Transplantation of neural stem cells for spinal cord injury]

Neural progenitor cells, including neural stem cells (NSCs), are an important potential graft material for cell therapeutics of damaged spinal cord. Here we used as a source of graft material a NSC-enriched population derived from human fetal spinal cord (Embryonic week 8-9) and expanded in vitro by neurosphere formation. NSCs labeled with BrdU (TP) or culture medium (CON) were transplanted into the adult marmoset spinal cord after contusion injury at C5 level. Grafted NSCs survived and migrated up to 7 mm far from the lesion epicenter. Double-staining with TuJ1 for neuron, GFAP for astrocyte, or CNPase for oligodendrocyte and BrdU revealed that grafted NSCs differentiated into neurons and oligodendrocytes 8 weeks after transplantation. More neurofilaments were observed in TP than those of CON. Furthermore, behavioral assessment of forelimb muscle strength using bar grip test and amount of spontaneous motor activity using infrared-rays monitoring revealed that the grafted NSCs significantly increased both of them compared to those of CON. These results indicate that in vitro expanded NSCs derived from human fetal spinal cord are useful sources for the therapeutics of spinal cord injury in primates.     

Transplantation of human neural stem cells for spinal cord injury in primates

Recent studies have shown that delayed transplantation of neural stem/progenitor cells (NSPCs) into the injured spinal cord can promote functional recovery in adult rats. Preclinical studies using nonhuman primates, however, are necessary before NSPCs can be used in clinical trials to treat human patients with spinal cord injury (SCI). Cervical contusion SCIs were induced in 10 adult common marmosets using a stereotaxic device. Nine days after injury, in vitro-expanded human NSPCs were transplanted into the spinal cord of five randomly selected animals, and the other sham-operated control animals received culture medium alone. Motor functions were evaluated through measurements of bar grip power and spontaneous motor activity, and temporal changes in the intramedullary signals were monitored by magnetic resonance imaging. Eight weeks after transplantation, all animals were sacrificed. Histologic analysis revealed that the grafted human NSPCs survived and differentiated into neurons, astrocytes, and oligodendrocytes, and that the cavities were smaller than those in sham-operated control animals. The bar grip power and the spontaneous motor activity of the transplanted animals were significantly higher than those of sham-operated control animals. These findings show that NSPC transplantation was effective for SCI in primates and suggest that human NSPC transplantation could be a feasible treatment for human SCI.     

Transplantation of embryonic spinal cord-derived neurospheres support growth of supraspinal projections and functional recovery after spinal cord injury in the neonatal rat

Great interest exists in using cell replacement strategies to repair the damaged central nervous system. Previous studies have shown that grafting rat fetal spinal cord into neonate or adult animals after spinal cord injury leads to improved anatomic growth/plasticity and functional recovery. It is clear that fetal tissue transplants serve as a scaffold for host axon growth. In addition, embryonic Day 14 (E14) spinal cord tissue transplants are also a rich source of neural-restricted and glial-restricted progenitors. To evaluate the potential of E14 spinal cord progenitor cells, we used in vitro-expanded neurospheres derived from embryonic rat spinal cord and showed that these cells grafted into lesioned neonatal rat spinal cord can survive, migrate, and differentiate into neurons and oligodendrocytes, but rarely into astrocytes. Synapses and partially myelinated axons were detected within the transplant lesion area. Transplanted progenitor cells resulted in increased plasticity or regeneration of corticospinal and brainstem-spinal fibers as determined by anterograde and retrograde labeling. Furthermore, transplantation of these cells promoted functional recovery of locomotion and reflex responses. These data demonstrate that progenitor cells when transplanted into neonates can function in a similar capacity as transplants of solid fetal spinal cord tissue.     

Neural stem cell, as a source of graft material for transplantation in neuronal disease

Self-renewing and multipotent neural stem cells are present in the adult human brain. We successfully harvested neural stem cells from mice and humans using misexpressed EGFP proteins under the control of the nestin second intron enhancer. High-level EGFP expressors derived from mouse embryos included a distinct subpopulation of cells that were self-renewable and multipotent. Further, we obtained that neural progenitor cells from rat fetal spinal cords using a neurosphere technique, and demonstrated their ability to divide and differentiate into neurons in vivo, where they were integrated into the host tissue in the injured rat spinal cord with resultant behavioral improvement of the recipient rat. We also harvested tyrosine hydroxylase-positive neurons from a transgenic mouse expressing GFP under the control of the tyrosine hydroxylase promoter, and successfully transplanted them into the striatum of rats with parkinsonism with marked improvement of the neurological symptoms. Since neural stem cells can adapt well in the host CNS, studies should focus on their application as a vector in gene therapy and on the introduction in vivo or ex vivo of genes to control their proliferation and differentiation. Neural stem cells are a potential, useful source for developing new therapy for CNS disorders.     

Multipotent embryonic spinal cord stem cells expanded by endothelial factors and Shh/RA promote functional recovery after spinal cord injury

Cell transplantation is a promising way to treat spinal cord injury and neurodegenerative disorders. Neural stem cells taken from the embryonic spinal cord are an appealing source of cells for transplantation because these cells are committed to making spinal cord progeny. However these stem cells are rare and require expansion in tissue culture to generate sufficient cells for transplantation. We have developed a novel method for expanding embryonic mouse spinal cord stem cells using a co-culture system with endothelial cells. This method improves neural stem cell survival and preserves their multipotency, including their ability to make motor neurons. Transplantation of endothelial-expanded neural stem cells that were treated with sonic hedgehog(Shh) and retinoic acid (RA) during the expansion phase, into an adult mouse SCI model resulted in significant recovery of sensory and motor function.     

Transplantation of neural cells derived from retinoic acid-treated cynomolgus monkey embryonic stem cells successfully improved motor function of hemiplegic mice with experimental brain injury

We induced neural cells by treating cynomolgus monkey embryonic stem (ES) cells with retinoic acid. The treated cells mainly expressed betaIIItubulin. They further differentiated into neurons expressing neurofilament middle chain (NFM) in elongated axons. Half of the cells differentiated into Islet1+ motoneurons in vitro. The monkey ES-derived neural cells were transplanted to hemiplegic mice with experimental brain injury mimicking stroke. The neural cells that had grafted into periventricular area of the mice distributed extensively over the injured cortex. Some of the transplanted cells expressed the neural stem/progenitor marker nestin 2 days after transplantation. The cells expressed markers characteristic of mature motoneurons 28 days after transplantation. Mice with the neural cell graft gradually recovered motor function, whereas control animals remained hemiplegic. This is the first demonstration that neural cells derived from nonhuman primate ES cells have the ability to restore motor function in an animal model of brain injury.     

Transplantation of Human Neural Precursor Cells Reverses Syrinx Growth in a Rat Model of Post-Traumatic Syringomyelia

Posttraumatic syringomyelia (PTS) is a serious condition of progressive expansion of spinal cord cysts, affecting patients with spinal cord injury years after injury. To evaluate neural cell therapy to prevent cyst expansion and potentially replace lost neurons, we developed a rat model of PTS. We combined contusive trauma with subarachnoid injections of blood, causing tethering of the spinal cord to the surrounding vertebrae, resulting in chronically expanding cysts. The cysts were usually located rostral to the injury, extracanalicular, lined by astrocytes. T2*-weighted magnetic resonance imaging (MRI) showed hyperintense fluid-filled cysts but also hypointense signals from debris and iron-laden macrophages/microglia. Two types of human neural stem/progenitor cells—fetal neural precursor cells (hNPCs) and neuroepithelial-like stem cells (hNESCs) derived from induced pluripotent stem cells—were transplanted to PTS cysts. Cells transplanted into cysts 10 weeks after injury survived at least 10 weeks, migrated into the surrounding parenchyma, but did not differentiate during this period. The cysts were partially obliterated by the cells, and cyst walls often merged with thin layers of cells in between. Cyst volume measurements with MRI showed that the volumes continued to expand in sham-transplanted rats by 102%, while the cyst expansion was effectively prevented by hNPCs and hNESCs transplantation, reducing the cyst volumes by 18.8% and 46.8%, respectively. The volume reductions far exceeded the volume of the added human cells. Thus, in an animal model closely mimicking the clinical situation, we provide proof-of-principle that transplantation of human neural stem/progenitor cells can be used as treatment for PTS.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13311-020-00987-3.Key Words: Spinal cord injury, Syringomyelia, Animal model, Transplantation, Human neural stem/progenitor cells     

Induced pluripotent stem cell-derived neural stem cell therapies for spinal cord injury

The greatest challenge to successful treatment of spinal cord injury is the limited regenerative capacity of the central nervous system and its inability to replace lost neurons and severed axons following injury. Neural stem cell grafts derived from fetal central nervous system tissue or embryonic stem cells have shown therapeutic promise by differentiation into neurons and glia that have the potential to form functional neuronal relays across injured spinal cord segments. However, implementation of fetal-derived or embryonic stem cell-derived neural stem cell therapies for patients with spinal cord injury raises ethical concerns. Induced pluripotent stem cells can be generated from adult somatic cells and differentiated into neural stem cells suitable for therapeutic use, thereby providing an ethical source of implantable cells that can be made in an autologous fashion to avoid problems of immune rejection. This review discusses the therapeutic potential of human induced pluripotent stem cell-derived neural stem cell transplantation for treatment of spinal cord injury, as well as addressing potential mechanisms, future perspectives and challenges.     

Lavendustin A enhances axon elongation in VHL gene-transfected neural stem cells

Axonal elongation is necessary for neuronal regeneration of the spinal cord after spinal injury. Recently neural stem cells have been proposed as hopeful graft donors for regeneration of the central nervous system. However, most grafted stem cells are not able to differentiate into neurons, and grafted stem cells cannot usually grow axons. Here, we show the effect of the protein tyrosine kinase inhibitor lavendustin A on axonal growth of neurons differentiated from neural stem cells obtained from adult rat hippocampal cells transfected with the von Hippel-Lindau (VHL) gene. Significantly greater axonal outgrowth was observed for the transfected cells treated with the inhibitor than for those not so treated. Thus, protein-tyrosine kinase inhibition is effective for axonal outgrowth of neurons differentiated from neural stem cells and may prove to be useful for neuronal regeneration via transplanted stem cells, particularly in the case of spinal cord injuries.     

Transplantation of placenta-derived mesenchymal stem cell-induced neural stem cells to treat spinal cord injury

Because of their strong proliferative capacity and multi-potency, placenta-derived mesenchymal stem cells have gained interest as a cell source in the field of nerve damage repair. In the present study, human placenta-derived mesenchymal stem ceils were induced to differentiate into neural stem cells, which were then transplanted into the spinal cord after local spinal cord injury in rats. The motor functional recovery and pathological changes in the injured spinal cord were observed for 3 successive weeks. The results showed that human placenta-derived mesenchymal stem cells can differentiate into neuron-like cells and that induced neural stem cells contribute to the restoration of injured spinal cord without causing transplant rejection. Thus, these cells promote the recovery of motor and sensory functions in a rat model of spinal cord injury. Therefore, human placenta-derived mesenchymal stem cells may be useful as seed cells during the repair of spinal cord injury.     

Motor neuron replacement therapy for amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis is a motor neuron degenerative disease that is also known as Lou Gehrig’s disease in the United States,Charcot’s disease in France,and motor neuron disease in the UK.The loss of motor neurons causes muscle wasting,paralysis,and eventually death,which is commonly related to respiratory failure,within 3-5 years after onset of the disease.Although there are a limited number of drugs approved for amyotrophic lateral sclerosis,they have had little success at treating the associated symptoms,and they cannot reverse the course of motor neuron degeneration.Thus,there is still a lack of effective treatment for this debilitating neurodegenerative disorder.Stem cell therapy for amyotrophic lateral sclerosis is a very attractive strategy for both basic and clinical researchers,particularly as transplanted stem cells and stem cell-derived neural progenitor/precursor cells can protect endogenous motor neurons and directly replace the lost or dying motor neurons.Stem cell therapies may also be able to re-establish the motor control of voluntary muscles.Here,we review the recent progress in the use of neural stem cells and neural progenitor cells for the treatment of amyotrophic lateral sclerosis.We focus on MN progenitor cells derived from fetal central nervous system tissue,embryonic stem cells,and induced pluripotent stem cells.In our recent studies,we found that transplanted human induced pluripotent stem cell-derived motor neuron progenitors survive well,differentiate into motor neurons,and extend axons into the host white matter,not only in the rostrocaudal direction,but also along motor axon tracts towards the ventral roots in the immunodeficient rat spinal cord.Furthermore,the significant motor axonal extension after neural progenitor cell transplantation in amyotrophic lateral sclerosis models demonstrates that motor neuron replacement therapy could be a promising therapeutic strategy for amyotrophic lateral sclerosis,particularly as a variety of stem cell derivatives,including induced pluripotent stem cells,are being considered for clinical trials for various diseases.     

Treatment of spinal cord injury by transplantation of fetal neural precursor cells engineered to express BMP inhibitor

Spontaneous recovery after spinal cord injury is limited. Transplantation of neural precursor cells (NPCs) into lesioned adult rat spinal cord results in only partial functional recovery, and most transplanted cells tend to differentiate predominantly into astrocytes. In order to improve functional recovery after transplantation, it is important that transplanted neural precursor cells appropriately differentiate into cell lineages required for spinal cord regeneration. In order to modulate the fate of transplanted cells, we advocate transplanting gene-modified neural precursor cells. We demonstrate that gene modification to inhibit bone morphogenetic protein (BMP) signaling by noggin expression promoted differentiation of neural precursor cells into neurons and oligodendrocytes, in addition to astrocytes after transplantation. Furthermore, functional recovery of the recipient mice with spinal cord injury was observed when noggin-expressing neural precursor cells were transplanted. These observations suggest that gene-modified neural precursor cells that express molecules involved in cell fate modulation could improve central nervous system (CNS) regeneration.     

Adult neural progenitor cell grafts survive after acute spinal cord injury and integrate along axonal pathways

The main rationale for cell-based therapies following spinal cord injury are: (i) replacement of degenerated spinal cord parenchyma by an axon growth supporting scaffold; (ii) remyelination of regenerating axons; and (iii), local delivery of growth promoting molecules. A potential source to meet these requirements is adult neural progenitor cells, which were examined in the present study. Fibroblast growth factor 2-responsive adult spinal cord-derived syngenic neural progenitor cells were either genetically modified in vitro to express green fluorescent protein (GFP) using retroviral vectors or prelabelled with bromodeoxyuridine (BrdU). Neural progenitor cells revealed antigenic properties of neurons and glial cells in vitro confirming their multipotency. This differentiation pattern was unaffected by retroviral transduction. GFP-expressing or BrdU-prelabelled neural progenitor cells were grafted as neurospheres directly into the acutely injured rat cervical spinal cord. Animals with lesions only served as controls. Three weeks postoperatively, grafted neural progenitor cells integrated along axonal profiles surrounding the lesion site. In contrast to observations in culture, grafted neural progenitor cells differentiated only into astro- and oligodendroglial lineages, supporting the notion that the adult spinal cord provides molecular cues for glial, but not for neuronal, differentiation. This study demonstrates that adult neural progenitor cells will survive after transplantation into the acutely injured spinal cord. The observed oligodendroglial and astroglial differentiation and integration along axonal pathways represent important prerequisites for potential remyelination and support of axonal regrowth.     

Transplanted hematopoietic stem cells from bone marrow differentiate into neural lineage cells and promote functional recovery after spinal cord injury in mice

Recovery in central nervous system disorders is hindered by the limited ability of the vertebrate central nervous system to regenerate lost cells, replace damaged myelin, and re-establish functional neural connections. Cell transplantation to repair central nervous system disorders is an active area of research, with the goal of reducing functional deficits. Recent animal studies showed that cells of the hematopoietic stem cell (HSC) fraction of bone marrow transdifferentiated into various nonhematopoietic cell lineages. We employed a mouse model of spinal cord injury and directly transplanted HSCs into the spinal cord 1 week after injury. We evaluated functional recovery using the hindlimb motor function score weekly for 5 weeks after transplantation. The data demonstrated a significant improvement in the functional outcome of mice transplanted with hematopoietic stem cells compared with control mice in which only medium was injected. Fluorescent in situ hybridization for the Y chromosome and double immunohistochemistry showed that transplanted cells survived 5 weeks after transplantation and expressed specific markers for astrocytes, oligodendrocytes, and neural precursors, but not for neurons. These results suggest that transplantation of HSCs from bone marrow is an effective strategy for the treatment of spinal cord injury.     

Differentiation of engrafted neuronal-restricted precursor cells is inhibited in the traumatically injured spinal cord

Differentiation of pluripotent neural stem cells engrafted into the adult normal and injured spinal cord is restricted to the glial lineage, suggesting that in vitro induction toward a neuronal lineage prior to transplantation and/or modification of the host environment may be necessary to initiate and increase the differentiation of neurons. In the present study, we investigated the differentiation of neuronal-restricted precursors (NRPs) grafted into the normal and contused adult rat spinal cord. NRPs proliferated through multiple passages in the presence of FGF2 and NT3 and differentiated into only neurons in vitro in the presence of retinoic acid and the absence of FGF2. Differentiated NRPs expressed GABA, glycine, glutamate, and ChAT. Two weeks to 2 months after engraftment of undifferentiated NRPs into adult normal spinal cord, large numbers of surviving cells were seen in all of the animals. The majority differentiated into betaIII-tubulin-positive neurons. Some transplanted NRPs expressed GABA and small numbers were glutamate- and ChAT-positive. NRPs were also transplanted into the epicenter of the contused adult rat spinal cord. Two weeks to 2 months after transplantation, some engrafted NRPs remained undifferentiated nestin-positive cells. Small numbers were MAP2- or betaIII-tubulin-positive neurons. However, the expression of GABA, glutamate, or ChAT was not observed. These results show that NRPs can differentiate into different types of neurons in the normal adult rat spinal cord, but that such differentiation is inhibited in the injured spinal cord. Manipulation of the microenvironment in the injured spinal cord will likely be necessary to facilitate neuronal replacement.     

Transplanting neural progenitors into a complete transection model of spinal cord injury

Neural progenitor cell (NPC) transplantation is a promising therapeutic strategy for spinal cord injury (SCI) because of the potential for cell replacement and restoration of connectivity. Our previous studies have shown that transplants of NPC, composed of neuron‐ and glia‐restricted progenitors derived from the embryonic spinal cord, survived well in partial lesion models and generated graft‐derived neurons, which could be used to form a functional relay. We have now examined the properties of a similar NPC transplant using a complete transection model in juvenile and adult rats. We found poor survival of grafted cells despite using a variety of lesion methods, matrices, and delays of transplantation. If, instead of cultured progenitor cells, the transplants were composed of segmental or dissociated segments of fetal spinal cord (FSC) derived from similar‐staged embryos, grafted cells survived and integrated well with host tissue in juvenile and adult rats. FSC transplants differentiated into neurons and glial cells, including astrocytes and oligodendrocytes. Graft‐derived neurons expressed glutaminergic and GABAergic markers. Grafted cells also migrated and extended processes into host tissue. Analysis of axon growth from the host spinal cord showed serotonin‐positive fibers and biotinylated dextran amine‐traced propriospinal axons growing into the transplants. These results suggest that in treating severe SCI, such as complete transection, NPC grafting faces major challenges related to cell survival and formation of a functional relay. Lessons learned from the efficacy of FSC transplants could be used to develop a therapeutic strategy based on neural progenitor cells for severe SCI. © 2014 Wiley Periodicals, Inc.     

Transplantation of erythropoietin gene-modified neural stem cells improves the repair of injured spinal cord

The protective effects of erythropoietin on spinal cord injury have not been well described. Here, the eukaryotic expression plasmid pc DNA3.1 human erythropoietin was transfected into rat neural stem cells cultured in vitro. A rat model of spinal cord injury was established using a free falling object. In the human erythropoietin-neural stem cells group, transfected neural stem cells were injected into the rat subarachnoid cavity, while the neural stem cells group was injected with non-transfected neural stem cells. Dulbecco's modified Eagle's medium/F12 medium was injected into the rats in the spinal cord injury group as a control. At 1–4 weeks post injury, the motor function in the rat lower limbs was best in the human erythropoietin-neural stem cells group, followed by the neural stem cells group, and lastly the spinal cord injury group. At 72 hours, compared with the spinal cord injury group, the apoptotic index and Caspase-3 gene and protein expressions were apparently decreased, and the bcl-2 gene and protein expressions were noticeably increased, in the tissues surrounding the injured region in the human erythropoietin-neural stem cells group. At 4 weeks, the cavities were clearly smaller and the motor and somatosensory evoked potential latencies were remarkably shorter in the human erythropoietin-neural stem cells group and neural stem cells group than those in the spinal cord injury group. These differences were particularly obvious in the human erythropoietin-neural stem cells group. More CM-Dil-positive cells and horseradish peroxidase-positive nerve fibers and larger amplitude motor and somatosensory evoked potentials were found in the human erythropoietin-neural stem cells group and neural stem cells group than in the spinal cord injury group. Again, these differences were particularly obvious in the human erythropoietin-neural stem cells group. These data indicate that transplantation of erythropoietin gene-modified neural stem cells into the subarachnoid cavity to help repair spinal cord injury and promote the recovery of spinal cord function better than neural stem cell transplantation alone. These findings may lead to significant improvements in the clinical treatment of spinal cord injuries.     

Optimal time for subarachnoid transplantation of neural progenitor cells in the treatment of contusive spinal cord injury

This study aimed to identify the optimal neural progenitor cell transplantation time for spinal cord injury in rats via the subarachnoid space. Cultured neural progenitor cells from 14-day embryonic rats, constitutively expressing enhanced green fluorescence protein, or media alone, were injected into the subarachnoid space of adult rats at 1 hour (acute stage), 7 days (subacute stage) and 28 days (chronic stage) after contusive spinal cord injury. Results showed that grafted neural progenitor cells migrated and aggregated around the blood vessels of the injured region, and infiltrated the spinal cord parenchyma along the tissue spaces in the acute stage transplantation group. However, this was not observed in subacute and chronic stage transplantation groups. O4- and glial fibrillary acidic protein-positive cells, representing oligodendrocytes and astrocytes respectively, were detected in the core of the grafted cluster attached to the cauda equina pia surface in the chronic stage transplantation group 8 weeks after transplantation. Both acute and subacute stage transplantation groups were negative for O4 and glial fibrillary acidic protein cells. Basso, Beattie and Bresnahan scale score comparisons indicated that rat hind limb locomotor activity showed better recovery after acute stage transplantation than after subacute and chronic transplantation. Our experimental findings suggest that the subarachnoid route could be useful for transplantation of neural progenitor cells at the acute stage of spinal cord injury. Although grafted cells survived only for a short time and did not differentiate into astrocytes or neurons, they were able to reach the parenchyma of the injured spinal cord and improve neurological function in rats. Transplantation efficacy was enhanced at the acute stage in comparison with subacute and chronic stages.     

神经干细胞移植修复鼠脊髓损伤的实验研究

目的 :观察神经干细胞移植治疗对鼠脊髓损伤后神经结构修复和功能恢复的作用并探讨其作用机制。方法 :制备鼠T10 脊髓损伤模型 ,体外培养、诱导鼠神经干细胞 ,定量评价神经干细胞移植对脊髓损伤后神经结构修复和功能恢复的影响。结果 :与对照组相比 ,神经干细胞移植组明显的增强了GAP 43mRNA的表达 ,促进了脊髓ChAT阳性的运动神经元的再生、结构的修复和下肢运动功能的恢复。结论 :神经干细胞移植促进了脊髓损伤后神经结构的修复和功能的恢复 ,是急性脊髓损伤一种有效的治疗方案