Spinal parenchymal occupation by neural stem cells after subpial delivery in adult immunodeficient rats.
ABSTRACT: Neural precursor cells (NSCs) hold great potential to treat a variety of neurodegenerative diseases and injuries to the spinal cord. However, current delivery techniques require an invasive approach in which an injection needle is advanced into the spinal parenchyma to deliver cells of interest. As such, this approach is associated with an inherent risk of spinal injury, as well as a limited delivery of cells into multiple spinal segments. Here, we characterize the use of a novel cell delivery technique that employs single bolus cell injections into the spinal subpial space. In immunodeficient rats, two subpial injections of human NSCs were performed in the cervical and lumbar spinal cord, respectively. The survival, distribution, and phenotype of transplanted cells were assessed 6-8 months after injection. Immunofluorescence staining and mRNA sequencing analysis demonstrated a near-complete occupation of the spinal cord by injected cells, in which transplanted human NSCs (hNSCs) preferentially acquired glial phenotypes, expressing oligodendrocyte (Olig2, APC) or astrocyte (GFAP) markers. In the outermost layer of the spinal cord, injected hNSCs differentiated into glia limitans-forming astrocytes and expressed human-specific superoxide dismutase and laminin. All animals showed normal neurological function for the duration of the analysis. These data show that the subpial cell delivery technique is highly effective in populating the entire spinal cord with injected NSCs, and has a potential for clinical use in cell replacement therapies for the treatment of ALS, multiple sclerosis, or spinal cord injury.
Project description:Effective in vivo use of adeno-associated virus (AAV)-based vectors to achieve gene-specific silencing or upregulation in the central nervous system has been limited by the inability to provide more than limited deep parenchymal expression in adult animals using delivery routes with the most clinical relevance (intravenous or intrathecal). Here, we demonstrate that the spinal pia membrane represents the primary barrier limiting effective AAV9 penetration into the spinal parenchyma after intrathecal AAV9 delivery. We develop a novel subpial AAV9 delivery technique and AAV9-dextran formulation. We use these in adult rats and pigs to show (i) potent spinal parenchymal transgene expression in white and gray matter including neurons, glial and endothelial cells after single bolus subpial AAV9 delivery; (ii) delivery to almost all apparent descending motor axons throughout the length of the spinal cord after cervical or thoracic subpial AAV9 injection; (iii) potent retrograde transgene expression in brain motor centers (motor cortex and brain stem); and (iv) the relative safety of this approach by defining normal neurological function for up to 6 months after AAV9 delivery. Thus, subpial delivery of AAV9 enables gene-based therapies with a wide range of potential experimental and clinical utilizations in adult animals and human patients.
Project description:In the adult brain, neural stem cells (NSC) must migrate to express their neuroplastic potential. The addition of recombinant reelin to human NSC (HNSC) cultures facilitates neuronal retraction in the neurospheroid. Because we detected reelin, alpha3-integrin receptor subunits, and disabled-1 immunoreactivity in HNSC cultures, it is possible that integrin-mediated reelin signal transduction is operative in these cultures. To investigate whether reelin is important in the regulation of NSC migration, we injected HNSCs into the lateral ventricle of null reeler and wild-type mice. Four weeks after transplantation, we detected symmetrical migration and extensive neuronal and glial differentiation of transplanted HNSCs in wild-type, but not in reeler mice. In reeler mice, most of the injected HNSCs failed to migrate or to display the typical differentiation pattern. However, a subpopulation of transplanted HNSCs expressing reelin did show a pattern of chain migration in the reeler mouse cortex. We also analyzed the endogenous NSC population in the reeler mouse using bromodeoxyuridine injections. In reeler mice, the endogenous NSC population in the hippocampus and olfactory bulb was significantly reduced compared with wild-type mice; in contrast, endogenous NSCs expressed in the subventricular zonewere preserved. Hence, it seems likely that the lack of endogenous reelin may have disrupted the migration of the NSCs that had proliferated in the SVZ. We suggest that a possible inhibition of NSC migration in psychiatric patients with a reelin deficit may be a potential problem in successful NSC transplantation in these patients.
Project description:AIMS:Neural stem cells (NSCs) in the adult mammalian spinal cord are activated in response to spinal cord injury (SCI); however, mechanisms modulating this process are not clear. Here, we noticed SCI elevated expression of vascular endothelial growth factor (VEGF) and we aimed to validate the roles of VEGF in NSCs activation after SCI and investigated the related signals during the process. METHODS:In vitro we detected whether VEGF promoted spinal cord NSCs proliferation and investigated the involved signals; In vivo, we injected VEGF into rat spinal cord to check the NSCs activation. RESULTS:In vitro, VEGF triggered spinal cord NSCs proliferation and maintained self-renewal. Further investigations demonstrated VEGF transactivated epidermal growth factor receptor (EGFR) through VEGF receptor 2 (VEGFR2) to promote spinal cord NSCs proliferation. In vivo, we injected VEGF into spinal cord by laminectomy to confirm the roles of VEGF-VEGFR2-EGFR signals in NSCs activation. VEGF significantly elevated the number of activated NSCs and increased EGFR phosphorylation. In contrast, intraspinal injection of specific inhibitors targeting EGFR and VEGFR2 decreased NSCs activation after SCI. Our results demonstrate that VEGF-VEGFR2-EGFR axis is important for NSCs activation after SCI, providing new insights into the mechanisms of spinal cord NSCs activation postinjury.
Project description:Survival and migration of transplanted neural stem cells (NSCs) are prerequisites for therapeutic benefits in spinal cord injury. We have shown that survival of NSC grafts declines after transplantation into the injured spinal cord, and that combining treadmill training (TMT) enhances NSC survival via insulin-like growth factor-1 (IGF-1). Here, we aimed to obtain genetic evidence that IGF-1 signaling in the transplanted NSCs determines the beneficial effects of TMT. We transplanted NSCs heterozygous (+/-) for Igf1r, the gene encoding IGF-1 receptor, into the mouse spinal cord after injury, with or without combining TMT. We analyzed the influence of genotype and TMT on locomotor recovery and survival and migration of NSC grafts. In vitro experiments were performed to examine the potential roles of IGF-1 signaling in the migratory ability of NSCs. Mice receiving +/- NSC grafts showed impaired locomotor recovery compared with those receiving wild-type (+/+) NSCs. Locomotor improvement by TMT was more pronounced with +/+ grafts. Deficiency of one allele of Igf1r significantly reduced survival and migration of the transplanted NSCs. Although TMT did not significantly influence NSC survival, it substantially enhanced the extent of migration for only +/+ NSCs. Cultured neurospheres exhibited dynamic motility with cytoplasmic protrusions, which was regulated by IGF-1 signaling. IGF-1 signaling in transplanted NSCs may be essential in regulating their survival and migration. Furthermore, TMT may promote NSC graft-mediated locomotor recovery via activation of IGF-1 signaling in transplanted NSCs. Dynamic NSC motility via IGF-1 signaling may be the cellular basis for the TMT-induced enhancement of migration.
Project description:Many studies have shown that bio-scaffolds have important value for promoting axonal regeneration of injured spinal cord. Indeed, cell transplantation and bio-scaffold implantation are considered to be effective methods for neural regeneration. This study was designed to fabricate a type of three-dimensional collagen/silk fibroin scaffold (3D-CF) with cavities that simulate the anatomy of normal spinal cord. This scaffold allows cell growth in vitro and in vivo. To observe the effects of combined transplantation of neural stem cells (NSCs) and 3D-CF on the repair of spinal cord injury. Forty Sprague-Dawley rats were divided into four groups: sham (only laminectomy was performed), spinal cord injury (transection injury of T10 spinal cord without any transplantation), 3D-CF (3D scaffold was transplanted into the local injured cavity), and 3D-CF + NSCs (3D scaffold co-cultured with NSCs was transplanted into the local injured cavity. Neuroelectrophysiology, imaging, hematoxylin-eosin staining, argentaffin staining, immunofluorescence staining, and western blot assay were performed. Apart from the sham group, neurological scores were significantly higher in the 3D-CF + NSCs group compared with other groups. Moreover, latency of the 3D-CF + NSCs group was significantly reduced, while the amplitude was significantly increased in motor evoked potential tests. The results of magnetic resonance imaging and diffusion tensor imaging showed that both spinal cord continuity and the filling of injury cavity were the best in the 3D-CF + NSCs group. Moreover, regenerative axons were abundant and glial scarring was reduced in the 3D-CF + NSCs group compared with other groups. These results confirm that implantation of 3D-CF combined with NSCs can promote the repair of injured spinal cord. This study was approved by the Institutional Animal Care and Use Committee of People's Armed Police Force Medical Center in 2017 (approval No. 2017-0007.2).
Project description:<h4>Background</h4>Mutation in the ubiquitously expressed cytoplasmic superoxide dismutase (SOD1) causes an inherited form of Amyotrophic Lateral Sclerosis (ALS). Mutant synthesis in motor neurons drives disease onset and early disease progression. Previous experimental studies have shown that spinal grafting of human fetal spinal neural stem cells (hNSCs) into the lumbar spinal cord of SOD1(G93A) rats leads to a moderate therapeutical effect as evidenced by local ?-motoneuron sparing and extension of lifespan. The aim of the present study was to analyze the degree of therapeutical effect of hNSCs once grafted into the lumbar spinal ventral horn in presymptomatic immunosuppressed SOD1(G93A) rats and to assess the presence and functional integrity of the descending motor system in symptomatic SOD1(G93A) animals.<h4>Methods/principal findings</h4>Presymptomatic SOD1(G93A) rats (60-65 days old) received spinal lumbar injections of hNSCs. After cell grafting, disease onset, disease progression and lifespan were analyzed. In separate symptomatic SOD1(G93A) rats, the presence and functional conductivity of descending motor tracts (corticospinal and rubrospinal) was analyzed by spinal surface recording electrodes after electrical stimulation of the motor cortex. Silver impregnation of lumbar spinal cord sections and descending motor axon counting in plastic spinal cord sections were used to validate morphologically the integrity of descending motor tracts. Grafting of hNSCs into the lumbar spinal cord of SOD1(G93A) rats protected ?-motoneurons in the vicinity of grafted cells, provided transient functional improvement, but offered no protection to ?-motoneuron pools distant from grafted lumbar segments. Analysis of motor-evoked potentials recorded from the thoracic spinal cord of symptomatic SOD1(G93A) rats showed a near complete loss of descending motor tract conduction, corresponding to a significant (50-65%) loss of large caliber descending motor axons.<h4>Conclusions/significance</h4>These data demonstrate that in order to achieve a more clinically-adequate treatment, cell-replacement/gene therapy strategies will likely require both spinal and supraspinal targets.
Project description:Neural stem cell (NSC) transplantation is a major focus of current research for treatment of spinal cord injury (SCI). However, it is very important to promote the survival and differentiation of NSCs into myelinating oligodendrocytes (OLs). In this study, myelin basic protein-activated T (MBP-T) cells were passively immunized to improve the SCI microenvironment. Olig2-overexpressing NSCs were infected with a lentivirus carrying the enhanced green fluorescent protein (GFP) reporter gene to generate Olig2-GFP-NSCs that were transplanted into the injured site to differentiate into OLs. Transferred MBP-T cells infiltrated the injured spinal cord, produced neurotrophic factors, and induced the differentiation of resident microglia and/or infiltrating blood monocytes into an "alternatively activated" anti-inflammatory macrophage phenotype by producing interleukin-13. As a result, the survival of transplanted NSCs increased fivefold in MBP-T cell-transferred rats compared with that of the vehicle-treated control. In addition, the differentiation of MBP-positive OLs increased 12-fold in Olig2-GFP-NSC-transplanted rats compared with that of GFP-NSC-transplanted controls. In the MBP-T cell and Olig2-GFP-NSC combined group, the number of OL-remyelinated axons significantly increased compared with those of all other groups. However, a significant decrease in spinal cord lesion volume and an increase in spared myelin and behavioral recovery were observed in Olig2-NSC- and NSC-transplanted MBP-T cell groups. Collectively, these results suggest that MBP-T cell adoptive immunotherapy combined with NSC transplantation has a synergistic effect on histological and behavioral improvement after traumatic SCI. Although Olig2 overexpression enhances OL differentiation and myelination, the effect on functional recovery may be surpassed by MBP-T cells.
Project description:BACKGROUND:A well-characterized method has not yet been established to reproducibly, efficiently, and safely isolate large numbers of clinical-grade multipotent human neural stem cells (hNSCs) from embryonic stem cells (hESCs). Consequently, the transplantation of neurogenic/gliogenic precursors into the CNS for the purpose of cell replacement or neuroprotection in humans with injury or disease has not achieved widespread testing and implementation. METHODS:Here, we establish an approach for the in vitro isolation of a highly expandable population of hNSCs using the manual selection of neural precursors based on their colony morphology (CoMo-NSC). The purity and NSC properties of established and extensively expanded CoMo-NSC were validated by expression of NSC markers (flow cytometry, mRNA sequencing), lack of pluripotent markers and by their tumorigenic/differentiation profile after in vivo spinal grafting in three different animal models, including (i) immunodeficient rats, (ii) immunosuppressed ALS rats (SOD1G93A), or (iii) spinally injured immunosuppressed minipigs. RESULTS:In vitro analysis of established CoMo-NSCs showed a consistent expression of NSC markers (Sox1, Sox2, Nestin, CD24) with lack of pluripotent markers (Nanog) and stable karyotype for more than 15 passages. Gene profiling and histology revealed that spinally grafted CoMo-NSCs differentiate into neurons, astrocytes, and oligodendrocytes over a 2-6-month period in vivo without forming neoplastic derivatives or abnormal structures. Moreover, transplanted CoMo-NSCs formed neurons with synaptic contacts and glia in a variety of host environments including immunodeficient rats, immunosuppressed ALS rats (SOD1G93A), or spinally injured minipigs, indicating these cells have favorable safety and differentiation characteristics. CONCLUSIONS:These data demonstrate that manually selected CoMo-NSCs represent a safe and expandable NSC population which can effectively be used in prospective human clinical cell replacement trials for the treatment of a variety of neurodegenerative disorders, including ALS, stroke, spinal traumatic, or spinal ischemic injury.
Project description:<h4>Background</h4>Spinal cord injury (SCI) is a common disease that results in motor and sensory disorders and even lifelong paralysis. The transplantation of stem cells, such as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs), or subsequently generated stem/progenitor cells, is predicted to be a promising treatment for SCI. In this study, we aimed to investigate effect of human iPSC-derived neural stem cells (hiPSC-NSCs) and umbilical cord-derived MSCs (huMSCs) in a mouse model of acute SCI.<h4>Methods</h4>Acute SCI mice model were established and were randomly treated as phosphate-buffered saline (PBS) (control group), repaired with 1?×?10<sup>5</sup> hiPSC-NSCs (NSC group), and 1?×?10<sup>5</sup> huMSCs (MSC group), respectively, in a total of 54 mice (n?=?18 each). Hind limb motor function was evaluated in open-field tests using the Basso Mouse Scale (BMS) at days post-operation (dpo) 1, 3, 5, and 7 after spinal cord injury, and weekly thereafter. Spinal cord and serum samples were harvested at dpo 7, 14, and 21. Haematoxylin-eosin (H&E) staining and Masson staining were used to evaluate the morphological changes and fibrosis area. The differentiation of the transplanted cells in vivo was evaluated with immunohistochemical staining.<h4>Results</h4>The hiPSC-NSC-treated group presented a significantly smaller glial fibrillary acidic protein (GFAP) positive area than MSC-treated mice at all time points. Additionally, MSC-transplanted mice had a similar GFAP+ area to mice receiving PBS. At dpo 14, the immunostained hiPSC-NSCs were positive for SRY-related high-mobility-group (HMG)-box protein-2 (SOX2). Furthermore, the transplanted hiPSC-NSCs differentiated into GFAP-positive astrocytes and beta-III tubulin-positive neurons, whereas the transplanted huMSCs differentiated into GFAP-positive astrocytes. In addition, hiPSC-NSC transplantation reduced fibrosis formation and the inflammation level. Compared with the control or huMSC transplanted group, the group with transplantation of hiPSC-NSCs exhibited significantly improved behaviours, particularly limb coordination.<h4>Conclusions</h4>HiPSC-NSCs promote functional recovery in mice with acute SCI by replacing missing neurons and attenuating fibrosis, glial scar formation, and inflammation.
Project description:In a previous study, we have shown that human neural stem cells (hNSCs) transplanted in brain of mouse intracerebral hemorrhage (ICH) stroke model selectively migrate to the ICH lesion and induce behavioral recovery. However, low survival rate of grafted hNSCs in the brain precludes long-term therapeutic effect. We hypothesized that hNSCs overexpressing Akt1 transplanted into the lesion site could provide long-term improved survival of hNSCs, and behavioral recovery in mouse ICH model. F3 hNSC was genetically modified with a mouse Akt1 gene using a retroviral vector. F3 hNSCs expressing Akt1 were found to be highly resistant to H(2)O(2)-induced cytotoxicity in vitro. Following transplantation in ICH mouse brain, F3.Akt1 hNSCs induced behavioral improvement and significantly increased cell survival (50-100% increase) at 2 and 8 weeks post-transplantation as compared to parental F3 hNSCs. Brain transplantation of hNSCs overexpressing Akt1 in ICH animals provided functional recovery, and survival and differentiation of grafted hNSCs. These results indicate that the F3.Akt1 human NSCs should be a great value as a cellular source for the cellular therapy in animal models of human neurological disorders including ICH.