Mesenchymal stem cell-derived exosomes promote neurogenesis and cognitive function recovery in a mouse model of Alzheimer's disease

Studies have shown that mesenchymal stem cell-derived exosomes can enhance neural plasticity and improve cognitive impairment. The purpose of this study was to investigate the effects of mesenchymal stem cell-derived exosomes on neurogenesis and cognitive capacity in a mouse model of Alzheimer's disease. Alzheimer's disease mouse models were established by injection of beta amyloid 1-42 aggregates into dentate gyrus bilaterally. Morris water maze and novel object recognition tests were performed to evaluate mouse cognitive deficits at 14 and 28 days after administration. Afterwards, neurogenesis in the subventricular zone was determined by immunofluorescence using doublecortin and PSA-NCAM antibodies. Results showed that mesenchymal stem cells-derived exosomes stimulated neurogenesis in the subventricular zone and alleviated beta amyloid 1-42-induced cognitive impairment, and these effects are similar to those shown in the mesenchymal stem cells. These findings provide evidence to validate the possibility of developing cell-free therapeutic strategies for Alzheimer's disease. All procedures and experiments were approved by Institutional Animal Care and Use Committee(CICUAL)(approval No. CICUAL2016-011) on April 25, 2016.     

Exosomes are released by cultured cortical neurones

Accumulating evidence shows that several cell types have the capacity to secrete membrane proteins by incorporating them into exosomes, which are small lipid vesicles derived from the intralumenal membranes of multivesicular bodies (MVBs) of the endocytic pathway. Exosomes are expelled in the extracellular space upon fusion of the MVB with the plasma membrane. Exosomal release is a way of secreting membrane proteins meant to be discarded, or to be passed on to other cells. Here, we demonstrate, using primary cortical cultures, that neurones and astrocytes can secrete exosomes. We find that exosomes released by cortical neurones contain the L1 cell adhesion molecule, the GPI-anchored prion protein, and the GluR2/3 but not the NR1 subunits of glutamate receptors. We also show that exosomal release is regulated by depolarisation. Our observation suggests that exosomes may have a regulatory function at synapses and could also allow intercellular exchange of membrane proteins within the brain.     

Exosomes Released from Bone-Marrow Stem Cells Ameliorate Hippocampal Neuronal Injury Through transferring miR-455-3p

BackgroundThe neuroprotective roles of mesenchymal stem cells (MSCs) in brain injury are elicited at least partially through the secretion exosomes containing microRNAs (miRNAs). We herein investigate the protective function of bone marrow MSCs (BMSCs)-derived exosomes harboring miR-455-3p against hippocampal neuronal injury in mouse and N2a cell damage model.MethodsFirst, BMSC surface markers were detected by flow cytometry, followed by extraction of BMSCs-derived exosomes (BMSCs-Exos). A mouse model of neuronal injury was induced by middle cerebral artery occlusion/reperfusion (MCAO/R), and N2a cells were exposed to oxygen-glucose deprivation/reoxygenation (OGD/R) for in vitro experiments. BMSCs-Exos were administrated in mice and N2a cells. We subsequently determined viability- and apoptosis-features using EdU staining, CCK-8, flow cytometry and Caspase-3 kits. Subsequently, we used RT-qPCR to assess miR-455-3p expression in brain tissues as well as N2a cells, and bioinformatic tools to predict the targeting mRNA of miR-455-3p, which was validated by dual-luciferase assays.ResultsBMSCs-Exos improved hippocampal neuronal injury in MCAO/R-treated mice and OGD/R-induced injury to N2a cells. BMSCs-Exos upregulated miR-455-3p expression in brain tissues of mice and OGD/R-treated N2a cells. miR-455-3p targeted and conversely regulated PDCD7 expression. The protective effect of BMSCs-Exos on OGD/R-treated N2a cells was markedly mitigated following miR-455-3p downregulation. Moreover, overexpression of miR-455-3p contributed to increased N2a cell activity and decreased apoptosis, while the rescue experiment results were opposite.ConclusionMSCs-derived exosomal miR-455-3p targeted PDCD7 to alleviate hippocampal neuronal injury in MCAO/R-treated mice and injury of OGD/R-treated N2a cells.     

Exosomes as mediators of neuron-glia communication in neuroinflammation

In recent years,a type of extracellular vesicles named exosomes has emerged that play an important role in intercellular communication under physiological and pathological conditions.These nanovesicles (30–150 nm) contain proteins,RNAs and lipids,and their internalization by bystander cells could alter their normal functions.This review focuses on recent knowledge about exosomes as messengers of neuron-glia communication and their participation in the physiological and pathological functions in the central nervous system.Special emphasis is placed on the role of exosomes under toxic or pathological stimuli within the brain,in which the glial exosomes containing inflammatory molecules are able to communicate with neurons and contribute to the pathogenesis of neuroinflammation and neurodegenerative disorders.Given the small size and characteristics of exosomes,they can cross the blood-brain barrier and be used as biomarkers and diagnosis for brain disorders and neuropathologies.Finally,although the application potential of exosome is still limited,current studies indicate that exosomes represent a promising strategy to gain pathogenic information to identify therapeutically targets and biomarkers for neurological disorders and neuroinflammation.     

Exosomes and their role in CNS viral infections

Exosomes are small membrane-bound vesicles that carry biological macromolecules from the site of production to target sites either in the microenvironment or at distant sites away from the origin. Exosomal content of cells varies with the cell type that produces them as well as environmental factors that alter the normal state of the cell such as viral infection. Human DNA and RNA viruses alter the composition of host proteins as well as incorporate their own viral proteins and other cargo into the secreted exosomes. While numerous viruses can infect various cell types of the CNS and elicit damaging neuropathologies, few have been studied for their exosomal composition, content, and function on recipient cells. Therefore, there is a pressing need to understand how DNA and RNA viral infections in CNS control exosomal release. Some of the more recent studies including HIV-1, HTLV-1, and EBV-infected B cells indicate that exosomes from these infections contain viral miRNAs, viral transactivators, and a host of cytokines that can control the course of infection. Finally, because exosomes can serve as vehicles for the cellular delivery of proteins and RNA and given that the blood-brain barrier is a formidable challenge in delivering therapeutics to the brain, exosomes may be able to serve as ideal vehicles to deliver protein or RNA-based therapeutics to the brain.     

Regeneration of the ischemic brain by engineered stem cells: Fuelling endogenous repair processes

After ischemic brain injury various cell types including neurons, glia and endothelial cells are damaged and lose their function. Effective regeneration of brain tissue requires that all these cell types have to be replenished and combined to form a new functional network. Recent advances in regenerative medicine show the ability of stem cells to differentiate into various cell lineages. Several types of stem cells have been used to treat ischemic brain injury in rodent models including neuronal stem cells, mesenchymal stem cells and hematopoietic stem cells. Although these studies show promising results, it remains to be determined whether the beneficial effect of cell-based therapies in ischemic brain injury results from direct replacement of damaged cells by the transplanted cells. On the basis of the current literature we propose that neuroprotection by activation of anti-apoptotic mechanisms as well as improvement of the trophic milieu necessary for endogenous repair processes may be more important mechanisms underlying the improved functional outcome after stem cell treatment. Transplantation of native unmodified stem cells as such may not be sufficient to boost repair mechanisms provided by the endogenous stem cell population. An important aim of this review is to discuss the literature on the possible enhancement of regenerative function by combining stem cell transplantation with gene transduction into stem cells to enhance their regenerative and neuroprotective therapeutic potential. Finally, we briefly discuss the possibility of translation of this therapy to the clinic.     

Current perspectives on potential role of albumin in neuroprotection

Albumin is the most abundant plasma protein synthesised mainly in the liver. It is also a major component of extracellular fluids including cerebrospinal fluid, interstitial fluid and lymph. Albumin has several biochemical properties including regulation of colloid osmotic pressure of plasma, transportation of hormones, fatty acids, drugs and metabolites across plasma, regulation of microvascular permeability, antioxidant activity, anti-thrombotic activity and anti-inflammatory activity. This multifunctional protein has been implicated in many neurological diseases owing to its ability to regulate hemodynamic properties of the brain circulation as well as the direct neuroprotective actions on neuronal and glial cells. In this review, we summarise various neuroprotective actions of the albumin in the brain. In experimental ischemic stroke, exogenous human serum albumin administration has been found to be neuroprotective via reducing brain swelling, prevention of post-ischemic thrombosis, anti-oxidant activity, hemodilution and increasing the perfusion to the ischemic tissue. Also, human serum albumin administration is currently under clinical trials for treatment of cerebral ischemia. In the experimental models of Alzheimer's disease, albumin has been implicated in neuroprotection by inhibiting polymerisation and enhancing the clearance of amyloid β. The direct neuroprotective actions on neuronal and glial cells are mediated via endogenously produced albumin or cellular uptake of blood derived albumin. These neuroprotective effects of albumin are partly attributed to anti-oxidant property and modulation of intracellular signalling of neuronal or glial cells. The recent finding of de novo synthesis of albumin in microglial cells directs us to explore newer roles of this endogenously produced multifunctional protein in normal as well as pathological conditions of the brain.     

Adult neurogenesis pharmacology in neurological diseases and disorders

With the confirmation that neurogenesis occurs in the adult brain and neural stem cells reside in the adult CNS, the focus of research has now shifted to the understanding of the function of newborn neuronal cells in the adult brain, and particularly in the pathologies of the nervous system. Neurogenesis has been reported to be modulated in a broad range of pathological conditions, including neurological diseases and disorders. More strikingly, studies have revealed that drugs currently used to treat neurological diseases and disorders, such as Alzheimer's disease and depression, increase adult neurogenesis, which may mediate their activities. However, some of these studies are the source of debates and controversies, and remain to be confirmed. Hence, the role and contribution of newly generated neuronal cells in neurological diseases and disorders, as well as the effect of drugs on adult neurogenesis and its significance remain to be elucidated and understood. This shows that adult neurogenesis is not only important for our understanding of development and therapy, but also for the physiopathology of the CNS and its pharmacology.     

外泌体对缺血性卒中的作用机制及研究进展

缺血性卒中是由于脑部相关区域血供中断，导致神经元缺血缺氧死亡和永久性神经功能损伤的一类脑血管疾病。外泌体是一种特殊的细胞外囊泡，其外膜为脂质双层结构，内含脂质、蛋 白质及DNA、RNA等物质，能够作为运输载体穿过血脑屏障，完成细胞间信息交流和遗传物质的转载， 这一过程对缺血性卒中后神经功能的恢复至关重要。其中微小核糖核酸（microRNA，miRNA/miR）作为缺血性卒中的标志物和治疗靶点，在缺血性卒中的诊断、治疗和预后中发挥重要作用。本文主要介绍了外泌体对缺血性卒中的作用机制、防治价值以及干细胞源性外泌体的治疗作用。     

Human neural stem cells genetically modified for brain repair in neurological disorders

Existence of multipotent neural stem cells (NSC) has been known in developing or adult mammalian CNS, including humans. NSC have the capacity to grow indefinitely and have multipotent potential to differentiate into three major cell types of CNS, neurons, astrocytes and oligodendrocytes. Stable clonal lines of human NSC have recently been generated from the human fetal telencephalon using a retroviral vector encoding v‐myc. One of the NSC lines, HB1.F3, carries normal human karyotype of 46XX and has the ability to self‐renew, differentiate into cells of neuronal and glial lineages, and integrate into the damaged CNS loci upon transplantation into the brain of animal models of Parkinson disease, HD, stroke and mucopolysaccharidosis. F3 human NSC were genetically engineered to produce L‐dihydroxyphenylalanine (L‐DOPA) by double transfection with cDNA for tyrosine hydroxylase and guanosine triphosphate cylohydrolase‐1, and transplantation of these cells in the brain of Parkinson disease model rats led to L‐DOPA production and functional recovery. Proactively transplanted F3 human NSC in rat striatum, supported the survival of host striatal neurons against neuronal injury caused by 3‐nitropro‐pionic acid in rat model of HD. Intravenously introduced through the tail vein, F3 human NSC were found to migrate into ischemic lesion sites, differentiate into neurons and glial cells, and improve functional deficits in rat stroke models. These results indicate that human NSC should be an ideal vehicle for cell replacement and gene transfer therapy for patients with neurological diseases. In addition to immortalized human NSC, immortalized human bone marrow mesenchymal stem cell lines have been generated from human embryonic bone marrow tissues with retroviral vectors encording v‐myc or teromerase gene. These immortalized cell lines of human bone marrow mesenchymal stem cells differentiated into neurons/glial cells, bone, cartilage and adipose tissue when they were grown in selective inducing media. There is further need for investigation into the neurogenic potential of the human bone marrow stem cell lines and their utility in animal models of neurological diseases.     

Stem cell therapy for Alzheimer's disease

Alzheimer's disease (AD) is a progressive neurodegenerative disorder which impairs the memory and intellectual abilities of the affected individuals. Loss of episodic as well as semantic memory is an early and principal feature. The basal forebrain cholinergic system is the population of neurons most affected by the neurodegenerative process. Extracellular as well as intracellular deposition of beta-amyloid or Abeta (Abeta) protein, intracellular formation of neurofibrillary tangles and neuronal loss are the neuropathological hallmarks of AD. In the last few years, hopes were raised that cell replacement therapy would provide cure by compensating the lost neuronal systems. Stem cells obtained from embryonic as well as adult tissue and grafted into the intact brain of mice or rats were mostly followed by their incorporation into the host parenchyma and differentiation into functional neural lineages. In the lesioned brain, stem cells exhibited targeted migration towards the damaged regions of the brain, where they engrafted, proliferated and matured into functional neurones. Neural precursor cells can be intravenously administered and yet migrate into brain damaged areas and induce functional recovery. Observations in animal models of AD have provided evidence that transplanted stem cells or neural precursor cells (NPCs) survive, migrate, and differentiate into cholinergic neurons, astrocytes, and oligodendrocytes with amelioration of the learning/memory deficits. Besides replacement of lost or damaged cells, stem cells stimulate endogenous neural precursors, enhance structural neuroplasticity, and down regulate proinflammatory cytokines and neuronal apoptotic death. Stem cells could also be genetically modified to express growth factors into the brain. In the last years, evidence indicated that the adult brain of mammals preserves the capacity to generate new neurons from neural stem/progenitor cells. Inefficient adult neurogenesis may contribute to the pathogenesis of AD and other neurodegenerative disorders. An attempt at mobilizing this endogenous pool of resident stem-like cells provides another attractive approach for the treatment of AD. Studies in patients with AD indicated decreased hippocampal volume derived by neurodegeneration. Intriguingly, many drugs including antidepressants, lithium, acetyl cholinesterase inhibitors, and ginkgo biloba, were able to enhance the impaired neurogenesis in this disease process. This paved the way towards exploring the possible pharmacological manipulation of neurogenesis which would offer an alternative approach for the treatment of AD.     

Cell therapy for central nervous system disorders: Current obstacles to progress

Cell therapy for disorders of the central nervous system has progressed to a new level of clinical application. Various clinical studies are underway for Parkinson's disease, stroke, traumatic brain injury, and various other neurological diseases. Recent biotechnological developments in cell therapy have taken advantage of the technology of induced pluripotent stem (iPS) cells. The advent of iPS cells has provided a robust stem cell donor source for neurorestoration via transplantation. Additionally, iPS cells have served as a platform for the discovery of therapeutics drugs, allowing breakthroughs in our understanding of the pathology and treatment of neurological diseases. Despite these recent advances in iPS, adult tissue‐derived mesenchymal stem cells remain the widely used donor for cell transplantation. Mesenchymal stem cells are easily isolated and amplified toward the cells' unique trophic factor‐secretion property. In this review article, the milestone achievements of cell therapy for central nervous system disorders, with equal consideration on the present translational obstacles for clinic application, are described.     

Clinical experience with stem cells and other cell therapies in neurological diseases

To overcome the limited capacity of the CNS for regeneration, the theoretical alternative would be to use stem cells for more effective management of chronic degenerative and inflammatory neurological conditions, and also of acute neuronal damage from injuries or cerebrovascular diseases. Although the adult brain contains small numbers of stem cells in restricted areas, this intrinsic stem cell repertoire is small and does not measurably contribute to functional recovery. Embryonic cells carrying pluripotent and self-renewal properties represent the stem cell prototype, but there are additional somatic stem cells that may be harvested and expanded from various tissues during adult life. Stem cell transplantation is based on the assumption that such cells may have the potential to regenerate or support the survival of the existing, partially damaged cells. This review summarizes the state-of-the-art and the clinical worldwide experience with the use of various types of stem cells in neurological diseases.     

Improvement of neuronal cell survival by astrocyte‐derived exosomes under hypoxic and ischemic conditions depends on prion protein

Prion protein (PrP) protects neural cells against oxidative stress, hypoxia, ischemia, and hypoglycemia. In the present study we confirm that cultured PrP‐deficient neurons are more sensitive to oxidative stress than wild‐type neurons and present the novel findings that wild‐type, but not PrP‐deficient astrocytes protect wild‐type cerebellar neurons against oxidative stress and that exosomes released from stressed wild‐type, but not from stressed PrP‐deficient astrocytes reduce neuronal cell death induced by oxidative stress. We show that neuroprotection by exosomes of stressed astrocytes depends on exosomal PrP but not on neuronal PrP and that astrocyte‐derived exosomal PrP enters into neurons, suggesting neuronal uptake of astrocyte‐derived exosomes. Upon exposure of wild‐type astrocytes to hypoxic or ischemic conditions PrP levels in exosomes were increased. By mass spectrometry and Western blot analysis, we detected increased levels of 37/67 kDa laminin receptor, apolipoprotein E and the ribosomal proteins S3 and P0, and decreased levels of clusterin/apolipoprotein J in exosomes from wild‐type astrocytes exposed to oxygen/glucose deprivation relative to exosomes from astrocytes maintained under normoxic conditions. The levels of these proteins were not altered in exosomes from stressed PrP‐deficient astrocytes relative to unstressed PrP‐deficient astrocytes. These results indicate that PrP in astrocytes is a sensor for oxidative stress and mediates beneficial cellular responses, e.g. release of exosomes carrying PrP and other molecules, resulting in improved survival of neurons under hypoxic and ischemic conditions. GLIA 2016;64:896–910     

Amyloid-beta peptide affects viability but not differentiation of embryonic and adult rat hippocampal progenitor cells

The neurological deficits that are characteristic of Alzheimer's Disease (AD) are ultimately a result of neuronal loss in distinct anatomical regions of the brain. This neuronal loss is thought to be due, in large part to the presence of the neurotoxic beta-amyloid (Abeta) deposits, that are characteristic of the AD brain. Transplantation therapy, in which neural stem cells (NSCs) or neural progenitor cells (NPCs) are introduced into damaged regions of the brain and induced to differentiate into replacement neurons, has been proposed as a possible therapeutic approach to treat AD. However, in the AD brain Abeta plaques, which remain in the area of neuronal degeneration, may affect the viability or differentiation potential of transplanted NSCs. Currently there is contradictory evidence concerning the effect of Abeta on NSCs. To further investigate the effect of Abeta on NSCs, we compared the mitochondrial function, proliferation and cellular differentiation of two populations of hippocampal NSCs (embryonic and adult derived) after Abeta exposure. Our results highlight the heterogeneity between different populations of NSCs even when derived from the same brain region. Our data also demonstrate that while mitochondrial function of NSCs is affected by Abeta, their proliferation and differentiation are not significantly influenced. Considered with previous studies, our results suggest that while NSCs do respond to the presence of Abeta, proliferation and differentiation of certain populations are not affected. Further study of the differences between susceptible vs. resistant populations of NSCs may provide crucial clues for the development of effective therapies to combat AD.     

Role of Exosomes Derived from miR-133b Modified MSCs in an Experimental Rat Model of Intracerebral Hemorrhage

Intracerebral hemorrhage (ICH) has poor outcomes due to high mortality and morbidity, but until now, the effective treatments remain limited. MicroRNAs (miRNAs) are vital regulators of gene expression and demonstrated to be linked to the pathogenesis of various central nervous system (CNS) diseases. Exosomes are considered as cell-to-cell communication vectors and secreted largely by mesenchymal stromal cells (MSCs). The present study investigated the role of miR-133b delivered by exosomes secreted from MSCs to brain tissues in rats after ICH. An autologous arterial blood ICH model in adult male Sprague–Dawley (SD) rats was used in this study. At 72 h after transfection with miR-133b mimics in MSCs, miR-133b-modified MSC-derived exosomes were collected from medium of MSCs and then injected to rats via tail vein. The levels of miR-133b in secreted exosomes and brain tissues of rats in various groups and the levels of RhoA, phosphorylations of extracellular signal regulating kinase (ERK1/2), and cAMP response element-binding protein (CREB) were detected by real-time PCR and western blot analysis, respectively. The effects of miR-133b on neuronal apoptosis and degeneration were respectively evaluated by TUNEL and fluoro-jade B staining. The miR-133b levels were reduced in brain tissues of rats at 24 h and peaked at 72 h after ICH. At 24 h after miR-133b-modified exosome administration, the level of miR-133b was significantly increased, while the apoptotic and neurodegenerative neurons were obviously reduced in brain tissues after ICH. The results of western blot analysis showed that miR-133b modified exosomes treatment remarkably suppressed RhoA expression and activated ERK1/2/CREB in brain tissues after ICH. Collectively, our investigation suggested that exosomes derived from miR-133b modified MSCs exhibited neuroprotective role for anti-apoptotic effect of miR-133b mediating RhoA and ERK1/2/CREB in rats after ICH.     

Collagen-chitosan scaffold impregnated with bone marrow mesenchymal stem cells for treatment of traumatic brain injury

Combinations of biomaterials and cells can effectively target delivery of cells or other therapeutic factors to the brain to rebuild damaged nerve pathways after brain injury.Porous collagen-chitosan scaffolds were prepared by a freeze-drying method based on brain tissue engineering.The scaffolds were impregnated with rat bone marrow mesenchymal stem cells.A traumatic brain injury rat model was established using the 300 g weight free fall impact method.Bone marrow mesenchymal stem cells/collagen-chitosan scaffolds were implanted into the injured brain.Modified neurological severity scores were used to assess the recovery of neurological function.The Morris water maze was employed to determine spatial learning and memory abilities.Hematoxylin-eosin staining was performed to measure pathological changes in brain tissue.Immunohistochemistry was performed for vascular endothelial growth factor and for 5-bromo-2-deoxyuridine(BrdU)/neuron specific enolase and BrdU/glial fibrillary acidic protein.Our results demonstrated that the transplantation of bone marrow mesenchymal stem cells and collagen-chitosan scaffolds to traumatic brain injury rats remarkably reduced modified neurological severity scores,shortened the average latency of the Morris water maze,increased the number of platform crossings,diminished the degeneration of damaged brain tissue,and increased the positive reaction of vascular endothelial growth factor in the transplantation and surrounding areas.At 14 days after transplantation,increased BrdU/glial fibrillary acidic protein expression and decreased BrdU/neuron specific enolase expression were observed in bone marrow mesenchymal stem cells in the injured area.The therapeutic effect of bone marrow mesenchymal stem cells and collagen-chitosan scaffolds was superior to stereotactic injection of bone marrow mesenchymal stem cells alone.To test the biocompatibility and immunogenicity of bone marrow mesenchymal stem cells and collagen-chitosan scaffolds,immunosuppressive cyclosporine was intravenously injected 12 hours before transplantation and 1-5 days after transplantation.The above indicators were similar to those of rats treated with bone marrow mesenchymal stem cells and collagen-chitosan scaffolds only.These findings indicate that transplantation of bone marrow mesenchymal stem cells in a collagen-chitosan scaffold can promote the recovery of neuropathological injury in rats with traumatic brain injury.This approach has the potential to be developed as a treatment for traumatic brain injury in humans.All experimental procedures were approved by the Institutional Animal Investigation Committee of Capital Medical University,China(approval No.AEEI-2015-035)in December 2015.     

Characterization of the secretome of human tooth germ stem cells (hTGSCs) reveals neuro-protection by fine-tuning micro-environment

Bone-marrow-derived mesenchymal stem cells (MSCs) demonstrate neuro-protective effects in several disease models. By producing growth-factors, cytokines and chemokines, they promote survival of neurons in damaged brain areas. Alternative MSC sources, such as human tooth germ stem cells (hTGSCs), have been investigated for their neuro-protective properties. They ameliorate effects of neuro-toxic agents by paracrine mechanisms, however these secreted bio-active molecules are not yet characterized. Therefore, the current study aimed to provide a detailed analysis of the secretome of hTGSCs. Brain cells were exposed to various toxic materials, including Alzheimer’s β-amyloid peptide (β-AP) and 6-hydroxy-dopamine (6-OHDA). When co-cultured with hTGSCs, the activity of a number of anti-oxidant enzymes (catalase, glutathione-s-transferase, glutathione-peroxidase, superoxide-dismutase) was increased and neuronal death/apoptosis was subsequently reduced. The composition of the secreted bio-active materials is influenced by various pre-existing factors such as oxygen and glucose deprivation and the age of cells (passage number). This report reveals for the first time that the neuro-protective secretome of hTGSCs and the micro-environment of cells have a mutual and dynamic impact on one another.