbilical-cord-blood derived cells (Chen and Ende, 2000; Ende et al., 2000; Garbuzova-Davis et al., 2003; Habisch et al., 2007), human neuron-like cells (Willing et al., 2001; GarbuzovaDavis et al., 2002), mouse bone marrow (mesenchymal) stem cells (Corti et al., 2004; Habisch et al., 2007), sertoli cells (Hemendinger et al., 2005) and neural stem cells (Corti et al., 2004). The ages of transplantation varied from 4 weeks (Corti et al., 2004) to 10 weeks (Corti et al., 2007). The administration routes included intravenous (Chen and Ende, 2000; Ende et al., 2000; Garbuzova-Davis et al., 2003), intraperitoneal (Corti et al., 2004), focal injection (L4–L5 segment, (Willing et al., 2001; Garbuzova-Davis et al., 2002; Corti et al., 2007), and intrathecal injection via cisterna magna (Habisch et al., 2007). In the present experiments, MSC transplantation only affected female mice. Sex differences have been observed previously in treatments of the G93A SOD1 mouse model of ALS. After transplantation of MSCs females, but not males, were more spontaneously active (although other clinical measures were not affected; Habisch et al., 2007). After treatment with clenbutol, a beta-2 adrenoceptor, the symptoms in females, but not males, improved (Teng et al., 2006), whereas treatment with cyclosporine A, a well-known immunosuppressant, had a robust effect on males and not females (Kirkinezos et al., 2004). These gender differences are so far without obvious explanation. In the present study, MSCs were shown to penetrate the
parenchyma of spinal cord and we speculate a possible neuroprotective effect or modulation of the neural environment could result. Previous results of transplantation experiments including ours indicate that required characteristics of donor cells might be their possession of neurotrophic effect and the ability to change neural environment, rather than necessarily being committed to neural lineage. Transplantation timing is also an elusive parameter. Transplantation is normally applied before the onset of ALS as a preventative therapy. In our experiment, the transplantation point was influenced by the fact that OECs survive at least as long as 100 days after transplantation with time-dependant depletion, and that early pathological abnormality in the spinal cord, such as reduction of motor neurons and reactive gliosis, appears before the appearance of clinical symptoms in transgenic SOD1-Leu126delTT mice (Watanabe et al., 2005). In human cases, disease duration is usually 3 to 5 years while only weeks or months in mice. Because our goal is to treat human ALS patients, we should keep in mind the difference, with regard to life span and natural course of the disease, between mouse and human. It is vital to understand how long the donor cells survive in the spinal cord and how we can prolong the survival of the donor cells. In any case, serial administration might be necessary for human treatment. With an intrathecal route of administration (via lumbar puncture in humans), repeated administration would be feasible. Administration route is important and indeed is a main theme of this report. Because of the large size of the human spinal cord, focal injection of donor cells, which is shown to be successful in some experiments on mice (Willing et al., 2001; Garbuzova-Davis et al., 2002), would not be so ideal for application in human. To overcome this problem, intrathecal diffusion appears a promising way to distribute the donor cells. With similar intent, human mesenchymal stem cells were transplanted into G93A SOD1 transgenic mice by
438
E. Morita et al. / Experimental Neurology 213 (2008) 431–438
intrathecal injection via cisterna magna (Habisch et al., 2007). That procedure did not affect the clinical outcome of the disease and it was speculated that the poor therapeutic potential was due to poor distribution of the transplanted cells into the spinal cord. In the present study, this was improved by injection into the fourth ventricle, rather than cisterna magna. As shown here, blue dye diffuses throughout the spinal cord in less than 3 h. The physiological CSF stream passes from the fourth ventricle caudally down the central canal of the spinal cord, whereas CSF in the cisterna magna is external to the spinal cord and circulates rostrally. This may explain why transplantation via the fourth ventricle is superior to via the cisterna magna with regard to delivering the cells to the spinal cord. There are other aspects, besides the parameters mentioned above, that could be optimized for successful therapy in the ALS mouse, and eventually human. For example, is there an optimum number of cells sufficient for cell therapy? Could the use of cell adhesion molecules facilitate the survival of donor cells? Would a combination of cell- and gene-therapy be more effective than either alone? There are reports about effectiveness of some neurotrophic factors in ALS patients. Ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF) and insulinlike growth factor (IGF-1) have demonstrated effect of protection and regeneration of motor neurons, and some have been> applied clinically (Ekestern, 2004). Such molecules could be delivered intrathecally by pump or by genetically modified cells that secrete neurotrophic factors. Normally the blood brain barrier prevents neurotrophic factors accessing directly to brain and spinal cord after intravenous injection or oral administration, although a recent report indicates that this barrier is compromised in G93A mice (GarbuzovaDavis et al., 2007). OECs are a candidate cell for transplantation after genetic modification because they can be engineered to produce neurotrophins (Ruitenberg et al., 2005), they can be safely harvested and autologously transplanted in humans (Feron et al., 2005), and they survive for long periods in the spinal cord after intrathecal injection, as shown here. Acknowledgments This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Watanabe Y), and by a Grant from the Research Committee of CNS Degenerative Diseases, Ministry of Health, Labour and Welfare of Japan (Nakashima K). References Bai, H., Suzuki, Y., Noda, T., Wu, S., Kataoka, K., Kitada, M., Ohta, M., Chou, H., Ide, C., 2003. Dissemination and proliferation of neural stem cells on the spinal cord by injection into the fourth ventricle of the rat: a method for cell transplantation. J. Neurosci. Methods 124, 181–187. Bianco, J.I., Perry, C., Harkin, D.G., Mackay-Sim, A., Feron, F., 2004. Neurotrophin 3 promotes purification and proliferation of olfactory ensheathing cells from human nose. Glia 45, 111–123. Chen, R., Ende, N., 2000. The potential for the use of mononuclear cells from human umbilical cord blood in the treatment of amyotrophic lateral sclerosis in SOD1 mice. J. Med. 31, 21–30. Cleveland, D.W., Rothstein, J.D., 2001. From Charcot to Lou Gehrig: deciphering selective motor neuron death in ALS. Nat. Rev. Neurosci. 2, 806–819. Corti, S., Locatelli, F., Donadoni, C., Guglieri, M., Papadimitriou, D., Strazzer, S., Del Bo, R., Comi, G.P., 2004. Wild-type bone marrow cells ameliorate the phenotype of SOD1G93A ALS mice and contribute to CNS, heart and skeletal muscle tissues. Brain 127, 2518–2532. Corti, S., Locatelli, F., Papadimitriou, D., Del Bo, R., Nizzardo, M., Nardini, M., Donadoni, C., Salani, S., Fortunato, F., Strazzer, S., Bresolin, N., Comi, G.P., 2007. Neural stem cells LewisX+ CXCR4+ modify disease progression in an amyotrophic lateral sclerosis model. Brain 130, 1289–1305. Cudkowicz, M.E., McKenna-Yasek, D., Sapp, P.E., Chin, W., Geller, B., Hayden, D.L., Schoenfeld, D.A., Hosler, B.A., Horvitz, H.R., Brown, R.H., 1997. Epidemiology of mutations in superoxide dismutase in amyotrophic lateral sclerosis. Ann. Neurol. 41, 210–221. Doucette, R., 1995. Olfactory ensheathing cells: potential for glial cell transplantation into areas of CNS injury. Histol. Histopathol. 10, 503–507.
Ekestern, E., 2004. Neurotrophic factors and amyotrophic lateral sclerosis. Neurodegener. Dis. 1, 88–100. Ende, N., Weinstein, F., Chen, R., Ende, M., 2000. Human umbilical cord blood effect on sod mice (amyotrophic lateral sclerosis). Life Sci. 67, 53–59. Feron, F., Perry, C., Cochrane, J., Licina, P., Nowitzke, A., Urquhart, S., Geraghty, T., Mackay-Sim, A., 2005. Autologous olfactory ensheathing cell transplantation in human spinal cord injury. Brain 128, 2951–2960. Garbuzova-Davis, S., Willing, A.E., Milliken, M., Saporta, S., Zigova, T., Cahill, D.W., Sanberg, P.R., 2002. Positive effect of transplantation of hNT neurons (NTera 2/D1 cell-line) in a model of familial amyotrophic lateral sclerosis. Exp. Neurol. 174, 169–180. Garbuzova-Davis, S., Willing, A.E., Zigova, T., Saporta, S., Justen, E.B., Lane, J.C., Hudson, J.E., Chen, N., Davis, C.D., Sanberg, P.R., 2003. Intravenous administration of human umbilical cord blood cells in a mouse model of amyotrophic lateral sclerosis: distribution, migration, and differentiation. J. Hematother. Stem Cell Res. 12, 255–270. Garbuzova-Davis, S., Haller, E., Saporta, S., Kolomey, I., Nicosia, S.V., Sanberg, P.R., 2007. Ultrastructure of blood-brain barrier and blood-spinal cord barrier in SOD1 mice modeling ALS. Brain Res. 1157, 126–137. Habisch, H.J., Janowski, M., Binder, D., Kuzma-Kozakiewicz, M., Widmann, A., Habich, A., Schwalenstocker, B., Hermann, A., Brenner, R., Lukomska, B., Domanska-Janik, K., Ludolph, A.C., Storch, A., 2007. Intrathecal application of neuroectodermally converted stem cells into a mouse model of ALS: limited intraparenchymal migration and survival narrows therapeutic effects. J. Neural Transm. 18, 18. Hakamata, Y., Tahara, K., Uchida, H., Sakuma, Y., Nakamura, M., Kume, A., Murakami, T., Takahashi, M., Takahashi, R., Hirabayashi, M., Ueda, M., Miyoshi, I., Kasai, N., Kobayashi, E., 2001. Green fluorescent protein-transgenic rat: a tool for organ transplantation research. Biochem. Biophys. Res. Commun. 286, 779–785. Hemendinger, R., Wang, J., Malik, S., Persinski, R., Copeland, J., Emerich, D., Gores, P., Halberstadt, C., Rosenfeld, J., 2005. Sertoli cells improve survival of motor neurons in SOD1 transgenic mice, a model of amyotrophic lateral sclerosis. Exp. Neurol. 196, 235–243. Jaarsma, D., Rognoni, F., van Duijn, W., Verspaget, H.W., Haasdijk, E.D., Holstege, J.C., 2001. CuZn superoxide dismutase (SOD1) accumulates in vacuolated mitochondria in transgenic mice expressing amyotrophic lateral sclerosis-linked SOD1 mutations. Acta Neuropathol. (Berl) 102, 293–305. Kirkinezos, I.G., Hernandez, D., Bradley, W.G., Moraes, C.T., 2004. An ALS mouse model with a permeable blood-brain barrier benefits from systemic cyclosporine A treatment. J. Neurochem. 88, 821–826. Li, Y., Field, P.M., Raisman, G., 1997. Repair of adult rat corticospinal tract by transplants of olfactory ensheathing cells. Science 277, 2000–2002. Mazzini, L., Mareschi, K., Ferrero, I., Vassallo, E., Oliveri, G., Boccaletti, R., Testa, L., Livigni, S., Fagioli, F., 2006. Autologous mesenchymal stem cells: clinical applications in amyotrophic lateral sclerosis. Neurol. Res. 28, 523–526. Nakashima, K., Watanabe, Y., Kuno, N., Nanba, E., Takahashi, K., 1995. Abnormality of Cu/Zn superoxide dismutase (SOD1) activity in Japanese familial amyotrophic lateral sclerosis with two base pair deletion in the SOD1 gene. Neurology 45, 1019–1020. Okabe, M., Ikawa, M., Kominami, K., Nakanishi, T., Nishimune, Y., 1997. ‘Green mice’ as a source of ubiquitous green cells. FEBS Lett. 407, 313–319. Oudega, M., 2007. Schwann cell and olfactory ensheathing cell implantation for repair of the contused spinal cord. Acta. Physiol. 189, 181–189. Pardo, A.C., Wong, V., Benson, L.M., Dykes, M., Tanaka, K., Rothstein, J.D., Maragakis, N.J., 2006. Loss of the astrocyte glutamate transporter GLT1 modifies disease in SOD1 (G93A) mice. Exp. Neurol. 201, 120–130. Peister, A., Mellad, J.A., Larson, B.L., Hall, B.M., Gibson, L.F., Prockop, D.J., 2004. Adult stem cells from bone marrow (MSCs) isolated from different strains of inbred mice vary in surface epitopes, rates of proliferation, and differentiation potential. Blood 103, 1662–1668 (Epub 2003 Oct 1630). Ramon-Cueto, A., Nieto-Sampedro, M., 1994. Regeneration into the spinal cord of transected dorsal root axons is promoted by ensheathing glia transplants. Exp. Neurol. 127, 232–244. Ramon-Cueto, A., Plant, G.W., Avila, J., Bunge, M.B., 1998. Long-distance axonal regeneration in the transected adult rat spinal cord is promoted by olfactory ensheathing glia transplants. J. Neurosci. 18, 3803–3815. Rosen, D.R., Siddique, T., Patterson, D., Figlewicz, D.A., Sapp, P., Hentati, A., Donaldson, D., Goto, J., O'Regan, J.P., Deng, H.X., 1993. Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature 362, 59–62. Ruitenberg, M.J., Levison, D.B., Lee, S.V., Verhaagen, J., Harvey, A.R., Plant, G.W., 2005. NT-3 expression from engineered olfactory ensheathing glia promotes spinal sparing and regeneration. Brain 128, 839–853. Teng, Y.D., Choi, H., Huang, W., Onario, R.C., Frontera, W.R., Snyder, E.Y., Sabharwal, S., 2006. Therapeutic effects of clenbuterol in a murine model of amyotrophic lateral sclerosis. Neurosci. Lett. 397, 155–158. Tohill, M., Mantovani, C., Wiberg, M., Terenghi, G., 2004. Rat bone marrow mesenchymal stem cells express glial markers and stimulate nerve regeneration. Neurosci. Lett. 362, 200–203. Watanabe, Y., Yasui, K., Nakano, T., Doi, K., Fukada, Y., Kitayama, M., Ishimoto, M., Kurihara, S., Kawashima, M., Fukuda, H., Adachi, Y., Inoue, T., Nakashima, K., 2005. Mouse motor neuron disease caused by truncated SOD1 with or without C-terminal modification. Brain Res. Mol. Brain Res. 135, 12–20. Willing, A.E., Garbuzova-Davis, S., Saporta, S., Milliken, M., Cahill, D.W., Sanberg, P.R., 2001. hNT neurons delay onset of motor deficits in a model of amyotrophic lateral sclerosis. Brain Res. Bull. 56, 525–530. Woodbury, D., Schwarz, E.J., Prockop, D.J., Black, I.B., 2000. Adult rat and human bone marrow stromal cells differentiate into neurons. J. Neurosci. Res. 61, 364–370.
Contents lists available at ScienceDirect
Experimental Neurology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y e x n r
A novel cell transplantation protocol and its application to an ALS mouse model Eri Morita a,b,1, Yasuhiro Watanabe a,⁎,1, Miho Ishimoto a,1, Toshiya Nakano a, Michio Kitayama a, Kenichi Yasui a, Yasuyo Fukada a, Koji Doi a, Asanka Karunaratne c, Wayne G Murrell c, Ratneswary Sutharsan c, Alan Mackay-Sim c, Yoshio Hata d, Kenji Nakashima a a
Department of Neurology, Institute of Neurological Sciences, Faculty of Medicine, Tottori University, 36-1 Nishico-cho, Yonago 683-8504, Japan Division of Neurobiology, School of Life Science, Tottori University Graduate School of Medical Science, Yonago 683-8504, Japan National Centre for Adult Stem Cell Research, Eskitis Institute for Cell and Molecular Therapies, Griffith University, Brisbane, Queensland 4111, Australia d Department of Integrative Bioscience, Tottori University Graduate School of Medical Science, Yonago 683-8504, Japan b c
a r t i c l e
i n f o
Article history: Received 13 February 2008 Revised 18 June 2008 Accepted 8 July 2008 Available online 22 July 2008 Keywords: Amyotrophic lateral sclerosis Transplantation Intrathecal Olfactory ensheathing cell Mesenchymal stem cell
a b s t r a c t Amyotrophic lateral sclerosis (ALS) is a lethal neurodegenerative disease, which selectively affects motor neurons throughout the central nervous system. The extensive distribution of motor neurons is an obstacle to applying cell transplantation therapy for the treatment of ALS. To overcome this problem, we developed a cell transplantation method via the fourth cerebral ventricle in mice. We used mouse olfactory ensheathing cells (OECs) and rat mesenchymal stem cells (MSCs) as donor cells. OECs are reported to promote regeneration and remyelination in the spinal cord, while MSCs have a capability to differentiate into several types of specific cells including neural cells. Furthermore both types of cells can be relatively easily obtained by biopsy in human. Initially, we confirmed the safety of the operative procedure and broad distribution of grafted cells in the spinal cord using wild-type mice. After transplantation, OECs distributed widely and survived as long as 100 days after transplantation, with a time-dependent depletion of cell number. In ALS model mice, OEC transplantation revealed no adverse effects but no significant differences in clinical evaluation were found between OEC-treated and non-transplanted animals. After MSC transplantation into the ALS model mice, females, but not males, showed a statistically longer disease duration than the non-transplanted controls. We conclude that intrathecal transplantation could be a promising way to deliver donor cells to the central nervous system. Further experiments to elucidate relevant conditions for optimal outcomes are required. © 2008 Elsevier Inc. All rights reserved.
Introduction Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder that clinically is characterized by skeletal muscle wasting and paralysis. Without respiratory assistance, death usually occurs less than 5 years after the clinical onset (Cleveland and Rothstein, 2001). ALS exhibits progressive loss of both upper and lower motor neurons in the cerebral cortex, brain stem and spinal cord. The cause of ALS is still elusive and no effective treatment exists so far. Approximately 90% of the disease is sporadic and about 10% is familial in form, called FALS. Twenty percent of FALS cases are associated with mutations of the Cu/Zn superoxide dismutase (SOD1) gene (Rosen et al., 1993; Cudkowicz et al., 1997). In recent years, ever-greater hopes have been placed in regenerative medicine using in-vitro-expanded cells to cure a damaged or degenerated central nervous system (CNS). As motor neurons distribute throughout the entire CNS, replenishment using stem cells by ⁎ Corresponding author. Fax: +81 859 38 6759. E-mail address: [email protected] (Y. Watanabe). 1 These authors contributed equally to this work. 0014-4886/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2008.07.011
focal injection would be impractical for the treatment of ALS. To make the transplanted cells distribute diversely, we developed a transplantation method for mice that allows even diffusion of donor cells throughout the CNS. Then we applied this protocol to cell transplant treatment in a mouse model of ALS. We chose olfactory ensheathing cells (OECs) and mesenchymal stem cells (MSCs) as suitable donor cells based on the following evidence. OECs, a specific glial cell type residing in the nose, assists axonal extension of olfactory sensory neurons from the peripheral nervous system to CNS (Doucette, 1995). The OEC promotes regeneration and remyelination of injured spinal pathways and enhances motor recovery in experimental animal models with spinal cord injury (Ramon-Cueto and Nieto-Sampedro, 1994; Li et al., 1997; RamonCueto et al., 1998). Indeed, OECs from patients with spinal cord lesion were grafted autologously as a clinical trial in Australia (Feron et al., 2005). On the other hand, MSCs can differentiate into various kinds of cells, including cardiomyocytes, neurons, and glia (Woodbury et al., 2000). Human MSC transplantation was reported to increase voluntary activity pre-symptomatically after transplantation into ALS model mice (Habisch et al., 2007). Importantly, both OECs and MSCs can be obtained by biopsy in human (Bianco et al., 2004; Mazzini et al., 2006),
432
E. Morita et al. / Experimental Neurology 213 (2008) 431–438
thereby making autologous transplantation feasible. Using these cells we attempted to alleviate clinical signs of motor neuron degeneration in a mouse model of ALS.
Table 1 Number of mice in different groups
Materials and methods
WT mice (n = 20)
Olfactory ensheathing cells (OECs)
Transgenic L126delTT (n = 29) Transgenic L126delTT (n = 34)
Nasal olfactory mucosae were collected from 5-week-old “green” mice: C57BL/6CR background, ubiquitously expressing green fluorescent protein (GFP) (Okabe et al., 1997). The tissues were incubated for 40 min at 37 °C in Dispase II (Roche, Mannheim, Germany) to isolate the lamina propria (LP) from the olfactory epithelium. The LP was then transferred into 0.25% collagenase type IA (Sigma, USA) in Dulbecco's modified Eagle medium/Ham's F12 (DMEM/F12) and incubated for 30 min at 37 °C. After centrifugation, the cell pellet was resuspended in the DMEM/F12 with 10% fetal bovine serum (FBS) and plated on poly-L-lysine (PLL)-coated dishes. At 80–90% confluence the cells were harvested and passaged with DMEM/F12 containing 1% insulin-transferrin-selenium supplement (ITS-X, Gibco, USA) and 50 ng/ml neurotrophin 3 (NT3, PeproTech EC Ltd, UK) onto a PLL-coated dish. Cells were maintained in this medium and passaged 5 to 6 times until OECs were purified to more than 95% (Bianco et al., 2004). Mesenchymal stem cells (MSCs) We isolated MSCs from GFP rats, because the technique to isolate and expand MSCs from mice is difficult especially from GFP transgenic mice (Peister et al., 2004). Rat MSCs were obtained from tibial and femoral bones of a 5-week-old LEW-GFP rat (Hakamata et al., 2001). Both ends of the bones were cut, and the bone marrow was extruded by using 21G needle with α-MEM (NK System, Japan) supplemented with 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin. After centrifugation the cell pellet was suspended in the medium. The cells were seeded on 75 cm2 flasks. Twenty-four hours after the seeding medium was refreshed to remove floating cells. At confluence the cells were passaged and re-plated. This cycle was repeated 3 times, after which the cells were preserved at −80 °C with cell banker (Uji field, Japan) until use (Tohill et al., 2004). As an additional marker, because the GFP fluorescence of rat MSCs proved to be too weak to identify transplanted cells in the host spinal cord, we also labeled them with dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) (Molecular Probes, USA). MSCs were incubated in the culture medium diluting 2 μg/ml DiI for 5 min at 37 °C, and then for an additional 15 min at 4 °C. After labeling, MSCs were washed twice with PBS and then transplanted. To provide immune suppression because MSCs were derived from rat, FK506 (3 mg/kg/day, kindly provided from Fujisawa Co, Japan) was administered orally to MSC-transplanted and sham-operated animals daily from 1 week before the operation for the duration of the study. Animals All the studies were carried out in accordance with the Guidelines for Animal Experimentation at the Faculty of Medicine, Tottori University. As the animal model of ALS, we used transgenic SOD1Leu126delTT mice with a mutated human SOD1 gene. This gene has a 2-bp deletion at codon 126 (Leu126delTT) found in a human FALS family (Nakashima et al., 1995). The mice begin to show clinical symptoms at around 20 to 21 weeks of age. After the disease duration of 1–2 weeks, mice die at 22–23 weeks (Watanabe et al., 2005). In these mice, there are no differences between genders with regard to ages of onset and death, and disease duration (Watanabe et al., 2005). The effects of transplantation on clinical outcomes were assessed by forming two groups of transgenic mice: littermates were dis-
OEC No operation OEC Sham-operated MSC Sham-operated
10-day survival
30-day survival
Endpoint
4 (4) 0 5 (5) 2 (2) 2 (2) 2 (2)
4 0 5 2 2 2
6 (6) 6 (6) 8 (4) 7 (4) 13 (8) 13 (7)
(4) (5) (2) (2) (2)
Clinical analysis was performed only on the endpoint groups. The number of animals used for histo-pathological analysis is shown in parentheses.
tributed equally into cell-transplanted and sham-operated mice, with the groups balanced with regard to date of birth, body weight, and gender. Littermate mice excess to these groups were divided into groups for histo-pathological analysis 10 or 30 days after the transplantation. The groups and numbers of mice used are listed in Table 1. Stereotaxic operation Cell transplantation was performed on wild-type (WT) C57BL/6CR mice and transgenic SOD1-Leu126delTT mice using sterotaxic coordinates (Bai et al., 2003). A hole 0.5 mm in diameter was made in the skull at the site 6 mm caudal to the lambda suture on the midline. A 30-G needle (Dentsply, Sankin, Japan) was then inserted into the fourth ventricle (3.75 mm depth from cerebellar surface) and 10 μl (3 to 4 × 105 cells) of suspension (OECs or MSCs) was injected slowly over 10 min. As sham controls, animals were injected with 10 μl phosphate-buffered saline (PBS) without cells. Prior to cell injection experiments, 5 μl pontamine sky blue (Sigma, USA) was injected using the same procedure, to confirm the accuracy of the operation. Clinical evaluation Clinical evaluation was performed every week from 1 week before the operation. This evaluation included body weight, hind limb extension score, foot print analysis, ages of onset and endpoint, and disease duration. A hind limb extension score was recorded as 0, 0.5, 1.0, 1.5 and 2.0 after evaluation of the reflex of lower limb according to published procedures (Garbuzova-Davis et al., 2003). Foot print analysis provided an indication of whether stride and gait were altered by the treatment. A composite stride-length measure was calculated in the following manner. The rear feet were dipped in ink and the animals were allowed to walk freely on paper. The distance between adjacent footprints was measured, starting with the first right–left pair. Then the distance from this left footprint to the next right footprint was measured. This was repeated for six adjacent consecutive footprints and the average distance between adjacent footprints was calculated, referred to here as “stride length”. Age of disease onset was determined when tremor or subtle weakness was identified during suspension by the tail and/or during voluntary walking. Endpoint was arbitrarily determined when mice were not able to get up from a lying position, or the eyes were highly infected (Jaarsma et al., 2001). These signs of impending death or actual death provided the experimental endpoint for survival and histological analysis. Preparation of tissue sections Wild-type C57BL/6CR mice transplanted with OECs, mice were histologically evaluated at 10, 30 and 100 days (an arbitrary endpoint based on the approximate age of the transgenic mice) after the transplantation (n = 4, 4, and 6 males at each time point, respectively)
E. Morita et al. / Experimental Neurology 213 (2008) 431–438
(Table 1). Wild-type C57BL/6CR mice without any operative procedure were also histologically evaluated for inflammation markers (male 3, female 3). Transgenic SOD1-Leu126delTT mice, transplanted with OECs or MSCs, were histologically evaluated at 10 days, 30 days and at the experimental endpoint. In the OEC-transplanted group, the following numbers of animals were analysed: 10 days, 3 male, 2 female; 30 days, 4 male, 1 female; endpoint, 4 male. In the (OEC) control group, the numbers were: 10 days, 2 female; 30 days, 1 male, 1 female; endpoint 3 male, 1 female. In the MSC-transplanted group the numbers were: 10 days, 2 female; 30 days, 2 female; endpoint, 4 male, 4 female. In the (MSC) control group the numbers were: 10 days, 2 female; 30 days, 1 male, 1 female; endpoint (4 male, 3 female). Animals were deeply anesthetized by intraperitoneal injection of pentobarbital sodium and transcardially perfused with normal saline solution, followed by 4% paraformaldehyde in 0.1 M phosphate-buffered solution. A lumbar spinal cord segment was immersed in the same fixative and cryoprotected in a series of sucrose solutions; 10%, 15% and 20% sucrose in PBS at 4 °C for 2 days. After tissue embedded in Tissue-Tec (Sakura, Japan) and frozen in liquid nitrogen cooled isopentane. Ten micrometer-thick sections were cut transversely using a cryostat. Histopathological and immunohistochemical staining Selected sections were stained with hematoxylin and eosin and with immunostaining for GF P, glial fibrillary acidic protein (GFAP), or Iba1. For immunohistochemical study, the sections were permeabilized with 0.2% Triton X-100 (Wako, Japan) and 4% paraformaldehyde, and blocked with 1 mg/ml bovine serum albumin (BSA, Sigma) and 1% normal goat serum (Funakoshi, Japan) in PBS. Primary antibodies used and their final dilutions were as follows: mouse anti-GFP monoclonal
433
antibody (1:250, MAB3580, Chemicon, USA), rabbit polyclonal antibody against GFP (1:250, MBL, Japan), rabbit polyclonal antibody against GFAP (prediluted, Dako, USA), and rabbit anti Iba1 (1:100, Wako, Japan). The secondary antibody was goat anti-mouse antibody IgG fluorescein isothiocyanate (FITC) (1:100, Santa Cruz Biotechnology, CA, USA), goat anti-rabbit antibody IgG FITC (1:100, Santa Cruz Biotechnology, CA, USA), and goat anti-rabbit antibody IgG Texas Red (TR) (1:100, Santa Cruz Biotechnology, CA, USA). Specimens were incubated with primary antibodies overnight at 4 °C, followed by incubation with secondary antibodies for an hour at room temperature. Immunoperoxidase staining The cryostat sections were first permeabilized with 0.2% Triton X-100 in PBS for 30 min, washed in PBS three times, and incubated 3% H2O2 for an hour at 37 °C. After washing in PBS three times, sections were blocked in 1 mg/ml BSA and 1% normal goat serum in PBS. The sections were then incubated with mouse anti-GFP antibody (1:250) and mouse anti-NeuN antibody (1:500, MAB377, Chemicon, USA) overnight at 4 °C. After washing in PBS three times, sections were incubated labeled polymer-horse radish peroxidase (HRP) anti-mouse (DAKO, USA) overnight at 4 °C. Subsequently, washed in PBS three times, the sections were reacted with 3,3′-diaminobenzidine tetrahydrochloride (DAB) for 15 min, followed by incubation with the same reagent containing 3% H2O2 for 5 min to visualized the antibody-bound HRP. Motor neuron quantification Serial cross-sections of the lumber spinal cords were stained with anti-NeuN antibodies. NeuN positive cells in the ventral gray matter
Fig. 1. Stereotaxic injection into the fourth ventricle in wild-type C57BL/6 mice. (A) Blue dye injected into the fourth ventricle diffuses throughout the surface of the spinal cord and cerebral base. (B) A small hemorrhage was observed resulting from the injection. The arrow indicates the injected region. The arrowhead indicates the fourth ventricle. (C) The spinal cord surface 30 days after GFP-OEC transplantation shown without fixation. (D–F) There were no differences between the OEC-transplanted and non-transplanted mice with regard to long-term motor function after the transplantation but there were minor differences in body weight (n = 6 each). The arrow indicates when the transplant was performed. ⁎P b 0.05. Scale bar = 1 mm in B; 500 μm in C.
434
E. Morita et al. / Experimental Neurology 213 (2008) 431–438
were counted in every fifth section and the results of at least 5 sections were averaged (Pardo et al., 2006). Statistical analysis The quantitative data were expressed as mean ± standard error of mean (SEM). Statistical analysis was performed with repeated measures ANO VA using Statview (Abacus Concept, USA). The statistical analysis of onset age and endpoint was performed with the Kaplan Meier method of SPSS 11.5J for Windows (SPSS inc, USA).
We then evaluated the perioperative and long-term safety of transplantation of OECs into the fourth ventricle in wild-type C57BL/ 6CR mice. Clinical assessments were carried out weekly for 9 weeks from 1 week before the transplantation until the mice were 200 days old. Similar assessments were made of sham-transplanted mice. There were no differences between OEC-transplanted (n = 6) and nontransplanted mice (n = 6) for all parameters (Figs. 1D–F). Examination of the spinal cords by fluorescent microscopy at 10, 30 and 100 days post-transplantation revealed OECs on the surface of the spinal cord for at least 100 days after transplantation while decreasing in a time-dependent manner (Fig. 1C).
Results OEC transplantation Transplantation procedure In an initial study, the brain and spinal cord were observed 3 h after injecting with pontamine sky blue via the fourth ventricle. The dye had diffused throughout the spinal cord and base of brain, with the surfaces of cerebellum and cerebrum free from dye at this time point (Fig. 1A). Microscopic observation revealed the presence of a small hemorrhagic scar in the cerebellum from the injection (Fig. 1B).
OECs derived from GFP mice were transplanted into transgenic SOD1-Leu126delTT animals (n = 8) at 13 weeks of age. Then we compared OEC-transplanted with control (PBS injected) groups (n = 7) by weight, hind limb extension score, stride length, ages of onset and endpoint, and disease duration. On the basis of clinical parameters there were no differences in any of these measures (Figs. 2A–E). Average ages of onset were 139.5 ± 4.5 days and 137.1 ± 6.4 days, and endpoints were 151.1 ± 6.0 days (OEC) and 156.1 ± 6.8 days (control).
Fig. 2. Effect of OEC transplantation in transgenic SOD1-Leu126delTT mice. (A–E) There were no significant differences between transplanted and sham-operated transgenic mice in body weight (A), hind limb extension (B), stride length (C). Kaplan Meier incidence (D) and survival (E) plots show no differences between the two groups — transplantation of OECs did not prolong the survival period of transgenic SOD1-Leu126delTT mice (OEC; n = 8, PBS; n = 7). (F) The surface of the spinal cord showing GFP-OECs 10 days after transplantation shown without fixation. (G) Section of the spinal cord of 30 days after GFP-OEC transplantation. No OECs were observed to enter the spinal cord parenchyma. (H) There was no difference between the OEC-transplanted and non-transplanted mice in the number of motor neurons in the ventral gray matter (n = 4 in each group). Scale bar= 0.33 mm in F; 100 μm in G.
E. Morita et al. / Experimental Neurology 213 (2008) 431–438
OECs reportedly have some beneficial characteristics for transplantation such as the ability to penetrate even to the glial scar area (Oudega., 2007) but our observations revealed that OECs did not invade into spinal cord parenchyma (Fig. 2G) remaining on the surface of the spinal cord (Fig. 2F). The number of motor neurons remaining at endpoint was similar between OEC-transplanted animals (17.1 ± 4.1/ hemi section; n = 4) and sham-operated animals (17.7 ± 2.2/hemi section; n = 4) (Fig. 2H). MSC transplantation MSCs were transplanted into transgenic SOD1-Leu126delTT mice (n = 13). As a control, PBS only was injected into transgenic SOD1Leu126delTT mice (n = 13). Because OEC transplantation at 13 weeks did not show any beneficial effect in transgenic SOD1-Leu126delTT animals, MSC transplantation in transgenic SOD1-Leu126delTT mice was performed at 14 weeks. Compared to controls the MSCtransplanted group did not show any difference on body weight,
435
extension score, and stride length (Figs. 3A–C). There were beneficial trends in ages of onset and endpoint, and disease duration in MSCtransplanted mice compared to control mice, however these did not reach statistical significance (Figs. 3D and E). Intriguingly, when we compared MSC-treated female mice with control female mice (n = 7 each), there was a statistically significant difference for disease duration between the two groups (MSC; 21.6 ± 5.4 days, control; 10.0 ± 2.2 days) (Fig. 3F). We observed that MSCs labeled with DiI stayed on the surface of the spinal cord of 10 and 30 days after transplantation (Figs. 4A and B), moreover some MSCs invaded into spinal cord parenchyma (Figs. 4C and D). In the brain, especially in the Purkinje cell layer in the cerebellum, we were able to observe survival and invasion of MSCs or their progeny (Figs. 4E and F). Using anti-GFP antibody staining, the GFP-positive MSC-derived cells still existed in sections of spinal cord at endpoint (Figs. 4G and H). The number of motor neurons remaining at endpoint was greater in the MSC group compared to the control group but this was not statistically significant (18.5 ± 3.8/hemi section,
Fig. 3. Effect of MSC transplantation in transgenic SOD1-Leu126delTT mice. (A–E) There were no significant differences between transplanted and sham-operated transgenic mice in body weight (A), hind limb extension (B), stride length (C). Kaplan Meier incidence (D) and survival (E) plots show no differences between the two groups — transplantation of MSCs did not prolong the survival period of transgenic SOD1-Leu126delTT mice (n = 13 in each group). (F) When sex is taken into account, disease duration of females treated with MSCs was significantly longer than control females (male; n = 6, female; n = 7 in each treatment group). ⁎P = 0.025. (G) There was no difference between the MSC-transplanted and nontransplanted mice in the number of motor neurons in the ventral gray matter (MSC; n = 8, PBS; n = 7).
436
E. Morita et al. / Experimental Neurology 213 (2008) 431–438
Fig. 4. Histopathological evaluations of the spinal cord and the brain after MSC transplantation. Spinal cord and brain after sham injections with phosphate-buffered saline (PBS: A, C, E, and G) or MSCs (MSC: B, D, F, and H). (B) 10 days after transplantation, many MSCs labeled with DiI survived on the surface of the spinal cord, shown here without fixation, and remained 30 days later (data not shown). (D) Section of the spinal cord 30 days after MSC transplantation. MSCs (DiI labeled, red) entered into spinal cord parenchyma. Astrocytes labeled with anti-GFAP antibody (green). (F) MSC also invaded into parenchyma of the brain, including into the Purkinje cell layer of the cerebellum: DiI labeled MSC (red), GFAP-labeled astrocytes (green). (H) MSCs were detected in sections of lumber spinal cord after fixation by anti-GFP antibody staining (brown). Scale bar = 500 μm in A and B; 100 μm in C–H.
n = 8, and 15.9 ± 3.9/hemi section, n = 7, respectively; Fig. 3G). There were no discernable differences in the numbers of remaining motor neurons in males compared to females. In order to assess the effect of MSC transplantation on suppressing inflammation, we employed immunohistochemical analyses for reactive astrogliosis (using antibodies to glial proteins, GFAP) and for activated microglia (using an antibody to Iba1). There was noticeable immunoreactivity for GFAP and Iba1 in MSC-transplanted animals at 10 days, 30 days and at experimental endpoint (Fig. 5). There was also immunostaining for both proteins in sham-operated control animal injections with PBS alone (Figs. 5C and H). In contrast, non-transgenic, unoperated, wild-type mice showed less immunoreactivity using the same antibodies and similar processing. These data were not
quantified but they suggest that the SOD1-Leu126delTT mice have increased “baseline” inflammation compared to wild-type controls which was not affected by MSC transplantation. Discussion In this study, we demonstrated that intrathecal cell transplantation via the fourth ventricle is feasible and safe in mouse. After transplantation in a mouse model of ALS (Watanabe et al., 2005), OECs showed no beneficial effect whereas MSCs prolonged the survival of female transgenic SOD1-Leu126delTT animals. Previously, a variety of cell transplantation experiments have been employed to treat a mouse model of ALS (the G93A SOD1 transgenic
E. Morita et al. / Experimental Neurology 213 (2008) 431–438
437
Fig. 5. Reactive astrogliosis and microglia activation after MSC transplantation. Spinal cord sections from MSC-transplanted, transgenic SOD1-Leu126delTT mice (A–C, F–H) and PBS injected, transgenic SOD1-Leu126delTT mice (D and I) 10 days, 30 days after transplantation and at experimental endpoint were immunoreactive for GFAP (A–D) and Iba1 (F–I). Less immunoreactivity was observed in non-transgenic mice (WT) of a similar age to the transgenic mice at experimental endpoint (E and J). Scale bar = 100 μm in A–J.
mouse). There is little consensus, however, on the effects of cell transplantation. Some experiments showed beneficial effects on animal clinical parameters (Chen and Ende, 2000; Ende et al., 2000; Willing et al., 2001; Garbuzova-Davis et al., 2002; 2003; Corti et al., 2004), while others showed no beneficial effects (Hemendinger et al., 2005; Habisch et al., 2007). It is difficult to generalize among these results because several parameters vary, namely, the cell type transplanted, the age at transplantation and the administration route. The donor cells included human umbilical-cord-blood derived cells (Chen and Ende, 2000; Ende et al., 2000; Garbuzova-Davis et al., 2003; Habisch et al., 2007), human neuron-like cells (Willing et al., 2001; GarbuzovaDavis et al., 2002), mouse bone marrow (mesenchymal) stem cells (Corti et al., 2004; Habisch et al., 2007), sertoli cells (Hemendinger et al., 2005) and neural stem cells (Corti et al., 2004). The ages of transplantation varied from 4 weeks (Corti et al., 2004) to 10 weeks (Corti et al., 2007). The administration routes included intravenous (Chen and Ende, 2000; Ende et al., 2000; Garbuzova-Davis et al., 2003), intraperitoneal (Corti et al., 2004), focal injection (L4–L5 segment, (Willing et al., 2001; Garbuzova-Davis et al., 2002; Corti et al., 2007), and intrathecal injection via cisterna magna (Habisch et al., 2007). In the present experiments, MSC transplantation only affected female mice. Sex differences have been observed previously in treatments of the G93A SOD1 mouse model of ALS. After transplantation of MSCs females, but not males, were more spontaneously active (although other clinical measures were not affected; Habisch et al., 2007). After treatment with clenbutol, a beta-2 adrenoceptor, the symptoms in females, but not males, improved (Teng et al., 2006), whereas treatment with cyclosporine A, a well-known immunosuppressant, had a robust effect on males and not females (Kirkinezos et al., 2004). These gender differences are so far without obvious explanation. In the present study, MSCs were shown to penetrate the
parenchyma of spinal cord and we speculate a possible neuroprotective effect or modulation of the neural environment could result. Previous results of transplantation experiments including ours indicate that required characteristics of donor cells might be their possession of neurotrophic effect and the ability to change neural environment, rather than necessarily being committed to neural lineage. Transplantation timing is also an elusive parameter. Transplantation is normally applied before the onset of ALS as a preventative therapy. In our experiment, the transplantation point was influenced by the fact that OECs survive at least as long as 100 days after transplantation with time-dependant depletion, and that early pathological abnormality in the spinal cord, such as reduction of motor neurons and reactive gliosis, appears before the appearance of clinical symptoms in transgenic SOD1-Leu126delTT mice (Watanabe et al., 2005). In human cases, disease duration is usually 3 to 5 years while only weeks or months in mice. Because our goal is to treat human ALS patients, we should keep in mind the difference, with regard to life span and natural course of the disease, between mouse and human. It is vital to understand how long the donor cells survive in the spinal cord and how we can prolong the survival of the donor cells. In any case, serial administration might be necessary for human treatment. With an intrathecal route of administration (via lumbar puncture in humans), repeated administration would be feasible. Administration route is important and indeed is a main theme of this report. Because of the large size of the human spinal cord, focal injection of donor cells, which is shown to be successful in some experiments on mice (Willing et al., 2001; Garbuzova-Davis et al., 2002), would not be so ideal for application in human. To overcome this problem, intrathecal diffusion appears a promising way to distribute the donor cells. With similar intent, human mesenchymal stem cells were transplanted into G93A SOD1 transgenic mice by
438
E. Morita et al. / Experimental Neurology 213 (2008) 431–438
intrathecal injection via cisterna magna (Habisch et al., 2007). That procedure did not affect the clinical outcome of the disease and it was speculated that the poor therapeutic potential was due to poor distribution of the transplanted cells into the spinal cord. In the present study, this was improved by injection into the fourth ventricle, rather than cisterna magna. As shown here, blue dye diffuses throughout the spinal cord in less than 3 h. The physiological CSF stream passes from the fourth ventricle caudally down the central canal of the spinal cord, whereas CSF in the cisterna magna is external to the spinal cord and circulates rostrally. This may explain why transplantation via the fourth ventricle is superior to via the cisterna magna with regard to delivering the cells to the spinal cord. There are other aspects, besides the parameters mentioned above, that could be optimized for successful therapy in the ALS mouse, and eventually human. For example, is there an optimum number of cells sufficient for cell therapy? Could the use of cell adhesion molecules facilitate the survival of donor cells? Would a combination of cell- and gene-therapy be more effective than either alone? There are reports about effectiveness of some neurotrophic factors in ALS patients. Ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF) and insulinlike growth factor (IGF-1) have demonstrated effect of protection and regeneration of motor neurons, and some have been> applied clinically (Ekestern, 2004). Such molecules could be delivered intrathecally by pump or by genetically modified cells that secrete neurotrophic factors. Normally the blood brain barrier prevents neurotrophic factors accessing directly to brain and spinal cord after intravenous injection or oral administration, although a recent report indicates that this barrier is compromised in G93A mice (GarbuzovaDavis et al., 2007). OECs are a candidate cell for transplantation after genetic modification because they can be engineered to produce neurotrophins (Ruitenberg et al., 2005), they can be safely harvested and autologously transplanted in humans (Feron et al., 2005), and they survive for long periods in the spinal cord after intrathecal injection, as shown here. Acknowledgments This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Watanabe Y), and by a Grant from the Research Committee of CNS Degenerative Diseases, Ministry of Health, Labour and Welfare of Japan (Nakashima K). References Bai, H., Suzuki, Y., Noda, T., Wu, S., Kataoka, K., Kitada, M., Ohta, M., Chou, H., Ide, C., 2003. Dissemination and proliferation of neural stem cells on the spinal cord by injection into the fourth ventricle of the rat: a method for cell transplantation. J. Neurosci. Methods 124, 181–187. Bianco, J.I., Perry, C., Harkin, D.G., Mackay-Sim, A., Feron, F., 2004. Neurotrophin 3 promotes purification and proliferation of olfactory ensheathing cells from human nose. Glia 45, 111–123. Chen, R., Ende, N., 2000. The potential for the use of mononuclear cells from human umbilical cord blood in the treatment of amyotrophic lateral sclerosis in SOD1 mice. J. Med. 31, 21–30. Cleveland, D.W., Rothstein, J.D., 2001. From Charcot to Lou Gehrig: deciphering selective motor neuron death in ALS. Nat. Rev. Neurosci. 2, 806–819. Corti, S., Locatelli, F., Donadoni, C., Guglieri, M., Papadimitriou, D., Strazzer, S., Del Bo, R., Comi, G.P., 2004. Wild-type bone marrow cells ameliorate the phenotype of SOD1G93A ALS mice and contribute to CNS, heart and skeletal muscle tissues. Brain 127, 2518–2532. Corti, S., Locatelli, F., Papadimitriou, D., Del Bo, R., Nizzardo, M., Nardini, M., Donadoni, C., Salani, S., Fortunato, F., Strazzer, S., Bresolin, N., Comi, G.P., 2007. Neural stem cells LewisX+ CXCR4+ modify disease progression in an amyotrophic lateral sclerosis model. Brain 130, 1289–1305. Cudkowicz, M.E., McKenna-Yasek, D., Sapp, P.E., Chin, W., Geller, B., Hayden, D.L., Schoenfeld, D.A., Hosler, B.A., Horvitz, H.R., Brown, R.H., 1997. Epidemiology of mutations in superoxide dismutase in amyotrophic lateral sclerosis. Ann. Neurol. 41, 210–221. Doucette, R., 1995. Olfactory ensheathing cells: potential for glial cell transplantation into areas of CNS injury. Histol. Histopathol. 10, 503–507.
Ekestern, E., 2004. Neurotrophic factors and amyotrophic lateral sclerosis. Neurodegener. Dis. 1, 88–100. Ende, N., Weinstein, F., Chen, R., Ende, M., 2000. Human umbilical cord blood effect on sod mice (amyotrophic lateral sclerosis). Life Sci. 67, 53–59. Feron, F., Perry, C., Cochrane, J., Licina, P., Nowitzke, A., Urquhart, S., Geraghty, T., Mackay-Sim, A., 2005. Autologous olfactory ensheathing cell transplantation in human spinal cord injury. Brain 128, 2951–2960. Garbuzova-Davis, S., Willing, A.E., Milliken, M., Saporta, S., Zigova, T., Cahill, D.W., Sanberg, P.R., 2002. Positive effect of transplantation of hNT neurons (NTera 2/D1 cell-line) in a model of familial amyotrophic lateral sclerosis. Exp. Neurol. 174, 169–180. Garbuzova-Davis, S., Willing, A.E., Zigova, T., Saporta, S., Justen, E.B., Lane, J.C., Hudson, J.E., Chen, N., Davis, C.D., Sanberg, P.R., 2003. Intravenous administration of human umbilical cord blood cells in a mouse model of amyotrophic lateral sclerosis: distribution, migration, and differentiation. J. Hematother. Stem Cell Res. 12, 255–270. Garbuzova-Davis, S., Haller, E., Saporta, S., Kolomey, I., Nicosia, S.V., Sanberg, P.R., 2007. Ultrastructure of blood-brain barrier and blood-spinal cord barrier in SOD1 mice modeling ALS. Brain Res. 1157, 126–137. Habisch, H.J., Janowski, M., Binder, D., Kuzma-Kozakiewicz, M., Widmann, A., Habich, A., Schwalenstocker, B., Hermann, A., Brenner, R., Lukomska, B., Domanska-Janik, K., Ludolph, A.C., Storch, A., 2007. Intrathecal application of neuroectodermally converted stem cells into a mouse model of ALS: limited intraparenchymal migration and survival narrows therapeutic effects. J. Neural Transm. 18, 18. Hakamata, Y., Tahara, K., Uchida, H., Sakuma, Y., Nakamura, M., Kume, A., Murakami, T., Takahashi, M., Takahashi, R., Hirabayashi, M., Ueda, M., Miyoshi, I., Kasai, N., Kobayashi, E., 2001. Green fluorescent protein-transgenic rat: a tool for organ transplantation research. Biochem. Biophys. Res. Commun. 286, 779–785. Hemendinger, R., Wang, J., Malik, S., Persinski, R., Copeland, J., Emerich, D., Gores, P., Halberstadt, C., Rosenfeld, J., 2005. Sertoli cells improve survival of motor neurons in SOD1 transgenic mice, a model of amyotrophic lateral sclerosis. Exp. Neurol. 196, 235–243. Jaarsma, D., Rognoni, F., van Duijn, W., Verspaget, H.W., Haasdijk, E.D., Holstege, J.C., 2001. CuZn superoxide dismutase (SOD1) accumulates in vacuolated mitochondria in transgenic mice expressing amyotrophic lateral sclerosis-linked SOD1 mutations. Acta Neuropathol. (Berl) 102, 293–305. Kirkinezos, I.G., Hernandez, D., Bradley, W.G., Moraes, C.T., 2004. An ALS mouse model with a permeable blood-brain barrier benefits from systemic cyclosporine A treatment. J. Neurochem. 88, 821–826. Li, Y., Field, P.M., Raisman, G., 1997. Repair of adult rat corticospinal tract by transplants of olfactory ensheathing cells. Science 277, 2000–2002. Mazzini, L., Mareschi, K., Ferrero, I., Vassallo, E., Oliveri, G., Boccaletti, R., Testa, L., Livigni, S., Fagioli, F., 2006. Autologous mesenchymal stem cells: clinical applications in amyotrophic lateral sclerosis. Neurol. Res. 28, 523–526. Nakashima, K., Watanabe, Y., Kuno, N., Nanba, E., Takahashi, K., 1995. Abnormality of Cu/Zn superoxide dismutase (SOD1) activity in Japanese familial amyotrophic lateral sclerosis with two base pair deletion in the SOD1 gene. Neurology 45, 1019–1020. Okabe, M., Ikawa, M., Kominami, K., Nakanishi, T., Nishimune, Y., 1997. ‘Green mice’ as a source of ubiquitous green cells. FEBS Lett. 407, 313–319. Oudega, M., 2007. Schwann cell and olfactory ensheathing cell implantation for repair of the contused spinal cord. Acta. Physiol. 189, 181–189. Pardo, A.C., Wong, V., Benson, L.M., Dykes, M., Tanaka, K., Rothstein, J.D., Maragakis, N.J., 2006. Loss of the astrocyte glutamate transporter GLT1 modifies disease in SOD1 (G93A) mice. Exp. Neurol. 201, 120–130. Peister, A., Mellad, J.A., Larson, B.L., Hall, B.M., Gibson, L.F., Prockop, D.J., 2004. Adult stem cells from bone marrow (MSCs) isolated from different strains of inbred mice vary in surface epitopes, rates of proliferation, and differentiation potential. Blood 103, 1662–1668 (Epub 2003 Oct 1630). Ramon-Cueto, A., Nieto-Sampedro, M., 1994. Regeneration into the spinal cord of transected dorsal root axons is promoted by ensheathing glia transplants. Exp. Neurol. 127, 232–244. Ramon-Cueto, A., Plant, G.W., Avila, J., Bunge, M.B., 1998. Long-distance axonal regeneration in the transected adult rat spinal cord is promoted by olfactory ensheathing glia transplants. J. Neurosci. 18, 3803–3815. Rosen, D.R., Siddique, T., Patterson, D., Figlewicz, D.A., Sapp, P., Hentati, A., Donaldson, D., Goto, J., O'Regan, J.P., Deng, H.X., 1993. Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature 362, 59–62. Ruitenberg, M.J., Levison, D.B., Lee, S.V., Verhaagen, J., Harvey, A.R., Plant, G.W., 2005. NT-3 expression from engineered olfactory ensheathing glia promotes spinal sparing and regeneration. Brain 128, 839–853. Teng, Y.D., Choi, H., Huang, W., Onario, R.C., Frontera, W.R., Snyder, E.Y., Sabharwal, S., 2006. Therapeutic effects of clenbuterol in a murine model of amyotrophic lateral sclerosis. Neurosci. Lett. 397, 155–158. Tohill, M., Mantovani, C., Wiberg, M., Terenghi, G., 2004. Rat bone marrow mesenchymal stem cells express glial markers and stimulate nerve regeneration. Neurosci. Lett. 362, 200–203. Watanabe, Y., Yasui, K., Nakano, T., Doi, K., Fukada, Y., Kitayama, M., Ishimoto, M., Kurihara, S., Kawashima, M., Fukuda, H., Adachi, Y., Inoue, T., Nakashima, K., 2005. Mouse motor neuron disease caused by truncated SOD1 with or without C-terminal modification. Brain Res. Mol. Brain Res. 135, 12–20. Willing, A.E., Garbuzova-Davis, S., Saporta, S., Milliken, M., Cahill, D.W., Sanberg, P.R., 2001. hNT neurons delay onset of motor deficits in a model of amyotrophic lateral sclerosis. Brain Res. Bull. 56, 525–530. Woodbury, D., Schwarz, E.J., Prockop, D.J., Black, I.B., 2000. Adult rat and human bone marrow stromal cells differentiate into neurons. J. Neurosci. Res. 61, 364–370.