Neural Stem Cell State Research Articles

SOX10 mediates glioblastoma cell-state plasticity

Phenotypic plasticity is a cause of glioblastoma therapy failure. We previously showed that suppressing the oligodendrocyte-lineage regulator SOX10 promotes glioblastoma progression. Here, we analyze SOX10-mediated phenotypic plasticity and exploit it for glioblastoma therapy design. We show that low SOX10 expression is linked to neural stem-cell (NSC)-like glioblastoma cell states and is a consequence of temozolomide treatment in animal and cell line models. Single-cell transcriptome profiling of Sox10-KD tumors indicates that Sox10 suppression is sufficient to induce tumor progression to an aggressive NSC/developmental-like phenotype, including a quiescent NSC-like cell population. The quiescent NSC state is induced by temozolomide and Sox10-KD and reduced by Notch pathway inhibition in cell line models. Combination treatment using Notch and HDAC/PI3K inhibitors extends the survival of mice carrying Sox10-KD tumors, validating our experimental therapy approach. In summary, SOX10 suppression mediates glioblastoma progression through NSC/developmental cell-state transition, including the induction of a targetable quiescent NSC state. This work provides a rationale for the design of tumor therapies based on single-cell phenotypic plasticity analysis.

SUMO-dependent transcriptional repression by Sox2 inhibits the proliferation of neural stem cells.

Sox2 is known for its roles in maintaining the stem cell state of embryonic stem cells and neural stem cells. In particular, it has been shown to slow the proliferation of these cell types. It is also known for its effects as an activating transcription factor. Despite this, analysis of published studies shows that it represses as many genes as it activates. Here, we identify a new set of target genes that Sox2 represses in neural stem cells. These genes are associated with centrosomes, centromeres and other aspects of cell cycle control. In addition, we show that SUMOylation of Sox2 is necessary for the repression of these genes and for its repressive effects on cell proliferation. Together, these data suggest that SUMO-dependent repression of this group of target genes is responsible for the role of Sox2 in regulating the proliferation of neural stem cells.

EPCO-41. MICRORNA-MEDIATED DIRECT NEURONAL REPROGRAMMING OF GLIOBLASTOMAS

Abstract Neuron-specific microRNAs miR-9/9* and miR-124 (miR-9/9*-124) are potent neurogenic molecules whose ectopic expression in non-neuronal somatic cells effectively induces direct cell fate conversion into a neuronal fate. Our recent work indicates that the fate transition occurs in distinct steps where miR-9/9*-124 first represses core gene regulatory networks to erase the original non-neuronal cell identity and subsequently activates the neuronal program to confer neuronal fate. Extending these findings, we reason that repression of similar gene regulatory networks by miR-9/9*-124 can also induce fate erasure in cancerous cells using glioblastoma as a paradigm. Glioblastoma multiforme (GBM) is a type of aggressive brain tumor with poor prognosis. Instead of having specific mutation profiles, GBMs present a global genetic network dysregulation which gives rise to a neural stem cell or progenitor cell-like state resembling embryonic neural development. Whereas miR-9/9*-124 plays a key role in inducing mitotic exit and orchestrating neurogenesis during development and neuronal reprogramming, we hypothesize that miR-9/9*-124 will instruct neuronal reprogramming of GBMs. Our results so far demonstrate that miR-9/9*-124 indeed exerts reprogramming activities in patient-derived GBM cell lines. Ectopic expression of miR-9/9*-124 in vitro readily induces GBM cells to exit cell cycle and promotes neuronal identity over time. In GBM intracerebral xenograft mouse model, mice receiving GBM cells overexpressing miR-9/9*-124 were able to survive longer than those with GBMs overexpressing non-specific microRNAs. We are currently carrying out transcriptomic and chromatin accessibility analyses to infer the epigenetic switch as GBM cells are reprogrammed to neurons. Based on these studies, we will further examine whether the GBM cells undergo irreversible fate erasure and are reprogrammed into a stable post-mitotic neuronal state. Our studies will provide insights into the plasticity of GBM fate that can be controlled by microRNAs and the key downstream regulatory nodes underlying fate transition of GBM to permanent post-mitotic state.

The Rb/E2F axis is a key regulator of the molecular signatures instructing the quiescent and activated adult neural stem cell state.

Long-term maintenance of the adult neurogenic niche depends on proper regulation of entry and exit from quiescence. Neural stem cell (NSC) transition from quiescence to activation is a complex process requiring precise cell-cycle control coordinated with transcriptional and morphological changes. How NSC fate transitions in coordination with the cell-cycle machinery remains poorly understood. Here we show that the Rb/E2F axis functions by linking the cell-cycle machinery to pivotal regulators of NSC fate. Deletion of Rb family proteins results in activation of NSCs, inducing a transcriptomic transition toward activation. Deletion of their target activator E2Fs1/3 results in intractable quiescence and cessation of neurogenesis. We show that the Rb/E2F axis mediates these fate transitions through regulation of factors essential for NSC function, including REST and ASCL1. Thus, the Rb/E2F axis is an important regulator of NSC fate, coordinating cell-cycle control with NSC activation and quiescence fate transitions.

Dormant state of quiescent neural stem cells links Shank3 mutation to autism development.

Autism spectrum disorders (ASDs) are common neurodevelopmental disorders characterized by deficits in social interactions and communication, restricted interests, and repetitive behaviors. Despite extensive study, the molecular targets that control ASD development remain largely unclear. Here, we report that the dormancy of quiescent neural stem cells (qNSCs) is a therapeutic target for controlling the development of ASD phenotypes driven by Shank3 deficiency. Using single-cell RNA sequencing (scRNA-seq) and transposase accessible chromatin profiling (ATAC-seq), we find that abnormal epigenetic features including H3K4me3 accumulation due to up-regulation of Kmt2a levels lead to increased dormancy of qNSCs in the absence of Shank3. This result in decreased active neurogenesis in the Shank3 deficient mouse brain. Remarkably, pharmacological and molecular inhibition of qNSC dormancy restored adult neurogenesis and ameliorated the social deficits observed in Shank3-deficient mice. Moreover, we confirmed restored human qNSC activity rescues abnormal neurogenesis and autism-like phenotypes in SHANK3-targeted human NSCs. Taken together, our results offer a novel strategy to control qNSC activity as a potential therapeutic target for the development of autism.

Selective translation of epigenetic modifiers affects the temporal pattern and differentiation of neural stem cells

The cerebral cortex is formed by diverse neurons generated sequentially from neural stem cells (NSCs). A clock mechanism has been suggested to underlie the temporal progression of NSCs, which is mainly defined by the transcriptome and the epigenetic state. However, what drives such a developmental clock remains elusive. We show that translational control of histone H3 trimethylation in Lys27 (H3K27me3) modifiers is part of this clock. We find that depletion of Fbl, an rRNA methyltransferase, reduces translation of both Ezh2 methyltransferase and Kdm6b demethylase of H3K27me3 and delays the progression of the NSC state. These defects are partially phenocopied by simultaneous inhibition of H3K27me3 methyltransferase and demethylase, indicating the role of Fbl in the genome-wide H3K27me3 pattern. Therefore, we propose that Fbl drives the intrinsic clock through the translational enhancement of the H3K27me3 modifiers that predominantly define the NSC state.

A Shared Transcriptional Identity for Forebrain and Dentate Gyrus Neural Stem Cells from Embryogenesis to Adulthood.

Adult neural stem cells (NSCs) reside in two distinct niches in the mammalian brain, the ventricular-subventricular zone (V-SVZ) of the forebrain lateral ventricles and the subgranular zone (SGZ) of the hippocampal dentate gyrus. They are thought to be molecularly distinct since V-SVZ NSCs produce inhibitory olfactory bulb (OB) interneurons and SGZ NSCs excitatory dentate granule neurons. Here, we have asked whether this is so by directly comparing V-SVZ and SGZ NSCs from embryogenesis to adulthood using single-cell transcriptional data. We show that the embryonic radial glial precursor (RP) parents of these two NSC populations are very similar, but differentially express a small cohort of genes involved in glutamatergic versus GABAergic neurogenesis. These different RPs then undergo a similar gradual transition to a dormant adult NSC state over the first three postnatal weeks. This dormancy state involves transcriptional shutdown of genes that maintain an active, proliferative, prodifferentiation state and induction of genes involved in sensing and regulating their niche environment. Moreover, when reactivated to generate adult-born progeny, both populations reacquire a development-like state and re-express proneurogenic genes. Thus, V-SVZ and SGZ NSCs share a common transcriptional state throughout their lifespans and transition into and out of dormancy via similar trajectories.

Dual activation of Shh and Notch signaling induces dramatic enlargement of neocortical surface area

The expansion of the neocortex represents a characteristic event over the course of mammalian evolution. Gyrencephalic mammals that have the larger brains with many folds (gyri and sulci) seem to have acquired higher intelligence, reflective of the enlargement of the neocortical surface area. In this process, germinal layers containing neural stem cells (NSCs) and neural progenitors expanded in number, leading to an increase in the total number of cortical neurons. In this study, we sought to expand neural stem/progenitor cells and enlarge the neocortical surface area by the dual activation of Shh and Notch signaling in transgenic (Tg) mice, promoting the proliferation of neural stem/progenitor cells by the Shh signaling effector while maintaining the undifferentiated state of NSCs by the Notch signaling effector. In the neocortical region of the Tg embryos, NSCs increased in number, and the ventricles, ventricular zone, and neocortical surface area were dramatically expanded. Furthermore, we observed that folds/wrinkles on the neocortical surface were progressively formed, accompanied by the vascular formation. These findings suggest that Shh and Notch signaling may be key regulators of mammalian brain evolution.

Identification of the gliogenic state of human neural stem cells to optimize in vitro astrocyte differentiation

BackgroundHuman preclinical models are crucial for advancing biomedical research. In particular consistent and robust protocols for astrocyte differentiation in the human system are rare. New methodWe performed a transcriptional characterization of human gliogenesis using embryonic H9- derived hNSCs. Based on these findings we established a fast and highly efficient protocol for the differentiation of mature human astrocytes. We could reproduce these results in induced pluripotent stem cell (iPSC)-derived NSCs. ResultsWe identified an increasing propensity of NSCs to give rise to astrocytes with repeated cell passaging. The gliogenic phenotype of NSCs was marked by a down-regulation of stem cell factors (e.g. SOX1, SOX2, EGFR) and an increase of glia-associated factors (e.g. NFIX, SOX9, PDGFRa). Using late passage NSCs, rapid and robust astrocyte differentiation can be achieved within 28 days. Comparison with existing method(s)In published protocols it usually takes around three months to yield in mature astrocytes. The difficulty, expense and time associated with generating astrocytes in vitro represents a major roadblock for glial cell research. We show that rapid and robust astrocyte differentiation can be achieved within 28 days. We describe here by an extensive sequential transcriptome analysis of hNSCs the characterization of the signature of a novel gliogenic stem cell population. The transcriptomic signature might serve to identify the proper divisional maturity. ConclusionsThis work sheds light on the factors associated with rapid NSC differentiation into glial cells. These findings contribute to understand human gliogenesis and to develop novel preclinical models that will help to study CNS disease such as Multiple Sclerosis.

Epigenetic plasticity and redox regulation of neural stem cell state and fate

The neural stem cells (NSCs) are essential for normal brain development and homeostasis. The cell state (i.e. quiescent versus activated) and fate (i.e. the cell lineage of choice upon differentiation) of NSCs are tightly controlled by various redox and epigenetic regulatory mechanisms. There is an increasing appreciation that redox and epigenetic regulations are intimately linked, but how this redox-epigenetics crosstalk affects NSC activity remains poorly understood. Another unresolved topic is whether the NSCs actually contribute to brain ageing and neurodegenerative diseases. In this review, we aim to 1) distill concepts that underlie redox and epigenetic regulation of NSC state and fate; 2) provide examples of the redox-epigenetics crosstalk in NSC biology; and 3) highlight potential redox- and epigenetic-based therapeutic opportunities to rescue NSC dysfunctions in ageing and neurodegenerative diseases.

Neural stem cell pools in the vertebrate adult brain: Homeostasis from cell-autonomous decisions or community rules?

Adult stem cell populations must coordinate their own maintenance with the generation of differentiated cell types to sustain organ physiology, in a spatially controlled manner and over long periods. Quantitative analyses of clonal dynamics have revealed that, in epithelia, homeostasis is achieved at the population rather than at the single stem cell level, suggesting that feedback mechanisms coordinate stem cell maintenance and progeny generation. In the central nervous system, however, little is known of the possible community processes underlying neural stem cell maintenance. Recent work, in part based on intravital imaging made possible in the adult zebrafish, conclusively highlights that homeostasis in neural stem cell pools may rely on population asymmetry and long-term spatiotemporal coordination of neural stem cell states and fates. These results suggest that neural stem cell assemblies in the vertebrate brain behave as self-organized systems, such that the stem cells themselves generate their own intrinsic niche.

The significance of gene expression dynamics in neural stem cell regulation.

Neural stem cells (NSCs) actively proliferate and generate neurons and glial cells (active state) in the embryonic brain, whereas they are mostly dormant (quiescent state) in the adult brain. The expression dynamics of Hes1 are different between active and quiescent NSCs. In active NSCs, Hes1 expression oscillates and periodically represses the expression of proneural genes such as Ascl1, thereby driving their oscillations. By contrast, in quiescent NSCs, Hes1 oscillations maintain expression at higher levels even at trough phases (thus continuous), thereby continuously suppressing proneural gene expression. High levels of Hes1 expression and the resultant suppression of Ascl1 promote the quiescent state of NSCs, whereas oscillatory Hes1 expression and the resultant oscillatory Ascl1 expression regulate their active state. Furthermore, in other developmental contexts, high, continuous Hes1 expression induces astrocyte differentiation or the formation of boundaries, which function as signaling centers. Thus, the expression dynamics of Hes1 are a key regulatory mechanism generating and maintaining various cell types in the nervous system.

Interaction of human IAPP and Aβ1-42 aggravated the AD-related pathology and impaired the cognition in mice

Alzheimer's disease (AD) and type 2 diabetes mellitus (T2DM) have a common pathology. Both diseases are characterized by local deposition of amyloid proteins in the brain or islet organ, but their phenotypes and clinical manifestation vary widely. Although the sources of islet amyloid polypeptide (IAPP) and amyloid beta (Aβ) are independent, their fibrillar sequences are highly homologous. The prevalence of AD in T2DM populations is considerably higher than that in the normal population, but a mechanistic linkage remains elusive. Therefore, the present study aimed to explore the effects of Aβ42 deposition in the brain on the persistently expression of human IAPP (hIAPP). Additionally, cognitive ability, synaptic plasticity, the state of neural stem cells and mitochondrial function were evaluated at 2 or 6 months after stereotaxically injected the oligomer Aβ1-42 into the dentate gyrus of hIAPP (−/+) mice or the wild-type littermates. We found that Aβ42 and amylin were co-located in hippocampus and Aβ42 levels increased when Aβ1-42 was injected in hIAPP transgenic mice compared with that of the wild-type littermates. Furthermore, at 6 months after Aβ1-42 injection in hIAPP (−/+) mice, it exhibits exacerbated AD-related pathologies including Aβ42 deposition, cognitive impairment, synapse reduction, neural stem cells exhaustion and mitochondrial dysfunction. Our present study suggested that hIAPP directly implicated the Aβ42 production and deposition as an important linkage between T2DM and AD.

Lysosomes and signaling pathways for maintenance of quiescence in adult neural stem cells.

Quiescence is a cellular strategy for maintaining somatic stem cells in a specific niche in a low metabolic state without senescence for a long period of time. During development, neural stem cells (NSCs) actively proliferate and self-renew, and their progeny differentiate into both neurons and glial cells to form mature brain tissues. On the other hand, most NSCs in the adult brain are quiescent and arrested in G0/G1 phase of the cell cycle. Quiescence is essential in order to avoid the precocious exhaustion of NSCs, ensuring a sustainable source of available stem cells in the brain throughout the lifespan. After receiving activation signals, quiescent NSCs reenter the cell cycle and generate new neurons. This switching between quiescence and proliferation is tightly regulated by diverse signaling pathways. Recent studies suggest significant involvement of cellular proteostasis (homeostasis of the proteome) in the quiescent state of NSCs. Proteostasis is the result of integrated regulation of protein synthesis, folding, and degradation. In this review, we discuss regulation of quiescence by multiple signaling pathways, especially bone morphogenetic protein and Notch signaling, and focus on the functional involvement of the lysosome, an organelle governing cellular degradation, in quiescence of adult NSCs.

A Multi-Channel System for Temperature Sensing of Neural Stem Cells in Adherent Culture.

Neural stem cells (NSCs) can gradually proliferate or differentiate during adherent culture. It is found that stem cells have different temperature characteristics in different physiological states. In order to detect the temperature of NSCs during adherent culture, in this study, we have designed a temperature monitoring system, in which a thin-film platinum resistor was used as the sensor. The NSCs were seeded on the sensor, and the data acquisition device was connected to the host computer via Bluetooth. Results indicate that there are about 5000 cells attached on the surface of each sensor, and the cell viability is maintained at about 90% after 24 h culture. An electrostatic force microscope (EFM) result proves that there is no electric field on the sensor surface to influence the activity of NSCs. This system can work continuously for more than 24 h with 0.05 °C detection sensitivity. Furthermore, the significant temperature change of NSCs is observed when stimulated by different concentrations of thyroid hormone, which demonstrates that the temperature change related to cell activity. Therefore, by detecting the temperature of the cell population, the fabricated system can provide reference information for studying the metabolic state of NSCs, as well as physiological responses of cells under various conditions in biomedical applications.

Regulation of active and quiescent somatic stem cells by Notch signaling.

Somatic stem/progenitor cells actively proliferate and give rise to different types of mature cells (active state) in embryonic tissues while they are mostly dormant (quiescent state) in many adult tissues. Notch signaling is known to regulate both active and quiescent states of somatic stem cells, but how it regulates these different states is unknown. Recent studies revealed that the Notch effector Hes1 is expressed differently during the active and quiescent states during neurogenesis and myogenesis: high in the quiescent state and oscillatory in the active state. When the Hes1 expression level is high, both Ascl1 and MyoD expression are continuously suppressed. By contrast, when Hes1 expression oscillates, it periodically represses expression of the neurogenic factor Ascl1 and the myogenic factor MyoD, thereby driving Ascl1 and MyoD oscillations. High levels of Hes1 and the resultant Ascl1 suppression promote the quiescent state of neural stem cells, while Hes1 oscillation‐dependent Ascl1 oscillations regulate their active state. Similarly, in satellite cells of muscles, known adult muscle stem cells, high levels of Hes1 and the resultant MyoD suppression seem to promote their quiescent state, while Hes1 oscillation‐dependent MyoD oscillations activate their proliferation and differentiation. Therefore, the expression dynamics of Hes1 is a key regulatory mechanism of generating and maintaining active/quiescent stem cell states.

Fully Synthetic Heparan Sulfate-Based Neural Tissue Construct That Maintains the Undifferentiated State of Neural Stem Cells

Heparin and heparan sulfate (HS) are attractive components for constructing biomaterials due to their ability to recruit and regulate the activity of growth factors. The structural and functional heterogeneity of naturally derived heparin and HS is, however, an impediment for the preparation of biomaterials for regenerative medicine. To address this problem, we have prepared hydrogels modified by well-defined synthetic HS-derived disaccharides. Human induced pluripotent cell-derived neural stem cells (HIP-NSCs) encapsulated in a polyethylene glycol-based hydrogel modified by a pendent HS disaccharide that is a known ligand for fibroblast growth factor-2 (FGF-2) exhibited a significant increase in proliferation and self-renewal. This observation is important because evidence is emerging that undifferentiated stems cells can yield significant therapeutic benefits via their paracrine signaling mechanisms. Our data indicate that the HS disaccharide protects FGF-2, which has a very short biological half-live, from degradation. It is anticipated that, by careful selection of a synthetic HS oligosaccharide, it will be possible to control retention and release of specific growth factor, which in turn will provide control over cell fate.

Detection and Monitoring of Stem Cell Differentiation Using Nanotechnology.

Stem cell differentiation and pluripotency are important stages in the regenerative medicine. The design and fabrication of user-friendly approach to save cost and time as well as monitor this process is crucial. Surface-enhanced Raman spectroscopy (SERS) is a sensitive technique to fabricate smart sensors for biological applications. In this technique, double enhancement effect on SERS signals is induced by utilizing graphene oxide (GO)-encapsulated gold nanoparticles (GNPs). The undifferentiated neural stem cells (NSCs) produced Raman peaks which were 3.5 times higher than those obtained from normal metal structures. The surface decorated using 3D GO-encapsulated GNPs was an effective, cost-saving, and nondestructive tool for distinguishing the differentiation state of NSCs. Moreover, the substrate composed of 3D GO-encapsulated gold nanoparticles was also effective for distinguishing the differentiation state of single NSC by using electrochemical and electrical techniques. Therefore, the recommended technique can be utilized as a powerful nondestructive in situ monitoring tool for the identification of the differentiation potential of various kinds of stem cells.

Dynamic Notch signalling regulates neural stem cell state progression in the Drosophila optic lobe

BackgroundNeural stem cells generate all of the neurons and glial cells in the central nervous system, both during development and in the adult to maintain homeostasis. In the Drosophila optic lobe, neuroepithelial cells progress through two transient progenitor states, PI and PII, before transforming into neuroblasts. Here we analyse the role of Notch signalling in the transition from neuroepithelial cells to neuroblasts.ResultsWe observed dynamic regulation of Notch signalling: strong activity in PI progenitors, low signalling in PII progenitors, and increased activity after neuroblast transformation. Ectopic expression of the Notch ligand Delta induced the formation of ectopic PI progenitors. Interestingly, we show that the E3 ubiquitin ligase, Neuralized, regulates Delta levels and Notch signalling activity at the transition zone. We demonstrate that the proneural transcription factor, Lethal of scute, is essential to induce expression of Neuralized and promote the transition from the PI progenitor to the PII progenitor state.ConclusionsOur results show dynamic regulation of Notch signalling activity in the transition from neuroepithelial cells to neuroblasts. We propose a model in which Lethal of scute activates Notch signalling in a non-cell autonomous manner by regulating the expression of Neuralized, thereby promoting the progression between different neural stem cell states.

Groucho related gene 5 (GRG5) is involved in embryonic and neural stem cell state decisions

Groucho related gene 5 (GRG5) is a multifunctional protein that has been implicated in late embryonic and postnatal mouse development. Here, we describe a previously unknown role of GRG5 in early developmental stages by analyzing its function in stem cell fate decisions. By both loss and gain of function approaches we demonstrate that ablation of GRG5 deregulates the Embryonic Stem Cell (ESC) pluripotent state whereas its overexpression leads to enhanced self-renewal and acquisition of cancer cell-like properties. The malignant characteristics of teratomas generated by ESCs that overexpress GRG5 reveal its pro-oncogenic potential. Furthermore, transcriptomic analysis and cell differentiation approaches underline GRG5 as a multifaceted signaling regulator that represses mesendodermal-related genes. When ESCs exit pluripotency, GRG5 promotes neuroectodermal specification via Wnt and BMP signaling suppression. Moreover, GRG5 promotes the neuronal reprogramming of fibroblasts and maintains the self-renewal of Neural Stem Cells (NSCs) by sustaining the activity of Notch/Hes and Stat3 signaling pathways. In summary, our results demonstrate that GRG5 has pleiotropic roles in stem cell biology functioning as a stemness factor and a neural fate specifier.