Points regarding cell transplantation for the treatment of spinal cord injur y

Transplantation of somatic cells, including bone marrow stromal cells (BMSCs), bone marrow mononu-clear cells (BMNCs), and choroid plexus epithelial cells (CPECs), enhances the outgrowth of regenerating axons and promotes locomotor improvements. They are not integrated into the host spinal cord, but disappear within 2-3 weeks after transplantation. Regenerating axons extend at the spinal cord lesion through the astrocyte-devoid area that is iflled with connective tissue matrices. Regenerating axons have characteristics of peripheral nerves:they are associated with Schwann cells, and embedded in connective tissue matrices. It has been suggested that neurotrophic factors secreted from BMSCs and CPECs promote“intrinsic”ability of the spinal cord to regenerate. Transplanted Schwann cells survive long-term, and are integrated into the host spinal cord, serving as an effective scaffold for the outgrowth of regenerating axons in the spinal cord. The disadvantage that axons are blocked to extend through the glial scar at the border of the lesion is overcome. Schwann cells have been approved for clinical applications. Neural stem/progenitor cells (NSPCs) survive long-term, proliferate, and differentiate into glial cells and/or neurons after trans-plantation. No method is available at present to manipulate and control the behaviors of NPSCs to allow them to appropriately integrate into the host spinal cord. NPSP transplantation is not necessarily effective for locomotor improvement.     

Thrombospondin 1 promotes synaptic formation in bone marrow-derived neuron-like cells

In this study, a combination of growth factors was used to induce bone marrow mesenchymal stem cells differentiation into neuron-like cells, in a broader attempt to observe the role of thrombospondin 1 in synapse formation. Results showed that there was no significant difference in the differentiation rate of neuron-like cells between bone marrow mesenchymal stem cells with thrombospondin induction and those without. However, the cell shape was more complex and the neurites were dendritic, with unipolar, bipolar or multipolar morphologies, after induction with thrombospondin 1. The induced cells were similar in morphology to normal neurites. Immunohistochemical staining showed that the number of positive cells for postsynaptic density protein 95 and synaptophysin 1 protein was significantly increased after induction with thrombospondin 1. These findings indicate that thrombospondin 1 promotes synapse formation in neuron-like cells that are differentiated from bone marrow mesenchymal stem cells.     

Neurogenesis in the adult olfactory bulb

Neurogenesis is the process by which cells divide,migrate,and subsequently differentiate into a neuronal phenotype.Significant rates of neurogenesis persist into adulthood in two brain regions,the subgranular zone of the dentate gyrus and the subventricular zone of the lateral ventricles.Cells of the subventricular zone divide and migrate via the rostral migratory stream to the olfactory bulb where they differentiate into granule and periglomerular cells.With the discovery of large-scale neurogenesis in the adult brain,there have been significant efforts to identify the mechanisms that control this process as well as the role of these cells in neuronal functioning.Although many questions remain unanswered,new insights appear daily about adult neurogenesis,regulatory mechanisms,and the fates of the progeny.In this review we highlight the main studies investigating factors that regulate neurogenesis in the subventricular zone,neuronal migration to the olfactory bulb,neuronal integration into the existing bulbar network and shortly discuss the functional meaning of this process.     

Growth factor- and cytokine-stimulated endothelial progenitor cells in post-ischemic cerebral neovascularization

Endothelial progenitor cells are resident in the bone marrow blood sinusoids and circulate in the peripheral circulation. They mobilize from the bone marrow after vascular injury and home to the site of injury where they differentiate into endothelial cells. Activation and mobilization of endothelial progenitor cells from the bone marrow is induced via the production and release of endothelial progenitor cell-activating factors and includes specific growth factors and cytokines in response to peripheral tissue hypoxia such as after acute ischemic stroke or trauma. Endotheli- al progenitor cells migrate and home to specific sites following ischemic stroke via growth factor/ cytokine gradients. Some growth factors are less stable under acidic conditions of tissue isch- emia, and synthetic analogues that are stable at low pH may provide a more effective therapeutic approach for inducing endothelial progenitor cell mobilization and promoting cerebral neovascularization following ischemic stroke.     

Lineage tracing of direct astrocyte-to-neuron conversion in the mouse cortex

Regenerating functional new neurons in the adult mammalian central nervous system has been proven to be very challenging due to the inability of neurons to divide and repopulate themselves after neuronal loss.Glial cells,on the other hand,can divide and repopulate themselves under injury or diseased conditions.We have previously reported that ectopic expression of NeuroD1 in dividing glial cells can directly convert them into neurons.Here,using astrocytic lineage-tracing reporter mice (Aldh1l1-CreER~(T2) mice crossing with Ai14 mice),we demonstrate that lineage-traced astrocytes can be successfully converted into NeuN-positive neurons after expressing NeuroD1 through adeno-associated viruses.Retroviral expression of NeuroD1 further confirms that dividing glial cells can be converted into neurons.Importantly,we demonstrate that for in vivo cell conversion study,using a safe level of adeno-associated virus dosage (10~(10)–10~(12) gc/mL,1μL) in the rodent brain is critical to avoid artifacts caused by toxic dosage,such as that used in a recent bioRxiv study(2×10~(13) gc/mL,1μL,mouse cortex).For therapeutic purpose under injury or diseased conditions,or for non-human primate studies,adeno-associated virus dosage needs to be optimized through a series of dose-finding experiments.Moreover,for future in vivo gliato-neuron conversion studies,we recommend that the adeno-associated virus results are further verified with retroviruses that mainly express transgenes in dividing glial cells in order to draw solid conclusions.The study was approved by the Laboratory Animal Ethics Committee of Jinan University,China (approval No.IACUC-20180330-06) on March 30,2018.     

Mash1 efficiently reprograms rat astrocytes into neurons简

To date, it remains poorly understood whether astrocytes can be easily reprogrammed into neurons. Mashl and Brn2 have been previously shown to cooperate to reprogram fibroblasts into neurons. In this study, we examined astrocytes from 2-month-old Sprague-Dawley rats, and found that Brn2 was expressed, but Mashl was not detectable. Thus, we hypothesized that Mashl alone could be used to reprogram astrocytes into neurons. We transfected a recombinant MSCV-MASH1 plasmid into astrocytes for 72 hours, and saw that all cells expressed Mashl. One week later, we observed the changes in morphology of astrocytes, which showed typical neuro- nal characteristics. Moreover, β-tubulin expression levels were significantly higher in astrocytes expressing Mashl than in control cells. These results indicate that Mashl alone can reprogram astrocytes into neurons.     

Rhesus monkey neural stem cell transplantation promotes neural regeneration in rats with hippocampal lesions

Rhesus monkey neural stem cells are capable of differentiating into neurons and glial cells.Therefore,neural stem cell transplantation can be used to promote functional recovery of the nervous system.Rhesus monkey neural stem cells(1×105 cells/μL) were injected into bilateral hippocampi of rats with hippocampal lesions.Confocal laser scanning microscopy demonstrated that green fluorescent protein-labeled transplanted cells survived and grew well.Transplanted cells were detected at the lesion site,but also in the nerve fiber-rich region of the cerebral cortex and corpus callosum.Some transplanted cells differentiated into neurons and glial cells clustering along the ventricular wall,and integrated into the recipient brain.Behavioral tests revealed that spatial learning and memory ability improved,indicating that rhesus monkey neural stem cells noticeably improve spatial learning and memory abilities in rats with hippocampal lesions.     

Platelet-rich plasma gel in combination with Schwann cells for repair of sciatic nerve injury

Bone marrow mesenchymal stem cells were isolated from New Zealand white rabbits, culture-expanded and differentiated into Schwann cell-like cells. Autologous platelet-rich plasma and Schwann cell-like cells were mixed in suspension at a density of 1 × 10 6 cells/mL, prior to introduction into a poly (lactic-co-glycolic acid) conduit. Fabricated tissue-engineered nerves were implanted into rabbits to bridge 10 mm sciatic nerve defects (platelet-rich plasma group). Controls were established using fibrin as the seeding matrix for Schwann cell-like cells at identical density to construct tissue-engineered nerves (fibrin group). Twelve weeks after implantation, toluidine blue staining and scanning electron microscopy were used to demonstrate an increase in the number of regenerating nerve fibers and thickness of the myelin sheath in the platelet-rich plasma group compared with the fibrin group. Fluoro-gold retrograde labeling revealed that the number of Fluo-ro-gold-positive neurons in the dorsal root ganglion and the spinal cord anterior horn was greater in the platelet-rich plasma group than in the fibrin group. Electrophysiological examination confirmed that compound muscle action potential and nerve conduction velocity were superior in the plate-let-rich plasma group compared with the fibrin group. These results indicate that autologous plate-let-rich plasma gel can effectively serve as a seeding matrix for Schwann cell-like cells to construct tissue-engineered nerves to promote peripheral nerve regeneration.     

Electroacupuncture in the repair of spinal cord injury:inhibiting the Notch signaling pathway and promoting neural stem cell proliferation

Electroacupuncture for the treatment of spinal cord injury has a good clinical curative effect, but the underlying mechanism is unclear. In our experiments, the spinal cord of adult Sprague-Dawley rats was clamped for 60 seconds. Dazhui(GV14) and Mingmen(GV4) acupoints of rats were subjected to electroacupuncture. Enzyme-linked immunosorbent assay revealed that the expression of serum inflammatory factors was apparently downregulated in rat models of spinal cord injury after electroacupuncture. Hematoxylin-eosin staining and immunohistochemistry results demonstrated that electroacupuncture contributed to the proliferation of neural stem cells in rat injured spinal cord, and suppressed their differentiation into astrocytes. Real-time quantitative PCR and western blot assays showed that electroacupuncture inhibited activation of the Notch signaling pathway induced by spinal cord injury. These findings indicate that electroacupuncture repaired the injured spinal cord by suppressing the Notch signaling pathway and promoting the proliferation of endogenous neural stem cells.     

Mash1 efficiently reprograms rat astrocytes into neurons

To date,it remains poorly understood whether astrocytes can be easily reprogrammed into neurons.Mash1 and Brn2 have been previously shown to cooperate to reprogram fibroblasts into neurons.In this study,we examined astrocytes from 2-month-old Sprague-Dawley rats,and found that Brn2 was expressed,but Mash1 was not detectable.Thus,we hypothesized that Mash1 alone could be used to reprogram astrocytes into neurons.We transfected a recombinant MSCV-MASH1 plasmid into astrocytes for 72 hours,and saw that all cells expressed Mash1.One week later,we observed the changes in morphology of astrocytes,which showed typical neuronal characteristics.Moreover,β-tubulin expression levels were significantly higher in astrocytes expressing Mash1 than in control cells.These results indicate that Mash1 alone can reprogram astrocytes into neurons.     

Retinal remodeling following photoreceptor degeneration causes retinal ganglion cell death

正The retina is the extension of the central nervous system that senses light.Cones and rods,situated in the outer retina,convert light into electrical signals that travel through intermediate neurons where these are further processed until they finally reach retinal ganglion cells(RGCs).The afferent neurons of the retina,the RGCs,send the     

Human umbilical cord Wharton’s jelly-derived oligodendrocyte precursor-like cells for axon and myelin sheath regeneration

Human umbilical mesenchymal stem cells from Wharton’s jelly of the umbilical cord were induced to differentiate into oligodendrocyte precursor-like cells in vitro. Oligodendrocyte precursor cells were transplanted into contused rat spinal cords. Immunofluorescence double staining indicated that transplanted cells survived in injured spinal cord, and differentiated into mature and immature oligodendrocyte precursor cells. Biotinylated dextran amine tracing results showed that cell transplantation promoted a higher density of the corticospinal tract in the central and caudal parts of the injured spinal cord. Luxol fast blue and toluidine blue staining showed that the volume of residual myelin was significantly increased at 1 and 2 mm rostral and caudal to the lesion epicenter after cell transplantation. Furthermore, immunofluorescence staining verified that the newly regenerated myelin sheath was derived from the central nervous system. Basso, Beattie and Bresnahan testing showed an evident behavioral recovery. These results suggest that human umbilical mesenchymal stem cell-derived oligodendrocyte precursor cells promote the regeneration of spinal axons and myelin sheaths.     

miR-21 promotes the differentiation of hair follicle-derived neural crest stem cells into Schwann cells

Hair follicle-derived neural crest stem cells can be induced to differentiate into Schwann cells in vivo and in vitro. However, the underlying regulatory mechanism during cell differentiation remains poorly understood. This study isolated neural crest stem cells from human hair follicles and induced them to differentiate into Schwann cells. Quantitative RT-PCR showed that microRNA(miR)-21 expression was gradually increased during the differentiation of neural crest stem cells into Schwann cells. After transfection with the miR-21 agonist(agomir-21), the differentiation capacity of neural crest stem cells was enhanced. By contrast, after transfection with the miR-21 antagonist(antagomir-21), the differentiation capacity was attenuated. Further study results showed that SOX-2 was an effective target of miR-21. Without compromising SOX2 mRNA expression, miR-21 can down-regulate SOX protein expression by binding to the 3′-UTR of miR-21 mRNA. Knocking out the SOX2 gene from the neural crest stem cells significantly reversed the antagomir-21 inhibition of neural crest stem cells differentiating into Schwann cells. The results suggest that miR-21 expression was increased during the differentiation of neural crest stem cells into Schwann cells and miR-21 promoted the differentiation through down-regulating SOX protein expression by binding to the 3′-UTR of SOX2 mRNA.     

Adipose-derived stem cells enhance myogenic differentiation in the mdx mouse model of muscular dystrophyvia paracrine signaling

Adipose-derived stem cells have been shown to promote peripheral nerve regeneration through the paracrine secretion of neurotrophic factors. However, it is unclear whether these cells can promote myogenic differentiation in muscular dystrophy. Adipose-derived stem cells(6 × 10~6) were injected into the gastrocnemius muscle of mdx mice at various sites. Dystrophin expression was found in the muscle fibers. Phosphorylation levels of Akt, mammalian target of rapamycin(mT OR), e IF-4E binding protein 1 and S6 kinase 1 were increased, and the Akt/mT OR pathway was activated. Simultaneously, myogenin levels were increased, whereas cleaved caspase 3 and vimentin levels were decreased. Necrosis and fibrosis were reduced in the muscle fibers. These findings suggest that adipose-derived stem cells promote the regeneration and survival of muscle cells by inhibiting apoptosis and fibrosis, thereby alleviating muscle damage in muscular dystrophy.     

Micro RNA-9 promotes the neuronal differentiation of rat bone marrow mesenchymal stem cells by activating autophagy

Micro RNA-9(mi R-9) has been shown to promote the differentiation of bone marrow mesenchymal stem cells into neuronal cells, but the precise mechanism is unclear. Our previous study confirmed that increased autophagic activity improved the efficiency of neuronal differentiation in bone marrow mesenchymal stem cells. Accumulating evidence reveals that mi RNAs adjust the autophagic pathways. This study used mi R-9-1 lentiviral vector and mi R-9-1 inhibitor to modulate the expression level of mi R-9. Autophagic activity and neuronal differentiation were measured by the number of light chain-3(LC3)-positive dots, the ratio of LC3-II/LC3, and the expression levels of the neuronal markers enolase and microtubule-associated protein 2. Results showed that LC3-positive dots, the ratio of LC3-II/LC3, and expression of neuron specific enolase and microtubule-associated protein 2 increased in the mi R-9+ group. The above results suggest that autophagic activity increased and bone marrow mesenchymal stem cells were prone to differentiate into neuronal cells when mi R-9 was overexpressed, demonstrating that mi R-9 can promote neuronal differentiation by increasing autophagic activity.     

基质细胞衍生因子-1诱导神经干细胞靶向性迁移的实验研究

Objective To investigate the effect ofstromal cell derived factor-1 (SDF-1) on the regulation of neural stem cells (NSCs) migration.Methods NSCs were obtained from the cerebral cortex of embryonic rats and cultured in serum-free medium,and their stem cell properties were assessed by means of induced differentiation in vitro into neurons and astrocytes.After in vitro cell culture,the purity of NSCs and the co-expression rate of CXCR4/nestin were detected by flow cytometry.Blind-well chambers were employed to detect the chemotactic effects of SDF-1 by counting the cells which had crossed a 8 μm pore membrane when confronted with varying concentrations of SDF-1 (0,1,10,50,100,500 and 1000 ng/mL),and the distribution of cells migrated out of the same neurosphere was overviewed by μ-slides in the persistent concentration gradient of SDF-1.Results Neurospheres were formed by persistent proliferation of NSCs, which were capable of differentiating into neurons (β-tubulin+) and astrocytes (GFAP+) in media without mitogens,and flow cytometry analyses showed that most of the cultured cells expressed nestin and the co-expression rate of CXCR4/nestin was nearly 80%.SDF-1 showed great chemotaxis to NSCs,and the amount of cells having migrated through the membrane in 500 ng/ml SDF-1 group was higher than that in other groups (P<0.05).When the cells were confronted with a linear concentration gradient (from 500 to 0 ng/mL),which was generated by diffusion and stable for at least 48 h,the cells migrated out ofa neruosphere could distribute irregularly with more cells locating in the region of higher concentration of SDF-1 and longer migration distance away from the center of the neurosphere than the opposite.Conclusion SDF-1 binding to its specific receptor CXCR4 was capable of inducing NSCs migrating directionally to the source of SDF-1.     

Long non-coding RNA GAS5 promotes PC12 cells differentiation into Tuj1-positive neuron-like cells and induces cell cycle arrest

Growth arrest-specific 5(GAS5) is an anti-oncogene that has been extensively studied in tumors. However, research on GAS5 in the context of nervous system disease is rare at present. This study aimed to investigate the role of the long non-coding RNA GAS5 in rat pheochromocytoma cells(PC12 cells). GAS5-overexpressing lentivirus was transfected into PC12 cells, and expression levels of GAS5 and C-myc were detected by real-time PCR. Ratios of cells in S phase were detected by 5-ethynyl-2′-deoxyuridine. Immunohistochemical staining was used to detect the immunoreactivity of neuron microtubule markers Tuj1, doublecortin, and microtubule-associated protein 2. Apoptosis was detected by flow cytometry, while expression of acetylcholine in cells was detected by western blot assay. We found that GAS5 can promote PC12 cells to differentiate into Tuj1-positive neuron-like cells with longer processes. In addition, cell proliferation and cell cycle were significantly suppressed by GAS5, whereas it had no effect on apoptosis of PC12 cells. Our results indicate that GAS5 could increase the expression of choline acetyltransferase and acetylcholine release. Thus, we speculate that GAS5 is beneficial to the recovery of neurons and the cholinergic nervous system.