Human mesenchymal stem cell transplantation extends survival, improves motor performance and decreases neuroinflammation in mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a lethal disease affecting motoneurons. In familial ALS, patients bear mutations in the superoxide dismutase gene (SOD1). We transplanted human bone marrow mesenchymal stem cells (hMSCs) into the lumbar spinal cord of asymptomatic SOD1(G93A) mice, an experimental model of ALS. hMSCs were found in the spinal cord 10 weeks after, sometimes close to motoneurons and were rarely GFAP- or MAP2-positive. In females, where progression is slower than in males, astrogliosis and microglial activation were reduced and motoneuron counts with the optical fractionator were higher following transplantation. Motor tests (Rotarod, Paw Grip Endurance, neurological examination) were significantly improved in transplanted males. Therefore hMSCs are a good candidate for ALS cell therapy: they can survive and migrate after transplantation in the lumbar spinal cord, where they prevent astrogliosis and microglial activation and delay ALS-related decrease in the number of motoneurons, thus resulting in amelioration of the motor performance.     

hNT neurons delay onset of motor deficits in a model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a degenerative motor neuron disease that manifests as a progressive muscular weakness leading to paralysis and death. Because of the diffuse nature of the motor neuron death, this disease is not considered a good candidate for treatment through neural transplantation. The purpose of this study was to show that transplantation of human neuron-like cells (hNT neurons) into the spinal cord of a transgenic ALS mouse model would improve motor deficits. The hNT neurons were transplanted bilaterally into L4-L5 spinal cord of the transgenic mice ( approximately 8 weeks of age), and the animals were evaluated on health and behavioral measures. The animals were perfused, and immunohistochemistry was performed to identify the transplanted cells. Transplantation of the hNT neurons into the spinal cord delayed the onset of motor behavioral symptoms. This was the first demonstration that even localized transplantation of neural cells directly into the parenchyma could improve motor function in an ALS model. Further study is needed to delineate the mechanism underlying these effects. This therapeutic approach has the potential to restore neural transmission, thereby improving quality of life for the ALS patient and possibly extend life expectancy.     

人骨髓间质干细胞经心脏移植治疗超氧化物歧化酶1-G93A转基因鼠的疗效

目的 研究经心脏移植入骨髓间质干细胞(hMSCs)对肌萎缩侧索硬化(ALS)模型鼠发病时间、生存期和病理的影响.方法 体外培养扩增hMSCs,流式细胞仪鉴定其性质及纯度.将3×10~6个第5代hMSCs经心脏移植入预放疗的8周龄超氧化物歧化酶1(SOD1)-G93A转基因鼠,用Weyd4分法评定移植鼠和未治疗鼠的生存期、发病时间,采取尼氏染色计数脊髓前角运动神经元,通过免疫荧光检测人特异性核抗原验证hMSCs在受体鼠中枢神经系统中的植入.结果 生存分析显示,经心脏移植hMSCs的ALS模型鼠平均发病时间为(172.85±3.82)d,比未治疗组[(156.56±3.60)d]延迟16 d,差异有统计学意义(x~2=10.888,P=0.001);hMSCs经心脏移植组平均生存期为(202.19±4.09)d,比未治疗组[(188.32±3.51)d]延长14 d,差异有统计学意义(x~2=3.917,P=0.04).尼氏染色显示在20周时移植鼠脊髓前角大运动神经元计数多于未治疗鼠;终末期hMSCs移植鼠中,在脑和脊髓前角病变区可检测到人特异性核抗原.结论 hMSCs可经心脏移植,在ALS模型鼠中可长期植入,延长生存期,延缓脊髓前角运动神经元的丢失.     

Introduction of the MASH1 gene into mouse embryonic stem cells leads to differentiation of motoneuron precursors lacking Nogo receptor expression that can be applicable for transplantation to spinal cord injury

ES cells transfected with the MASH1 gene yielded purified spinal motoneuron precursors expressing HB9 and Islet1. The cells lacked the expression of Nogo receptor that was of great advantage for axon growth after transplantation to an injured spinal cord. After transplantation, mice with the complete transection of spinal cord exhibited excellent improvement of the motor functions. Electrophysiological assessment confirmed the quantitative recovery of motor-evoked potential in the transplanted spinal cord. In the grafted spinal cord, gliosis was inhibited and Nogo receptor expression was scarcely detected. The transplanted cells labeled with GFP showed extensive outgrowth of axons positive for neurofilament middle chain, connected to each other and expressed Synaptophysin, Lim1/2 and Islet1. Thus, the in vivo differentiation into mature spinal motoneurons and the reconstitution of neuronal pathways were suggested. The grafted cell population was purified for neurons and was free from teratoma development. These therapeutic strategies may contribute to a potent treatment for spinal cord injury in future.     

Positive effect of transplantation of hNT neurons (NTera 2/D1 cell-line) in a model of familial amyotrophic lateral sclerosis

Transplantation of hNT Neurons derived from the human teratocarcinoma cell-line (NTera2/D1) has been shown to ameliorate motor dysfunction in a number of injury or disease models in which the deficits are fairly localized. However, these cells have not been used before in a model with more extensive neurodegeneration. The aim of this study is to determine the effects of hNT Neuron transplants on motor neuron function in a mouse model of familial amyotrophic lateral sclerosis (FALS) in which there is a substitution of Alanine for Glycine at position 93 of the human SOD1 gene (G93A). Amyotrophic lateral sclerosis is a fatal degenerative motor neuron disease affecting the spinal cord, brainstem, and cortex. This disease clinically manifests as progressive muscular weakness and atrophy, leading to paralysis and death within 3-5 years of diagnosis. The FALS represents 10-13% of all cases. A range of behavioral tests was used to examine spontaneous locomotor activity, coordination, and muscle strength of mice. Long-term (10-11 weeks) transplantation of hNT Neurons into the L(4)-L(5) segments of the ventral horn spinal cord of FALS(G93A) mice at 7 weeks of age (before onset of overt behavioral symptoms of disease) delayed the onset of motor dysfunction for at least 3 weeks. The average lifespan of the transplanted mice was 128 days compared to 106 days for media-injected group. The last mouse in the hNT Neuron transplanted group was euthanized at 135 days of age when it display partial paralysis of the hindlimbs. Immunohistochemical analysis of the implanted spinal cords demonstrated the survival of grafted hNT Neurons and showed many healthy-appearing motor neurons near the implant site. These results suggest that hNT Neuron transplantation may be a promising therapeutic strategy for ALS.     

Brain and spinal cord affected by amyotrophic lateral sclerosis induce differential growth factors expression in rat mesenchymal and neural stem cells

C. Nicaise, D. Mitrecic and R. Pochet (2011) Neuropathology and Applied Neurobiology 37, 179–188
Brain and spinal cord affected by amyotrophic lateral sclerosis induce differential growth factors expression in rat mesenchymal and neural stem cells Stem cell research raises hopes for incurable neurodegenerative diseases. In amyotrophic lateral sclerosis (ALS), affecting the motoneurones of the central nervous system (CNS), stem cell‐based therapy aims to replace dying host motoneurones by transplantation of cells in disease‐affected regions. Moreover, transplanted stem cells can serve as a source of trophic factors providing neuroprotection, slowing down neuronal degeneration and disease progression. Aim: To determine the profile of seven trophic factors expressed by mesenchymal stem cells (MSC) and neural stem cells (NSC) upon stimulation with CNS protein extracts from SOD1‐linked ALS rat model. Methods: Culture of rat MSC, NSC and fibroblasts were incubated with brain and spinal cord extracts from SOD1(G93A) transgenic rats and mRNA expression of seven growth factors was measured by quantitative PCR. Results: MSC, NSC and fibroblasts exhibited different expression patterns. Nerve growth factor and brain‐derived neurotropic factor were significantly upregulated in both NSC and MSC cultures upon stimulation with SOD1(G93A) CNS extracts. Fibroblast growth factor 2, insulin‐like growth factor and glial‐derived neurotropic factor were upregulated in NSC, while the same factors were downregulated in MSC. Vascular endothelial growth factor A upregulation was restricted to MSC and fibroblasts. Surprisingly, SOD1(G93A) spinal cord, but not the brain extract, upregulated brain‐derived neurotropic factor in MSC and glial‐derived neurotropic factor in NSC. Conclusions: These results suggest that inherent characteristics of different stem cell populations define their healing potential and raise the concept of ALS environment in stem cell transplantation.     

Transplantation of neurotrophin-3-transfected bone marrow mesenchymal stem cells for the repair of spinal cord injury

Bone marrow mesenchymal stem cell transplantation has been shown to be therapeutic in the repair of spinal cord injury. However, the low survival rate of transplanted bone marrow mesen- chymal stem cells in vivo remains a problem. Neurotrophin-3 promotes motor neuron survival and it is hypothesized that its transfection can enhance the therapeutic effect. We show that in vitro transfection of neurotrophin-3 gene increases the number of bone marrow mesenchymal stem cells in the region of spinal cord injury. These results indicate that neurotrophin-3 can promote the survival of bone marrow mesenchymal stem cells transplanted into the region of spinal cord injury and potentially enhance the therapeutic effect in the repair of spinal cord injury.     

Acute glial activation by stab injuries does not lead to overt damage or motor neuron degeneration in the G93A mutant SOD1 rat model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease where motor neurons within the brain and spinal cord are lost, leading to paralysis and death. Recently, a correlation between head trauma and the incidence of ALS has been reported. Furthermore, new invasive neurosurgical studies are being planned which involve inserting needles directly to the spinal cord. We therefore tested whether acute trauma to the spinal cord via a knife wound injury would lead to accelerated disease progression in rodent models of ALS (SOD1^G93A rats). A longitudinal stab injury using a small knife was performed within the lumbar spinal cord region of presymptomatic SOD1^G93A rats. Host glial activation was detected in the lumbar area surrounding a micro-knife lesion at 2 weeks after surgery in both wild type and SOD1^G93A animals. However, there was no sign of motor neuron loss in the injured spinal cord of any animal and normal motor function was maintained in the ipsilateral limb. These results indicate that motor neurons in presymptomatic G93A animals are not affected by an invasive puncture wound injury involving reactive astrocytes. Furthermore, acute trauma alone does not accelerate disease onset or progression in this ALS model which is important for future strategies of gene and cell therapies directly targeting the spinal cord of ALS patients.     

Stem cell factor‐activated bone marrow ameliorates amyotrophic lateral sclerosis by promoting protective microglial migration

Amyotrophic lateral sclerosis (ALS) is a progressive disease associated with motor neuron death. Several experimental treatments, including cell therapy using hematopoietic or neuronal stem cells, have been tested in ALS animal models, but therapeutic benefits have been modest. Here we used a new therapeutic strategy, bone marrow transplantation (BMT) with stem cell factor (SCF)‐ or FMS‐like tyrosine kinase 3 (flt3)‐activated bone marrow (BM) cells for the treatment of hSOD1(G93A) transgenic mice. Motor function and survival showed greater improvement in the SCF group than in the group receiving BM cells that had not been activated (BMT alone group), although no improvement was shown in the flt3 group. In addition, larger numbers of BM‐derived cells that expressed the microglia marker Iba1 migrated to the spinal cords of recipient mice compared with the BMT‐alone group. Moreover, after SCF activation, but not flt3 activation or no activation, the migrating microglia expressed glutamate transporter‐1 (GLT‐1). In spinal cords in the SCF group, inflammatory cytokines tumor necrosis factor‐α and interleukin‐1β were suppressed and the neuroprotective molecule insulin‐like growth factor‐1 increased relative to nontreatment hSOD1(G93A) transgenic mice. Therefore, SCF activation changed the character of the migrating donor BM cells, which resulted in neuroprotective effects. These studies have identified SCF‐activated BM cells as a potential new therapeutic agent for the treatment of ALS. © 2014 Wiley Periodicals, Inc.     

Radionuclide imaging of mesenchymal stem cells transplanted into spinal cord

In vivo tracking of stem cells implanted to spinal cord by radionuclide imaging was investigated. The high expression of transferrin receptor on human mesenchymal stem cells (hMSCs) was verified by flow cytometry, radioligand binding and immunofluorescence. Radiolabelled transferrin was chosen as a tracer for scintigraphic imaging of the hMSCs transplanted into spinal cord of rabbits. Comparative experiments with radiolabelled human serum albumin as tracer and PBS as graft as well as ex vivo autoradiography demonstrated the specific uptake of radiolabelled transferrin of hMSCs. hMSCs could be detected in vivo with radiolabelled transferrin targeting at cellular transferrin receptors at an early stage after transplantation into spinal cord.     

Fragmentation of the Golgi apparatus of Betz cells in patients with amyotrophic lateral sclerosis

The Golgi apparatus (GA) of the large pyramidal motor neurons in the cerebral cortex (Betz cells), was examined in sixteen patients with sporadic amyotrophic lateral sclerosis (ALS), in one patient with familial ALS (FALS), and in ten non-ALS age matched controls including one patient with Huntington's disease and one patient with a brain infarct. The GA was immunostained with an antibody against the MG-160 protein, a conserved sialoglycoprotein of the medial cisternae of the organelle. In ALS, 13.2% of counted Betz cells had fragmented GA in contrast to 0.6% in the ten non-ALS controls. The fragmentation of the GA of Betz cells was identical to that previously reported in spinal cord motor neurons from patients with sporadic ALS and in transgenic mice expressing the G93A mutation of the gene encoding the Cu/Zn superoxide dismutase. The striking morphological similarity between the fragmentation of the GA observed in Betz cells and in spinal cord motor neurons suggests that a similar pathogenic mechanism is responsible for both, and that the fragmentation of the GA of the spinal cord motor neurons is not a consequence of deafferentation due to the degeneration of the Betz cells.     

Mesenchymal stem cell transplantation in amyotrophic lateral sclerosis: A Phase I clinical trial

Amyotrophic Lateral Sclerosis (ALS) is a devastating incurable disease. Stem-cell-based therapies represent a new possible strategy for ALS clinical research. The objectives of this Phase 1 clinical study were to assess the feasibility and toxicity of mesenchymal stem cell transplantation and to test the impact of a cell therapy in ALS patients. The trial was approved and monitored by the National Institute of Health and by the Ethics Committees of all participating Institutions. Autologous MSCs were isolated from bone marrow, expanded in vitro and analyzed according to GMP conditions. Expanded MSCs were suspended in the autologous cerebrospinal fluid (CSF) and directly transplanted into the spinal cord at a high thoracic level with a surgical procedure. Ten ALS patients were enrolled and regularly monitored before and after transplantation by clinical, psychological, neuroradiological and neurophysiological assessments. There was no immediate or delayed transplant-related toxicity. Clinical, laboratory, and radiographic evaluations of the patients showed no serious transplant-related adverse events. Magnetic resonance images (MRI) showed no structural changes (including tumor formation) in either the brain or the spinal cord. However the lack of post mortem material prevents any definitive conclusion about the vitality of the MSCs after transplantation. In conclusion, this study confirms that MSC transplantation into the spinal cord of ALS patients is safe and that MSCs might have a clinical use for future ALS cell based clinical trials.     

Combining growth factor and stem cell therapy for amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease where motor neurons within the brain and spinal cord are lost, leading to paralysis and death. Certain growth factors should, in principle, be able to protect dying motor neurons. However, targeted delivery to the spinal cord or brain has been a constant problem. There is also accumulating evidence that glial cells might play a crucial role in maintaining motor neuron function and survival in ALS. Stem cells isolated and expanded in culture can be modified to release growth factors and generate glial cells following transplantation into the spinal cord or brain. As such, they might be able to both detoxify the local environment around dying motor neurons and deliver trophic factors. Here we examine the feasibility of translating these findings into new treatments for ALS patients.     

Transplanted CG4 Cells (an Oligodendrocyte Progenitor Cell Line) Survive, Migrate, and Contribute to Repair of Areas of Demyelination in X-Irradiated and Damaged Spinal Cord but Not in Normal Spinal Cord

In this study, we have examined the behavior of alac-Z-transfected O-2A progenitor cell line, CG4, following transplantation into normal and X-irradiated adult rat spinal cord, and we have also addressed the issue of whether CG4 cells transplanted remotely from ethidium bromide-induced demyelinating lesions in both X-irradiated and nonirradiated spinal cord are able to contribute to their repair. Following transplantation into X-irradiated spinal cord, CG4 cells survive, divide, and migrate extensively. The migration occurs mainly within the parenchymal tissue of the cord without preference for white or gray matter. Moreover, CG4 cells migrating away from their point of introduction are able to enter areas of demyelination and remyelinate the demyelinated axons therein. In contrast, when CG4 cells are transplanted into nonirradiated spinal cord, their survival is limited to areas of damage created by the injection procedure. The CG4 cells do not survive in undamaged, nonirradiated spinal cord. When transplanted remotely from areas of demyelination they are unable to traverse intervening areas of normal white matter, although they may enter lesions if transplanted into their close vicinity. These results have important implications for the development of potential therapeutic strategies for the treatment of multifocal demyelinating disorders that are based on glial cell transplantation.     

A novel cell transplantation protocol and its application to an ALS mouse model

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.     

局部注射骨髓间充质干细胞治疗大鼠脊髓损伤：运动功能有改善吗？

背景：目前研究多为骨髓间充质干细胞的体外培养及细胞移植对颅内疾病的治疗,对植入细胞在损伤脊髓中的成活、分化、迁移、结构重建等了解有限。 目的：探讨局部骨髓间充质干细胞移植在脊髓损伤修复中的作用和骨髓间充质干细胞替代治疗的可行性。 方法：成年健康雌性SD大鼠随机分为细胞移植组和对照组,建立SD大鼠脊髓横断损伤模型,伤后即刻分别向损伤区局部移植大鼠骨髓间充质干细胞悬液或无钙镁磷酸缓冲液。在术前和术后1 d,1周,2周,3周,4周和8周进行BBB评分,观测大鼠的运动功能,并于移植后1周免疫组织化学染色法观察BrdU标记的骨髓间充质干细胞在脊髓损伤处的存活情况,移植后4周进行损伤脊髓的大体观察和组织学检测。 结果与结论：移植后第1~8周细胞移植组BBB评分均髙于对照组;术后1周免疫组织化学染色结果显示在细胞移植组大鼠脊髓远端检测到BrdU阳性细胞,术后4周脊髓损伤处发现有神经纤维。证实通过损伤后立即局部注射的方式将骨髓间充质干细胞移植进大鼠脊髓损伤区,细胞可在损伤区存活;存活的骨髓间充质干细胞可分化为神经元,在损伤局部形成神经元通路,从而促进脊髓神经纤维传导功能的恢复,并促进高位脊髓损伤后大鼠后肢运动功能恢复。     

Propofol injection combined with bone marrow mesenchymal stem cell transplantation better improves electrophysiological function in the hindlimb of rats with spinal cord injury than monotherapy

The repair effects of bone marrow mesenchymal stem cell transplantation on nervous system damage are not satisfactory. Propofol has been shown to protect against spinal cord injury. Therefore, this study sought to explore the therapeutic effects of their combination on spinal cord injury. Rat models of spinal cord injury were established using the weight drop method. Rats were subjected to bone marrow mesenchymal stem cell transplantation via tail vein injection and/or propofol injection via tail vein using an infusion pump. Four weeks after cell transplantation and/or propofol treatment, the cavity within the spinal cord was reduced. The numbers of PKH-26-positive cells and horseradish peroxidase-positive nerve fibers apparently increased in the spinal cord. Latencies of somatosensory evoked potentials and motor evoked potentials in the hindlimb were noticeably shortened, amplitude was increased and hindlimb motor function was obviously improved. Moreover, the combined effects were better than cell transplantation or propofol injection alone. The above data suggest that the combination of propofol injection and bone marrow mesenchymal stem cell transplantation can effectively improve hindlimb electrophysiological function, promote the recovery of motor funtion, and play a neuroprotective role in spinal cord injury in rats.     

Transplantation of in vitro-expanded fetal neural progenitor cells results in neurogenesis and functional recovery after spinal cord contusion injury in adult rats

Neural progenitor cells, including neural stem cells, are a potential expandable source of graft material for transplantation aimed at repairing the damaged CNS. Here we present the first evidence that in vitro-expanded fetus-derived neurosphere cells were able to generate neurons in vivo and improve motor function upon transplantation into an adult rat spinal-cord-contusion injury model. As the source of graft material, we used a neural stem cell-enriched population that was derived from rat embryonic spinal cord (E14.5) and expanded in vitro by neurosphere formation. Nine days after contusion injury, these neurosphere cells were transplanted into adult rat spinal cord at the injury site. Histological analysis 5 weeks after the transplantation showed that mitotic neurogenesis occurred from the transplanted donor progenitor cells within the adult rat spinal cord, a nonneurogenic region; that these donor-derived neurons extended their processes into the host tissues; and that the neurites formed synaptic structures. Furthermore, analysis of motor behavior using a skilled reaching task indicated that the treated rats showed functional recovery. These results indicate that in vitro-expanded neurosphere cells derived from the fetal spinal cord are a potential source for transplantable material for treatment of spinal cord injury.