Survival Of Neural Stem Cells Research Articles

Effects of the iron oxide nanoparticle Molday ION Rhodamine B on the viability and regenerative function of neural stem cells: relevance to clinical translation.

An essential component of developing successful neural stem cell (NSC)-based therapies involves the establishment of methodologies to noninvasively monitor grafted NSCs within brain tissues in real time. In this context, ex vivo labeling with ultrasmall superparamagnetic iron oxide (USPIO) particles has been shown to enable efficient tracking of transplanted NSCs via magnetic resonance imaging (MRI). However, whether and how USPIO labeling affects the intrinsic biology of NSCs is not thoroughly understood, and remains an active area of investigation. Here, we perform a comprehensive examination of rat NSC survival and regenerative function upon labeling with the USPIO, Molday ION Rhodamine B (MIRB), which allows for dual magnetic resonance and optical imaging. After optimization of labeling efficiency, two specific doses of MIRB (20 and 50 μg/mL) were chosen and were followed for the rest of the study. We observed that both MIRB doses supported the robust detection of NSCs, over an extended period of time in vitro and in vivo after transplantation into the striata of host rats, using MRI and post hoc fluorescence imaging. Both in culture and after neural transplantation, the higher 50 μg/mL MIRB dose significantly reduced the survival, proliferation, and differentiation rate of the NSCs. Interestingly, although the lower 20 μg/mL MIRB labeling did not produce overtly negative effects, it increased the proliferation and glial differentiation of the NSCs. Additionally, application of this dose also changed the morphological characteristics of neurons and glia produced after NSC differentiation. Importantly, the transplantation of NSCs labeled with either of the two MIRB doses upregulated the immune response in recipient animals. In particular, in animals receiving the 50 μg/mL MIRB-labeled NSCs, this immune response consisted of an increased number of CD68+-activated microglia, which appeared to have phagocytosed MIRB particles and cells contributing to an exaggerated MRI signal dropout in the animals. Overall, these results indicate that although USPIO particles, such as MIRB, may have advantageous labeling and magnetic resonance-sensitive features for NSC tracking, a further examination of their effects might be necessary before they can be used in clinical scenarios of cell-based transplantation.

Hes3 expression in the adult mouse brain is regulated during demyelination and remyelination

Hes3 is a component of the STAT3-Ser/Hes3 Signaling Axis controlling the growth and survival of neural stem cells and other plastic cells. Pharmacological activation of this pathway promotes neuronal rescue and behavioral recovery in models of ischemic stroke and Parkinson's disease. Here we provide initial observations implicating Hes3 in the cuprizone model of demyelination and remyelination. We focus on the subpial motor cortex of mice because we detected high Hes3 expression. This area is of interest as it is impacted both in human demyelinating diseases and in the cuprizone model. We report that Hes3 expression is reduced at peak demyelination and is partially restored within 1 week after cuprizone withdrawal. This raises the possibility of Hes3 involvement in demyelination/remyelination that may warrant additional research. Supporting a possible role of Hes3 in the maintenance of oligodendrocyte markers, a Hes3 null mouse strain shows lower levels of myelin basic protein in undamaged adult mice, compared to wild-type controls. We also present a novel method for culturing the established oligodendrocyte progenitor cell line oli-neu in a manner that maintains Hes3 expression as well as its self-renewal and differentiation potential, offering an experimental tool to study Hes3. Based upon this approach, we identify a Janus kinase inhibitor and dbcAMP as powerful inducers of Hes3 gene expression. We provide a new biomarker and cell culture method that may be of interest in demyelination/remyelination research.

Valosin-containing protein is a key mediator between autophagic cell death and apoptosis in adult hippocampal neural stem cells following insulin withdrawal.

BackgroundProgrammed cell death (PCD) plays essential roles in the regulation of survival and function of neural stem cells (NSCs). Abnormal regulation of this process is associated with developmental and degenerative neuronal disorders. However, the mechanisms underlying the PCD of NSCs remain largely unknown. Understanding the mechanisms of PCD in NSCs is crucial for exploring therapeutic strategies for the treatment of neurodegenerative diseases.ResultWe have previously reported that adult rat hippocampal neural stem (HCN) cells undergo autophagic cell death (ACD) following insulin withdrawal without apoptotic signs despite their normal apoptotic capabilities. It is unknown how interconnection between ACD and apoptosis is mediated in HCN cells. Valosin-containing protein (VCP) is known to be essential for autophagosome maturation in mammalian cells. VCP is abundantly expressed in HCN cells compared to hippocampal tissue and neurons. Pharmacological and genetic inhibition of VCP at basal state in the presence of insulin modestly impaired autophagic flux, consistent with its known role in autophagosome maturation. Of note, VCP inaction in insulin-deprived HCN cells significantly decreased ACD and down-regulated autophagy initiation signals with robust induction of apoptosis. Overall autophagy level was also substantially reduced, suggesting the novel roles of VCP at initial step of autophagy.ConclusionTaken together, these data demonstrate that VCP may play an essential role in the initiation of autophagy and mediation of crosstalk between ACD and apoptosis in HCN cells when autophagy level is high upon insulin withdrawal. This is the first report on the role of VCP in regulation of NSC cell death. Elucidating the mechanism by which VCP regulates the crosstalk of ACD and apoptosis will contribute to understanding the molecular mechanism of PCD in NSCs.

Pretreatment of Mouse Neural Stem Cells with Carbon Monoxide-Releasing Molecule-2 Interferes with NF-κB p65 Signaling and Suppresses Iron Overload-Induced Apoptosis.

Neural stem cell (NSC) transplantation is a promising approach to repair the damaged brain after hemorrhagic stroke; however, it is largely limited by the poor survival of donor cells. Breakdown products of the hematoma and subsequent iron overload contribute to the impairment of survival of neural cells. There is little information regarding the mechanism involved in the death of grafted cells. Furthermore, therapeutic research targeted to improving the survival of grafted neural stem cells (NSCs) is strikingly lacking. Here, we showed that iron overload induced apoptosis of C17.2 cells, a cell line originally cloned from mouse NSCs and immortalized by v-myc. Pretreatment with carbon monoxide-releasing molecule-2 (CORM-2) markedly protected C17.2 cells against iron overload in a dose-dependent manner. Moreover, CORM-2 interfered with NF-κB signaling, including inhibition of nuclear translocation and down-regulation of NF-κB p65. TUNEL staining showed that preconditioning C17.2 cells with CORM-2 enhanced their resistance to apoptosis induced by iron overload, which was concomitant with down-regulation of the pro-apoptotic proteins (Bax and cleaved caspase-3) and up-regulation of the anti-apoptotic protein Bcl2. The protective effect of CORM-2 could be simulated by BAY11-7082, a special inhibitor of NF-κB p65. These results provide a novel and effective strategy to enhance the survival of NSCs after transplantation and, therefore, their efficacy in repairing brain injury due to hemorrhagic stroke.

Targeted Inhibition of Leucine-Rich Repeat and Immunoglobulin Domain-Containing Protein 1 in Transplanted Neural Stem Cells Promotes Neuronal Differentiation and Functional Recovery in Rats Subjected to Spinal Cord Injury.

Leucine-rich repeat and immunoglobulin domain-containing protein (LINGO)-1 is expressed in neural stem cells, and its neutralization results in sustained neuronal immaturity. Thus, targeted inhibition of LINGO-1 via RNA interference may enhance transplanted neural stem cell survival and neuronal differentiation in vivo. Furthermore, LINGO-1 RNA interference in neural stem cells represents a potential therapeutic strategy for spinal cord injury. Department of Spine Surgery, First Affiliated Hospital of Sun Yat-sen University. Translational Medicine Center Research Laboratory, First Affiliated Hospital of Sun Yat-sen University. Female Sprague-Dawley rats. The animals were divided into three groups that underwent laminectomy and complete spinal cord transection accompanied by transplantation of control-RNA interference-treated or LINGO-1-RNA interference-treated neural stem cells at the injured site in vivo. In vitro, neural stem cells were divided into four groups for the following treatments: control, control RNA interference lentivirus, LINGO-1 RNA interference lentivirus and LINGO-1 complementary DNA lentivirusand the Key Projects of the Natural Science Foundation of Guangdong Province (No. S2013020012818). Neural stem cells in each treatment group were examined for cell survival and neuronal differentiation in vitro and in vivo via immunofluorescence and Western blot analysis. Axonal regeneration and tissue repair were assessed via retrograde tracing using Fluorogold, electron microscopy, hematoxylin-eosin staining and MRI. Rats were also examined for functional recovery based on the measurement of spinal cord-evoked potentials and the Basso-Beattie-Bresnahan score. LINGO-1-RNA interference-treated neural stem cell transplantation increased tissue repair and functional recovery of the injured spinal cord in rats. Similarly, LINGO-1 RNA interference increased neural stem cell survival and neuronal differentiation in vitro. The mechanism underlying the effect of LINGO-1 RNA interference on the injured rat spinal cord may be that the significant inhibition of LINGO-1 expression in neural stem cells inactivated the RhoA and Notch signaling pathways, which act downstream of LINGO-1. Our findings indicate that transplantation of LINGO-1-RNA interference-treated neural stem cells facilitates functional recovery after spinal cord injury and represents a promising potential strategy for the repair of spinal cord injury.

Effects of Panax notoginseng saponins on proliferation and differentiation of rat embryonic cortical neural stem cells

BackgroundWe aimed to study the effect of Panax notoginseng saponins (PNS) on the proliferation, differentiation, self-renewal, and expressions of basic fibroblast growth factor (bFGF) and brain-derived neurotrophic factor (BDNF) in rat embryonic neural stem cells (NSCs). MethodsCortical stem cells were isolated from rat embryos on Embryonic Day 17 (E17) and identified by nestin expression. Subsequently, primary culture, subculturing, and single cell cloning were performed on the cells. After the first cell passage (P1), the cells were resuspended and divided into a control group and a treatment group. Control cells were cultured in serum-free basal culture medium with B27 and dulbecco's modified eagle medium (DMEM)/F12. The same medium supplemented with PNS (100 μg/mL) was used to culture cells in the treatment group. Both groups were incubated at 37°C in a 5% CO2 incubator. Immunocytochemistry was performed 4 days after incubation. ResultsPrimary, P1, and P2 cells in the treatment group formed neurospheres, as did single cell clones of the P1 cells in this group. After being cultured for 4 days, the number of nestin-, proliferating cell nuclear antigen (PCNA)-, Tuj-1-, neurofilament (NF)-, vimentin-, glial fibrillary acidic protein (GFAP)-, bFGF-, and BDNF-positive cells significantly increased in the treatment group in comparison to the control group. All positively stained cells could form clear clusters. ConclusionPNS can promote rat embryonic cortical NSC survival, self-renewal, proliferation, and differentiation through neurotrophic factors by autocrine or paracrine signaling.

Neurogenic Effects of Cell-Free Extracts of Adipose Stem Cells.

Stem-cell-based therapies are regarded as promising treatments for neurological disorders, and adipose-derived stem cells (ASCs) are a feasible source of clinical application of stem cell. Recent studies have shown that stem cells have a therapeutic potential for use in the treatment of various illnesses through paracrine action. To examine the effects of cell components of ASCs on neural stem cells (NSCs), we treated cell-free extracts of ASCs (CFE-ASCs) containing various components with brain-derived NSCs. To elucidate the effects of CFE-ASCs in NSC proliferation, we treated mouse subventricular zone-derived cultured NSCs with various doses of CFE-ASCs. As a result, CFE-ASCs were found to induce the proliferation of NSCs under conditions of growth factor deprivation in a dose-dependent manner (p<0.01). CFE-ASCs increase the expression of neuron and astrocyte differentiation markers including Tuj-1 (p<0.05) and glial fibrillary acidic protein (p<0.01) without altering the cell’s fate in differentiating NSCs. In addition, treatment with CFE-ASCs induces an increase in neurite numbers (p<0.01) and lengths of NSCs (p<0.05). Furthermore, CFE-ASCs rescue the hydrogen peroxide-induced reduction of NSCs’ viability (p<0.05) and neurite branching (p<0.01). Findings from our study indicate that CFE-ASCs support the survival, proliferation and differentiation of NSCs accompanied with neurite outgrowth, suggesting that CFE-ASCs can modulate neurogenesis in the central nervous system.

Abstracts from The 12th Symposium of the International Neurotrauma Society February 1-4, 2016 Cape Town, South Africa.

Abstracts from The 12th Symposium of the International Neurotrauma Society February 1-4, 2016 Cape Town, South Africa.

Abstract 129: Optogenetic Inhibition of Striatal Neurons Improves the Survival of Implanted Neural Stem Cell and Neurological Outcomes After Ischemic Stroke

Background: Administration of neural stem cell (NSC) into peri-infarct region is a promising treatment to improve the outcome after brain ischemia. However, whether the survival of implanted NSC is influenced by endogenous neuronal activity remains unclear. Using optogenetic technique, we regulated the activity of striatal neuron and investigated whether the excitation or the inhibition of striatal neuronal activity could improve NSC survival and overall neurological outcome. Methods: Adeno-assosiated virus carrying ChR2 or ArchT gene under the control of promoter CaMKII was injected into mice striatum two weeks before the surgery of 60 minutes transient middle cerebral artery occlusion (tMCAO). NSCs cultured from transgenic mice expressing fluorescent protein or isometric phosphate buffer saline was transplanted into peri-infarct region at striatum at 4 days after tMCAO. The striatal neurons were stimulated 15 minutes daily by 473nm pulse or 530nm constant laser via implanted optical fiber from 7 to 13 days after tMCAO. Neurological severity score (NSS) was assessed at 3 and 14 days. After sacrificing the animals at 14 days after tMCAO, brain infarct volume was measured with cresyl violet staining. The fluorescence intensity and the diffuse area of implanted NSC were calculated to evaluate the survival of NSC. Results: Mice received NSC transplantation and optogenetic inhibition showed smaller infarct volume compared to that only received NSC transplantation or PBS (p&lt;0.05). The fluorescence diffuse area of transplanted NSC was larger in mice with optogenetic inhibition than that without optogenetic inhibition (p&lt;0.05). Mice received NSC transplantation and optogenetic excitation had larger infarct volume and higher NSS than mice only received NSC transplantation (p&lt;0.05), but showed no differences compared to mice received PBS (p&gt;0.05). The fluorescence diffuse area and the mean optical density of transplanted NSC was smaller in mice with optogenetic excitation than that without optogenetic excitation (p&lt;0.05). Conclusion: Inhibition of striatal neuronal activity at sub-acute phase after ischemia can improve the treatment of implanted NSC. To the opposite, excitation of striatal neuronal activity can reduce the NSC treatment.

Effect of controlled release of brain-derived neurotrophic factor and neurotrophin-3 from collagen gel on neural stem cells.

This study aimed to examine the effect of controlled release of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) from collagen gel on rat neural stem cells (NSCs). With three groups of collagen gel, BDNF/collagen gel, and NT-3/collagen gel as controls, BDNF and NT-3 were tested in the BDNF-NT-3/collagen gel group at different time points. The enzyme-linked immunosorbent assay results showed that BDNF and NT-3 were steadily released from collagen gels for 10 days. The cell viability test and the bromodeoxyuridine incorporation assay showed that BDNF-NT-3/collagen gel supported the survival and proliferation of NSCs. The results also showed that the length of processes was markedly longer and differentiation percentage from NSCs into neurons was much higher in the BDNF-NT-3/collagen gel group than those in the collagen gel, BDNF/collagen gel, and NT-3/collagen gel groups. These findings suggest that BDNF-NT-3/collagen gel could significantly improve the ability of NSCs proliferation and differentiation.

Inhibition of HSP90 Promotes Neural Stem Cell Survival from Oxidative Stress through Attenuating NF-κB/p65 Activation.

Stem cell survival after transplantation determines the efficiency of stem cell treatment, which develops as a novel potential therapy for several central nervous system (CNS) diseases in recent decades. The engrafted stem cells face the damage of oxidative stress, inflammation, and immune response at the lesion point in host. Among the damaging pathologies, oxidative stress directs stem cells to apoptosis and even death through several signalling pathways and DNA damage. However, the in-detail mechanism of stem cell survival from oxidative stress has not been revealed clearly. Here, in this study, we used hydrogen peroxide (H2O2) to induce the oxidative damage on neural stem cells (NSCs). The damage was in consequence demonstrated involving the activation of heat shock protein 90 (HSP90) and NF-κB/p65 signalling pathways. Further application of the pharmacological inhibitors, respectively, targeting at each signalling indicated an upper-stream role of HSP90 upon NF-κB/p65 on NSCs survival. Preinhibition of HSP90 with the specific inhibitor displayed a significant protection on NSCs against oxidative stress. In conclusion, inhibition of HSP90 would attenuate NF-κB/p65 activation by oxidative induction and promote NSCs survival from oxidative damage. The HSP90/NF-κB mechanism provides a new evidence on rescuing NSCs from oxidative stress and also promotes the stem cell application on CNS pathologies.

Retinoic acid-loaded polymeric nanoparticles enhance vascular regulation of neural stem cell survival and differentiation after ischaemia.

Stroke is one of the leading causes of death and disability worldwide. However, current therapies only reach a small percentage of patients and may cause serious side effects. We propose the therapeutic use of retinoic acid-loaded nanoparticles (RA-NP) to safely and efficiently repair the ischaemic brain by creating a favourable pro-angiogenic environment that enhances neurogenesis and neuronal restitution. Our data showed that RA-NP enhanced endothelial cell proliferation and tubule network formation and protected against ischaemia-induced death. To evaluate the effect of RA-NP on vascular regulation of neural stem cell (NSC) survival and differentiation, endothelial cell-conditioned media (EC-CM) were collected. EC-CM from healthy RA-NP-treated cells reduced NSC death and promoted proliferation while EC-CM from ischaemic RA-NP-treated cells decreased cell death, increased proliferation and neuronal differentiation. In parallel, human endothelial progenitor cells (hEPC), which are part of the endogenous repair response to vascular injury, were collected from ischaemic stroke patients. hEPC treated with RA-NP had significantly higher proliferation, which further highlights the therapeutic potential of this formulation. To conclude, RA-NP protected endothelial cells from ischaemic death and stimulated the release of pro-survival, proliferation-stimulating factors and differentiation cues for NSC. RA-NP were shown to be up to 83-fold more efficient than free RA and to enhance hEPC proliferation. These data serve as a stepping stone to use RA-NP as vasculotrophic and neurogenic agents for vascular disorders and neurodegenerative diseases with compromised vasculature.

Perspectives of TRPV1 Function on the Neurogenesis and Neural Plasticity.

The development of new strategies to renew and repair neuronal networks using neural plasticity induced by stem cell graft could enable new therapies to cure diseases that were considered lethal until now. In adequate microenvironment a neuronal progenitor must receive molecular signal of a specific cellular context to determine fate, differentiation, and location. TRPV1, a nonselective calcium channel, is expressed in neurogenic regions of the brain like the subgranular zone of the hippocampal dentate gyrus and the telencephalic subventricular zone, being valuable for neural differentiation and neural plasticity. Current data show that TRPV1 is involved in several neuronal functions as cytoskeleton dynamics, cell migration, survival, and regeneration of injured neurons, incorporating several stimuli in neurogenesis and network integration. The function of TRPV1 in the brain is under intensive investigation, due to multiple places where it has been detected and its sensitivity for different chemical and physical agonists, and a new role of TRPV1 in brain function is now emerging as a molecular tool for survival and control of neural stem cells.

Effects of Triclosan on Neural Stem Cell Viability and Survival.

Triclosan is an antimicrobial or sanitizing agent used in personal care and household products such as toothpaste, soaps, mouthwashes and kitchen utensils. There are increasing evidence of the potentially harmful effects of triclosan in many systemic and cellular processes of the body. In this study, we investigated the effects of triclosan in the survivability of cultured rat neural stem cells (NSCs). Cortical cells from embryonic day 14 rat embryos were isolated and cultured in vitro. After stabilizing the culture, triclosan was introduced to the cells with concentrations ranging from 1 μM to 50 μM and in varied time periods. Thereafter, cell viability parameters were measured using MTT assay and PI staining. TCS decreased the cell viability of treated NSC in a concentration-dependent manner along with increased expressions of apoptotic markers, cleaved caspase-3 and Bax, while reduced expression of Bcl2. To explore the mechanisms underlying the effects of TCS in NSC, we measured the activation of MAPKs and intracellular ROS. TCS at 50 μM induced the activations of both p38 and JNK, which may adversely affect cell survival. In contrast, the activities of ERK, Akt and PI3K, which are positively correlated with cell survival, were inhibited. Moreover, TCS at this concentration augmented the ROS generation in treated NSC and depleted the glutathione activity. Taken together, these results suggest that TCS can induce neurodegenerative effects in developing rat brains through mechanisms involving ROS activation and apoptosis initiation.

BDNF Pretreatment of Human Embryonic-Derived Neural Stem Cells Improves Cell Survival and Functional Recovery after Transplantation in Hypoxic–Ischemic Stroke

Intra-arterial neural stem cell (NSC) therapy has the potential to improve long-term outcomes after stroke. Here we evaluate if pretreatment of NSCs with brain-derived neurotrophic factor (BDNF) prior to transplantation improves cell engraftment and functional recovery following hypoxic-ischemic (HI) stroke. Human embryonic-derived NSCs with or without BDNF pretreatment (1 h, 100 ng/ml) were transplanted 3 days after HI stroke. Functional recovery was assessed using the horizontal ladder test. Cell engraftment was evaluated using bioluminescence imaging (BLI) and histological counts of SC121(+) cells. Fluoro-Jade C (FJC) and NeuN stains were used to evaluate neuroprotection. The effect of BDNF on NSCs was analyzed using a migration assay, immunocytochemistry, Luminex proteomic assay, and RT-qPCR.BLI analysis demonstrated significantly higher photon flux in the BDNF-treated NSC group compared to untreated NSC (p = 0.049) and control groups (p = 0.0021) at 1 week after transplantation. Immunohistochemistry confirmed increased transplanted cell survival in the cortex (p = 0.0126) and hippocampus (p = 0.0098) of animals injected with BDNF-treated NSCs compared to untreated NSCs. Behavioral testing revealed that the BDNF-treated NSC group demonstrated increased sensorimotor recovery compared to the untreated NSC and control groups (p < 0.001) over the 1-month period (p < 0.001) following transplantation. A significant improvement in performance was found in the BDNF-treated NSC group compared to the control group at 14, 21, and 28 (p < 0.05) days after transplantation. The cortex and hippocampus of the BDNF-treated NSC group had significantly more SC121(+) NSCs (p = 0.0125, p = 0.0098), fewer FJC(+) neurons (p = 0.0370, p = 0.0285), and a higher percentage of NeuN(+) expression (p = 0.0354) in the cortex compared to the untreated NSC group. BDNF treatment of NSCs resulted in significantly greater migration to SDF-1, secretion of M-CSF, VEGF, and expression of CXCR4, VCAM-1, Thrombospondins 1 and 2, and BDNF. BDNF pretreatment of NSCs results in higher initial NSC engraftment and survival, increased neuroprotection, and greater functional recovery when compared to untreated NSCs.

Ethosuximide Induces Hippocampal Neurogenesis and Reverses Cognitive Deficits in an Amyloid-β Toxin-induced Alzheimer Rat Model via the Phosphatidylinositol 3-Kinase (PI3K)/Akt/Wnt/β-Catenin Pathway

Neurogenesis involves generation of new neurons through finely tuned multistep processes, such as neural stem cell (NSC) proliferation, migration, differentiation, and integration into existing neuronal circuitry in the dentate gyrus of the hippocampus and subventricular zone. Adult hippocampal neurogenesis is involved in cognitive functions and altered in various neurodegenerative disorders, including Alzheimer disease (AD). Ethosuximide (ETH), an anticonvulsant drug is used for the treatment of epileptic seizures. However, the effects of ETH on adult hippocampal neurogenesis and the underlying cellular and molecular mechanism(s) are yet unexplored. Herein, we studied the effects of ETH on rat multipotent NSC proliferation and neuronal differentiation and adult hippocampal neurogenesis in an amyloid β (Aβ) toxin-induced rat model of AD-like phenotypes. ETH potently induced NSC proliferation and neuronal differentiation in the hippocampus-derived NSC in vitro. ETH enhanced NSC proliferation and neuronal differentiation and reduced Aβ toxin-mediated toxicity and neurodegeneration, leading to behavioral recovery in the rat AD model. ETH inhibited Aβ-mediated suppression of neurogenic and Akt/Wnt/β-catenin pathway gene expression in the hippocampus. ETH activated the PI3K·Akt and Wnt·β-catenin transduction pathways that are known to be involved in the regulation of neurogenesis. Inhibition of the PI3K·Akt and Wnt·β-catenin pathways effectively blocked the mitogenic and neurogenic effects of ETH. In silico molecular target prediction docking studies suggest that ETH interacts with Akt, Dkk-1, and GSK-3β. Our findings suggest that ETH stimulates NSC proliferation and differentiation in vitro and adult hippocampal neurogenesis via the PI3K·Akt and Wnt·β-catenin signaling.

The effect of chronic concurrent intake of alcohol and cocaine on neural stem cell survival and differentiation

The effect of chronic concurrent intake of alcohol and cocaine on neural stem cell survival and differentiation

OP043D CULTURE OF MURINE NEURAL STEM CELLS ON DECELLULARIZED MOUSE BRAIN SECTIONS

INTRODUCTION: Their inherent tumour tropism renders neural stem cells (NSC) intriguing as tumour-targeting gene therapy vehicles for brain cancers. However, effective cell therapy relies on the intrinsic capacity of cellular grafts to overcome hypoxic and/or immunological barriers during transplantation. In this context, it is hypothesized that structural support for grafted NSC - in the form of a 3D biocompatible matrix - will be of utmost importance. METHOD: A protocol to decellularize 1.5 mm thick mouse brain sections was designed and validated using molecular analysis and microscopy. Next, the resulting extracellular matrix (ECM) scaffolds were seeded with murine NSC expressing both eGFP and luciferase. Bioluminescence imaging was used to monitor survival and growth. Phenotype was evaluated using confocal microscopy and compared to standard 2D cultured NSC. RESULTS: We optimised and validated a novel decellularization protocol for 1.5 mm thick mouse brain sections, resulting in the generation of acellular 3D brain tissue-derived ECM scaffolds. These 3D ECM scaffolds supported long-term ex vivo survival and growth of NSC during a 2-13 week monitoring period. Confocal microscopy revealed the 3D organisation of scaffold-supported NSC cultures. Under mitogenic stimuli, most cells in this 3D culture retained the NSC phenotype, with only a small minority undergoing spontaneous differentiation. CONCLUSION: The development of this new 3D NSC culture system will become useful for new research on the development of neural networks, brain tumour remodelling, drug testing and brain cell transplantation research. Moreover, it may offer a new modality for 3D in vitro brain research when using brain cancer stem cells.

Overexpression of mitochondrial Hsp75 protects neural stem cells against microglia-derived soluble factor-induced neurotoxicity by regulating mitochondrial permeability transition pore opening in vitro

Microglia (MG)-induced neurotoxicity, a major determinant of Alzheimer's disease, is closely related to the survival of neural stem cells (NSCs). Heat shock protein 75 (Hsp75) has been reported to exert protective effects against environmental stresses; however, whether or not it protects NSCs against MG-derived soluble factor-induced neurotoxicity remains unclear. In the present study, we constructed NSCs that overexpressed human Hsp75 protein and established a co-culture system in order to elucidate the role of Hsp75 in NSC-MG interactions. The results obtained indicated that Hsp75 expression increased after 12 h of soluble factor induction and continued to increase for up to 36 h of treatment. The overexpression of Hsp75 decreased NSC apoptosis and preserved mitochondrial membrane potential. Further experiments revealed that the overexpression of Hsp75 inhibited the formation of cyclophilin D (CypD)-dependent mitochondrial permeability transition pore (mPTP) involvement in neurotoxicity-mediated mitochondrial dysfunction and suppressed the activation of the mitochondrial apoptotic cascade, as demonstrated by the inhibition of the release of cytochrome c (Cytc) and the activation of caspase-3. The findings of this study demonstrate that Hsp75 overexpression prevents the impairment of NSCs induced by MG-derived soluble factors by regulating the opening of mPTP. Thus, Hsp75 warrants further investigation as a potential candidate for protection against neurotoxicity.

DOT1L Activity Promotes Proliferation and Protects Cortical Neural Stem Cells from Activation of ATF4-DDIT3-Mediated ER Stress In Vitro

Growing evidence suggests that the lysine methyltransferase DOT1L/KMT4 has important roles in proliferation, survival, and differentiation of stem cells in development and in disease. We investigated the function of DOT1L in neural stem cells (NSCs) of the cerebral cortex. The pharmacological inhibition and shRNA-mediated knockdown of DOT1L impaired proliferation and survival of NSCs. DOT1L inhibition specifically induced genes that are activated during the unfolded protein response (UPR) in the endoplasmic reticulum (ER). Chromatin-immunoprecipitation analyses revealed that two genes encoding for central molecules involved in the ER stress response, Atf4 and Ddit3 (Chop), are marked with H3K79 methylation. Interference with DOT1L activity resulted in transcriptional activation of both genes accompanied by decreased levels of H3K79 dimethylation. Although downstream effectors of the UPR, such as Ppp1r15a/Gadd34, Atf3, and Tnfrsf10b/Dr5 were also transcriptionally activated, this most likely occurred in response to increased ATF4 expression rather than as a direct consequence of altered H3K79 methylation. While stem cells are particularly vulnerable to stress, the UPR and ER stress have not been extensively studied in these cells yet. Since activation of the ER stress program is also implicated in directing stem cells into differentiation or to maintain a proliferative status, the UPR must be tightly regulated. Our and published data suggest that histone modifications, including H3K4me3, H3K14ac, and H3K79me2, are implicated in the control of transcriptional activation of ER stress genes. In this context, the loss of H3K79me2 at the Atf4- and Ddit3-promoters appears to mark a point-of-no-return that activates the death program in NSCs.