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Human mesenchymal stromal cells ameliorate the phenotype of SOD1-G93A ALS mice
Cytotherapy (2007) Vol. 9, No. 5, 414 426
Human mesenchymal stromal cells ameliorate the phenotype of SOD1-G93A ALS mice C-P Zhao1, C Zhang1,2, S-N Zhou3, Y-M Xie4, Y-H Wang5, H Huang1, Y-C Shang1, W-Y Li2, C Zhou1, M-J Yu2 and S-W Feng1 1
Department of Neurology, First Affiliated Hospital, Sun Yat-sen University, Guangzhou, PR China, 2Stem Cells and Tissue Engineering Research Center of Sun Yat-sen University, Guangzhou, PR China, 3Department of Neurology, Qi-Lu Hospital, ShanDong University, JiNan, PR China, 4 Department of Neurology, University of North Carolina at Chapel Hill, North Carolina, USA and 5Department of Neurosurgery, Affiliated Hospital, Yang Zhou University, Yang Zhou, PR China
Background Amyotrophic lateral sclerosis (ALS) is a progressive, lethal, neurodegenerative disease, currently without any effective therapy. Multiple advantages make mesenchymal stromal cells (MSC) a good candidate for cellular therapy in many intractable diseases such as stroke and brain injury. Until now, no irrefutable evidence exists regarding the outcome of MSC transplantation in the mouse model of ALS. The present study was designed to investigate the therapeutic potential of human MSC (hMSC) in the mouse model of ALS (SOD1-G93A mice). Methods hMSC were isolated from iliac crest aspirates from healthy donors and kept in cell cultures. hMSC of the fifth passage were delivered intravenously into irradiated pre-symptomatic SOD1-G93A mice. Therapeutic effects were analyzed by survival analysis, rotarod test, motor neuron count in spinal cord and electrophysiology. The engraftment and in vivo differentiation of hMSC were examined in the brain and spinal cord of hMSC-transplanted mice.
Introduction Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the loss of motor neurons in the spinal cord, brain stem and cerebral cortex, resulting in weakness, spasticity, paralysis and dysphagia. About 10% of cases are dominantly inherited and 20% of familial ALS patients arise from mutations in the gene for Cu/Zn superoxide dismutase (SOD1). The SOD1-G93A mice express a mutant SOD1 carrying the Gly93Ala missense mutation and develop ALS-like motor neuron
Results After intravenous injection into irradiated pre-symptomatic SOD1G93A mice, hMSC survived more than 20 weeks in recipient mice, migrated into the parenchyma of brain and spinal cord and showed neuroglia differentiation. Moreover, hMSC-transplanted mice showed significantly delayed disease onset (14 days), increased lifespan (18 days) and delayed disease progression compared with untreated mice. Discussion Our data document the positive effects of hMSC transplantation in the mouse model of ALS. It may signify the potential use of hMSC in treatment of ALS. Keywords amyotrophic lateral sclerosis, mesenchymal stromal cells, SOD1-G93A mice, therapy, transplantation.
disease [1], serving as a conventional tool for testing new therapeutic strategies. Although a large number of laboratory advances have been made, including studies on neurotrophic factors, antioxidants, anti-inflammatory agents and drugs modifying excitable amino acids, there is still no therapy available that has proven efficient in the treatment of ALS. Novel therapeutic strategies are directed at replacing or repairing the damaged motor neurons [2]. Cellular therapy holds the potential answer to this problem.
Correspondence to: Cheng Zhang, Department of Neurology, First Affiliated Hospital, and Stem Cells and Tissue Engineering Research Center, Sun Yat-sen University, Guangzhou 510080, PR China. E-mail: [email protected] – 2007 ISCT
DOI: 10.1080/14653240701376413
hMSC ameliorate the phenotype of ALS mice
There have been several reports on stem cell transplantation in the ALS animal model during the last 5 years. Positive effects were observed after hNT neurons (derived from human teratocarcinoma cell line NTera2/D1) were transplanted into the ventral horn of the spinal cord of SOD1-G93A mice [3]. Human UC blood and wild-type mice BM have been reported to increase the SOD1-G93A mouse lifespan [4,5]. Little is known about the dynamic changes of the motor neuron in ventral horns and axonal degeneration after stem cell transplantation in the mouse model of ALS. Among the variety of stem cells, adult BM mesenchymal stromal cells (MSC) are a promising source for cellular therapy. MSC can be expanded in number within a short time and cryopreserved for long periods without losing their potentiality. Because of low immunogenicity, the use of MSC overcomes the problem of limited donor cell number and high immunorejection incidence, and opens up the possibility of employing autologous sources. MSC can differentiate not only into mesodermal cells but also endodermal and ectodermal cells (neurons, astrocytes and oligodendrocytes) [6]. MSC have been reported to survive, proliferate and migrate into damaged tissue with positive functional effects in animal models of stroke [7], spinal cord injury [8] and Parkinson’s disease [9]. Even after being injected into the blood stream, MSC have been shown to migrate to the central nervous system (CNS) and develop into cells that appear to be neurons, astrocytes [10] or myelinating cells in the spinal cord [11]. MSC can also produce many trophic factors in vitro and in vivo, which play important roles in brain development and remodeling. Moreover, MSC can be used as vectors of cytokines, trophic factors and other pharmacologic molecules to prevent cell death, tissue inflammation and damage. The evidence highlights the potential use of MSC for therapy in ALS. One clinical report describes transplanting autologous MSC into the spinal cord of ALS patients as a safe approach [12]. Because of the small number of treated patients (seven) in this trial and the limitations of detecting the transplanted cells, it is impossible to confirm whether direct delivery of MSC to the spinal cord could allow MSC to survive in the microenvironment for long periods of time [13] or delay the disease progression in ALS. This present study was aimed at investigating the therapeutic potential of intravenous administration of human MSC (hMSC) in the mouse model of ALS
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(SOD1-G93A mice). Dynamic changes of the motor neurons of the lumbar ventral horn and axonal degeneration after hMSC transplantation were observed. The engraftment and in vivo differentiation of hMSC in the brain and spinal cord were examined in the hMSC-transplanted mice. The results of our investigation advance the understanding of the transplantation effects and risk benefit ratio of hMSC transplantation as a treatment for ALS.
Methods Animals Transgenic male mice, B6SJL-TgN (SOD1-G93A) 1GUR (No. 002726), were purchased from The Jackson Laboratories (Bar Harbor, ME, USA), which overexpress human SOD1 and carried the Gly93Ala mutation [1]. A colony was derived from them and maintained by breeding male transgenic mice to naive (B6 SJL/J)F1 dams. Offspring were genotyped for the SOD1-G93A transgene using PCR of genomic DNA extracted from the blood of tail veins, as outlined by Jackson Laboratories. Animals were housed one to five to a cage, with a 12-h light/dark cycle, and fed standard murine chow and water ad libitum. The room temperature was 238C. All animal experiments were performed according to institutional guidelines that are in compliance with national and international law and policies.
Preparation of hMSC Heparinized BM cells were obtained from iliac crest aspirates of healthy human volunteers after informed consent, and were used in accordance with the procedures approved by the human experimentation and ethics committees of China. Mononuclear cells were separated by centrifugation in a Ficoll Hypaque gradient (1.077 g/mL; Sigma, St Louis, MO, USA) and washed twice with PBS. Cells were suspended in DMEM with a low glucose content (1 g/L; L-DMEM; Gibco Invitrogen Co., Carlsbad, CA, USA) supplemented with 10% FBS and seeded in T 25-cm2 culture flasks at a concentration of 1 106 cells/cm2. Cultures were maintained at 378C in a humidified atmosphere containing 5% carbon dioxide. The culture medium was replaced every 3 days and non-adherent cells were discarded. When the culture flasks became nearly confluent (80 90%), the adherent cells were detached with 2.5g/L trypsin in 1.0 mM sodium EDTA (Na2-EDTA; Invitrogen). The hMSC was rinsed twice and re-seeded at a 1:3 dilution in T 25-cm2 culture flasks. The above manipulation was repeated up to the fifth
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passage in order to obtain high-purity hMSC and acquire enough cells for transplantation.
Analysis of hMSC by FCM To validate the identity of the cells, flow cytometry (FCM) was performed to detect the cell-surface Ag markers specific for hMSC. The hMSC of the fifth passage were harvested with 0.25% trypsin and resuspended in PBS at 2 104 cells/reaction tube. The hMSC were fixed in 4% cold paraformaldehyde for 30 min and washed with PBS containing 2% FBS. Cells were incubated with mouse antihuman CD29 (1:1000; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), anti-CD34 (1:1000; Santa Cruz), antiCD44 (1:1000; Santa Cruz), anti-CD45 (1:1000; Santa Cruz) Ab and FITC-labeled goat anti-mouse secondary Ab (1:100; Santa Cruz). The samples were characterized by FCM.
hMSC transplantation The SOD1-G93A mice were distributed randomly into each group and all were evaluated up to end-stage. One to 3 days before transplantation, the SOD1-G93A mice (8 weeks of age) were irradiated with 6.0 Gy from a60Co source (0.454 Gy/min). hMSC (3 106) resuspended in 0.3mL L-DMEM, or 0.3 mL L-DMEM only, was injected into the tail vein of each recipient mouse. Before and after infusion, mice were kept under pathogen-free conditions and provided with fresh water. The groups were as follows. (1) hMSC-transplanted group: SOD1-G93A mice transplanted with hMSC after irradiation (n 29). (2) Untreated group: SOD1-G93A mice injected with only 0.3mL L-DMEM (n 34). (3) Normal group: wild-type littermates (SOD1-G93A negative) (n 30). (4) Control group: SOD1-G93A mice injected with 0.3mL L-DMEM after irradiation (n 12). Three mice of the hMSC-transplanted group, untreated group and normal group were killed at 12, 16 and 20 weeks of age for motor neuron count and CMAP assessment. Independent investigators were blinded to the animal’s post-transplant status to avoid subjective bias in all experiments.
Motor assessment and survival All SOD1-G93A mice were monitored daily for disease progression and survival by investigators blinded to the condition of the treatment. The mice were evaluated for signs of motor deficit with 4-point scoring system according to Weydt et al. [14]. The rotarod test was used to assess
motor co-ordination, strength and balance [15]. Fifteen mice of the hMSC-transplanted group, 14 mice of the untreated group and 10 mice of the normal group were tested weekly from 8 weeks of age (age and sex of mice were matched in three groups). The mice were tested on the rod rotating at a constant speed of 16 r.p.m. The longest latency was recorded and 180 seconds was chosen as the arbitrary cut-off time. Mortality was scored as the age at death [16].
Electrophysiologic recording (CMAP recording) For compound muscle action potential (CMAP) analysis, three mice from each group were anesthetized with 10% chloral hydrate (0.3 mL/100g, i.p.) at 12, 16 and 20 weeks of age. Electrophysiologic recordings across the nerve segment were made using an electromyogram apparatus (BL-420E; ChengDu, SiChuan, China). The sciatic nerve was stimulated at the paraspinal site. The stimulation consisted of single 1-millisecond 1-Hz supramaximal pulses through a needle electrode. A ground needle electrode was placed on the back of the mouse; an anode needle was inserted at the base of the tail; reference needle electrode was placed near the Achilles’s tendon. The evoked CMAP was recorded from the medial part of the gastrocnemius muscle with a unipolar needle electrode [17]. An initial negative deflection indicated recording at the motor point. In order to assess whether the stimulation was supramaximal and electrical stimulation and the response were reproducible, the stimulation intensity was increased gradually until the response would not increase any more or increase in a quantal fashion. At this time the maximal potential (CMAP) was recorded and analyzed (measured peak-to-peak) [18] with software (BL-NewCentury 2.0; ChengDu). Both the right and left hind limbs were studied.
Motor neuron count For motor neuron counts, three mice from each group were killed at the ages of 12, 16 and 20 weeks. The mice were anesthetized with 10% chloral hydrate (0.3 mL/100 g, i.p.) and perfused transcardially with 0.9% NaCl solution followed by ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2). The lumbar enlargements of each spinal cord were removed and post-fixed in the same fixative for 2 24 h at 48C. After fixation, the spinal cords were dehydrated with increasing concentrations of ethanol and embedded in paraffin. Serial transverse sections (5 mm) were cut through the lumbar enlargements. Every first section from one in five series
hMSC ameliorate the phenotype of ALS mice
was sampled using a random starting point of L3 L5 segments, mounted on a poly-L-lysine (Boster Co., WuHan, China) glass slide and stained with cresyl violet (Sigma; eight sections for each animal). On each section, at 200 magnification, the area ventral to the most dorsal extent of the central canal, including laminae VII, VIII and IX, was designated the ventral horn. Only motor neurons with a diameter 20 mm and bearing a clearly identifiable nucleus, nucleolus and cytoplasm with at least one thick process were counted by investigators blinded to the condition of the treatment [19]. The count and analysis were done with a computer-based image analysis system (Image-Pro-Plus, MediaCybernetics Inc., Silver Spring, MD, USA).
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(1:5000; Sigma) and examined under a fluorescent microscope (Olympus BX51, Olympus Co., Tokyo, Japan). HuNu-positive cells were counted on an average of three random sections of brain and lumbar spinal cord per mouse (at 400 magnification).
Statistical analysis Descriptive data are presented as the mean9SEM. Evaluations of survival and onset of the symptoms were performed by Kaplan Meier analysis. The rotarod test, motor neuron count and CAMP analysis were analyzed by ANOVA (analysis of variance) followed by a Student Newman Keuls post hoc analysis for multiple comparisons.
Results Tissue immunofluorescence analysis When the progression of disease symptoms reached endstage, six mice were killed (anesthesia and fixation methods as for the motor neuron count). The brains, brain stems, spinal cords, hearts, lungs, kidneys, spleens and muscles of hMSC-transplanted and untreated mice were removed. The organs were post-fixed in 4% paraformaldehyde for 6 h and dehydrated in 20% and 30% sucrose in 0.1 M phosphate buffer (pH 7.2) at 48 C for 1 2 days. Then blocks were frozen in Tissue-Tek OCT compound and cut transversely at a thickness of 7 mm in a cryostat (LeicaCM1850, Leica, Nussloch, Germany). Every first section from one in five series was sampled and mounted on poly-L-lysine glass slides. The slides were washed with deionized water to remove the freezing medium, rinsed three times in PBS and permeabilized with 0.3% Triton X100 in PBS at room temperature. Non-specific Ab binding was blocked with incubation in 10% normal goat serum (Boster Co.) for 1 h at room temperature. The slides were incubated overnight at 48C with MAb against human nuclei (HuNu, 1:50; Chemicon, Termecula, CA, USA) followed by 2 h of incubation with secondary Ab conjugated to FITC (1:100; Santa Cruz). Slides were rinsed and incubated overnight at 48C in the second series of primary Ab: monoclonal anti-human nestin (1:50; Santa Cruz), monoclonal anti-human GFAP (1:50; GeneTech, ShangHai, China), b-tubulin III (TuJ1, 1:100; Chemicon) and rabbit polyclonal Ab anti-human NSE (1:200; Neomarkers, Fremont, CA, USA). Then the slides were incubated in secondary Ab conjugated to Cy3 (1:200; Santa Cruz) for 1 h at room temperature. After rinsing them in PBS, the slides were counterstained with DAPI
After four serial passages of adherent cells, a homogeneous population of bipolar fibroblast-like cells were attained (Figure 1A). In order to verify the nature of the cells, surface markers were analyzed using FCM. As shown in Figure 1, hMSC expressed CD29 (98.8%), CD44 (99.7%) but not CD34 (a hematopoietic stem cell marker) or CD45 (leukocyte common Ag), indicating these cells were of mesenchymal origin with high purity.
Effect of hMSC transplantation on disease onset, survival and neuromuscular function Thirty-four untreated SOD1-G93A mice were observed in this study. The onset of symptoms appeared at 156.5599 3.598 days of age and the average age at end-stage was 188.32493.513 days. In order to obtain successful engraftment of hMSC, irradiation is necessary. In order to avoid impairments resulting from high-dose irradiation [20] and because of the low immunogenicity of MSC, a lower irradiation dose, 6.0 Gy, was used compared to the 8.0 Gy in Corti et al. ’s [4] study and 9.5 11.0 Gy in Solomon et al. ’s [21] study. About 20% of mice in the control group (SOD1-G93A mice undergoing 6.0 Gy irradiation but injected with medium only) died within 30 days. The survivors became very weak and pale in the first 1 2 months after irradiation and then recovered gradually from the damage of irradiation. The onset of ALS type symptoms and the lifespan of the irradiated survivors were similar to those of the untreated group (153.66794.907 days vs.156.55993.598 days for onset, x2 1.140, P 0.286, and 183.88996.129 days vs. 188.32493.513 days for lifespan, x2 0.7, P 0.403). A 6.0 Gy dose of irradiation had little effect on the onset or
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Figure 1. Morphology and FCM analysis of hMSC at the fifth passage. (A) Phase-contrast images of the fibroblast-like morphology of hMSC at the fifth passage (200 magnification). (C F) FCM analysis of hMSC. hMSC expressed CD29 and CD44 (C, E) but not CD34 or CD45 (D, F). (B) Control.
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lifespan of this disease in this mouse strain. hMSC transplantation delayed the onset of ALS type symptoms about 14 days (x2 6.549, P 0.01 B0.05) and prolonged the lifespan about 18 days compared with the untreated SOD1-G93A littermates (x2 9.445, P 0.002 B0.01) (Table 1). The survival and onset (Figure 2A, B) indicated that there was a great delay of onset and an increase of lifespan. The longest lifespan was 232 days in untreated mice and 246 days in hMSC-transplanted mice. Neuromuscular motor function tests were performed by rotarod test on the hMSC-transplanted group, untreated group and normal group. Untreated mice showed a marked decrease in performance between 22 weeks and 27 weeks of age, whereas the hMSC-transplanted mice showed a 3-week delay of motor function loss compared with untreated mice (Figure 2C).
Effect of hMSC transplantation on motor neuron loss Mean ventral horn motor neuron numbers in untreated mice, hMSC-transplanted mice and normal mice were quantitated at 12, 16 and 20 weeks of age (n 3 at each time point in each group). In the lumbar ventral horn of both untreated mice and hMSC-transplanted mice, a progressive decrease of motor neurons was observed from 12 to 20 weeks of age. The numbers of motor neurons in untreated mice and hMSC-transplanted mice were significantly different from those of the normal group (P B0.001 at 12, 16 and 20 weeks, respectively). The loss of motor neurons in untreated mice was much faster and more severe than in hMSC-transplanted mice. At 12 weeks of age, mean motor neurons of both groups were similar (12.3192.14 in untreated mice vs. 12.3591.98 in hMSCtransplanted mice, P 0.05) but at 16 and 20 weeks motor neurons of untreated mice were significantly fewer than those of transplanted mice (8.8191.58 in untreated mice vs. 10.8392.14 in hMSC-transplanted mice at 16 weeks, P B0.001, and 5.6590.97 in untreated mice vs. 8.2191.64
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in hMSC-transplanted mice at 20 weeks, P B0.001) (Figures 3 and 4A).
Effect of hMSC transplantation on axonal degeneration To evaluate the effect of hMSC transplantation on axonal degeneration in SOD1-G93A mice, the CMAP of the sciatic nerve was recorded in normal mice, untreated mice and hMSC-transplanted mice at 12, 16 and 20 weeks of age (six CMAP recordings at each time point in each group were analyzed). As demonstrated in Figure 5, the response increased slowly with increasing stimulation intensity, until the response increased in a quantal fashion at the end and the supramaximal response was obtained. The CMAP amplitudes of untreated mice and hMSC-transplanted mice decreased gradually with aging and the level attained at 20 weeks of age was very low (note the scale). As shown in Figure 4B, the CMAP amplitudes of untreated mice and hMSC-transplanted mice were significantly different from those of the normal group at 16 and 20 weeks, respectively (P B0.001) but not at 12 weeks (P 0.05). There was no significant difference of CMAP amplitudes recorded in the gastrocnemius muscle between untreated mice and hMSCtransplanted mice at 12 and 16 weeks, respectively. However, the CMAP amplitude of untreated mice at 20 weeks was significant lower than that of hMSC-transplanted mice at the same time point (0.53090.390 mV in untreated mice and 1.39990.736 mV in hMSC-transplanted mice, P B0.05).
Survival and phenotype of hMSC in CNS In immunohistofluorescence analysis, hMSC were detected (identified by the human-specific marker HuNu) in many peripheral tissues, such as lung, liver, spleen and BM. HuNu-positive cells appeared to survive in the brain, brain stem and spinal cord even at the end-stage. While some HuNu-positive cells were detected in or near blood vessels, the majority were found in the parenchyma of gray matter and white matter far away from vessels in the brain and
Table 1. Onset time and survival of untreated mice, hMSC-transplanted mice and control mice Group
n
Mean days to onset
Mean survival
SOD1 untreated SOD1 hMSC-transplanted SOD1control mice
34 29 9
156.55993.598 170.62193.814* 153.66794.907
188.32493.513 206.72493.852** 183.88996.129
*P B0.05, **P B0.01. There was a great delay of onset and an increase of lifespan in hMSC-transplanted mice compared with untreated mice. There was no significant difference between untreated mice and control mice.
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Figure 2. Characteristics of survival, onset and neuromuscular function in untreated and hMSC-transplanted SOD1-G93A mice. (A) Survival (Kaplan Meier) analysis of untreated mice (red), hMSC-transplanted mice (green) and control mice (blue). (B) Kaplan Meier analysis of onset of untreated mice (red), hMSC-transplanted mice (green) and control mice (blue). (C) Rotarod performance of untreated mice, hMSC-transplanted mice and normal mice. * Indicates there was a significant difference between untreated and normal mice; pentagon, indicates there was a significant difference between transplanted and normal mice; triangle, indicates transplanted mice performed significantly better than untreated mice.
spinal cord. By quantification analysis, there were about 4.4790.75 cells/field at 400 magnification staining positive for HuNu in the brain and about 1.3190.54 cells in the ventral horn of lumbar spinal cord. This suggested that hMSC enters CNS via blood vessels and migrates into parenchyma. Using immunofluorescence double-staining, hMSC gave rise to cells expressing neuronal and astrocyte markers. In the brain, some HuNu-positive cells were detected expressing the immature neuron maker b-tubulin III and astrocyte marker GFAP (Figure 6E H); in the spinal cord there were a few HuNu-positive cells expressing either the mature neuron-specific marker NSE or immature
neuron maker b-tubulin III (Figure 6I L). hMSC staining positive for the neuron marker assumed a round or polygonal shape. It was also found that most of the HuNu-positive hMSC did not express any of the above neural markers.
Discussion The major findings in our present study were: (1) hMSC has therapeutic potential in SOD1-G93A mice; (2) intravenous transplantation of hMSC delayed the disease onset of SOD1-G93A mice by about 14 days and prolonged the lifespan by on average 18 days; (3)
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Figure 3. Histologic evaluation of surviving motor neurons in lumbar spinal cord at 12, 16 and 20 weeks of age.(A) Normal mice at 12 weeks. (B D)untreated mice at 12, 16 and 20 weeks. (E G) hMSC-transplanted mice at 12, 16 and 20 weeks. Bar 100 mm. N, normal mice; A, untreated mice; A T, hMSC-transplanted mice.
administration of hMSC ameliorated the phenotype of SOD1-G93A mice and delayed motor neuron loss and axonal degeneration; (4) a tissue chimerism was acquired with evidence that HuNu-positive cells are detected in many tissues, including peripheral tissue and CNS; (5) hMSC migrated into the brain and spinal cord, penetrated
Figure 4. Quantification of surviving motor neurons in lumbar spinal cord and CMAP in the gastrocnemius muscle at 12, 16 and 20 weeks of age. (A) At 16 and 20 weeks, motor neurons of hMSC-transplanted mice were significantly more than untreated mice (*PB0.001). (B) The CMAP amplitude of untreated mice at 20 weeks was significantly lower than that of hMSC-transplanted mice(**PB0.05).
parenchyma and expressed neuronal specific markers (b-tubulin III and NSE) and an astrocytic marker (GFAP). Stem cell therapy is being actively and enthusiastically considered for many intractable CNS diseases. MSC are suitable and promising for human therapies because of their potential for differentiation and easy access. The powerful proliferation ability and extensive differentiation potentiality of MSC make them similar to embryonic stem cells [22]. In addition, the use of MSC overcomes the need for immunologic matching between donor and recipient, the problem of limited donor cell number and reveals the novel possibility of using autologous non-neuronal sources. Positive effects of MSC transplantation on function restoration have been reported in cerebral ischemic models [23], brain injury [24], spinal cord injury [25], Parkinson [9] and other CNS diseases. ALS should be a good candidate for MSC transplantation because of the lack of efficient therapy and the severity of the symptoms. This present study first demonstrated that intravenous administration of hMSC into pre-symptomatic SOD1G93A mice delayed disease onset by about 14 days and prolonged the lifespan on average 18 days. hMSC sustained neuromuscular motor function until 25 weeks of age, while motor function in untreated mice began to deteriorate at 22 weeks of age. This positive effect was verified further by histopathologic and electrophysiologic analysis. Although motor neuron loss from 12 weeks was not prevented in hMSC-transplanted mice (compared with the normal group, P B0.001), hMSC transplantation slowed the pace of progression of neuron loss (there was a significance
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Figure 5. Recordings of evoked CMAP in the gastrocnemius muscle after stimulation of the sciatic nerve in untreated mice and hMSCtransplanted mice. (A) CMAP tracings of normal mice at 12 weeks. (B, D, F) CMAP tracings of untreated mice at 12, 16 and 20 weeks. (C, E, G) CMAP tracings of hMSC-transplanted mice at 12, 16 and 20 weeks. N, normal mice; A, untreated mice; A T, transplanted SOD1-G93A ALS mice.
difference of motor neuron numbers in ventral horns at 16 and 20 weeks of age between untreated mice and hMSCtransplanted mice). It is documented that primary motor neuron loss and secondary degeneration of peripheral motor nerves take place in SOD1-G93A mice [1,26,27]. Both denervation and reinnervation exist in muscle at an early stage [17,26]. The CMAP amplitudes of untreated mice and hMSC-transplanted mice at 12 weeks were not significantly different from those of the normal group. These data suggest sprouting and reinneravation compensated for the loss of motor neuron at this time. At 16 weeks, reinnervation might partially compensate for the motor neuron loss. At 20 weeks, the amplitude of the CMAP of hMSC-transplanted mice was significant higher than that
of untreated mice, suggesting hMSC transplantation delayed the secondary degeneration and extended the period of reinnervation of peripheral motor nerves. This lifespan increase (about 10%) was comparable with that obtained in previous studies using BM, human NT neurons and human UC blood [3 5]. The hMSC intravenous transplantation protocol was more feasible and less invasive, with less side-effects. This positive effect is based on the engraftment and long-term survival of transplanted hMSC in the recipient mice. In histoimmunofluoresce analysis, the HuNu-positive cells were detected in many tissues, such as lung, liver, spleen, brain and spinal cord, at end-stage. A tissue chimerism was acquired in hMSC-transplanted mice. Further investigation showed
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Figure 6. Immunohistofluorescence analysis of hMSC in CNS and lung. (A, B) HuNu-positive cells were found in lumbar spinal cord (green); counterstaining with DAPI (blue) (red arrow). (C, D) A few HuNu-positive cells were found in lung (green); counterstaining with DAPI (blue) (red arrow). (E, F) Double-staining with b-tubulin III (red)/HuNu (green) hMSC was found outside blood vessels in the brain; counterstaining with DAPI (blue)(white arrow). (G, H) Double-staining with GFAP (red)/HuNu (green) hMSC was found in the parenchyma of brain, assuming a phenotype of astrocytes (white arrow). (I, J) Double-staining with b-tubulin III (red)/HuNu (green) hMSC was near a blood vessel. (K, L) Double-staining with NSE (red)/HuNu (green) hMSC appeared in the parenchyma in the lumbar spinal cord (white arrow). Many HuNupositive cells (red arrow) were not positive to neural markers in the CNS.
that a few cells were trapped in microvasculature, in particular of lungs, on the first pass of intravenous infusion. This result is consistent with other studies [28 30]. It is believed that a more efficient transplantation route will promote more stem cell engrafting in lesions of the CNS and more benefits will be obtained in the future. Previous studies have indicated that MSC can find their way into the brain and spinal cord when delivered intravenously in animal models of stroke and CNS trauma [10,11,31,32]. The migration mechanism needs to be clarified further. MCP-1, MIP-1, IL-8 and ischemic cerebral tissue could enhance hMSC migration, indicating that hMSC are targeted by inflammatory chemotactic agents and cytokines [33]. In ALS, microglias and astrocytes are activated and a large amount of cytokines, chemokines are released, including IL-1a, IL-1b, IL1RA, IL-2, IL-3, IL-4, TNF-a, TGF-b, M-CSF and MCP-1 [34,35]. It is possible that these molecules serve as signals inducing cell migration. Another problem, the
blood brain barrier (BBB) of SOD1-G93A mice, is relatively intact compared with that of stroke and trauma animal models. But in the present study, the hMSCtransplanted mice were pre-conditioned with irradiation. It is documented [36] that irradiation induces an increase in engraftment levels of hMSC in the brain, heart, BM and muscles in NOD-SCID mice. Francois et al. [37] attribute this migration and homing of cells to cytokines and chemokines resulting from the injury by irradiation. It is reported that endothelial cells undergo apoptosis, the endothelial cell density decreases and BBB breaks down at an early stage after single-dose brain irradiation [37 40]. Moreover, irradiation-induced permeability is accompanied by an increase in cell adhesion in the BBB [41,42] that triggers the disorganization of endothelial cell-to-cell adherens junctions, thereby facilitating cells transmigration [43]. A possible mechanism in our study was that hMSC were chemotactic by these signals, crossed the damaged BBB and settled down in the parenchyma of CNS.
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Although a number of cells showed features suggestive of neurons and glia in the brain and spinal cord, the functional improvement could not be explained by neurogenesis alone. It is much more likely that combined effects were operative. The etiopathology of ALS is very complex, including oxidative stress and glutamate excitoxicity [44], autoimmune mechanisms [45], abnormal accumulations of neurofilaments [46], altered glial function [47], mitochondrial dysfunction and impairment of trophic support [48]. hMSC transplantation might delay the progress of ALS through multiple pathways. Most of the hMSC detected in CNS in our study did not express any markers of neurons and astrocytes. hMSC might influence the microenvironment in CNS through a nonneuron replacement mechanism. MSC can produce many trophic factors in vitro and in vivo, such as IGF-1, BDNF, NGF, bFGF and VEGF [49,50]. Mechanisms proposed for the neuroprotective effect of these agents include free radical scavenging, anti-apoptotic activity, anti-inflammatory activity and anti-glutamate excitotoxicity [50 53]. Recently, it has been demonstrated that MSC reduce cell death and apoptosis and increase the DNA proliferation rate in astrocyte post-ischemia [54]. Co-culture of MSC with hippocampal slices results in reduced cell death after oxygen-glucose deprivation, suggesting that MSC are able to promote neuron survival [55]. Indeed, there is a report introducing the concept that stem cells may exert a therapeutic influence on abnormal hosts by exerting a protective ‘chaperone’ effect [56], which is consistent with the concept that in ALS non-neuronal cells could ameliorate degeneration and survival of SOD1 mice [57].We do not exclude the possibility that the benefit of hMSC transplantation results from combined effects, including cell replacement (neuron and neuroglia), neuroprotection and scavenging toxic agents. This study has demonstrated that hMSC engrafted, survived, migrated and penetrated into brain and spinal cord and showed some neuroglia differentiation after system delivery in SOD1-G93A mice. What was more important was that intravenous transplantation of hMSC delayed disease progression and extended the survival of SOD1-G93A mice. Although the biologic mechanisms involved in ameliorating the phenotype of disease with transplantation of hMSC needs to be clarified, the present findings, in the context of the rapidly expanding field of stem cell biology, have pointed to the BM MSC as an invaluable resource for neurodegenerative disease.
Acknowledgements The authors would like to gratefully acknowledge and thank Dr Ye-Chun Ruan in the Life Science College of Sun Yat-sen University for electrophysiologic recording. This work is supported by grants from the National Natural Science Foundation of China (30370510, 30170337), the CMB Fund (4209347) and the Key Project of the State Ministry of Public Health (2001321).
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Human mesenchymal stromal cells ameliorate the phenotype of SOD1-G93A ALS mice C-P Zhao1, C Zhang1,2, S-N Zhou3, Y-M Xie4, Y-H Wang5, H Huang1, Y-C Shang1, W-Y Li2, C Zhou1, M-J Yu2 and S-W Feng1 1
Department of Neurology, First Affiliated Hospital, Sun Yat-sen University, Guangzhou, PR China, 2Stem Cells and Tissue Engineering Research Center of Sun Yat-sen University, Guangzhou, PR China, 3Department of Neurology, Qi-Lu Hospital, ShanDong University, JiNan, PR China, 4 Department of Neurology, University of North Carolina at Chapel Hill, North Carolina, USA and 5Department of Neurosurgery, Affiliated Hospital, Yang Zhou University, Yang Zhou, PR China
Background Amyotrophic lateral sclerosis (ALS) is a progressive, lethal, neurodegenerative disease, currently without any effective therapy. Multiple advantages make mesenchymal stromal cells (MSC) a good candidate for cellular therapy in many intractable diseases such as stroke and brain injury. Until now, no irrefutable evidence exists regarding the outcome of MSC transplantation in the mouse model of ALS. The present study was designed to investigate the therapeutic potential of human MSC (hMSC) in the mouse model of ALS (SOD1-G93A mice). Methods hMSC were isolated from iliac crest aspirates from healthy donors and kept in cell cultures. hMSC of the fifth passage were delivered intravenously into irradiated pre-symptomatic SOD1-G93A mice. Therapeutic effects were analyzed by survival analysis, rotarod test, motor neuron count in spinal cord and electrophysiology. The engraftment and in vivo differentiation of hMSC were examined in the brain and spinal cord of hMSC-transplanted mice.
Introduction Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the loss of motor neurons in the spinal cord, brain stem and cerebral cortex, resulting in weakness, spasticity, paralysis and dysphagia. About 10% of cases are dominantly inherited and 20% of familial ALS patients arise from mutations in the gene for Cu/Zn superoxide dismutase (SOD1). The SOD1-G93A mice express a mutant SOD1 carrying the Gly93Ala missense mutation and develop ALS-like motor neuron
Results After intravenous injection into irradiated pre-symptomatic SOD1G93A mice, hMSC survived more than 20 weeks in recipient mice, migrated into the parenchyma of brain and spinal cord and showed neuroglia differentiation. Moreover, hMSC-transplanted mice showed significantly delayed disease onset (14 days), increased lifespan (18 days) and delayed disease progression compared with untreated mice. Discussion Our data document the positive effects of hMSC transplantation in the mouse model of ALS. It may signify the potential use of hMSC in treatment of ALS. Keywords amyotrophic lateral sclerosis, mesenchymal stromal cells, SOD1-G93A mice, therapy, transplantation.
disease [1], serving as a conventional tool for testing new therapeutic strategies. Although a large number of laboratory advances have been made, including studies on neurotrophic factors, antioxidants, anti-inflammatory agents and drugs modifying excitable amino acids, there is still no therapy available that has proven efficient in the treatment of ALS. Novel therapeutic strategies are directed at replacing or repairing the damaged motor neurons [2]. Cellular therapy holds the potential answer to this problem.
Correspondence to: Cheng Zhang, Department of Neurology, First Affiliated Hospital, and Stem Cells and Tissue Engineering Research Center, Sun Yat-sen University, Guangzhou 510080, PR China. E-mail: [email protected] – 2007 ISCT
DOI: 10.1080/14653240701376413
hMSC ameliorate the phenotype of ALS mice
There have been several reports on stem cell transplantation in the ALS animal model during the last 5 years. Positive effects were observed after hNT neurons (derived from human teratocarcinoma cell line NTera2/D1) were transplanted into the ventral horn of the spinal cord of SOD1-G93A mice [3]. Human UC blood and wild-type mice BM have been reported to increase the SOD1-G93A mouse lifespan [4,5]. Little is known about the dynamic changes of the motor neuron in ventral horns and axonal degeneration after stem cell transplantation in the mouse model of ALS. Among the variety of stem cells, adult BM mesenchymal stromal cells (MSC) are a promising source for cellular therapy. MSC can be expanded in number within a short time and cryopreserved for long periods without losing their potentiality. Because of low immunogenicity, the use of MSC overcomes the problem of limited donor cell number and high immunorejection incidence, and opens up the possibility of employing autologous sources. MSC can differentiate not only into mesodermal cells but also endodermal and ectodermal cells (neurons, astrocytes and oligodendrocytes) [6]. MSC have been reported to survive, proliferate and migrate into damaged tissue with positive functional effects in animal models of stroke [7], spinal cord injury [8] and Parkinson’s disease [9]. Even after being injected into the blood stream, MSC have been shown to migrate to the central nervous system (CNS) and develop into cells that appear to be neurons, astrocytes [10] or myelinating cells in the spinal cord [11]. MSC can also produce many trophic factors in vitro and in vivo, which play important roles in brain development and remodeling. Moreover, MSC can be used as vectors of cytokines, trophic factors and other pharmacologic molecules to prevent cell death, tissue inflammation and damage. The evidence highlights the potential use of MSC for therapy in ALS. One clinical report describes transplanting autologous MSC into the spinal cord of ALS patients as a safe approach [12]. Because of the small number of treated patients (seven) in this trial and the limitations of detecting the transplanted cells, it is impossible to confirm whether direct delivery of MSC to the spinal cord could allow MSC to survive in the microenvironment for long periods of time [13] or delay the disease progression in ALS. This present study was aimed at investigating the therapeutic potential of intravenous administration of human MSC (hMSC) in the mouse model of ALS
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(SOD1-G93A mice). Dynamic changes of the motor neurons of the lumbar ventral horn and axonal degeneration after hMSC transplantation were observed. The engraftment and in vivo differentiation of hMSC in the brain and spinal cord were examined in the hMSC-transplanted mice. The results of our investigation advance the understanding of the transplantation effects and risk benefit ratio of hMSC transplantation as a treatment for ALS.
Methods Animals Transgenic male mice, B6SJL-TgN (SOD1-G93A) 1GUR (No. 002726), were purchased from The Jackson Laboratories (Bar Harbor, ME, USA), which overexpress human SOD1 and carried the Gly93Ala mutation [1]. A colony was derived from them and maintained by breeding male transgenic mice to naive (B6 SJL/J)F1 dams. Offspring were genotyped for the SOD1-G93A transgene using PCR of genomic DNA extracted from the blood of tail veins, as outlined by Jackson Laboratories. Animals were housed one to five to a cage, with a 12-h light/dark cycle, and fed standard murine chow and water ad libitum. The room temperature was 238C. All animal experiments were performed according to institutional guidelines that are in compliance with national and international law and policies.
Preparation of hMSC Heparinized BM cells were obtained from iliac crest aspirates of healthy human volunteers after informed consent, and were used in accordance with the procedures approved by the human experimentation and ethics committees of China. Mononuclear cells were separated by centrifugation in a Ficoll Hypaque gradient (1.077 g/mL; Sigma, St Louis, MO, USA) and washed twice with PBS. Cells were suspended in DMEM with a low glucose content (1 g/L; L-DMEM; Gibco Invitrogen Co., Carlsbad, CA, USA) supplemented with 10% FBS and seeded in T 25-cm2 culture flasks at a concentration of 1 106 cells/cm2. Cultures were maintained at 378C in a humidified atmosphere containing 5% carbon dioxide. The culture medium was replaced every 3 days and non-adherent cells were discarded. When the culture flasks became nearly confluent (80 90%), the adherent cells were detached with 2.5g/L trypsin in 1.0 mM sodium EDTA (Na2-EDTA; Invitrogen). The hMSC was rinsed twice and re-seeded at a 1:3 dilution in T 25-cm2 culture flasks. The above manipulation was repeated up to the fifth
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passage in order to obtain high-purity hMSC and acquire enough cells for transplantation.
Analysis of hMSC by FCM To validate the identity of the cells, flow cytometry (FCM) was performed to detect the cell-surface Ag markers specific for hMSC. The hMSC of the fifth passage were harvested with 0.25% trypsin and resuspended in PBS at 2 104 cells/reaction tube. The hMSC were fixed in 4% cold paraformaldehyde for 30 min and washed with PBS containing 2% FBS. Cells were incubated with mouse antihuman CD29 (1:1000; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), anti-CD34 (1:1000; Santa Cruz), antiCD44 (1:1000; Santa Cruz), anti-CD45 (1:1000; Santa Cruz) Ab and FITC-labeled goat anti-mouse secondary Ab (1:100; Santa Cruz). The samples were characterized by FCM.
hMSC transplantation The SOD1-G93A mice were distributed randomly into each group and all were evaluated up to end-stage. One to 3 days before transplantation, the SOD1-G93A mice (8 weeks of age) were irradiated with 6.0 Gy from a60Co source (0.454 Gy/min). hMSC (3 106) resuspended in 0.3mL L-DMEM, or 0.3 mL L-DMEM only, was injected into the tail vein of each recipient mouse. Before and after infusion, mice were kept under pathogen-free conditions and provided with fresh water. The groups were as follows. (1) hMSC-transplanted group: SOD1-G93A mice transplanted with hMSC after irradiation (n 29). (2) Untreated group: SOD1-G93A mice injected with only 0.3mL L-DMEM (n 34). (3) Normal group: wild-type littermates (SOD1-G93A negative) (n 30). (4) Control group: SOD1-G93A mice injected with 0.3mL L-DMEM after irradiation (n 12). Three mice of the hMSC-transplanted group, untreated group and normal group were killed at 12, 16 and 20 weeks of age for motor neuron count and CMAP assessment. Independent investigators were blinded to the animal’s post-transplant status to avoid subjective bias in all experiments.
Motor assessment and survival All SOD1-G93A mice were monitored daily for disease progression and survival by investigators blinded to the condition of the treatment. The mice were evaluated for signs of motor deficit with 4-point scoring system according to Weydt et al. [14]. The rotarod test was used to assess
motor co-ordination, strength and balance [15]. Fifteen mice of the hMSC-transplanted group, 14 mice of the untreated group and 10 mice of the normal group were tested weekly from 8 weeks of age (age and sex of mice were matched in three groups). The mice were tested on the rod rotating at a constant speed of 16 r.p.m. The longest latency was recorded and 180 seconds was chosen as the arbitrary cut-off time. Mortality was scored as the age at death [16].
Electrophysiologic recording (CMAP recording) For compound muscle action potential (CMAP) analysis, three mice from each group were anesthetized with 10% chloral hydrate (0.3 mL/100g, i.p.) at 12, 16 and 20 weeks of age. Electrophysiologic recordings across the nerve segment were made using an electromyogram apparatus (BL-420E; ChengDu, SiChuan, China). The sciatic nerve was stimulated at the paraspinal site. The stimulation consisted of single 1-millisecond 1-Hz supramaximal pulses through a needle electrode. A ground needle electrode was placed on the back of the mouse; an anode needle was inserted at the base of the tail; reference needle electrode was placed near the Achilles’s tendon. The evoked CMAP was recorded from the medial part of the gastrocnemius muscle with a unipolar needle electrode [17]. An initial negative deflection indicated recording at the motor point. In order to assess whether the stimulation was supramaximal and electrical stimulation and the response were reproducible, the stimulation intensity was increased gradually until the response would not increase any more or increase in a quantal fashion. At this time the maximal potential (CMAP) was recorded and analyzed (measured peak-to-peak) [18] with software (BL-NewCentury 2.0; ChengDu). Both the right and left hind limbs were studied.
Motor neuron count For motor neuron counts, three mice from each group were killed at the ages of 12, 16 and 20 weeks. The mice were anesthetized with 10% chloral hydrate (0.3 mL/100 g, i.p.) and perfused transcardially with 0.9% NaCl solution followed by ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2). The lumbar enlargements of each spinal cord were removed and post-fixed in the same fixative for 2 24 h at 48C. After fixation, the spinal cords were dehydrated with increasing concentrations of ethanol and embedded in paraffin. Serial transverse sections (5 mm) were cut through the lumbar enlargements. Every first section from one in five series
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was sampled using a random starting point of L3 L5 segments, mounted on a poly-L-lysine (Boster Co., WuHan, China) glass slide and stained with cresyl violet (Sigma; eight sections for each animal). On each section, at 200 magnification, the area ventral to the most dorsal extent of the central canal, including laminae VII, VIII and IX, was designated the ventral horn. Only motor neurons with a diameter 20 mm and bearing a clearly identifiable nucleus, nucleolus and cytoplasm with at least one thick process were counted by investigators blinded to the condition of the treatment [19]. The count and analysis were done with a computer-based image analysis system (Image-Pro-Plus, MediaCybernetics Inc., Silver Spring, MD, USA).
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(1:5000; Sigma) and examined under a fluorescent microscope (Olympus BX51, Olympus Co., Tokyo, Japan). HuNu-positive cells were counted on an average of three random sections of brain and lumbar spinal cord per mouse (at 400 magnification).
Statistical analysis Descriptive data are presented as the mean9SEM. Evaluations of survival and onset of the symptoms were performed by Kaplan Meier analysis. The rotarod test, motor neuron count and CAMP analysis were analyzed by ANOVA (analysis of variance) followed by a Student Newman Keuls post hoc analysis for multiple comparisons.
Results Tissue immunofluorescence analysis When the progression of disease symptoms reached endstage, six mice were killed (anesthesia and fixation methods as for the motor neuron count). The brains, brain stems, spinal cords, hearts, lungs, kidneys, spleens and muscles of hMSC-transplanted and untreated mice were removed. The organs were post-fixed in 4% paraformaldehyde for 6 h and dehydrated in 20% and 30% sucrose in 0.1 M phosphate buffer (pH 7.2) at 48 C for 1 2 days. Then blocks were frozen in Tissue-Tek OCT compound and cut transversely at a thickness of 7 mm in a cryostat (LeicaCM1850, Leica, Nussloch, Germany). Every first section from one in five series was sampled and mounted on poly-L-lysine glass slides. The slides were washed with deionized water to remove the freezing medium, rinsed three times in PBS and permeabilized with 0.3% Triton X100 in PBS at room temperature. Non-specific Ab binding was blocked with incubation in 10% normal goat serum (Boster Co.) for 1 h at room temperature. The slides were incubated overnight at 48C with MAb against human nuclei (HuNu, 1:50; Chemicon, Termecula, CA, USA) followed by 2 h of incubation with secondary Ab conjugated to FITC (1:100; Santa Cruz). Slides were rinsed and incubated overnight at 48C in the second series of primary Ab: monoclonal anti-human nestin (1:50; Santa Cruz), monoclonal anti-human GFAP (1:50; GeneTech, ShangHai, China), b-tubulin III (TuJ1, 1:100; Chemicon) and rabbit polyclonal Ab anti-human NSE (1:200; Neomarkers, Fremont, CA, USA). Then the slides were incubated in secondary Ab conjugated to Cy3 (1:200; Santa Cruz) for 1 h at room temperature. After rinsing them in PBS, the slides were counterstained with DAPI
After four serial passages of adherent cells, a homogeneous population of bipolar fibroblast-like cells were attained (Figure 1A). In order to verify the nature of the cells, surface markers were analyzed using FCM. As shown in Figure 1, hMSC expressed CD29 (98.8%), CD44 (99.7%) but not CD34 (a hematopoietic stem cell marker) or CD45 (leukocyte common Ag), indicating these cells were of mesenchymal origin with high purity.
Effect of hMSC transplantation on disease onset, survival and neuromuscular function Thirty-four untreated SOD1-G93A mice were observed in this study. The onset of symptoms appeared at 156.5599 3.598 days of age and the average age at end-stage was 188.32493.513 days. In order to obtain successful engraftment of hMSC, irradiation is necessary. In order to avoid impairments resulting from high-dose irradiation [20] and because of the low immunogenicity of MSC, a lower irradiation dose, 6.0 Gy, was used compared to the 8.0 Gy in Corti et al. ’s [4] study and 9.5 11.0 Gy in Solomon et al. ’s [21] study. About 20% of mice in the control group (SOD1-G93A mice undergoing 6.0 Gy irradiation but injected with medium only) died within 30 days. The survivors became very weak and pale in the first 1 2 months after irradiation and then recovered gradually from the damage of irradiation. The onset of ALS type symptoms and the lifespan of the irradiated survivors were similar to those of the untreated group (153.66794.907 days vs.156.55993.598 days for onset, x2 1.140, P 0.286, and 183.88996.129 days vs. 188.32493.513 days for lifespan, x2 0.7, P 0.403). A 6.0 Gy dose of irradiation had little effect on the onset or
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Figure 1. Morphology and FCM analysis of hMSC at the fifth passage. (A) Phase-contrast images of the fibroblast-like morphology of hMSC at the fifth passage (200 magnification). (C F) FCM analysis of hMSC. hMSC expressed CD29 and CD44 (C, E) but not CD34 or CD45 (D, F). (B) Control.
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lifespan of this disease in this mouse strain. hMSC transplantation delayed the onset of ALS type symptoms about 14 days (x2 6.549, P 0.01 B0.05) and prolonged the lifespan about 18 days compared with the untreated SOD1-G93A littermates (x2 9.445, P 0.002 B0.01) (Table 1). The survival and onset (Figure 2A, B) indicated that there was a great delay of onset and an increase of lifespan. The longest lifespan was 232 days in untreated mice and 246 days in hMSC-transplanted mice. Neuromuscular motor function tests were performed by rotarod test on the hMSC-transplanted group, untreated group and normal group. Untreated mice showed a marked decrease in performance between 22 weeks and 27 weeks of age, whereas the hMSC-transplanted mice showed a 3-week delay of motor function loss compared with untreated mice (Figure 2C).
Effect of hMSC transplantation on motor neuron loss Mean ventral horn motor neuron numbers in untreated mice, hMSC-transplanted mice and normal mice were quantitated at 12, 16 and 20 weeks of age (n 3 at each time point in each group). In the lumbar ventral horn of both untreated mice and hMSC-transplanted mice, a progressive decrease of motor neurons was observed from 12 to 20 weeks of age. The numbers of motor neurons in untreated mice and hMSC-transplanted mice were significantly different from those of the normal group (P B0.001 at 12, 16 and 20 weeks, respectively). The loss of motor neurons in untreated mice was much faster and more severe than in hMSC-transplanted mice. At 12 weeks of age, mean motor neurons of both groups were similar (12.3192.14 in untreated mice vs. 12.3591.98 in hMSCtransplanted mice, P 0.05) but at 16 and 20 weeks motor neurons of untreated mice were significantly fewer than those of transplanted mice (8.8191.58 in untreated mice vs. 10.8392.14 in hMSC-transplanted mice at 16 weeks, P B0.001, and 5.6590.97 in untreated mice vs. 8.2191.64
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in hMSC-transplanted mice at 20 weeks, P B0.001) (Figures 3 and 4A).
Effect of hMSC transplantation on axonal degeneration To evaluate the effect of hMSC transplantation on axonal degeneration in SOD1-G93A mice, the CMAP of the sciatic nerve was recorded in normal mice, untreated mice and hMSC-transplanted mice at 12, 16 and 20 weeks of age (six CMAP recordings at each time point in each group were analyzed). As demonstrated in Figure 5, the response increased slowly with increasing stimulation intensity, until the response increased in a quantal fashion at the end and the supramaximal response was obtained. The CMAP amplitudes of untreated mice and hMSC-transplanted mice decreased gradually with aging and the level attained at 20 weeks of age was very low (note the scale). As shown in Figure 4B, the CMAP amplitudes of untreated mice and hMSC-transplanted mice were significantly different from those of the normal group at 16 and 20 weeks, respectively (P B0.001) but not at 12 weeks (P 0.05). There was no significant difference of CMAP amplitudes recorded in the gastrocnemius muscle between untreated mice and hMSCtransplanted mice at 12 and 16 weeks, respectively. However, the CMAP amplitude of untreated mice at 20 weeks was significant lower than that of hMSC-transplanted mice at the same time point (0.53090.390 mV in untreated mice and 1.39990.736 mV in hMSC-transplanted mice, P B0.05).
Survival and phenotype of hMSC in CNS In immunohistofluorescence analysis, hMSC were detected (identified by the human-specific marker HuNu) in many peripheral tissues, such as lung, liver, spleen and BM. HuNu-positive cells appeared to survive in the brain, brain stem and spinal cord even at the end-stage. While some HuNu-positive cells were detected in or near blood vessels, the majority were found in the parenchyma of gray matter and white matter far away from vessels in the brain and
Table 1. Onset time and survival of untreated mice, hMSC-transplanted mice and control mice Group
n
Mean days to onset
Mean survival
SOD1 untreated SOD1 hMSC-transplanted SOD1control mice
34 29 9
156.55993.598 170.62193.814* 153.66794.907
188.32493.513 206.72493.852** 183.88996.129
*P B0.05, **P B0.01. There was a great delay of onset and an increase of lifespan in hMSC-transplanted mice compared with untreated mice. There was no significant difference between untreated mice and control mice.
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Figure 2. Characteristics of survival, onset and neuromuscular function in untreated and hMSC-transplanted SOD1-G93A mice. (A) Survival (Kaplan Meier) analysis of untreated mice (red), hMSC-transplanted mice (green) and control mice (blue). (B) Kaplan Meier analysis of onset of untreated mice (red), hMSC-transplanted mice (green) and control mice (blue). (C) Rotarod performance of untreated mice, hMSC-transplanted mice and normal mice. * Indicates there was a significant difference between untreated and normal mice; pentagon, indicates there was a significant difference between transplanted and normal mice; triangle, indicates transplanted mice performed significantly better than untreated mice.
spinal cord. By quantification analysis, there were about 4.4790.75 cells/field at 400 magnification staining positive for HuNu in the brain and about 1.3190.54 cells in the ventral horn of lumbar spinal cord. This suggested that hMSC enters CNS via blood vessels and migrates into parenchyma. Using immunofluorescence double-staining, hMSC gave rise to cells expressing neuronal and astrocyte markers. In the brain, some HuNu-positive cells were detected expressing the immature neuron maker b-tubulin III and astrocyte marker GFAP (Figure 6E H); in the spinal cord there were a few HuNu-positive cells expressing either the mature neuron-specific marker NSE or immature
neuron maker b-tubulin III (Figure 6I L). hMSC staining positive for the neuron marker assumed a round or polygonal shape. It was also found that most of the HuNu-positive hMSC did not express any of the above neural markers.
Discussion The major findings in our present study were: (1) hMSC has therapeutic potential in SOD1-G93A mice; (2) intravenous transplantation of hMSC delayed the disease onset of SOD1-G93A mice by about 14 days and prolonged the lifespan by on average 18 days; (3)
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Figure 3. Histologic evaluation of surviving motor neurons in lumbar spinal cord at 12, 16 and 20 weeks of age.(A) Normal mice at 12 weeks. (B D)untreated mice at 12, 16 and 20 weeks. (E G) hMSC-transplanted mice at 12, 16 and 20 weeks. Bar 100 mm. N, normal mice; A, untreated mice; A T, hMSC-transplanted mice.
administration of hMSC ameliorated the phenotype of SOD1-G93A mice and delayed motor neuron loss and axonal degeneration; (4) a tissue chimerism was acquired with evidence that HuNu-positive cells are detected in many tissues, including peripheral tissue and CNS; (5) hMSC migrated into the brain and spinal cord, penetrated
Figure 4. Quantification of surviving motor neurons in lumbar spinal cord and CMAP in the gastrocnemius muscle at 12, 16 and 20 weeks of age. (A) At 16 and 20 weeks, motor neurons of hMSC-transplanted mice were significantly more than untreated mice (*PB0.001). (B) The CMAP amplitude of untreated mice at 20 weeks was significantly lower than that of hMSC-transplanted mice(**PB0.05).
parenchyma and expressed neuronal specific markers (b-tubulin III and NSE) and an astrocytic marker (GFAP). Stem cell therapy is being actively and enthusiastically considered for many intractable CNS diseases. MSC are suitable and promising for human therapies because of their potential for differentiation and easy access. The powerful proliferation ability and extensive differentiation potentiality of MSC make them similar to embryonic stem cells [22]. In addition, the use of MSC overcomes the need for immunologic matching between donor and recipient, the problem of limited donor cell number and reveals the novel possibility of using autologous non-neuronal sources. Positive effects of MSC transplantation on function restoration have been reported in cerebral ischemic models [23], brain injury [24], spinal cord injury [25], Parkinson [9] and other CNS diseases. ALS should be a good candidate for MSC transplantation because of the lack of efficient therapy and the severity of the symptoms. This present study first demonstrated that intravenous administration of hMSC into pre-symptomatic SOD1G93A mice delayed disease onset by about 14 days and prolonged the lifespan on average 18 days. hMSC sustained neuromuscular motor function until 25 weeks of age, while motor function in untreated mice began to deteriorate at 22 weeks of age. This positive effect was verified further by histopathologic and electrophysiologic analysis. Although motor neuron loss from 12 weeks was not prevented in hMSC-transplanted mice (compared with the normal group, P B0.001), hMSC transplantation slowed the pace of progression of neuron loss (there was a significance
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Figure 5. Recordings of evoked CMAP in the gastrocnemius muscle after stimulation of the sciatic nerve in untreated mice and hMSCtransplanted mice. (A) CMAP tracings of normal mice at 12 weeks. (B, D, F) CMAP tracings of untreated mice at 12, 16 and 20 weeks. (C, E, G) CMAP tracings of hMSC-transplanted mice at 12, 16 and 20 weeks. N, normal mice; A, untreated mice; A T, transplanted SOD1-G93A ALS mice.
difference of motor neuron numbers in ventral horns at 16 and 20 weeks of age between untreated mice and hMSCtransplanted mice). It is documented that primary motor neuron loss and secondary degeneration of peripheral motor nerves take place in SOD1-G93A mice [1,26,27]. Both denervation and reinnervation exist in muscle at an early stage [17,26]. The CMAP amplitudes of untreated mice and hMSC-transplanted mice at 12 weeks were not significantly different from those of the normal group. These data suggest sprouting and reinneravation compensated for the loss of motor neuron at this time. At 16 weeks, reinnervation might partially compensate for the motor neuron loss. At 20 weeks, the amplitude of the CMAP of hMSC-transplanted mice was significant higher than that
of untreated mice, suggesting hMSC transplantation delayed the secondary degeneration and extended the period of reinnervation of peripheral motor nerves. This lifespan increase (about 10%) was comparable with that obtained in previous studies using BM, human NT neurons and human UC blood [3 5]. The hMSC intravenous transplantation protocol was more feasible and less invasive, with less side-effects. This positive effect is based on the engraftment and long-term survival of transplanted hMSC in the recipient mice. In histoimmunofluoresce analysis, the HuNu-positive cells were detected in many tissues, such as lung, liver, spleen, brain and spinal cord, at end-stage. A tissue chimerism was acquired in hMSC-transplanted mice. Further investigation showed
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Figure 6. Immunohistofluorescence analysis of hMSC in CNS and lung. (A, B) HuNu-positive cells were found in lumbar spinal cord (green); counterstaining with DAPI (blue) (red arrow). (C, D) A few HuNu-positive cells were found in lung (green); counterstaining with DAPI (blue) (red arrow). (E, F) Double-staining with b-tubulin III (red)/HuNu (green) hMSC was found outside blood vessels in the brain; counterstaining with DAPI (blue)(white arrow). (G, H) Double-staining with GFAP (red)/HuNu (green) hMSC was found in the parenchyma of brain, assuming a phenotype of astrocytes (white arrow). (I, J) Double-staining with b-tubulin III (red)/HuNu (green) hMSC was near a blood vessel. (K, L) Double-staining with NSE (red)/HuNu (green) hMSC appeared in the parenchyma in the lumbar spinal cord (white arrow). Many HuNupositive cells (red arrow) were not positive to neural markers in the CNS.
that a few cells were trapped in microvasculature, in particular of lungs, on the first pass of intravenous infusion. This result is consistent with other studies [28 30]. It is believed that a more efficient transplantation route will promote more stem cell engrafting in lesions of the CNS and more benefits will be obtained in the future. Previous studies have indicated that MSC can find their way into the brain and spinal cord when delivered intravenously in animal models of stroke and CNS trauma [10,11,31,32]. The migration mechanism needs to be clarified further. MCP-1, MIP-1, IL-8 and ischemic cerebral tissue could enhance hMSC migration, indicating that hMSC are targeted by inflammatory chemotactic agents and cytokines [33]. In ALS, microglias and astrocytes are activated and a large amount of cytokines, chemokines are released, including IL-1a, IL-1b, IL1RA, IL-2, IL-3, IL-4, TNF-a, TGF-b, M-CSF and MCP-1 [34,35]. It is possible that these molecules serve as signals inducing cell migration. Another problem, the
blood brain barrier (BBB) of SOD1-G93A mice, is relatively intact compared with that of stroke and trauma animal models. But in the present study, the hMSCtransplanted mice were pre-conditioned with irradiation. It is documented [36] that irradiation induces an increase in engraftment levels of hMSC in the brain, heart, BM and muscles in NOD-SCID mice. Francois et al. [37] attribute this migration and homing of cells to cytokines and chemokines resulting from the injury by irradiation. It is reported that endothelial cells undergo apoptosis, the endothelial cell density decreases and BBB breaks down at an early stage after single-dose brain irradiation [37 40]. Moreover, irradiation-induced permeability is accompanied by an increase in cell adhesion in the BBB [41,42] that triggers the disorganization of endothelial cell-to-cell adherens junctions, thereby facilitating cells transmigration [43]. A possible mechanism in our study was that hMSC were chemotactic by these signals, crossed the damaged BBB and settled down in the parenchyma of CNS.
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Although a number of cells showed features suggestive of neurons and glia in the brain and spinal cord, the functional improvement could not be explained by neurogenesis alone. It is much more likely that combined effects were operative. The etiopathology of ALS is very complex, including oxidative stress and glutamate excitoxicity [44], autoimmune mechanisms [45], abnormal accumulations of neurofilaments [46], altered glial function [47], mitochondrial dysfunction and impairment of trophic support [48]. hMSC transplantation might delay the progress of ALS through multiple pathways. Most of the hMSC detected in CNS in our study did not express any markers of neurons and astrocytes. hMSC might influence the microenvironment in CNS through a nonneuron replacement mechanism. MSC can produce many trophic factors in vitro and in vivo, such as IGF-1, BDNF, NGF, bFGF and VEGF [49,50]. Mechanisms proposed for the neuroprotective effect of these agents include free radical scavenging, anti-apoptotic activity, anti-inflammatory activity and anti-glutamate excitotoxicity [50 53]. Recently, it has been demonstrated that MSC reduce cell death and apoptosis and increase the DNA proliferation rate in astrocyte post-ischemia [54]. Co-culture of MSC with hippocampal slices results in reduced cell death after oxygen-glucose deprivation, suggesting that MSC are able to promote neuron survival [55]. Indeed, there is a report introducing the concept that stem cells may exert a therapeutic influence on abnormal hosts by exerting a protective ‘chaperone’ effect [56], which is consistent with the concept that in ALS non-neuronal cells could ameliorate degeneration and survival of SOD1 mice [57].We do not exclude the possibility that the benefit of hMSC transplantation results from combined effects, including cell replacement (neuron and neuroglia), neuroprotection and scavenging toxic agents. This study has demonstrated that hMSC engrafted, survived, migrated and penetrated into brain and spinal cord and showed some neuroglia differentiation after system delivery in SOD1-G93A mice. What was more important was that intravenous transplantation of hMSC delayed disease progression and extended the survival of SOD1-G93A mice. Although the biologic mechanisms involved in ameliorating the phenotype of disease with transplantation of hMSC needs to be clarified, the present findings, in the context of the rapidly expanding field of stem cell biology, have pointed to the BM MSC as an invaluable resource for neurodegenerative disease.
Acknowledgements The authors would like to gratefully acknowledge and thank Dr Ye-Chun Ruan in the Life Science College of Sun Yat-sen University for electrophysiologic recording. This work is supported by grants from the National Natural Science Foundation of China (30370510, 30170337), the CMB Fund (4209347) and the Key Project of the State Ministry of Public Health (2001321).
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