Substrate patterning: an emerging technology for the study of neuronal behavior

Extracellular matrix (ECM) proteins and cell-cell adhesion molecules (CAM) play important roles in neuronal development and differentiation. In the investigation of these roles, patterned substrates have proven to be a notably useful tool. Photolithographic and microprinting techniques can be used to make patterns of ECMs, CAMs, amino acids, and organofunctional groups for culturing neurons and other cell types. Experiments performed using these substrates have provided unique insights into the roles of cell-substratum adhesion, cell shape, and ECM composition on important cell functions, including survival, migration, neurite outgrowth, and development of polarity. Patterns may also be designed to localize cell bodies and confine their processes to predetermined areas of a substrate. Finally, the behavior of neurons on patterned substrates may prove helpful in the design of scaffoldings and nerve guides tailored for regeneration and repair of the nervous system.     

神经干细胞分化为神经元的基因调控

神经干细胞的研究进展为神经退行性病变和神经系统损伤的功能重建带来了令人振奋的希望。促使神经干细胞定向分化成所需神经元是实现神经干细胞临床应用的重要关键。目前对神经干细胞分化的分子机制还没有完全阐明。神经干细胞的分化与一系列因素促进转录因子表达有关,这些因子启动中枢神经系统基因表达程序从而导致神经干细胞向不同谱系分化。本文对神经干细胞分化为神经元过程中的基因调控的最新研究进展作一综述。     

核因子-κB在神经干细胞增殖和分化中的作用

核因子-κB（NF-κB）是一种可诱导的转录因子。在神经系统中，NF-κB由神经元、神经胶质细胞和神经干细胞表达。神经干细胞具有强大的增殖能力和多向分化潜能，在神经系统损伤的修复中起重要作用。近年来的研究发现，NF-κB信号系统参与了中枢神经系统神经干细胞增殖和分化的调控，通过对其机制的深入研究，有望为脑缺血、脑炎、神经变性等疾病的治疗开辟新的途径。文章对NF-κB在神经干细胞增殖和分化中的多重作用进行了综述。     

Induced pluripotent stem cell-derived neural stem cell therapies for spinal cord injury

The greatest challenge to successful treatment of spinal cord injury is the limited regenerative capacity of the central nervous system and its inability to replace lost neurons and severed axons following injury. Neural stem cell grafts derived from fetal central nervous system tissue or embryonic stem cells have shown therapeutic promise by differentiation into neurons and glia that have the potential to form functional neuronal relays across injured spinal cord segments. However, implementation of fetal-derived or embryonic stem cell-derived neural stem cell therapies for patients with spinal cord injury raises ethical concerns. Induced pluripotent stem cells can be generated from adult somatic cells and differentiated into neural stem cells suitable for therapeutic use, thereby providing an ethical source of implantable cells that can be made in an autologous fashion to avoid problems of immune rejection. This review discusses the therapeutic potential of human induced pluripotent stem cell-derived neural stem cell transplantation for treatment of spinal cord injury, as well as addressing potential mechanisms, future perspectives and challenges.     

Neonatal mouse cortical but not isogenic human astrocyte feeder layers enhance the functional maturation of induced pluripotent stem cell‐derived neurons in culture

Human induced pluripotent stem (iPS) cell‐derived neurons and astrocytes are attractive cellular tools for nervous system disease modeling and drug screening. Optimal utilization of these tools requires differentiation protocols that efficiently generate functional cell phenotypes in vitro. As nervous system function is dependent on networked neuronal activity involving both neuronal and astrocytic synaptic functions, we examined astrocyte effects on the functional maturation of neurons from human iPS cell‐derived neural stem cells (NSCs). We first demonstrate human iPS cell‐derived NSCs can be rapidly differentiated in culture to either neurons or astrocytes with characteristic cellular, molecular and physiological features. Although differentiated neurons were capable of firing multiple action potentials (APs), few cells developed spontaneous electrical activity in culture. We show spontaneous electrical activity was significantly increased by neuronal differentiation of human NSCs on feeder layers of neonatal mouse cortical astrocytes. In contrast, co‐culture on feeder layers of isogenic human iPS cell‐derived astrocytes had no positive effect on spontaneous neuronal activity. Spontaneous electrical activity was dependent on glutamate receptor‐channel function and occurred without changes in I_Na, I_K, V_m, and AP properties of iPS cell‐derived neurons. These data demonstrate co‐culture with neonatal mouse cortical astrocytes but not human isogenic iPS cell‐derived astrocytes stimulates glutamatergic synaptic transmission between iPS cell‐derived neurons in culture. We present RNA‐sequencing data for an immature, fetal‐like status of our human iPS cell‐derived astrocytes as one possible explanation for their failure to enhance synaptic activity in our co‐culture system.     

Hippocampal stem cells differentiate into excitatory and inhibitory neurons

Stem cell technology promises new and rapid advances in cell therapy and drug discovery. Clearly, the value of this approach will be limited by the differentiated functions displayed by the progeny of stem cells. The foetal and adult central nervous system (CNS) harbour stem cells that can be expanded in vitro and differentiate into immature neurons and glia. Surprisingly, we do not know if neurons derived from stem cells form synapses, a definitive feature of neuronal function. Neuronal differentiation is a complex process and in this paper we establish conditions that permit extensive maturation of neurons in the presence of neurotrophins. These conditions permit the differentiation of rat hippocampal stem cells into both excitatory (glutamatergic) and inhibitory (GABAergic) neurons. The proportion of excitatory and inhibitory synapses was strongly influenced by specific neurotrophins, and these responses reflect the region of origin of the stem cells in the brain. These data show that stem cells can be used to study mechanisms of excitation and inhibition in the nervous system.     

Regulation of adult olfactory neurogenesis by insulin-like growth factor-I

Insulin-like growth factor-I (IGF-I) has multiple effects within the developing nervous system but its role in neurogenesis in the adult nervous system is less clear. The adult olfactory mucosa is a site of continuing neurogenesis that expresses IGF-I, its receptor and its binding proteins. The aim of the present study was to investigate the roles of IGF-I in regulating proliferation and differentiation in the olfactory mucosa. The action of IGF-I was assayed in serum-free culture combined with bromodeoxyuridine-labelling of proliferating cells and immunochemistry for specific cell types. IGF-I and its receptor were expressed by globose basal cells (the neuronal precursor) and by olfactory neurons. IGF-I reduced the numbers of proliferating neuronal precursors, induced their differentiation into neurons and promoted morphological differentiation of neurons. The evidence suggests that IGF-I is an autocrine and/or paracrine signal that induces neuronal precursors to differentiate into olfactory sensory neurons. These effects appear to be similar to the cellular effects of IGF-I in the developing nervous system.     

Leukemia inhibitory factor is a proliferative factor for olfactory sensory neurons

Neuropoietic cytokines are known to play crucial roles in neuronal development. Among them, leukemia inhibitory factor (LIF) has been implicated in various processes of neuronal development, such as neuronal differentiation, survival and neurogenesis. Moreover, LIF is highly expressed in regions of the central nervous system where adult neurogenesis occurs. LIF was tested for its efficacy in promoting postnatal neurogenesis using LIF-null mice and dissociated cultures of early postnatal rat olfactory sensory neurons. Our results indicate that LIF promoted proliferation of olfactory sensory neuron precursors both in vivo and in vitro. In addition, LIF did not affect proliferation of non-neuronal cells. LIF may therefore be useful when developing stem cell therapy to replace damaged olfactory sensory neurons as well as a therapeutic agent to treat some anosmic symptoms.     

A role for nuclear PTEN in neuronal differentiation.

Mutations of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a protein and lipid phosphatase, have been associated with gliomas, macrocephaly, and mental deficiencies. We have assessed PTEN's role in the nervous system and find that PTEN is expressed in mouse brain late in development, starting at approximately postnatal day 0. In adult brain, PTEN is preferentially expressed in neurons and is especially evident in Purkinje neurons, olfactory mitral neurons, and large pyramidal neurons. To analyze the function of PTEN in neuronal differentiation, we used two well established model systems-pheochromocytoma cells and cultured CNS stem cells. PTEN is expressed during neurotrophin-induced differentiation and is detected in both the nucleus and cytoplasm. Suppression of PTEN levels with antisense oligonucleotides does not block initiation of neuronal differentiation. Instead, PTEN antisense leads to death of the resulting, immature neurons, probably during neurite extension. In contrast, PTEN is not required for astrocytic differentiation. These observations indicate that PTEN acts at multiple sites in the cell, regulating the transition of differentiating neuroblasts to postmitotic neurons.     

Oligodendrocyte and Extracellular Matrix Contributions to Central Nervous System Motor Function: Implications for Dystonia

The quest to elucidate nervous system function and dysfunction in disease has focused largely on neurons and neural circuits. However, fundamental aspects of nervous system development, function, and plasticity are regulated by nonneuronal elements, including glial cells and the extracellular matrix (ECM). The rapid rise of genomics and neuroimaging techniques in recent decades has highlighted neuronal–glial interactions and ECM as a key component of nervous system development, plasticity, and function. Abnormalities of neuronal–glial interactions have been understudied but are increasingly recognized to play a key role in many neurodevelopmental disorders. In this report, we consider the role of myelination and the ECM in the development and function of central nervous system motor circuits and the neurodevelopmental disease dystonia. © 2022 International Parkinson and Movement Disorder Society     

Ectopic expression of the immune adaptor protein CD3zeta in neural stem/progenitor cells disrupts cell-fate specification

Immune signaling and neuroinflammatory mediators have recently emerged as influential variables that regulate neural precursor/stem cell (NPC) behavior and function. In this study, we investigated whether the signaling adaptor protein CD3ζ, a transmembrane protein involved in T cell differentiation and function and recently shown to regulate neuronal development in the central nervous system (CNS), may have a role in NPC differentiation. We analyzed the expression profile of CD3ζ in embryonic rat brain during neurogenic periods and in neurosphere-derived neural cells, and we investigated the action of CD3ζ on cell differentiation. We found that CD3ζ expression coincided with neuronal commitment, but its forced expression in NPCs prevented the production of neurons and oligodendrocytes, but not astroglial cells. This blockade of neuronal differentiation was operated through an ITAM-independent mechanism, but required the Asp36 of the CD3ζ transmembrane domain involved in membrane receptor interaction. Together, our findings show that ectopic CD3ζ expression in NPCs impaired their normal cell-fate specification and suggest that variations of CD3ζ expression in the developing CNS might result in neurodevelopmental anomalies.     

Surface protein patterns govern morphology, proliferation, and expression of cellular markers but have no effect on physiological properties of cortical precursor cells

The ability to differentiate and give rise to neurons, astrocytes, and oligodendrocytes is an inherent feature of neural stem cells, which raises hopes for cell-based therapies of neurodegenerative diseases. However, there are many hurdles to cross before such regimens can be applied clinically. A considerable challenge is to elucidate the factors that contribute to neural differentiation. In this study, we evaluated the possibility of steering neuronal maturation by growing cortical precursor cells on microscale surface patterns of extracellular matrix (ECM) proteins. When the cells were encouraged to extend processes along lines of ECM proteins, they displayed a much more mature morphology, less proliferation capacity, and greater expression of a neuronal marker in comparison with cells grown in clusters on ECM dots. This implied that the growth pattern alone could play a crucial role for neural differentiation. However, in spite of the strikingly different morphology, when performing whole-cell patch-clamp experiments, we never observed any differences in the functional properties between cells grown on the two patterns. These results clearly demonstrate that morphological appearances are not representative measures of the functional phenotype or grade of neuronal maturation, stressing the importance of complementary electrophysiological evidence. To develop successful transplantation therapies, increased cell survival is critical. Because process-bearing neurons are sensitive and break easily, it would be of clinical interest to explore further the differentiating capacity of the cells cultured on the ECM dot pattern, described in this article, which are devoid of processes but display the same functional properties as neurons with mature morphology.     

Selective expression of an endogenous lactose-binding lectin gene in subsets of central and peripheral neurons

Cellular interactions in a variety of vertebrate non-neural tissues are thought to be mediated by cell surface carbohydrate structures. The detection of cell-specific surface carbohydrates and carbohydrate-binding proteins within the embryonic nervous system has raised the possibility that carbohydrate recognition may also contribute to the interactions of developing neurons. Soluble lactose-binding lectins constitute one class of carbohydrate-binding proteins expressed in the vertebrate nervous system. We describe here the isolation of cDNAs from rat brain libraries encoding one of these lectins, RL-14.5, and demonstrate that this protein is not only homologous to other soluble lectins, but also identical in primary sequence to a lectin present in at least one non-neural tissue. RNA blot analysis and in situ hybridization reveal a restricted pattern of expression of RL-14.5 mRNA within the rat nervous system. High levels of RL-14.5 mRNA are present in primary sensory neurons and motoneurons in the spinal cord and brain stem. Moreover, expression of RL-14.5 mRNA in sensory and motoneurons is detectable soon after neuronal differentiation. These findings, together with previous studies demonstrating the selective expression of oligosaccharide ligands for RL-14.5 on the same neurons, are consistent with the idea that carbohydrate-mediated interactions contribute to the development of this subset of mammalian neurons.     

Ischemia-induced neural stem/progenitor cells express pyramidal cell markers

Adult brain-derived neural stem cells have acquired a lot of interest as an endurable neuronal cell source that can be used for central nervous system repair in a wide range of neurological disorders such as ischemic stroke. Recently, we identified injury-induced neural stem/progenitor cells in the poststroke murine cerebral cortex. In this study, we show that, after differentiation in vitro, injury-induced neural stem/progenitor cells express pyramidal cell markers Emx1 and CaMKIIα, as well as mature neuron markers MAP2 and Tuj1. 5-bromo-2-deoxyuridinine-positive neurons in the peristroke cortex also express such pyramidal markers. The presence of newly regenerated pyramidal neurons in the poststroke brain might provide a noninvasive therapeutic strategy for stroke treatment with functional recovery.     

Expression of α5 integrin rescues fibronectin responsiveness in NT2N CNS neuronal cells

The extracellular matrix protein fibronectin is implicated in neuronal regeneration in the peripheral nervous system. In the central nervous system (CNS), fibronectin is up‐regulated at sites of penetrating injuries and stroke; however, CNS neurons down‐regulate the fibronectin receptor α5β1 integrin during differentiation and generally respond poorly to fibronectin. NT2N CNS neuron‐like cells (derived from NT2 precursor cells) have been used in preclinical and clinical studies for treatment of stroke and a variety of CNS injury and disease models. Here we show that, like primary CNS neurons, NT2N cells down‐regulate α5β1 integrin during differentiation and respond poorly to fibronectin. The poor neurite outgrowth by NT2N cells on fibronectin can be rescued by transducing NT2 precursors with a retroviral vector expressing α5 integrin under the control of the murine stem cell virus 5′ long terminal repeat. Sustained α5 integrin expression is compatible with the CNS‐like neuronal differentiation of NT2N cells and does not prevent robust neurite outgrowth on other integrin ligands. Thus, α5 integrin expression in CNS neuronal precursor cells may provide a strategy for enhancing the outgrowth and survival of implanted cells in cell‐replacement therapies for CNS injury and disease. © 2009 Wiley‐Liss, Inc.     

Extracellular matrix effects on neurosphere cell motility

There is a paucity of information on the roles of extracellular matrix (ECM) and substrate molecules in general with regard to the growth and differentiation of neural stem and progenitor cells. There are well-established findings of a dense, presumably astrocyte-derived ECM in the persistently neurogenic subependymal zone and its migratory extension the rostral migratory stream. Cells cultured from this region, as well as from early postnatal cerebellum, generate multipotent neurospheres, but at present there is little information as to the ECM regulation of these neural stem cell populations. The present study examined the behavior of cerebellar-derived neurospheres on the matrix components laminin, fibronectin, and chondroitin sulfate proteoglycan. The results showed that laminin and fibronectin significantly increase cell migration velocity as compared to CSPG. Fibronectin effected a maximal velocity after 48 h, whereas maximal velocity on laminin and CSPG was not reached until 72 h. Both laminin and fibronectin were very permissive substrates for cellular outgrowth. Chondroitin sulfate proteoglcyan showed a significant inhibition of migratory outgrowth and velocity. These ECM molecules did not appear to affect the fate choice of neurons and glia, thus their role in neuropoietic structures may be to facilitate or deter cell movement and process outgrowth.     

Advances in human stem cell therapies:pre-clinical studies and the outlook for central nervous system regeneration

Cell transplantation has come to the forefront of regenerative medicine alongside the discovery and application of stem cells in both research and clinical settings. There are several types of stem cells currently being used for pre-clinical regenerative therapies, each with unique characteristics, benefits and limitations. This brief review will focus on recent basic science advancements made with embryonic stem cells and induced pluripotent stem cells. Both embryonic stem cells and induced pluripotent stem cells provide platforms for new neurons to replace dead and/or dying cells following injury. Due to their capacity for reprogramming and differentiation into any neuronal type, research in preclinical rodent models has shown that embryonic stem cells and induced pluripotent stem cells can integrate, survive and form connections in the nervous system similar to de novo cells. Going forward however, there are some limitations to consider with the use of either stem cell type. Ethically, embryonic stem cells are not an ideal source of cells, genetically, induced pluripotent stem cells are not ideal in terms of personalized treatment for those with certain genetic diseases the latter of which may guide regenerative medicine away from personalized stem cell based therapies and into optimized stem cell banks. Nonetheless, the potential of these stem cells in central nervous system regenerative therapy is only beginning to be appreciated. For example, through genetic modification, stem cells serve as ideal platforms to reintroduce missing or downregulated molecules into the nervous system to further induce regenerative growth. In this review, we highlight the limitations of stem cell based therapies whilst discussing some of the means of overcoming these limitations.