Neural Stem Cell Maintenance Research Articles

In-vitro differentiation of human embryonic stem cells into spinal cord neural stem cells.

In-vitro differentiation of human embryonic stem cells into spinal cord neural stem cells (NSCs) can help researchers better understand the cellular processes associated with spinal cord development and regeneration, and provide therapeutic strategies for spinal cord disorders. However, effective and consistent methods for the generation of human spinal cord NSCs are rare. Objective of the study is to establish methods for the in-vitro induction and long-term maintenance of human spinal cord NSCs. H9 cells were treated with neural induction medium for 10 days under single-cell seeding condition, followed by treatment with neural maintenance medium and replacement with NSC medium after five passages. The identity of the generated cells was determined by immunofluorescence, immunoblotting, and cleavage under targets and tagmentation (CUT&Tag) assays. After the neural induction, OCT4, an embryonic stem cell marker, was significantly reduced, whereas NESTIN and PAX6, two NSC markers, were clearly increased. After the neural maintenance, most of the H9-derived cells consistently expressed NESTIN and PAX6 together with SOX1 and HOXC9, two spinal cord markers. The Homer known motif enrichment results of the CUT&Tag assay confirmed the expression of HOXC9 in the H9-derived spinal cord NSCs, which can be maintained for more than 40 days under an in vitro culture system. This study sheds new light on effective induction and maintenance of human spinal cord NSCs.

MicroRNA-582-5p Contributes to the Maintenance of Neural Stem Cells Through Inhibiting Secretory Protein FAM19A1.

Epigenetic regulations on the maintenance of neural stem cells (NSCs) are complicated and far from been fully understood. Our previous findings have shown that after blocking Notch signaling in NSCs in vivo, the stemness of NSCs decreases, accompanied by the downregulated expression of miR-582-5p. In the current study, we further investigated the function and mechanism of miR-582-5p in the maintenance of NSCs in vitro and in vivo. After transfecting a mimic of miR-582-5p, the formation of neurospheres and proliferation of NSCs and intermediate progenitor cells (NS/PCs) were enhanced, and the expression of stemness markers such as Sox2, Nestin, and Pax6 also increased. The results were reversed after transfection of an inhibitor of miR-582-5p. We further generated miR-582 knock-out (KO) mice to investigate its function in vivo, and we found that the number of NSCs in the subventricular zone (SVZ) region decreased and the number of neuroblasts increased in miR-582 deficient mice, indicating reduced stemness and enhanced neurogenesis of NSCs. Moreover, RNA-sequencing and molecular biological analysis revealed that miR-582-5p regulates the stemness and proliferation of NSCs by inhibiting secretory protein FAM19A1. In summary, our research uncovered a new epigenetic mechanism that regulates the maintenance of NSCs, therefore providing novel targets to amplify NSCs in vitro and to promote neurogenesis in vivo during brain pathology and aging.

Zebrafish: A New Promise to Study the Impact of Metabolic Disorders on the Brain.

Zebrafish has become a popular model to study many physiological and pathophysiological processes in humans. In recent years, it has rapidly emerged in the study of metabolic disorders, namely, obesity and diabetes, as the regulatory mechanisms and metabolic pathways of glucose and lipid homeostasis are highly conserved between fish and mammals. Zebrafish is also widely used in the field of neurosciences to study brain plasticity and regenerative mechanisms due to the high maintenance and activity of neural stem cells during adulthood. Recently, a large body of evidence has established that metabolic disorders can alter brain homeostasis, leading to neuro-inflammation and oxidative stress and causing decreased neurogenesis. To date, these pathological metabolic conditions are also risk factors for the development of cognitive dysfunctions and neurodegenerative diseases. In this review, we first aim to describe the main metabolic models established in zebrafish to demonstrate their similarities with their respective mammalian/human counterparts. Then, in the second part, we report the impact of metabolic disorders (obesity and diabetes) on brain homeostasis with a particular focus on the blood–brain barrier, neuro-inflammation, oxidative stress, cognitive functions and brain plasticity. Finally, we propose interesting signaling pathways and regulatory mechanisms to be explored in order to better understand how metabolic disorders can negatively impact neural stem cell activity.

Stemness Maintenance and Massproduction of Neural Stem Cells on Poly L-Lactic Acid Nanofibrous Membrane Based on Piezoelectriceffect.

Neural stem cells (NSCs) therapy is promising for treating neurodegenerative disorders and neural injuries. However, the limited in vitro expansion, spontaneous differentiation, and decrease in stemness obstruct the acquisition of high quantities of NSCs, restricting the clinical application of cell-based therapies and tissue engineering. This article reports a facile method of promoting NSCs expansion and maintaining stemness using wireless electrical stimulation triggered by piezoelectric nanomaterials. A nanofibrous membrane of poly L-lactic acid (PLLA) is prepared by electrostatic spinning, and the favorable piezoelectric property of PLLA facilitates the freeing of electrons after transformation. These self-powered electric signals generated by PLLA significantly enhance NSCs proliferation. Further, an undifferentiated cellular state is maintained in the NSCs cultured on the surfaces of PLLA nanofibers exposed to ultrasonic vibration. In addition, the neural differentiation potencies and functions of NSCs expanded by piezoelectric-driven localized electricity are not attenuated. Moreover, cell stemness can be maintained by wireless electric stimulation. Taken together, the electronic signals mediated by PLLA nanofibers facilitate NSCs proliferation. This efficient and simple strategy can maintain the stemness of NSCs during proliferation, which is essential for their clinical application, and opens up opportunities for the mass production of NSCs for use in cell therapy.

BuMPing Into Neurogenesis: How the Canonical BMP Pathway Regulates Neural Stem Cell Divisions Throughout Space and Time.

Bone morphogenetic proteins (BMPs) are secreted factors that contribute to many aspects of the formation of the vertebrate central nervous system (CNS), from the initial shaping of the neural primordium to the maturation of the brain and spinal cord. In particular, the canonical (SMAD1/5/8-dependent) BMP pathway appears to play a key role during neurogenesis, its activity dictating neural stem cell fate decisions and thereby regulating the growth and homeostasis of the CNS. In this mini-review, I summarize accumulating evidence demonstrating how the canonical BMP activity promotes the amplification and/or maintenance of neural stem cells at different times and in diverse regions of the vertebrate CNS, and highlight findings suggesting that this function is evolutionarily conserved.

ATM modulates subventricular zone neural stem cell maintenance and senescence through Notch signaling pathway.

Ataxia telangiectasia mutated (ATM) plays an essential role in DNA damage response and the maintenance of genomic stability. However, the role of ATM in regulating the function of adult neural stem cells (NSCs) remains unclear. Here we report that ATM deficiency led to accumulated DNA damage and decreased DNA damage repair capacity in neural progenitor cells. Moreover, we observed ATM ablation lead to the short-term increase of proliferation of neural progenitor cells, resulting in the depletion of the NSC pool over time, and this loss of NSC quiescence resulted in accelerated cell senescence. We further apply RNA sequencing to unravel that ATM knockout significantly affected Notch signaling pathway, furthermore, notch activation inhibit the abnormal increased proliferation of ATM-/- NSCs. Taken together, these findings indicate that ATM can serve as a key regulator for the normal function of adult NSCs by maintaining their stemness and preventing cellular senescence primarily through Notch signaling pathway.

Roles of ERRα and TGF-β signaling in stemness enhancement induced by 1 µM bisphenol A exposure via human neural stem cells.

Bisphenol A (BPA) is a common industrial chemical widely used to produce various plastics and is known to impair neural stem cells (NSCs). However, the effects of low-dose BPA exposure on the stemness maintenance and differentiation fate of NSCs remain unclear in the infant brain. The present study demonstrated that 1 µM BPA promoted human NSC proliferation and stemness, without significantly increasing apoptosis. The Chip-seq experiments demonstrated that both the cell cycle and the TGF-β signaling pathway were accelerated after treatment with 1 µM BPA. Subsequently, estrogen-related receptor α (ERRα) gene knockout cell lines were constructed using CRISPR/Cas9. Further western blotting and chromatin immunoprecipitation-PCR experiments demonstrated that BPA maintained cell stemness by binding to an EERα receptor and activating the TGF-β1 signaling pathway, including the downstream factors Aurora kinases B and Id2. In conclusion, the stemness of NSCs could be maintained by BPA at 1 µM through the activation of the ERRα and TGF-β1 signaling pathways and could restrain the differentiation of NSCs into neurons. The present research further clarified the mechanism of BPA toxicity on NSCs from the novel perspective of ERRα and TGF-β1 signaling pathways regulated by BPA and provided insights into potential novel methods of prevention and therapy for neurogenic diseases.

IL2 Neural stem cell regulation and brain development

Quiescent neural stem cells (NSCs) in the adult mouse brain are the source of neurogenesis that regulates innate and adaptive behaviors. Adult NSCs in the subventricular zone (SVZ) are derived from a subpopulation of embryonic neural stem-progenitor cells (NPCs) that is characterized by a slower cell cycle relative to the more abundant rapid cycling NPCs that build the brain. We have previously shown that slow cell cycle can cause the establishment of adult NSCs at the SVZ, although the underlying mechanism remains unknown. We found that Notch and an effector Hey1 form a module that is upregulated by cell cycle arrest in slowly dividing NPCs. In contrast to the oscillatory expression of the Notch effectors Hes1 and Hes5 in fast cycling progenitors, Hey1 displays a non-oscillatory stationary expression pattern and contributes to the long-term maintenance of NSCs. These findings reveal a novel division of labor in Notch effectors where cell cycle rate biases effector selection and cell fate. I will also discuss the heterogeneity of slowly dividing embryonic NPCs and the lineage relationship between adult NSCs and ependymal cells, which together form the niche for adult neurogenesis at the SVZ.

Non-canonical function of FIP200 is required for neural stem cell maintenance and differentiation by limiting TBK1 activation and p62 aggregate formation

FIP200 is an essential autophagy gene implicated in the regulation of postnatal neural progenitor/stem cells (NSCs). However, the contribution of FIP200’s canonical-autophagy function and its non-canonical functions to postnatal NSC maintenance remains unclear. Utilizing a recently generated Fip200-4A allele that specifically impairs FIP200’s canonical-autophagy function, we found that non-canonical functions of FIP200 was required for regulation of mouse NSC maintenance and neurogenesis in vivo. Ablating the non-canonical functions of FIP200, but not its autophagy function, increased TBK1 activation and p62 phosphorylation at S403 in NSCs. Phosphorylation of p62 was dependent on TBK1 kinase activity and increased the propensity of p62 aggregate formation specifically in FIP200-null NSCs. Accordingly, inhibition of TBK1 by amlexanox reduced p62 aggregates and restored NSC maintenance and differentiation in Fip200hGFAP cKO mice. These results reveal a mechanism for the non-canonical functions of FIP200 in NSC maintenance and differentiation by limiting TBK1 activation and subsequently, p62 aggregate formation.

The Fine Art of Writing a Message: RNA Metabolism in the Shaping and Remodeling of the Nervous System.

Neuronal morphogenesis, integration into circuits, and remodeling of synaptic connections occur in temporally and spatially defined steps. Accordingly, the expression of proteins and specific protein isoforms that contribute to these processes must be controlled quantitatively in time and space. A wide variety of post-transcriptional regulatory mechanisms, which act on pre-mRNA and mRNA molecules contribute to this control. They are thereby critically involved in physiological and pathophysiological nervous system development, function, and maintenance. Here, we review recent findings on how mRNA metabolism contributes to neuronal development, from neural stem cell maintenance to synapse specification, with a particular focus on axon growth, guidance, branching, and synapse formation. We emphasize the role of RNA-binding proteins, and highlight their emerging roles in the poorly understood molecular processes of RNA editing, alternative polyadenylation, and temporal control of splicing, while also discussing alternative splicing, RNA localization, and local translation. We illustrate with the example of the evolutionary conserved Musashi protein family how individual RNA-binding proteins are, on the one hand, acting in different processes of RNA metabolism, and, on the other hand, impacting multiple steps in neuronal development and circuit formation. Finally, we provide links to diseases that have been associated with the malfunction of RNA-binding proteins and disrupted post-transcriptional regulation.

T-Cell Factors as Transcriptional Inhibitors: Activities and Regulations in Vertebrate Head Development.

Since its first discovery in the late 90s, Wnt canonical signaling has been demonstrated to affect a large variety of neural developmental processes, including, but not limited to, embryonic axis formation, neural proliferation, fate determination, and maintenance of neural stem cells. For decades, studies have focused on the mechanisms controlling the activity of β-catenin, the sole mediator of Wnt transcriptional response. More recently, the spotlight of research is directed towards the last cascade component, the T-cell factor (TCF)/Lymphoid-Enhancer binding Factor (LEF), and more specifically, the TCF/LEF-mediated switch from transcriptional activation to repression, which in both embryonic blastomeres and mouse embryonic stem cells pushes the balance from pluri/multipotency towards differentiation. It has been long known that Groucho/Transducin-Like Enhancer of split (Gro/TLE) is the main co-repressor partner of TCF/LEF. More recently, other TCF/LEF-interacting partners have been identified, including the pro-neural BarH-Like 2 (BARHL2), which belongs to the evolutionary highly conserved family of homeodomain-containing transcription factors. This review describes the activities and regulatory modes of TCF/LEF as transcriptional repressors, with a specific focus on the functions of Barhl2 in vertebrate brain development. Specific attention is given to the transcriptional events leading to formation of the Organizer, as well as the roles and regulations of Wnt/β-catenin pathway in growth of the caudal forebrain. We present TCF/LEF activities in both embryonic and neural stem cells and discuss how alterations of this pathway could lead to tumors.

Cell cycle arrest determines adult neural stem cell ontogeny by an embryonic Notch-nonoscillatory Hey1 module

Quiescent neural stem cells (NSCs) in the adult mouse brain are the source of neurogenesis that regulates innate and adaptive behaviors. Adult NSCs in the subventricular zone are derived from a subpopulation of embryonic neural stem-progenitor cells (NPCs) that is characterized by a slower cell cycle relative to the more abundant rapid cycling NPCs that build the brain. Yet, how slow cell cycle can cause the establishment of adult NSCs remains largely unknown. Here, we demonstrate that Notch and an effector Hey1 form a module that is upregulated by cell cycle arrest in slowly dividing NPCs. In contrast to the oscillatory expression of the Notch effectors Hes1 and Hes5 in fast cycling progenitors, Hey1 displays a non-oscillatory stationary expression pattern and contributes to the long-term maintenance of NSCs. These findings reveal a novel division of labor in Notch effectors where cell cycle rate biases effector selection and cell fate.

Neuron-Radial Glial Cell Communication via BMP/Id1 Signaling Is Key to Long-Term Maintenance of the Regenerative Capacity of the Adult Zebrafish Telencephalon.

The central nervous system of adult zebrafish displays an extraordinary neurogenic and regenerative capacity. In the zebrafish adult brain, this regenerative capacity relies on neural stem cells (NSCs) and the careful management of the NSC pool. However, the mechanisms controlling NSC pool maintenance are not yet fully understood. Recently, Bone Morphogenetic Proteins (BMPs) and their downstream effector Id1 (Inhibitor of differentiation 1) were suggested to act as key players in NSC maintenance under constitutive and regenerative conditions. Here, we further investigated the role of BMP/Id1 signaling in these processes, using different genetic and pharmacological approaches. Our data show that BMPs are mainly expressed by neurons in the adult telencephalon, while id1 is expressed in NSCs, suggesting a neuron-NSC communication via the BMP/Id1 signaling axis. Furthermore, manipulation of BMP signaling by conditionally inducing or repressing BMP signaling via heat-shock, lead to an increase or a decrease of id1 expression in the NSCs, respectively. Induction of id1 was followed by an increase in the number of quiescent NSCs, while knocking down id1 expression caused an increase in NSC proliferation. In agreement, genetic ablation of id1 function lead to increased proliferation of NSCs, followed by depletion of the stem cell pool with concomitant failure to heal injuries in repeatedly injured mutant telencephala. Moreover, pharmacological inhibition of BMP and Notch signaling suggests that the two signaling systems cooperate and converge onto the transcriptional regulator her4.1. Interestingly, brain injury lead to a depletion of NSCs in animals lacking BMP/Id1 signaling despite an intact Notch pathway. Taken together, our data demonstrate how neurons feedback on NSC proliferation and that BMP1/Id1 signaling acts as a safeguard of the NSC pool under regenerative conditions.

Optimal Extracellular Matrix Niches for Neurogenesis: Identifying Glycosaminoglycan Chain Composition in the Subventricular Neurogenic Zone.

In the adult mammalian brain, new neurons are generated in a restricted region called the neurogenic niche, which refers to the specific regulatory microenvironment of neural stem cells (NSCs). Among the constituents of neurogenic niches, the extracellular matrix (ECM) has emerged as a key player in NSC maintenance, proliferation, and differentiation. In particular, heparan sulfate (HS) proteoglycans are capable of regulating various growth factor signaling pathways that influence neurogenesis. In this review, we summarize our current understanding of the ECM niche in the adult subventricular zone (SVZ), with a special focus on basement membrane (BM)-like structures called fractones, and discuss how fractones, particularly their composition of glycosaminoglycans (GAGs), may influence neurogenesis.

Effects of deubiquitylases on the biological behaviors of neural stem cells.

New neurons are generated throughout life in distinct regions of the mammalian brain due to the proliferation and differentiation of neural stem cells (NSCs). Ubiquitin, a post-translational modification of cellular proteins, is an important factor in regulating neurogenesis. Deubiquitination is a biochemical process that mediates the removal of ubiquitin moieties from ubiquitin-conjugated substrates. Recent studies have provided growing evidence that deubiquitylases (DUBs) which reverse ubiquitylation process play critical roles in NSCs maintenance, differentiation and maturation. This review mainly focused on the relationship of DUBs and NSCs, and further summarized recent advances in our understanding of DUBs on regulating NSCs biological behaviors.

A Shift in Tissue Stiffness During Hippocampal Maturation Correlates to the Pattern of Neurogenesis and Composition of the Extracellular Matrix.

Aging changes the mechanical properties of brain tissue, such as stiffness. It has been proposed that the maintenance and differentiation of neural stem cells (NSCs) are regulated in accordance with extracellular stiffness. Neurogenesis is observed in restricted niches, including the dentate gyrus (DG) of the hippocampus, throughout mammalian lifetimes. However, profiles of tissue stiffness in the DG in comparison with the activity of NSCs from the neonatal to the matured brain have rarely been addressed so far. Here, we first applied ultrasound-based shear-wave elasticity imaging (SWEI) in living animals to assess shear modulus as in vivo brain stiffness. To complement the assay, atomic force microscopy (AFM) was utilized to determine the Young’s modulus in the hippocampus as region-specific stiffness in the brain slice. The results revealed that stiffness in the granule cell layer (GCL) and the hilus, including the subgranular zone (SGZ), increased during hippocampal maturation. We then quantified NSCs and immature neural cells in the DG with differentiation markers, and verified an overall decrease of NSCs and proliferative/immature neural cells along stages, showing that a specific profile is dependent on the subregion. Subsequently, we evaluated the amount of chondroitin sulfate proteoglycans (CSPGs), the major extracellular matrix (ECM) components in the premature brain by CS-56 immunoreactivity. We observed differential signal levels of CSPGs by hippocampal subregions, which became weaker during maturation. To address the contribution of the ECM in determining tissue stiffness, we manipulated the function of CSPGs by enzymatic digestion or supplementation with chondroitin sulfate, which resulted in an increase or decrease of stiffness in the DG, respectively. Our results illustrate that stiffness in the hippocampus shifts due to the composition of ECM, which may affect postnatal neurogenesis by altering the mechanical environment of the NSC niche.

FOS Rescues Neuronal Differentiation of Sox2-Deleted Neural Stem Cells by Genome-Wide Regulation of Common SOX2 and AP1(FOS-JUN) Target Genes

The transcription factor SOX2 is important for brain development and for neural stem cells (NSC) maintenance. Sox2-deleted (Sox2-del) NSC from neonatal mouse brain are lost after few passages in culture. Two highly expressed genes, Fos and Socs3, are strongly downregulated in Sox2-del NSC; we previously showed that Fos or Socs3 overexpression by lentiviral transduction fully rescues NSC’s long-term maintenance in culture. Sox2-del NSC are severely defective in neuronal production when induced to differentiate. NSC rescued by Sox2 reintroduction correctly differentiate into neurons. Similarly, Fos transduction rescues normal or even increased numbers of immature neurons expressing beta-tubulinIII, but not more differentiated markers (MAP2). Additionally, many cells with both beta-tubulinIII and GFAP expression appear, indicating that FOS stimulates the initial differentiation of a “mixed” neuronal/glial progenitor. The unexpected rescue by FOS suggested that FOS, a SOX2 transcriptional target, might act on neuronal genes, together with SOX2. CUT&RUN analysis to detect genome-wide binding of SOX2, FOS, and JUN (the AP1 complex) revealed that a high proportion of genes expressed in NSC are bound by both SOX2 and AP1. Downregulated genes in Sox2-del NSC are highly enriched in genes that are also expressed in neurons, and a high proportion of the “neuronal” genes are bound by both SOX2 and AP1.

The tweety Gene Family: From Embryo to Disease.

The tweety genes encode gated chloride channels that are found in animals, plants, and even simple eukaryotes, signifying their deep evolutionary origin. In vertebrates, the tweety gene family is highly conserved and consists of three members—ttyh1, ttyh2, and ttyh3—that are important for the regulation of cell volume. While research has elucidated potential physiological functions of ttyh1 in neural stem cell maintenance, proliferation, and filopodia formation during neural development, the roles of ttyh2 and ttyh3 are less characterized, though their expression patterns during embryonic and fetal development suggest potential roles in the development of a wide range of tissues including a role in the immune system in response to pathogen-associated molecules. Additionally, members of the tweety gene family have been implicated in various pathologies including cancers, particularly pediatric brain tumors, and neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease. Here, we review the current state of research using information from published articles and open-source databases on the tweety gene family with regard to its structure, evolution, expression during development and adulthood, biochemical and cellular functions, and role in human disease. We also identify promising areas for further research to advance our understanding of this important, yet still understudied, family of genes.

Nuclear isoform of FGF13 regulates post-natal neurogenesis in the hippocampus through an epigenomic mechanism.

The hippocampus is one of two niches in the mammalian brain with persistent neurogenesis into adulthood. The neurogenic capacity of hippocampal neural stem cells (NSCs) declines with age, but the molecular mechanisms of this process remain unknown. In this study, we find that fibroblast growth factor 13 (FGF13) is essential for the post-natal neurogenesis in mouse hippocampus, and FGF13 deficiency impairs learning and memory. In particular, we find that FGF13A, the nuclear isoform of FGF13, is involved in the maintenance of NSCs and the suppression of neuronal differentiation during post-natal hippocampal development. Furthermore, we find that FGF13A interacts with ARID1B, a unit of Brahma-associated factor chromatin remodeling complex, and suppresses the expression of neuron differentiation-associated genes through chromatin modification. Our results suggest that FGF13A is an important regulator for maintaining the self-renewal and neurogenic capacity of NSCs in post-natal hippocampus, revealing an epigenomic regulatory function of FGFs in neurogenesis.

Strategy for Designing a Cell Scaffold to Enable Wireless Electrical Stimulation for Enhanced Neuronal Differentiation of Stem Cells.

Electrical stimulation (ES) offers significant advantages in modulating the behavior of stem cells on conductive scaffolds for neural tissue engineering. However, it is necessary to realize wireless ES to avoid the use of external wires in tissues. Thus, herein, a strategy is reported to develop a stem cell scaffold that allows wireless ES. A conductive annular graphene substrate is designed and grown by chemical vapor deposition; this substrate is used as a secondary coil to achieve wireless ES via electromagnetic induction in the presence of a primary coil. The substrate shows excellent biocompatibility for the culture of neural stem cells (NSCs). The results indicate that the applied wireless ES enhances neuronal differentiation, facilitates the formation of neurites, and does not substantially affect the viability and stemness maintenance of NSCs. Collectively, this system provides a strategy for achieving synergy between wireless ES and conductive scaffolds for neural regenerative medicine, which can be further utilized for the regeneration of other tissues.