Therapeutic neuronal stem cells: patents at the forefront

Background: Neural stem cells (NSCs) hold the promise to cure a broad range of neurological diseases and injuries, particularly neurodegenerative diseases and spinal cord injuries. The recent confirmation that neurogenesis occurs in the adult brain and that NSCs reside in the adult CNS opens new opportunities and avenues for cellular therapy. Objectives: To provide an overview of the current patent situation related to NSCs and to highlight the limitations and challenges of bringing NSC research to therapy. Method: Reviewing the early studies and patents in NSC research. Conclusion: NSCs are in clinical trials for Batten and Parkinson's diseases. However, clinical development and other limitations will make it difficult for pharmaceutical/biotech companies to break even with these early patents.     

Epigallocatechin-3-gallate rescues LPS-impaired adult hippocampal neurogenesis through suppressing the TLR4-NF-κB signaling pathway in mice

Adult hippocampal dentate granule neurons are generated from neural stem cells (NSCs) in the mammalian brain, and the fate specification of adult NSCs is precisely controlled by the local niches and environment, such as the subventricular zone (SVZ), dentate gyrus (DG), and Toll-like receptors (TLRs). Epigallocatechin-3-gallate (EGCG) is the main polyphenolic flavonoid in green tea that has neuroprotective activities, but there is no clear understanding of the role of EGCG in adult neurogenesis in the DG after neuroinflammation. Here, we investigate the effect and the mechanism of EGCG on adult neurogenesis impaired by lipopolysaccharides (LPS). LPS-induced neuroinflammation inhibited adult neurogenesis by suppressing the proliferation and differentiation of neural stem cells in the DG, which was indicated by the decreased number of Bromodeoxyuridine (BrdU)-, Doublecortin (DCX)- and Neuronal Nuclei (NeuN)-positive cells. In addition, microglia were recruited with activatingTLR4-NF-κB signaling in the adult hippocampus by LPS injection. Treating LPS-injured mice with EGCG restored the proliferation and differentiation of NSCs in the DG, which were decreased by LPS, and EGCG treatment also ameliorated the apoptosis of NSCs. Moreover, pro-inflammatory cytokine production induced by LPS was attenuated by EGCG treatment through modulating the TLR4-NF-κB pathway. These results illustrate that EGCG has a beneficial effect on impaired adult neurogenesis caused by LPSinduced neuroinflammation, and it may be applicable as a therapeutic agent against neurodegenerative disorders caused by inflammation.     

Neural stem cells: the basic biology and prospects for brain repair]

Neural stem cells (NSCs) are multipotential progenitor cells that have self-renewal activities. A single NSC is capable of generating various kinds of cells within the CNS, including neurons, astrocytes, and oligodendrocytes. Because of these characteristics, there is an increasing interest in NSCs and neural progenitor cells from the aspects of both basic developmental biology and therapeutic applications for damaged brain. By understanding the nature of NSCs present in the CNS, extracellular factors and signal transduction cascades involved in the differentiation and maintenance of NSCs, population dynamics and localization of NSCs in embryonic and adult brains, prospective identification and isolation of NSCs, and induction of NSCs into particular neuronal phenotypes, it would be possible to develop a feasible strategy to manipulate cells in situ to treat damaged brain.     

Novel strategies for discovering inhibitors of Dengue and Zika fever

Neurogenesis occurs in discrete regions of the adult brain, particularly the hippocampus. It is enhanced in the hippocampus of animal models and patients with neurological diseases and disorders, such as Alzheimer's disease (AD) and epilepsy. Adult hippocampal neurogenesis is modulated by drugs used for treating AD and depression, particularly galantamine, memantine and fluoxetine. This reveals that adult neurogenesis and newly generated neuronal cells of the adult hippocampus are involved in neurological diseases and disorders and that adult neurogenesis and neural stem cells (NSCs) of the adult hippocampus are the target of drugs used for treating AD and depression. Hence, adult neurogenesis and NSCs open new opportunities for our understanding of the pathology of the nervous system and new avenues to discover and develop novel drugs for treating neurogical diseases and disorders; drugs that would target specifically the NSCs of the neurogenic regions in the adult brain, or neurogenic drugs, and that would reverse or compensate deficits and impairments associated with neurological diseases and disorders, particularly those associated with the hippocampus. Adult NSCs represent a model to discover and develop novel drugs for treating neurological diseases and disorders. These drugs may also have potential for regenerative medicine and the treatment of brain tumors.     

Functional Multipotency of Stem Cells: A Conceptual Review of Neurotrophic Factor-Based Evidence and Its Role in Translational Research

We here propose an updated concept of stem cells (SCs), with an emphasis on neural stem cells (NSCs). The conventional view, which has touched principally on the essential property of lineage multipotency (e.g., the ability of NSCs to differentiate into all neural cells), should be broadened to include the emerging recognition of biofunctional multipotency of SCs to mediate systemic homeostasis, evidenced in NSCs in particular by the secretion of neurotrophic factors. Under this new conceptual context and taking the NSC as a leading example, one may begin to appreciate and seek the “logic” behind the wide range of molecular tactics the NSC appears to serve at successive developmental stages as it integrates into and prepares, modifies, and guides the surrounding CNS micro- and macro-environment towards the formation and self-maintenance of a functioning adult nervous system. We suggest that embracing this view of the “multipotency” of the SCs is pivotal for correctly, efficiently, and optimally exploiting stem cell biology for therapeutic applications, including reconstitution of a dysfunctional CNS.     

Adult neurogenesis and neural stem cells as a model for the discovery and development of novel drugs

Neurogenesis occurs in discrete regions of the adult brain, particularly the hippocampus. It is enhanced in the hippocampus of animal models and patients with neurological diseases and disorders, such as Alzheimer's disease (AD) and epilepsy. Adult hippocampal neurogenesis is modulated by drugs used for treating AD and depression, particularly galantamine, memantine and fluoxetine. This reveals that adult neurogenesis and newly generated neuronal cells of the adult hippocampus are involved in neurological diseases and disorders and that adult neurogenesis and neural stem cells (NSCs) of the adult hippocampus are the target of drugs used for treating AD and depression. Hence, adult neurogenesis and NSCs open new opportunities for our understanding of the pathology of the nervous system and new avenues to discover and develop novel drugs for treating neurogical diseases and disorders; drugs that would target specifically the NSCs of the neurogenic regions in the adult brain, or neurogenic drugs, and that would reverse or compensate deficits and impairments associated with neurological diseases and disorders, particularly those associated with the hippocampus. Adult NSCs represent a model to discover and develop novel drugs for treating neurological diseases and disorders. These drugs may also have potential for regenerative medicine and the treatment of brain tumors.     

Click chemistry extracellular vesicle/peptide/chemokine nanocarriers for treating central nervous system injuries

Central nervous system (CNS) injuries, including stroke, traumatic brain injury, and spinal cord injury, are essential causes of death and long-term disability and are difficult to cure, mainly due to the limited neuron regeneration and the glial scar formation. Herein, we apply extracellular vesicles (EVs) secreted by M2 microglia to improve the differentiation of neural stem cells (NSCs) at the injured site, and simultaneously modify them with the injured vascular targeting peptide (DA7R) and the stem cell recruiting factor (SDF-1) on their surface via copper-free click chemistry to recruit NSCs, inducing their neuronal differentiation, and serving as the nanocarriers at the injured site (Dual-EV). Results prove that the Dual-EV could target human umbilical vascular endothelial cells (HUVECs), recruit NSCs, and promote the neuronal differentiation of NSCs in vitro. Furthermore, 10 miRNAs are found to be upregulated in Dual-M2-EVs compared to Dual-M0-EVs via bioinformatic analysis, and further NSC differentiation experiment by flow cytometry reveals that among these miRNAs, miR30b-3p, miR-222-3p, miR-129-5p, and miR-155-5p may exert effect of inducing NSC to differentiate into neurons. In vivo experiments show that Dual-EV nanocarriers achieve improved accumulation in the ischemic area of stroke model mice, potentiate NSCs recruitment, and increase neurogenesis. This work provides new insights for the treatment of neuronal regeneration after CNS injuries as well as endogenous stem cells, and the click chemistry EV/peptide/chemokine and related nanocarriers for improving human health.     

Untangling the functional potential of PSA-NCAM-expressing cells in CNS development and brain repair strategies

Central nervous system (CNS) neural stem cells (NSCs), which are mostly defined by their ability to self-renew and to generate the three main cell lineages of the CNS, were isolated from discrete regions of the adult mammalian CNS including the subventricular zone (SVZ) of the lateral ventricle and the dentate gyrus in the hippocampus. At early stages of CNS cell fate determination, NSCs give rise to progenitors that express the polysialylated form of the neural cell adhesion molecule (PSA-NCAM). PSA-NCAM(+) cells persist in adult brain regions where neuronal plasticity and sustained formation of new neurons occur. PSA-NCAM has been shown to be involved in the regulation of CNS myelination as well as in changes of cell morphology that are necessary for motility, axonal guidance, synapse formation, and functional plasticity in the CNS. Although being preferentially committed to a restricted either glial or neuronal fate, cultured PSA-NCAM(plus) progenitors do preserve a relative degree of multipotentiality. Considering that PSA-NCAM(+) cells can be neatly used for brain repair purposes, there is much interest for studying signaling factors regulating their development. With this regard, it is noteworthy that neurotransmitters, which belong to the micro-environment of neural cells in vivo, regulate morphogenetic events preceding synaptogenesis such as cell proliferation, migration, differentiation and death. Consistently, several ionotropic but also G-protein-coupled neurotransmitter receptors were found to be expressed in CNS embryonic and postnatal progenitors. In the present review, we outlined the ins and outs of PSA-NCAM(plus) cells addressing to what extent our understanding of extrinsic and in particular neurotransmitter-mediated signaling in these CNS precursor cells might represent a new leading track to develop alternative strategies to stimulate brain repair.     

Neurogenesis in the adult brain

Neurogenesis is a process that involves cell proliferation, migration and differentiation. Adult neurogenesis has been discovered by Altman in the mid 1960s. It is known now that neurogenesis occurs in two main neurogenic areas of the adult mammalian brain: the olfactory bulb and the hippocampal dentate gyrus, although other brain regions, such as cortex or substantia nigra cannot be excluded. The rate of neurogenesis can be regulated in a positive and negative manner by several factors like, age, growth factors, hormones, environmental or pharmacological stimuli. Functional significance of adult neurogenesis is still under investigation, however, several evidences suggest involvement of newly generated neurons in cognitive processes. There are also several findings indicating that the impairment of adult neurogenesis may be involved in the pathophysiology of some brain diseases, like depression, epilepsy, ischemia or neurodegenerative disorders. It appears that alterations in the rate of neurogenesis may have important functional and therapeutic implications.     

A symphony of signals conducts early and late stages of adult neurogenesis

Neurogenesis is continually occurring in two regions within the mammalian central nervous system (CNS) and increasing evidence suggests that it is important for selective learning and memory. How this plasticity is maintained in isolated niches within mature networks has been extensively studied in recent years, and a large body of evidence has accumulated describing many different regulatory factors and points of regulation. In this review, we attempt to organize the current research by summarizing findings affecting early neurogenesis: during proliferation, fate commitment and migration, versus late neurogenesis: including dendritic development, synaptic integration, and survival. We discuss the roles of three different classes of factors regulating early and late phases of neurogenesis: intrinsic factors, extrinsic factors, and neurotransmitters. Finally, we suggest that neurotransmitters may act upstream from extracellular other factors and cell-intrinsic mechanisms by coupling network activity to the niche microenvironment and intracellular machinery to ultimately regulate neurogenesis.     

Neurogenic drugs and compounds to treat CNS diseases and disorders

Neurological diseases and related conditions affect an estimated 1 billion of individuals worldwide [1]. There is still no cure for neurological diseases and disorders, barely a few treatments more or less efficient. This mandates the design and development of novel paradigms and strategies, to discover and develop new treatments and cures for these diseases. Neurogenesis occurs in the adult brain of mammals primarily in two regions, the dentate gyrus (DG) of the hippocampus and the subventricular zone, in various species including in humans. Neural progenitor and stem cells have been isolated, propagated and characterized in vitro from the adult brain of mammals, including humans. The confirmation that neurogenesis occurs in the adult brain and neural stem cells (NSCs) reside in the adult central nervous system (CNS) of mammals, opens new avenues and opportunities for treating neurological diseases and injuries [2].     

An Aminopropyl Carbazole Derivative Induces Neurogenesis by Increasing Final Cell Division in Neural Stem Cells

P7C3 and its derivatives, 1-(3,6-dibromo-9H-carbazol-9-yl)-3-(p-tolylamino)propan-2-ol (1) and N-(3-(3,6-dibromo-9H-carbazol-9-yl)-2-hydroxypropyl)-N-(3-methoxyphenyl)-4-methylbenzenesulfonamide (2), were previously reported to increase neurogenesis in rat neural stem cells (NSCs). Although P7C3 is known to increase neurogenesis by protecting newborn neurons, it is not known whether its derivatives also have protective effects to increase neurogenesis. In the current study, we examined how 1 induces neurogenesis. The treatment of 1 in NSCs increased numbers of cells in the absence of epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2), while not affecting those in the presence of growth factors. Compound 1 did not induce astrocytogenesis during NSC differentiation. 5-Bromo-2′-deoxyuridine (BrdU) pulsing experiments showed that 1 significantly enhanced BrdU-positive neurons. Taken together, our data suggest that 1 promotes neurogenesis by the induction of final cell division during NSC differentiation.     

Neural stem cells as tools for drug discovery: novel platforms and approaches

Introduction: Neural stem cells catalyze strong interests for the development of systems to screen for effective drugs to treat neurodegenerative conditions and/or improve neurogenesis, fields where the classical approaches have so far failed in discovering successful drugs.

Areas covered: The authors review the known biology of NSCs, their normal function in development, the adult brain, and in vitro culture systems. The authors also discuss the scientific and technological progress which will aid wider applications of NSCs for drug screening/development purposes. The authors base this article on literature searches performed through PubMed and Google Scholar.

Expert opinion: NSC systems present unique opportunities that are starting to be successfully explored for genetic and chemical screening. These systems provide the possibility of identifying and optimizing molecules/drugs that could lead to the tighter control in self-renewal and lineage specification of NSCs as well as their functional maturation. This could be crucial in moving forward NSC-based therapies. It is expected that recent advances in the method of producing NSCs from patient-specific human induced pluripotent stem (iPS) cells and in the technologies to grow them in vitro, while preserving their full developmental potential, will allow a full exploitation of NSCs both in drug discovery programs and in predictive toxicology studies.     

Reducing histone acetylation rescues cognitive deficits in mouse model of fragile X syndrome

Fragile X syndrome(FXS) is the most prevalent inherited intellectual disability, resulting from a loss of fragile X mental retardation protein(FMRP). Patients with FXS suffer lifelong cognitive disabilities, but the function of FMRP in the adult brain and the mechanism underlying age-related cognitive decline in FXS is not fully understood. Here, we report that a loss of FMRP results in increased protein synthesis of histone acetyltransferase EP300 and ubiquitinationmediated degradation of histone deacetylase HDAC1 in adult hippocampal neural stem cells(NSCs). Consequently, FMRPdeficient NSCs exhibit elevated histone acetylation and age-related NSC depletion, leading to cognitive impairment in mature adult mice. Reducing histone acetylation rescues both neurogenesis and cognitive deficits in mature adult FMRPdeficient mice. Our work reveals a role for FMRP and histone acetylation in cognition and presents a potential novel therapeutic strategy for treating adult FXS patients.     

Adult neural stem cell therapy: expansion in vitro, tracking in vivo and clinical transplantation

Neural stem cells (NSCs) are present not only in the developing nervous systems, but also in the adult human central nervous system (CNS). It is long thought that the subventricular zone of the lateral ventricles and the dentate gyrus of the hippocampus are the main sources of human adult NSCs, which are considered to be a reservoir of new neural cells. Recently adult NSCs with potential neural capacity have been isolated from white matter and inferior prefrontal subcortex in the human brain. Rapid advances in the stem cell biology have raised appealing possibilities of replacing damaged or lost neural cells by transplantation of in vitro-expanded stem cells and/or their neuronal progeny. However, sources of stem cells, large scale expansion, control of the differentiations, and tracking in vivo represent formidable challenges. In this paper we review the characteristics of the adult human NSCs, their potentiality in terms of proliferation and differentiation capabilities, as well as their large scale expansion for clinical needs. This review focuses on the major advances in brain stem cell-based therapy from the clinical perspective, and summarizes our work in clinical phase I-II trials with autologuous transplantation of adult NSCs for patients with open brain trauma. It also describes multiple approaches to monitor adult human NSCs labeled superparamagnetic nanoparticles after transplantation and explores the intriguing possibility of stem cell transplantation.     

Neuron stem cell NLRP6 sustains hippocampal neurogenesis to resist stress-induced depression

Neurogenesis decline in hippocampal dentate gyrus (DG) participates in stress-induced depressive-like behaviors, but the underlying mechanism remains poorly understood. Here, we observed low-expression of NOD-like receptor family pyrin domain containing 6 (NLRP6) in hippocampus of stress-stimulated mice, being consistent with high corticosterone level. NLRP6 was found to be abundantly expressed in neural stem cells (NSCs) of DG. Both Nlrp6 knockout (Nlrp6^−/−) and NSC-conditional Nlrp6 knockout (Nlrp6CKO) mice were susceptible to stress, being more likely to develop depressive-like behaviors. Interestingly, NLRP6 was required for NSC proliferation in sustaining hippocampal neurogenesis and reinforcing stress resilience during growing up. Nlrp6 deficiency promoted esophageal cancer-related gene 4 (ECRG4) expression and caused mitochondrial dysfunction. Corticosterone as a stress factor significantly down-regulated NLRP6 expression, damaged mitochondrial function and suppressed cell proliferation in NSCs, which were blocked by Nlrp6 overexpression. ECRG4 knockdown reversed corticosterone-induced NSC mitochondrial function and cell proliferation disorders. Pioglitazone, a well-known clinical drug, up-regulated NLRP6 expression to inhibit ECRG4 expression in its protection against corticosterone-induced NSC mitochondrial dysfunction and proliferation restriction. In conclusion, this study demonstrates that NLRP6 is essential to maintain mitochondrial homeostasis and proliferation in NSCs, and identifies NLRP6 as a promising therapeutic target for hippocampal neurogenesis decline linked to depression.     

The Neural Cell Adhesion Molecule-Derived (NCAM)-Peptide FG Loop (FGL) Mobilizes Endogenous Neural Stem Cells and Promotes Endogenous Regenerative Capacity after Stroke

The neural cell adhesion molecule (NCAM)-derived peptide FG loop (FGL) modulates synaptogenesis, neurogenesis, and stem cell proliferation, enhances cognitive capacities, and conveys neuroprotection after stroke. Here we investigated the effect of subcutaneously injected FGL on cellular compartments affected by degeneration and regeneration after stroke due to middle cerebral artery occlusion (MCAO), namely endogenous neural stem cells (NSC), oligodendrocytes, and microglia. In addition to immunohistochemistry, we used non-invasive positron emission tomography (PET) imaging with the tracer [¹⁸F]-fluoro-L-thymidine ([¹⁸F]FLT) to visualize endogenous NSC in vivo. FGL significantly increased endogenous NSC mobilization in the neurogenic niches as evidenced by in vivo and ex vivo methods, and it induced remyelination. Moreover, FGL affected neuroinflammation. Extending previous in vitro results, our data show that the NCAM mimetic peptide FGL mobilizes endogenous NSC after focal ischemia and enhances regeneration by amplifying remyelination and modulating neuroinflammation via affecting microglia. Results suggest FGL as a promising candidate to promote recovery after stroke.     

Adult hippocampal neural stem/progenitor cells in vitro are vulnerable to the mycotoxin ochratoxin-A.

The common mycotoxin ochratoxin-A (OTA) accumulates in brain, causes oxidative stress, and elicits a DNA repair response that varies across brain regions and neuronal populations. Neural stem/progenitor cells (NSCs) prepared from hippocampus of adult mouse brain were tested for their vulnerability to the toxin in vitro. The following measurements were made in NSC cell cultures in both proliferation and differentiation media: (1) viability (trypan blue exclusion), (2) proliferative activity ([(3)H]-thymidine uptake), (3) the DNA repair response (oxyguanosine glycosylase activity), and (4) antioxidative response (superoxide dismutase). Cells that had proliferated to 90-100% confluency in the presence of epidermal growth factor and basic fibroblast growth factor were induced to differentiate by removal of the growth factors. OTA, added to the cultures in concentrations of 0.01-100 microg/ml, caused a dose- and time-dependent (6-72 h) decrease in viability of both proliferating and differentiating NSC. Proliferating NSC exhibited a greater vulnerability to the toxin than differentiated neurons despite robust DNA repair and antioxidative responses. Preconditioning of NSC with a 12-h incubation with the pro-oxidant diethyl maleate increased DNA repair activity and, subsequently, provided a moderate degree of neuroprotection. Overall, these results lead to speculation that OTA exposure may contribute to impaired hippocampal neurogenesis in vivo, resulting in depression and memory deficits, conditions reported to be linked to mycotoxin exposure in humans.     

嗅成鞘细胞与神经干细胞共移植对阿尔茨海默病大鼠海马生长相关蛋白-43表达的影响

目的探讨嗅成鞘细胞(OECs)与神经干细胞(NSCs)共移植对阿尔茨海默病(AD)大鼠海马生长相关蛋白(GAP)-43表达的影响。方法雄性SD大鼠双侧穹隆-海马伞切断,建立AD大鼠模型。实验动物分为4组:NSCs移植组、NSCs与OECs共移植组、AD模型组、正常对照组,每组30只。从SD胎鼠(孕16～18d)取材,在体外分别培养和共培养OECs与NSCs,并将共培养的OECs与NSCs移植于AD大鼠大脑侧脑室;观察各组大鼠行为学、宿主脑海马GAP-43和NOS阳性神经元表达的变化。结果①NSCs与OECs共移植组潜伏期明显缩短、通过平台次数明显增加,与NSCs移植组、AD模型组差异有统计学意义(P<0.05)。②反转录聚合酶链反应(RT-PCR)与Western Blot分析,NSCs与OECs共移植组宿主脑海马GAP-43mRNA和GAP-43蛋白的表达量高于NSCs移植组、AD模型组(P<0.05)。③NSCs与OECs共移植组NOS阳性细胞(22.2±1.7)较NSCs移植组(13.1±1.9)、AD模型组(7.1±0.9)明显增多(P<0.05)。结论嗅成鞘细胞与神经干细胞共移植能促进宿主脑海马GAP-43的表达,其可能在AD大鼠中枢神经系统损伤时具有促进神经再生和修复的作用。     

Fibroblast Growth Factor-2 Signaling in Neurogenesis and Neurodegeneration

Fibroblast growth factor-2 (FGF2), also known as basic FGF, is a multi-functional growth factor. One of the 22-member FGF family, it signals through receptor tyrosine kinases encoding FGFR1-4. FGF2 activates FGFRs in cooperation with heparin or heparin sulfate proteoglycan to induce its pleiotropic effects in different tissues and organs, which include potent angiogenic effects and important roles in the differentiation and function of the central nervous system (CNS). FGF2 is crucial to development of the CNS, which explains its importance in adult neurogenesis. During development, high levels of FGF2 are detected from neurulation onwards. Moreover, developmental expression of FGF2 and its receptors is temporally and spatially regulated, concurring with development of specific brain regions including the hippocampus and substantia nigra pars compacta. In adult neurogenesis, FGF2 has been implicated based on its expression and regulation of neural stem and progenitor cells in the neurogenic niches, the subventricular zone (SVZ) and the subgranular zone (SGZ) of the hippocampal dentate gyrus. FGFR1 signaling also modulates inflammatory signaling through the surface glycoprotein CD200, which regulates microglial activation. Because of its importance in adult neurogenesis and neuroinflammation, manipulation of FGF2/FGFR1 signaling has been a focus of therapeutic development for neurodegenerative disorders, such as Alzheimer’s disease, multiple sclerosis, Parkinson’s disease and traumatic brain injury. Novel strategies include intranasal administration of FGF2, administration of an NCAM-derived FGFR1 agonist, and chitosan-based nanoparticles for the delivery of FGF2 in pre-clinical animal models. In this review, we highlight current research towards therapeutic interventions targeting FGF2/FGFR1 in neurodegenerative disorders.