Neural stem cells: involvement in adult neurogenesis and CNS repair

Recent advances in stem cell research, including the selective expansion of neural stem cells (NSCs) in vitro, the induction of particular neural cells from embryonic stem cells in vitro, the identification of NSCs or NSC-like cells in the adult brain and the detection of neurogenesis in the adult brain (adult neurogenesis), have laid the groundwork for the development of novel therapies aimed at inducing regeneration in the damaged central nervous system (CNS). There are two major strategies for inducing regeneration in the damaged CNS: (i) activation of the endogenous regenerative capacity and (ii) cell transplantation therapy. In this review, we summarize the recent findings from our group and others on NSCs, with respect to their role in insult-induced neurogenesis (activation of adult NSCs, proliferation of transit-amplifying cells, migration of neuroblasts and survival and maturation of the newborn neurons), and implications for therapeutic interventions, together with tactics for using cell transplantation therapy to treat the damaged CNS.     

Regeneration of the central nervous system using endogenous repair mechanisms

Recent advances in developmental and stem cell biology have made regeneration-based therapies feasible as therapeutic strategies for patients with damaged central nervous systems (CNSs), including those with spinal cord injuries, Parkinson disease, or stroke. These strategies can be classified into two approaches: (i) the replenishment of lost neural cells and (ii) the induction of axonal regeneration. The first approach includes the activation of endogenous neural stem cells (NSCs) in the adult CNS and cell transplantation therapy. Endogenous NSCs have been shown to give rise to new neurons after insults, including ischemia, have been sustained; this form of neurogenesis followed by the migration and functional maturation of neuronal cells, as well as the responses of glial cells and the vascular system play crucial roles in endogenous repair mechanisms in damaged CNS tissue. In this review, we will summarize the recent advances in regeneration-based therapeutic approaches using endogenous NSCs, including the results of our own collaborative groups.     

Neural stem cell therapy for brain disease

Brain diseases, including brain tumors, neurodegenerative disorders, cerebrovascular diseases, and traumatic brain injuries, are among the major disorders influencing human health, currently with no effective therapy. Due to the low regeneration capacity of neurons, insufficient secretion of neurotrophic factors, and the aggravation of ischemia and hypoxia after nerve injury, irreversible loss of functional neurons and nerve tissue damage occurs. This damage is difficult to repair and regenerate the central nervous system after injury. Neural stem cells (NSCs) are pluripotent stem cells that only exist in the central nervous system. They have good self-renewal potential and ability to differentiate into neurons, astrocytes, and oligodendrocytes and improve the cellular microenvironment. NSC transplantation approaches have been made for various neurodegenerative disorders based on their regenerative potential. This review summarizes and discusses the characteristics of NSCs, and the advantages and effects of NSCs in the treatment of brain diseases and limitations of NSC transplantation that need to be addressed for the treatment of brain diseases in the future.     

Immunomodulation: The next target of mesenchymal stem cell-derived exosomes in the context of ischemic stroke

Ischemic stroke (IS) is the most prevalent form of brain disease, characterized by high morbidity, disability, and mortality. However, there is still a lack of ideal prevention and treatment measures in clinical practice. Notably, the transplantation therapy of mesenchymal stem cells (MSCs) has been a hot research topic in stroke. Nevertheless, there are risks associated with this cell therapy, including tumor formation, coagulation dysfunction, and vascular occlusion. Also, a growing number of studies suggest that the therapeutic effect after transplantation of MSCs is mainly attributed to MSC-derived exosomes (MSC-Exos). And this cell-free mediated therapy appears to circumvent many risks and difficulties when compared to cell therapy, and it may be the most promising new strategy for treating stroke as stem cell replacement therapy. Studies suggest that suppressing inflammation via modulation of the immune response is an additional treatment option for IS. Intriguingly, MSC-Exos mediates the inflammatory immune response following IS by modulating the central nervous system, the peripheral immune system, and immunomodulatory molecules, thereby promoting neurofunctional recovery after stroke. Thus, this paper reviews the role, potential mechanisms, and therapeutic potential of MSC-Exos in post-IS inflammation in order to identify new research targets.     

Purification of immature neuronal cells from neural stem cell progeny

Large-scale proliferation and multi-lineage differentiation capabilities make neural stem cells (NSCs) a promising renewable source of cells for therapeutic applications. However, the practical application for neuronal cell replacement is limited by heterogeneity of NSC progeny, relatively low yield of neurons, predominance of astrocytes, poor survival of donor cells following transplantation and the potential for uncontrolled proliferation of precursor cells. To address these impediments, we have developed a method for the generation of highly enriched immature neurons from murine NSC progeny. Adaptation of the standard differentiation procedure in concert with flow cytometry selection, using scattered light and positive fluorescent light selection based on cell surface antibody binding, provided a near pure (97%) immature neuron population. Using the purified neurons, we screened a panel of growth factors and found that bone morphogenetic protein-4 (BMP-4) demonstrated a strong survival effect on the cells in vitro, and enhanced their functional maturity. This effect was maintained following transplantation into the adult mouse striatum where we observed a 2-fold increase in the survival of the implanted cells and a 3-fold increase in NeuN expression. Additionally, based on the neural-colony forming cell assay (N-CFCA), we noted a 64 fold reduction of the bona fide NSC frequency in neuronal cell population and that implanted donor cells showed no signs of excessive or uncontrolled proliferation. The ability to provide defined neural cell populations from renewable sources such as NSC may find application for cell replacement therapies in the central nervous system.     

Treatment of multiple sclerosis by transplantation of neural stem cells derived from induced pluripotent stem cells

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS), with focal T lymphocytic infiltration and damage of myelin and axons. The underlying mechanism of pathogenesis remains unclear and there are currently no effective treatments. The development of neural stem cell (NSC) transplantation provides a promising strategy to treat neurodegenerative disease. However, the limited availability of NSCs prevents their application in neural disease therapy. In this study, we generated NSCs from induced pluripotent stem cells (iPSCs) and transplanted these cells into mice with experimental autoimmune encephalomyelitis (EAE), a model of MS. The results showed that transplantation of iPSC-derived NSCs dramatically reduced T cell infiltration and ameliorated white matter damage in the treated EAE mice. Correspondingly, the disease symptom score was greatly decreased, and motor ability was dramatically rescued in the iPSC-NSC-treated EAE mice, indicating the effectiveness of using iPSC-NSCs to treat MS. Our study provides pre-clinical evidence to support the feasibility of treating MS by transplantation of iPSC-derived NSCs.     

Neural stem cells as biological minipumps: a faster route to cell therapy for the CNS?

One strategy for the use of neural stem cells (NSCs) in treating neurological disorders is as transplantable "biological minipumps", in which genetically engineered neural stem cells serve as sources of secreted therapeutic (neuroprotective or tumoricidal) agents. Neural stem cells are highly mobile within the brain and demonstrate a tropism for various types of central nervous system (CNS) pathology, making them promising candidates for targeted gene delivery vehicles. Although neural stem cells have also been proposed as a potential source of replacement neurons and astrocytes to repopulate injured or degenerating neural circuits, the challenges involved in rebuilding damaged brain architecture are substantial and remain an active area of investigation. In contrast, the use of NSCs as biological minipumps does not rely on neuronal differentiation, axonal targeting, or synaptogenesis. This strategy may be a faster route to cell-based therapy of the CNS and is poised to move into human clinical trials. This review considers two types of neurologic disease that may be suitable targets for this alternative approach to NSC therapy: glial brain tumors and traumatic brain injury. We examine some of the key scientific and technical issues that must be addressed for the successful use of NSCs as minipumps.     

Stem cells act through multiple mechanisms to benefit mice with neurodegenerative metabolic disease

Intracranial transplantation of neural stem cells (NSCs) delayed disease onset, preserved motor function, reduced pathology and prolonged survival in a mouse model of Sandhoff disease, a lethal gangliosidosis. Although donor-derived neurons were electrophysiologically active within chimeric regions, the small degree of neuronal replacement alone could not account for the improvement. NSCs also increased brain beta-hexosaminidase levels, reduced ganglioside storage and diminished activated microgliosis. Additionally, when oral glycosphingolipid biosynthesis inhibitors (beta-hexosaminidase substrate inhibitors) were combined with NSC transplantation, substantial synergy resulted. Efficacy extended to human NSCs, both to those isolated directly from the central nervous system (CNS) and to those derived secondarily from embryonic stem cells. Appreciating that NSCs exhibit a broad repertoire of potentially therapeutic actions, of which neuronal replacement is but one, may help in formulating rational multimodal strategies for the treatment of neurodegenerative diseases.     

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.     

Neural stem cells traffic functional mitochondria via extracellular vesicles

Neural stem cell (NSC) transplantation induces recovery in animal models of central nervous system (CNS) diseases. Although the replacement of lost endogenous cells was originally proposed as the primary healing mechanism of NSC grafts, it is now clear that transplanted NSCs operate via multiple mechanisms, including the horizontal exchange of therapeutic cargoes to host cells via extracellular vesicles (EVs). EVs are membrane particles trafficking nucleic acids, proteins, metabolites and metabolic enzymes, lipids, and entire organelles. However, the function and the contribution of these cargoes to the broad therapeutic effects of NSCs are yet to be fully understood. Mitochondrial dysfunction is an established feature of several inflammatory and degenerative CNS disorders, most of which are potentially treatable with exogenous stem cell therapeutics. Herein, we investigated the hypothesis that NSCs release and traffic functional mitochondria via EVs to restore mitochondrial function in target cells. Untargeted proteomics revealed a significant enrichment of mitochondrial proteins spontaneously released by NSCs in EVs. Morphological and functional analyses confirmed the presence of ultrastructurally intact mitochondria within EVs with conserved membrane potential and respiration. We found that the transfer of these mitochondria from EVs to mtDNA-deficient L929 Rho⁰ cells rescued mitochondrial function and increased Rho⁰ cell survival. Furthermore, the incorporation of mitochondria from EVs into inflammatory mononuclear phagocytes restored normal mitochondrial dynamics and cellular metabolism and reduced the expression of pro-inflammatory markers in target cells. When transplanted in an animal model of multiple sclerosis, exogenous NSCs actively transferred mitochondria to mononuclear phagocytes and induced a significant amelioration of clinical deficits. Our data provide the first evidence that NSCs deliver functional mitochondria to target cells via EVs, paving the way for the development of novel (a)cellular approaches aimed at restoring mitochondrial dysfunction not only in multiple sclerosis, but also in degenerative neurological diseases.

This study shows that neural stem cells are able to transfer functional mitochondria via extracellular vesicles to target cells both in vitro and in vivo, suggesting that functional mitochondrial transfer via extracellular vesicles is a signaling mechanism used by neural stem cells to modulate the physiology and metabolism of target cells.     

Folic acid stimulates proliferation of transplanted neural stem cells after focal cerebral ischemia in rats

Folic acid (FA) stimulates neural stem cell (NSC) proliferation in vitro and enhances hippocampal neurogenesis in rats after middle cerebral artery occlusion (MCAO). The effect of FA supplementation on exogenous NSCs transplanted in MCAO rats was observed to determine if FA can stimulate NSC replacement after focal cerebral ischemia. Rats were randomly assigned to 3 groups: MCAO; MCAO and exogenous NSC transplantation (MCAO+NSCs); and MCAO, NSC transplantation and FA (MCAO+NSCs+FA). FA (0.8 mg/kg) or vehicle was administered by gavage daily for 28 days before MCAO and 23 days afterward. NSCs were labeled with superparamagnetic iron oxide (SPIO) and bromodeoxyuridine (BrdU) prior to transplantation into the striatum, contralateral to the ischemic zone, at 2 days post-MCAO. Magnetic resonance imaging tracking and fluorescent immunohistochemistry, as well as measurement of serum folate concentration, were performed at intervals up to 21 days after transplantation. FA supplementation caused sustained increases of 400–600% in serum folate concentration. Magnetic resonance images indicated that SPIO-labeled NSCs were more abundant at the transplantation and ischemic brain sites in MCAO+NSCs+FA rats than in MCAO+NSCs rats. Similarly, immunohistochemistry showed that the numbers of Sox-2/BrdU double positive cells at the transplantation and ischemic sites were higher in the rats that received FA. In conclusion, after focal cerebral ischemia, FA supplementation stimulates transplanted NSCs to proliferate and migrate to ischemic sites.     

Microenvironmental determinants of adult neural stem cell proliferation and lineage commitment in the healthy and injured central nervous system

The discovery of neural stem cells (NSC) which ensure continuous neurogenesis in the adult mammalian brain, has led to a conceptual revolution in basic neuroscience and to high hopes for clinical nervous tissue repair. However, several research issues remain to address before neural stem cells can be harnessed for regenerative therapies. The presence of NSC in a nervous structure is demonstrated in vitro by primary culture of dissociated adult nervous tissue in the presence of the specific mitogens EGF and bFGF. This leads to spherical masses of proliferating cells endowed with capacities for self-renewal and, after growth factor removal, differentiation into the three characteristic cell types of nervous tissue (neurons, astrocytes, oligodendrocytes). In vivo, neurogenesis per se, i.e. production of new neurons, occurs only in a small subset of NSC-endowed structures. The production of oligodendrocytes, i.e. myelinating glial cells, is similarly restricted. Such in vivo restrictions were formally demonstrated to arise from the tissular microenvironnement, which led to the emerging concept of "neurogenic niche". In this context, major challenges now consist in identifying the nature of tissue-specific extracellular signals that determine lineage commitment of NSC progeny, understanding why NSCs display weak in vivo reactivity to lesions compared to other stem cell types in adults, and identifying the factors behind the very high resistance to tumorigenesis displayed by NSCs. Altogether, the current data offer hope for the future use of adult NSCs in regenerative therapies, provided that tissue-specific signals are identified in view of counteracting the intrinsic repression of new cell genesis and/or stimulating endogenous NSC recruitment to lesion sites.     

Beta 1 integrin-mediated effects of tenascin-R domains EGFL and FN6-8 on neural stem/progenitor cell proliferation and differentiation in vitro

Neural stem/progenitor cells (NSCs) have the capacity for self-renewal and differentiation into major classes of central nervous system cell types, such as neurons, astrocytes, and oligodendrocytes. The determination of fate of NSCs appears to be regulated by both intrinsic and extrinsic factors. Mounting evidence has shown that extracellular matrix molecules contribute to NSC proliferation and differentiation as extrinsic factors. Here we explore the effects of the epidermal growth factor-like (EGFL) and fibronectin type III homologous domains 6-8 (FN6-8) of the extracellular matrix molecule tenascin-R on NSC proliferation and differentiation. Our results show that domain FN6-8 inhibited NSC proliferation and promoted NSCs differentiation into astrocytes and less into oligodendrocytes or neurons. The EGFL domain did not affect NSC proliferation, but promoted NSC differentiation into neurons and reduced NSC differentiation into astrocytes and oligodendrocytes. Treatment of NSCs with beta 1 integrin function-blocking antibody resulted in attenuation of inhibition of the effect of FN6-8 on NSC proliferation. The influence of EGFL or FN6-8 on NSCs differentiation was inhibited by beta 1 integrin antibody application, implicating beta 1 integrin in proliferation and differentiation induced by EGFL and FN6-8 mediated triggering of NSCs.     

Melatonin antagonizes interleukin‐18‐mediated inhibition on neural stem cell proliferation and differentiation

Neural stem cells (NSCs) are self‐renewing, pluripotent and undifferentiated cells which have the potential to differentiate into neurons, oligodendrocytes and astrocytes. NSC therapy for tissue regeneration, thus, gains popularity. However, the low survivals rate of the transplanted cell impedes its utilities. In this study, we tested whether melatonin, a potent antioxidant, could promote the NSC proliferation and neuronal differentiation, especially, in the presence of the pro‐inflammatory cytokine interleukin‐18 (IL‐18). Our results showed that melatonin per se indeed exhibited beneficial effects on NSCs and IL‐18 inhibited NSC proliferation, neurosphere formation and their differentiation into neurons. All inhibitory effects of IL‐18 on NSCs were significantly reduced by melatonin treatment. Moreover, melatonin application increased the production of both brain‐derived and glial cell‐derived neurotrophic factors (BDNF, GDNF) in IL‐18‐stimulated NSCs. It was observed that inhibition of BDNF or GDNF hindered the protective effects of melatonin on NSCs. A potentially protective mechanism of melatonin on the inhibition of NSC's differentiation caused IL‐18 may attribute to the up‐regulation of these two major neurotrophic factors, BNDF and GNDF. The findings indicate that melatonin may play an important role promoting the survival of NSCs in neuroinflammatory diseases.     

Getting the right stuff： controlling neural stem cell state and fate in vivo and in vitro with biomaterials

Stem cell therapy holds great promises in medical treatment by, e.g., replacing lost cells, re-constitute healthy cell populations and also in the use of stem cells as vehicles for factor and gene delivery. Embryonic stem cells have rightfully attracted a large interest due to their proven capacity of differentiating into any cell type in the embryo in vivo. Tissue-specific stem ceils are however already in use in medical practice, and recently the first systematic medical trials involving human neural stem cell （NSC） therapy have been launched. There are yet many obstacles to overcome and procedures to improve. To ensure progress in the medical use of stem cells increased basic knowledge of the molecular mechanisms that govern stem cell characteristics is necessary. Here we provide a review of the literature on NSCs in various aspects of cell therapy, with the main focus on the potential of using biomaterials to control NSC characteristics, differentiation, and delivery. We summarize results from studies on the characteristics of endogenous and transplanted NSCs in rodent models of neurological and cancer diseases, and highlight recent advancements in polymer compatibility and applicability in regulating NSC state and fate. We suggest that the development of specially designed polymers, such as hydrogels, is a crucial issue to improve the outcome of stem cell therapy in the central nervous system.     

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.     

Tauroursodeoxycholic acid increases neural stem cell pool and neuronal conversion by regulating mitochondria-cell cycle retrograde signaling

The low survival and differentiation rates of stem cells after either transplantation or neural injury have been a major concern of stem cell-based therapy. Thus, further understanding long-term survival and differentiation of stem cells may uncover new targets for discovery and development of novel therapeutic approaches. We have previously described the impact of mitochondrial apoptosis-related events in modulating neural stem cell (NSC) fate. In addition, the endogenous bile acid, tauroursodeoxycholic acid (TUDCA) was shown to be neuroprotective in several animal models of neurodegenerative disorders by acting as an anti-apoptotic and anti-oxidant molecule at the mitochondrial level. Here, we hypothesize that TUDCA might also play a role on NSC fate decision. We found that TUDCA prevents mitochondrial apoptotic events typical of early-stage mouse NSC differentiation, preserves mitochondrial integrity and function, while enhancing self-renewal potential and accelerating cell cycle exit of NSCs. Interestingly, TUDCA prevention of mitochondrial alterations interfered with NSC differentiation potential by favoring neuronal rather than astroglial conversion. Finally, inhibition of mitochondrial reactive oxygen species (mtROS) scavenger and adenosine triphosphate (ATP) synthase revealed that the effect of TUDCA is dependent on mtROS and ATP regulation levels. Collectively, these data underline the importance of mitochondrial stress control of NSC fate decision and support a new role for TUDCA in this process.     

Regulation of neural stem cell by bone morphogenetic protein (BMP) signaling during brain development

Neurogenesis is the process in which neurons are generated from neural stem/progenitor cells (NSCs/NPCs). It involves the proliferation and neuronal fate specification/differentiation of NSCs, as well as migration, maturation and functional integration of the neuronal progeny into neuronal network. NSCs exhibit the two essential properties of stem cells: self-renewal and multipotency. Contrary to previous dogma that neurogenesis happens only during development, it is generally accepted now that neurogenesis can take place throughout life in mammalian brains. This raises a new therapeutic potential of applying stem cell therapy for stroke, neurodegenerative diseases and other diseases. However, the maintenance and differentiation of NSCs/NPCs are tightly controlled by the extremely intricate molecular networks. Uncovering the underlying mechanisms that drive the differentiation, migration and maturation of specific neuronal lineages for use in regenerative medicine is, therefore, crucial for the application of stem cell for clinical therapy as well as for providing insight into the mechanisms of human neurogenesis. Here, we focus on the role of bone morphogenetic protein (BMP) signaling in NSCs during mammalian brain development.