Neural Stem Cells Group Research Articles

Neural stem cell transplantation improves neurological function in focal cerebral ischemia through GDNF/PI3K/AKT axis.

The aim of this experiment was to analyze the ameliorating effect of neural stem cells (NSCs) on focal cerebral ischemia (FCI) through GDNF/PI3K/AKT axis, so as to provide evidence for future clinical application of NSCs. In this study, the 15 Sprague-Dawley (SD) male rats were modeled for middle cerebral artery occlusion (MCAO)-induced FCI and then grouped: NSCs group was treated with NSC transplantation, GDNF/NSCs group was transplanted with recombinant adenovirus pAdEasy-1-pAdTrackCMV-GDNF-transfedcted NSCs, and the blank group was treated with normal saline transplantation. Rats were tested by rotarod and corner turn tests at 1 week and 4 weeks after NSC transplantation, and the levels of tumor necrosis factor-α (TNF-α), interleukin-6/8 (IL-6/8), superoxide dismutase (SOD) and malondialdehyde (MDA) were quantified. Then all rats were killed and their brain tissues were HE stained for the determination of and GDNF/PI3K/AKT axis-associated protein expression. The results of the experiment showed that: at the 1st and 4th week after transplantation, the time on the rod, number of turnings and SOD were the lowest in the blank group among the three groups, while IL-6, IL-8, TNF-α and MDA were the highest (P<0.05). Increased time on the rod, number of turnings and SOD, as well as decreased IL-6, IL-8, TNF-α and MDA were observed in NSCs and GDNF/NSCs groups after transplantation, with better performance in GDNF/NSCs group (P<0.05). Based on HE staining of brain tissue, GDNF/NSCs group had the most significant improvement in tissue injury and the highest GDNF, PI3K, AKT and p-AKT protein expression among the three groups (P<0.05). In conclusions, NSC transplantation can ameliorate neurological function in MCAO-induced FCI rats through the GDNF/PI3K/AKT axis.

Combined treatment of high-intensity interval training with neural stem cell generation on contusive model of spinal cord injury in rats.

Spinal cord injury (SCI) leads to inflammation, axonal degeneration, and gliosis. A combined treatment of exercise and neural stem cells (NSC) has been proposed to improve neural repair. This study evaluated a combined treatment of high-intensity interval training (HIIT) with NSC generation from adipose-derived stem cells (ADSCs) on a contusive model of SCI in rats. In vitro, rat ADSCs were isolated from the perinephric regions of Sprague-Dawley rats using enzymatic digestion. The ADSCs were transdifferentiated into neurospheres using B27, EGF, and bFGF. After production of NSC, they were labeled using green fluorescent protein (GFP). For the in vivo study, rats were divided into eight groups: control group, sham operation group, sham operation + HIIT group, sham operation + NSC group, SCI group, SCI + HIIT group, SCI + NSC group, and SCI/HIIT/NSC group. Laminectomy was carried out at the T12 level using the impactor system. HIIT was performed three times per week. To assess behavioral function, the Basso-Beattie-Bresnahan (BBB) locomotor test and H-reflex was carried out once a week for 12 weeks. We examined glial fibrillary acidic protein (GFAP), S100β, and NF200 expression. NSC transplantation, HIIT and combined therapy with NSC transplantation, and the HIIT protocol improved locomotor function with decreased maximum H to maximum M reflexes (H/M ratio) and increased the Basso-Beattie-Bresnahan score. Combined therapy in contused rats using the HIIT protocol and neurosphere-derived NSC transplantation improves functional and histological outcomes.

A novel stem cell type at the basal side of the subventricular zone maintains adult neurogenesis

According to the current consensus, murine neural stem cells (NSCs) apically contacting the lateral ventricle generate differentiated progenitors by rare asymmetric divisions or by relocating to the basal side of the ventricular–subventricular zone (V‐SVZ). Both processes will ultimately lead to the generation of adult‐born olfactory bulb (OB) interneurons. In contrast to this view, we here find that adult‐born OB interneurons largely derive from an additional NSC‐type resident in the basal V‐SVZ. Despite being both capable of self‐renewal and long‐term quiescence, apical and basal NSCs differ in Nestin expression, primary cilia extension and frequency of cell division. The expression of Notch‐related genes also differs between the two NSC groups, and Notch activation is greatest in apical NSCs. Apical downregulation of Notch‐effector Hes1 decreases Notch activation while increasing proliferation across the niche and neurogenesis from apical NSCs. Underscoring their different roles in neurogenesis, lactation‐dependent increase in neurogenesis is paralleled by extra activation of basal but not apical NSCs. Thus, basal NSCs support OB neurogenesis, whereas apical NSCs impart Notch‐mediated lateral inhibition across the V‐SVZ.

Transplantation of bioengineered Reelin-loaded PLGA/PEG micelles can accelerate neural tissue regeneration in photothrombotic stroke model of mouse.

Ischemic stroke is characterized by extensive neuronal loss, glial scar formation, neural tissue degeneration that leading to profound changes in the extracellular matrix, neuronal circuitry, and long‐lasting functional disabilities. Although transplanted neural stem cells (NSCs) can recover some of the functional deficit after stroke, retrieval is not complete and repair of lost tissue is negligible. Therefore, the current challenge is to use the combination of NSCs with suitably enriched biomaterials to retain these cells within the infarct cavity and accelerate the formation of a de novo tissue. This study aimed to test the regenerative potential of polylactic‐co‐glycolic acid‐polyethylene glycol (PLGA‐PEG) micelle biomaterial enriched with Reelin and embryonic NSCs on photothrombotic stroke model of mice to gain appropriate methods in tissue engineering. For this purpose, two sets of experiments, either in vitro or in vivo models, were performed. In vitro analyses exhibited PLGA‐PEG plus Reelin‐induced proliferation rate (Ki‐67+ NSCs) and neurite outgrowth (axonization and dendritization) compared to PLGA‐PEG + NSCs and Reelin + NSCs groups (p < 0.05). Besides, neural differentiation (Map‐2+ cells) was high in NSCs cultured in the presence of Reelin‐loaded PLGA‐PEG micelles (p < 0.05). Double immunofluorescence staining showed that Reelin‐loaded PLGA‐PEG micelles increased the number of migrating neural progenitor cells (DCX+ cells) and mature neurons (NeuN+ cells) around the lesion site compared to the groups received PLGA‐PEG and Reelin alone after 1 month (p < 0.05). Immunohistochemistry results showed that the PLGA/PEG plus Reelin significantly decreased the astrocytic gliosis and increased local angiogenesis (vWF‐positive cells) relative to the other groups. These changes led to the reduction of cavity size in the Reelin‐loaded PLGA‐PEG+NSCs group. Neurobehavioral tests indicated Reelin‐loaded PLGA‐PEG+NSCs promoted neurological outcome and functional recovery (p < 0.05). These results indicated that Reelin‐loaded PLGA‐PEG is capable of promoting NSCs dynamic growth, neuronal differentiation, and local angiogenesis following ischemic injury via providing a desirable microenvironment. These features can lead to neural tissue regeneration and functional recovery.

Effect of combination therapy with neural stem cell transplantation and teramethylpyrazine in rats following acute spinal cord injury.

This study was to explore the effects of teramethylpyrazine (TMP) administered in conjunction with neural stem cell transplantation on motor function, pathological lesions and the Janus kinase (JAK)2/signal transducer and activator of transcription 3 signal transduction pathway in rats following acute spinal cord injury (SCI). Female Sprague-Dawley rats were randomly divided into sham, model, neural stem cells (NSCs) and NSCs+TMP groups. Motor function was evaluated using the Basso, Beattie, Bresnahan scale. Spinal cord neuropathies and neuron apoptosis were observed by HE and TUNEL staining. The brain-derived neurotrophic factor (BDNF), Nogo-A, JAK2 and p-JAK2 protein levels were measured by western blot analysis. NSCs+TMP significantly improved rat motor function, attenuated impaired spinal cords, and decreased cellular apoptosis, compared with NSCs therapy alone (P < 0.05). In addition, expression of BDNF protein was significantly higher in NSCs+TMP rats compared with other groups regardless of time postinjury (P < 0.05). The highest expression levels of Nogo-A protein were observed in the model group. The expression of p-JAK2 in the NSCs+TMP group was relatively lower than the model and NSCs groups (P < 0.05). In rats with SCI, NSCs+TMP effectively improved motor function and offered spinal cord protection by increasing BDNF and decreasing Nogo-A levels, as well as inhibiting the JAK2/STAT3 signal transduction pathway, suggesting that TMP could be a useful agent in NSCs transplantation in the treatment of SCI.

Influence of moxibustion on NR2B and PKMζ after neural stem cell transplantation in the rats with vascular dementia: 艾灸对血管性痴呆大鼠神经干细胞移植后海马NR2B和PKMζ的影响

To observe the influence of moxibustion on learning and memory ability in the rats with vascular dementia (VD) and explore the potential effect mechanism. A total of 80 rats, screened by Morris water maze, were randomly divided into a sham-operation group, a model group, a neural stem cells (NSCs) group, a NSCs + piracetam group and a NSCs + moxibustion group, 16 rats in each group. After corresponding treatments, Morris water maze and immunofluorescence technique were adopted to evaluate the therapeutic effect respectively. Comparison among groups after modeling: compared with the sham-operation group, the escape latency was longer (P < 0.01) and the times of crossing platform were reduced (P < 0.01) in the rats of the model group. Comparison among groups after treatment: compared with the model group, the escape latency was shortened (P < 0.01) and the times of crossing platform were increased (P < 0.05) in the rats of the NSCs group. Compared with the NSCs group, the escape latency was shorter and the expressions of the hippocampus NR2B/EGFP and PKMζ/EGFP expression level increased (all P < 0.05) in the rats of the NSCs + piracetam group and the NSCs + moxibustion group (all P < 0.05). Compared with the NSCs + piracetam group, the escape latency was shorter and the expressions of the hippocampus NR2B/EGFP and PKMζ/EGFP expression level were higher in the rats of the moxibustion + NSCs group (all P < 0.05) Moxibustion improves the spatial learning and memory ability of the VD rats and promotes the reconstruction of neurogenesis and synaptic function, which may be related to the up-regulation of the expressions of hippocampus NR2B and PKMζ expressions.

Intracerebroventricular Administration of hNSCs Improves Neurological Recovery after Cardiac Arrest in Rats.

Irreversible brain injury and neurological dysfunction induced by cardiac arrest (CA) have long been a clinical challenge due to lack of effective therapeutic interventions to reverse neuronal loss and prevent secondary reperfusion injury. The neuronal regenerative potential of neural stem cells (NSCs) provides a possible solution to this clinical deficit. We investigated the neuronal recovery potential of human neural stem cells (hNSCs) via intracerebroventricular (ICV) xenotransplantation after CA in rats and the effects of transplanted NSCs on the proliferation and migration of endogenous NSCs. Outcome measures included neurological functional recovery measured by neurological deficit score (NDS), electrophysiologic analysis of EEG, and assessment of proliferation and migration at the cellular level and the Wnt/β-catenin pathway at the molecular level. Neurological functional assessment based on aggregate neurological deficit score (NDS) showed better recovery of function after hNSCs therapy (P < 0.05). Tracking of stem cells' proliferation with Ki67 antibody suggested that the NSCs group had more prominent proliferation compared to control group (number of Ki67+ cells, Control VS. NSC: 89.0 ± 31.6 VS. 352.7 ± 97.3, P < 0.05). In addition, cell migration tracked by Dcx antibody showed more Dcx + cells migrated to the far distance zone from SVZ in the treatment group (P < 0.05). Further immunofluorescence staining confirmed that the expression of the Wnt signaling pathway protein (β-catenin) was upregulated in the NSC group (P < 0.05). ICV delivery of hNSCs promotes endogenous NSC proliferation and migration and ultimately enhances neuronal survival and neurological functional recovery. Wnt/β-catenin pathway may be involved in the initiation and maintenance of this enhancement.Graphical abstract.

Effects of neural stem cells in combination with nerve neurotrophic factor 3 gene modified olfactory ensheathing cell co-transplants on functional recovery after traumatic brain injury in the adult rats

Objective To observe the effects of neural stem cells (NSCs) combined with neurotrophin-3 gene modified olfactory ensheathing cells (OECs-NT-3) co-transplants on functional recovery after traumatic brain injury in the adult rats. Methods NSCs and OECs were cultured from hippocampus and olfactory bulb of neonatal rats and then identified. OECs-NT-3 was constructed by lentiviral vector containing NT-3 gene. Male SD rats weighing (200±20) g were randomly divided into sham group, model group, OECs-NT-3 transplantation group, NSCs transplantation group and OECs-NT-3 combined NSCs transplantation group. The rat brain injury model was established. The corresponding cells were transplanted. At day 1, 7, 14 and 28 after transplantation, the modified neurological severity score (mNSS) was used to evaluate neurological function, and brain tissues were taken at each time point for paraffin sections. Hematoxylin-eosin staining was performed. Cell apoptosis was detected by terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) method. Immunohistochemical staining of BrdU and p75 was done. Results At day 28 of transplantation, the mNSS scores in rats in the combined group (4.00±1.05) were significantly different from those in the model group (8.50±1.43), the OECs-NT-3 group (5.50±1.35) and the NSCs group (6.00±1.49) (t=-7.997, -2.764, -3.464, P 0.05). Hematoxylin and eosin (HE) staining revealed that the cell density in the sham group, model group, OECs-NT-3 group, NSCs group and the combined group was (220±22), (299±47), (347±35), (390±72) and (523±55) cells, respectively. As compared with the model group, OECs-NT-3 group and NSCs group, the cell density in the combined group was significantly increased (t=7.246, 5.996, 3.373, P<0.05), and the tissue structure was relatively regular. The p75 and BrdU immunohistochemistry showed that the two kinds of transplanted cells could survive continuously and were widely distributed around the lesion. At day 7 after transplantation, the apoptotic index in the sham group, model group, OECs-NT-3 group, NSCs group and combined group was (1.67±0.17)%, (47.49±7.92)%, (31.22±3.97)%, (34.48±4.69)% and (18.17±3.13)% respectively. The apoptosis rate in the combined transplantation group was significantly lower than that in the model group, OECs-NT-3 group and NSCs group (t=5.963, 4.470, 5.012, P<0.05). Conclusion Transplantation of NSCs combined with OECs-NT-3 can alleviate neuronal apoptosis after brain injury in rats, repair tissue defects, and promote the recovery of nerve function. Key words: Neural stem cell; Neurotrophin-3 gene; Gene modify; Olfactory ensheathing cells; Combined transplantation; Traumatic brain injury

Neurotoxic role of interleukin-17 in neural stem cell differentiation after intracerebral hemorrhage.

Interleukin 17 (IL-17) and its main producer, T cell receptor γδ cells, have neurotoxic effects in the pathogenesis of intracerebral hemorrhage (ICH), aggravating brain injuries. To investigate the correlation between IL-17 and ICH, we dynamically screened serum IL-17 concentrations using enzyme-linked immunosorbent assay and explored the clinical values of IL-17 in ICH patients. There was a significant negative correlation between serum IL-17 level and neurological recovery status in ICH patients (r = -0.498, P < 0.01). To study the neurotoxic role of IL-17, C57BL/6 mice were used to establish an ICH model by injecting autologous blood into the caudate nucleus. Subsequently, the mice were treated with mouse neural stem cells (NSCs) and/or IL-17 neutralizing antibody for 72 hours. Flow cytometry, brain water content detection, Nissl staining, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling results indicated that NSC transplantation significantly reduced IL-17 expression in peri-hematoma tissue, but there was no difference in T cell receptor γδ cells. Compared with the ICH group, there were fewer apoptotic bodies and more Nissl bodies in the ICH + NSC group and the ICH + NSC + IL-17 group. To investigate the potential effect of IL-17 on directional differentiation of NSCs, we cultured mouse NSCs (NE-4C) alone or co-cultured them with T cell receptor γδ cells, which were isolated from mouse peripheral blood mononuclear cells, for 7 days. The results of western blot assays revealed that IL-17 secreted by T cell receptor γδ cells reduced the differentiation of NSCs into astrocytes and neurons, while IL-17 neutralization relieved the inhibition of directional differentiation into astrocytes rather than neurons. In conclusion, serum IL-17 levels were elevated in the early stage of ICH and were negatively correlated with outcome in ICH patients. Animal experiments and cytological investigations therefore demonstrated that IL-17 probably has neurotoxic roles in ICH because of its inhibitory effects on the directional differentiation of NSCs. The application of IL-17 neutralizing antibody may promote the directional differentiation of NSCs into astrocytes. This study was approved by the Clinical Research Ethics Committee of Anhui Medical University of China (For human study: Approval No. 20170135) in December 2016. All animal handling and experimentation were reviewed and approved by the Institutional Animal Care and Use Committee of Anhui Medical University (approval No. 20180248) in December 2017.

Three-dimensional bioprinting collagen/silk fibroin scaffold combined with neural stem cells promotes nerve regeneration after spinal cord injury.

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).

PET imaging of metabolic changes after neural stem cells and GABA progenitor cells transplantation in a rat model of temporal lobe epilepsy.

Stem cell transplantation is promising for temporal lobe epilepsy (TLE) treatment. This study aimed to use PET imaging for the investigation of dynamic metabolic changes after transplantation of human neural stem cells (NSCs) and human GABA progenitor cells (GPCs) in a rat model of TLE. 18F-FDG PET imaging, video-electroencephalography (EEG), whole-cell patch-clamp recordings and immunostaining were performed after transplantation of NSCs and GPCs. PET imaging demonstrated that glucose metabolism was gradually improved in the NSCs group, but decreased in GPCs and the control. Video-EEG manifested that seizures were suppressed after NSCs or GPCs transplantation; whole-cell patch-clamp confirmed increased inhibitory response of GPC-derived cells; immunostaining studies verified that the transplanted NSCs and GPCs could survive, migrate and differentiate into mature neuronal subtypes. 18F-FDG PET imaging could be a distinguishing approach for evaluation of dynamic glycolytic metabolic changes after transplantation of NSCs and GPCs in TLE. Whole-cell patch-clamp provides evidence for functional maturation and integration of transplanted stem cells within host circuits.

Neural Stem Cell Transplantation Improves Locomotor Function in Spinal Cord Transection Rats Associated with Nerve Regeneration and IGF-1 R Expression

Transplantation of neural stem cells (NSCs) is a potential strategy for the treatment of spinal cord transection (SCT). Here we investigated whether transplanted NSCs would improve motor function of rats with SCT and explored the underlying mechanism. First, the rats were divided into sham, SCT, and NSC groups. Rats in the SCT and NSC groups were all subjected to SCT in T10, and were administered with media and NSC transplantation into the lesion site, respectively. Immunohistochemistry was used to label Nestin-, TUNEL-, and NeuN-positive cells and reveal the expression and location of type I insulin-like growth factor receptor (IGF-1 R). Locomotor function of hind limbs was assessed by Basso, Beattie, Bresnahan (BBB) score and inclined plane test. The conduction velocity and amplitude of spinal nerve fibers were measured by electrophysiology and the anatomical changes were measured using magnetic resonance imaging. Moreover, expression of IGF-1 R was determined by real-time polymerase chain reaction and Western blotting. The results showed that NSCs could survive and differentiate into neurons in vitro and in vivo. SCT-induced deficits were reduced by NSC transplantation, including increase in NeuN-positive cells and decrease in apoptotic cells. Moreover, neurophysiological profiles indicated that the latent period was decreased and the peak-to-peak amplitude of spinal nerve fibers conduction was increased in transplanted rats, while morphological measures indicated that fractional anisotropy and the number of nerve fibers in the site of spinal cord injury were increased after NSC transplantation. In addition, mRNA and protein level of IGF-1 R were increased in the rostral segment in the NSC group, especially in neurons. Therefore, we concluded that NSC transplantation promotes motor function improvement of SCT, which might be associated with activated IGF-1 R, especially in the rostral site. All of the above suggests that this approach has potential for clinical treatment of spinal cord injury.

Transplanting GABAergic Neurons Differentiated from Neural Stem Cells into Hippocampus Inhibits Seizures and Epileptiform Discharges in Pilocarpine-Induced Temporal Lobe Epilepsy Model

This study aimed to explore whether intrahippocampal transplantation of GABAergic neurons generated invitro ameliorated seizures and epileptiform discharges via increasing γ-aminobutyric acid (GABA)-associated inhibition mediated by the addition of new GABAergic neurons. Neural stem cells (NSCs) isolated from newborn rats were induced and differentiated into GABAergic neurons. A total of 36 Pilocarpine-induced pharmacoresistant epileptic rats were divided into 3 groups: PBS (phosphate-buffered saline) group, NSCs group, and GABAergic neurons group (GABA group), with an additional 10 normal rats used (normal rat control group). The effects of grafting on spontaneous recurrent seizures (SRS) were examined and hippocampal GABA content was measured after grafting. In the GABA group, the frequency of electroencephalography decreased significantly compared with the PBS group (P < 0.001), but there was no significant difference between the GABA group and NSCs group. Compared with the PBS group, the overall frequency and duration of SRS significantly decreased in the transplantation group, especially in the GABA group (P < 0.01). The number of GABAergic neurons was highest in the GABA group compared with the other groups (P < 0.001). Furthermore, hippocampal GABA concentrations significantly increased in the GABA group. We show that GABAergic neurons generated invitro from NSCs and grafted into the hippocampi of chronically epileptic rats can significantly reduce the frequency of electroencephalography and frequency and duration of SRS via increasing GABA-associated inhibition mediated by the addition of new GABAergic neurons.

Embryonic intraventricular transplantation of neural stem cells augments inflammation-induced prenatal brain injury

ObjectivePrenatal brain injury results from undesirable circumstances during the embryonic development. Current endeavors for treating this complication are basically excluded to postnatal therapeutic approaches. Neural stem cell therapy has shown great promise for treating neurodevelopmental disorders. To our knowledge, this is the first study that investigates the therapeutic effect of in utero transplantation of neural stem cells (NSCs) in inflammation model of prenatal brain injury. MethodsTo induce prenatal injury, time-mated C57BL6J mice were intraperitoneally injected with 50 μg/kg lipopolysaccharide (LPS(on the day 15 of gestation. In the treatment group, NSCs were transplanted into the lateral ventricle of embryos on day 17 of gestation. The expression of GFAP, Iba-1, Olig2, and NeuN were assessed by real time PCR and immunohistochemistry. Changes in IL-6, TNF-α and IL-10 cytokines level, and caspase 3 activity were evaluated in the cortex of pups. ResultsIntrauterine transplanted NSCs homed to the brain cortex of offspring. Brain levels of pro-inflammatory cytokines showed a significant downward trend in the NSCs group. Furthermore, NSCs ameliorated inflammation-induced reactive microgliosis and astrogliosis as well as cellular degeneration. Apoptosis inhibition in the treated group was demonstrated by the decline in the caspase 3 activity and dark neurons. ConclusionThis study suggests a promising prospect to initiate the treatment of prenatal brain injury before birth by intrauterine transplantation of NSCs.

Experimental study of lentivirus-mediated Nogo extracellular peptide residues 1-40 gene and neurotrophin 3 gene co-transduction in neural stem cells

To explore the feasibility of co-transduction and co-expression of Nogo extracellular peptide residues 1-40 (NEP1-40) gene and neurotrophin 3 (NT-3) gene into neural stem cells (NSCs). NSCs were derived from the cortex tissue of Sprague Dawley rat embryo. The experiment included 5 groups: no-load lentiviral vector transducted NSCs (group A), NEP1-40 transducted NSCs (group B), NT-3 transducted NSCs (group C), NEP1-40 and NT-3 corporately transducted NSCs (group D), and blank control (group E). Target genes were transducted into NSCs by lentiviral vectors of different multiplicity of infection (MOI; 5, 10, 15) for different time (24, 48, 72 hours). Fluorescent microscope was used to observe the expression of fluorescence protein and acquire the optimum MOI and optimum collection time. Real-time fluorescence quantitative PCR and Western blot tests were utilized to evaluate the gene expressions of NEP1-40 and NT-3 in NSCs and protein expressions of NEP1-40 and NT-3 in NSCs and in culture medium. The optimum MOI for both target gene was 10 and the optimum collection time was 48 hours. The real-time fluorescence quantitative PCR and Western blot results showed that the mRNA and protein relative expressions of NEP1-40 in groups B and D were significantly higher than those in groups A and C ( P<0.05), but no significant difference was found between groups B and D, and between groups A and C ( P>0.05). The mRNA and protein relative expressions of NT-3 in groups C and D were significantly higher than those in groups A and B ( P<0.05), but no significant difference was found between groups A and B, and between groups C and D ( P>0.05). NEP1-40 and NT-3 gene can be successfully co-transducted into NSCs by the mediation of lentiviral vector. The expressions of the two target genes are stable and have no auxo-action or antagonism between each other.

Recombinant PEP-1-SOD1 improves functional recovery after neural stem cell transplantation in rats with traumatic brain injury.

The transplantation of neural stem cells (NSCs) has been demonstrated as a potential treatment strategy for traumatic brain injury (TBI). Cu, Zn-superoxide dismutase (SOD1) is an important antioxidant enzyme that detoxifies intracellular reactive oxygen species, thereby protecting cells from oxidative damage. PEP-1, a peptide carrier, is able to deliver full-length native peptides or proteins into cells. Therefore, the current study investigated the effect of the transplantation of NSCs in combination with PEP-1-SOD1 for the treatment of experimental TBI in rats. Initially, the effect of PEP-1-SOD1 on the proliferation of NSCs was evaluated by MTT assay. PEP-1-SOD1 (0.5, 2.5 and 4.5 µM) significantly increased the proliferation rates of NSCs at 24, 48 and 72 h in a dose-dependent manner. PEP-1-SOD1 also promoted the differentiation of NSCs in vitro. The in vivo experiment showed that PEP-1-SOD1 in combination with NSC transplantation significantly improved the functional recovery of rats following TBI compared with NSC transplantation alone. A significant increase in brain aquaporin-4 (AQP4) mRNA and protein expression levels was observed 4 days post-TBI in PEP-1-SOD1, NSCs and PEP-1-SOD1 + NSCs groups compared with the saline group. The PEP-1-SOD1 + NSCs group showed a further increase of AQP4 mRNA and protein expression levels compared with the NSCs and PEP-1-SOD1 groups. In conclusion, the current data suggests that PEP-1-SOD1 may promote the proliferation and differentiation of NSCs, and thereby improve the functional recovery of TBI model rats following NSCs transplantation through upregulating the expression of AQP4.

Identification of gene expression signatures across different types of neural stem cells with the Monte-Carlo feature selection method.

Adult neural stem cells (NSCs) are a group of multi-potent, self-renewing progenitor cells that contribute to the generation of new neurons and oligodendrocytes. Three subtypes of NSCs can be isolated based on the stages of the NSC lineage, including quiescent neural stem cells (qNSCs), activated neural stem cells (aNSCs) and neural progenitor cells (NPCs). Although it is widely accepted that these three groups of NSCs play different roles in the development of the nervous system, their molecular signatures are poorly understood. In this study, we applied the Monte-Carlo Feature Selection (MCFS) method to identify the gene expression signatures, which can yield a Matthews correlation coefficient (MCC) value of 0.918 with a support vector machine evaluated by ten-fold cross-validation. In addition, some classification rules yielded by the MCFS program for distinguishing above three subtypes were reported. Our results not only demonstrate a high classification capacity and subtype-specific gene expression patterns but also quantitatively reflect the pattern of the gene expression levels across the NSC lineage, providing insight into deciphering the molecular basis of NSC differentiation.

Adjudin-preconditioned neural stem cells enhance neuroprotection after ischemia reperfusion in mice

BackgroundTransplantation of neural stem cells (NSCs) has been proposed as a promising therapeutic strategy for the treatment of ischemia/reperfusion (I/R)-induced brain injury. However, existing evidence has also challenged this therapy on its limitations, such as the difficulty for stem cells to survive after transplantation due to the unfavorable microenvironment in the ischemic brain. Herein, we have investigated whether preconditioning of NSCs with adjudin, a small molecule compound, could enhance their survivability and further improve the therapeutic effect for NSC-based stroke therapy.MethodWe aimed to examine the effect of adjudin pretreatment on NSCs by measuring a panel of parameters after their transplantation into the infarct area of ipsilateral striatum 24 hours after I/R in mice.ResultsWe found that pretreatment of NSCs with adjudin could enhance the viability of NSCs after their transplantation into the stroke-induced infarct area. Compared with the untreated NSC group, the adjudin-preconditioned group showed decreased infarct volume and neurobehavioral deficiency through ameliorating blood–brain barrier disruption and promoting the expression and secretion of brain-derived neurotrophic factor. We also employed H2O2-induced cell death model in vitro and found that adjudin preconditioning could promote NSC survival through inhibition of oxidative stress and activation of Akt signaling pathway.ConclusionThis study showed that adjudin could be used to precondition NSCs to enhance their survivability and improve recovery in the stroke model, unveiling the value of adjudin for stem cell-based stroke therapy.

Schwann cells promote the capability of neural stem cells to differentiate into neurons and secret neurotrophic factors.

The present study investigated whether co-culturing Schwann cells (SCs) with neural stem cells (NSCs) improves viability, direction of differentiation and secretion of brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor (GDNF) in NSCs. The three groups assessed were as follows: SCs, NSCs, and a co-culture of SCs and NSCs. Cellular morphological changes were observed under an inverted phase contrast microscope and quantified. Cells were identified by immunofluorescence staining: S100 for SCs, Nestin for NSCs, microtubule associated protein (Map) 2 and NeuN for neurons and glial fibrillary acidic protein for astrocytes. Cell viability was evaluated by MTT assay. Secretion of BDNF and GDNF was quantified; mRNA expression was quantified by reverse transcription-quantitative polymerase chain reaction. The majority of NSCs in the co-cultured group differentiated into neurons. The cell survival rate of the co-culture group was significantly higher than the other groups on days 3, 5 and 10 (P<0.01). The secretion of BDNF in the co-culture group was significantly higher than NSCs on days 3, 5 and 7 (P<0.05), while the amount of GDNF in co-culture was significantly higher than both NSCs and SCs on day 1 (P<0.05). BDNF and GDNF gene expression in the co-culture group was significantly higher than SCs (P<0.01). Gene expression of Map2 in co-culture group was also significantly higher than both NSC and SC groups (P<0.01). Therefore, co-cultured SCs and NSCs promote differentiation of NSCs into neurons and secrete higher levels of neurotropic factors including BDNF and GDNF.

The protective effect of human umbilical cord mesenchymal stem cells-induced neural stem cells in the vitreous on the blood-retinal barrier in diabetic rats

Objective: To investigate the effect of intravitreal injection of neural stem cells (NSC) induced from human umbilical cord mesenchymal stem cells, and to provide a theoretical basis for the clinical treatment of blood-retinal barrier damage due to diabetic retinopathy (DR). Methods: Experimental study. Sixty male Sprague-Dawley rats were randomly divided into control group, DR group and NSC group. Diabetic rats were induced by injection of streptozotocin, and the control rats were injected with an equal volume of solvent. Three months after the establishment of diabetic models, the NSC group was injected with 2 μl of NSC in the right vitreous, and the DR group was injected with 2 μl of phosphate-buffered saline. One month later, all the rats were sacrificed. The retinal vessels and leakage were examined with flat-mounted retinas. Vascular permeability was quantified by analyzing albumin leakage using the Evans blue (EB) method. Retina was examined by hematoxylin and eosin staining. Results: Retinal blood vessels of the control rats were normal, with no EB leakage outside the vessels. The background fluorescence was enhanced and focal leakage and focal dilated vessels were detected in the DR group. In the NSC group, background fluorescence was enhanced slightly and EB leakage area decreased significantly compared with the DR group. The average EB in control group, DR group and NSC group were (9.91±1.53), (24.67±2.26) and (12.85±2.58)μg/g, The EB leakage in the NSC group decreased significantly compared with the DR group (q=9.748, P<0.05). Pathological hematoxylin and eosin staining showed that the retinal layer structure was normal and clear in the control group, the retina was thin, the cell arrangement was in disorder and the nucleus was swelling in the DR group, the status of the NSC group was between the other two groups. Conclusions: Transferring human umbilical cord mesenchymal stem cells-induced NSC in vitro to diabetic rat models by intravitreal injection could reduce leakage of blood vessels and attenuate blood-retinal barrier breakdown induced by diabetes. (Chin J Ophthalmol, 2017, 53: 53-58).