Activation Of Neural Stem Cells Research Articles

Constructing Electron-Rich Ru Clusters on Non-Stoichiometric Copper Hydroxide for Superior Biocatalytic ROS Scavenging to Treat Inflammatory Spinal Cord Injury.

Traumatic spinal cord injury (SCI) represents a complex neuropathological challenge that significantly impacts the well-being of affected individuals. The quest for efficacious antioxidant and anti-inflammatory therapies is both a compelling necessity and a formidable challenge. Here, in this work, the innovative synthesis of electron-rich Ru clusters on non-stoichiometric copper hydroxide that contain oxygen vacancy defects (Ru/def-Cu(OH)2), which can function as a biocatalytic reactive oxygen species (ROS) scavenger for efficiently suppressing the inflammatory cascade reactions and modulating the endogenous microenvironments in SCI, is introduced. The studies reveal that the unique oxygen vacancies promote electron redistribution and amplify electron accumulation at Ru clusters, thus enhancing the catalytic activity of Ru/def-Cu(OH)2 in multielectron reactions involving oxygen-containing intermediates. These advancements endow the Ru/def-Cu(OH)2 with the capacity to mitigate ROS-mediated neuronal death and to foster a reparative microenvironment by dampening inflammatory macrophage responses, meanwhile concurrently stimulating the activity of neural stem cells, anti-inflammatory macrophages, and oligodendrocytes. Consequently, this results in a robust reparative effect on traumatic SCI. It is posited that the synthesized Ru/def-Cu(OH)2 exhibits unprecedented biocatalytic properties, offering a promising strategy to develop ROS-scavenging and anti-inflammatory materials for the management of traumatic SCI and a spectrum of other diseases associated with oxidative stress.

CRISPR–Cas9 screens reveal regulators of ageing in neural stem cells

Ageing impairs the ability of neural stem cells (NSCs) to transition from quiescence to proliferation in the adult mammalian brain. Functional decline of NSCs results in the decreased production of new neurons and defective regeneration following injury during ageing1–4. Several genetic interventions have been found to ameliorate old brain function5–8, but systematic functional testing of genes in old NSCs—and more generally in old cells—has not been done. Here we develop in vitro and in vivo high-throughput CRISPR–Cas9 screening platforms to systematically uncover gene knockouts that boost NSC activation in old mice. Our genome-wide screens in primary cultures of young and old NSCs uncovered more than 300 gene knockouts that specifically restore the activation of old NSCs. The top gene knockouts are involved in cilium organization and glucose import. We also establish a scalable CRISPR–Cas9 screening platform in vivo, which identified 24 gene knockouts that boost NSC activation and the production of new neurons in old brains. Notably, the knockout of Slc2a4, which encodes the GLUT4 glucose transporter, is a top intervention that improves the function of old NSCs. Glucose uptake increases in NSCs during ageing, and transient glucose starvation restores the ability of old NSCs to activate. Thus, an increase in glucose uptake may contribute to the decline in NSC activation with age. Our work provides scalable platforms to systematically identify genetic interventions that boost the function of old NSCs, including in vivo, with important implications for countering regenerative decline during ageing.

A cholinergic signaling pathway underlying cortical circuit activation of quiescent neural stem cells in the lateral ventricle.

Neural stem cells (NSCs) in the subventricular zone (SVZ) located along the lateral ventricles (LVs) of the mammalian brain continue to self-renew to produce new neurons after birth and into adulthood. Quiescent LV cells, which are situated close to the ependymal cells lining the LVs, are activated by choline acetyltransferase-positive (ChAT+) neurons within the subependymal (subep) region of the SVZ when these neurons are stimulated by projections from the anterior cingulate cortex (ACC). Here, we uncovered a signaling pathway activated by the ACC-subep-ChAT+ circuit responsible for the activation and proliferation of quiescent LV NSCs specifically in the ventral area of the SVZ. This circuit activated muscarinic M3 receptors on quiescent LV NSCs, which subsequently induced signaling mediated by the inositol 1,4,5-trisphosphate receptor type 1 (IP3R1). Downstream of IP3R1 activation, which would be expected to increase intracellular Ca2+, Ca2+-/calmodulin-dependent protein kinase II δ and the MAPK10 signaling pathway were stimulated and required for the proliferation of quiescent LV NSCs in the SVZ. These findings reveal the mechanisms that regulate quiescent LV NSCs and underscore the critical role of projections from the ACC in promoting their proliferative activity within the ventral SVZ.

EphA4 Targeting Peptide-Conjugated Extracellular Vesicles Rejuvenates Adult Neural Stem Cells and Exerts Therapeutic Benefits in Aging Rats.

Aging and various neurodegenerative diseases cause significant reduction in adult neurogenesis and simultaneous increase in quiescent neural stem cells (NSCs), which impact the brain's regenerative capabilities. To deal with this challenging issue, current treatments involve stem cell transplants or prevention of neurodegeneration; however, the efficacy or success of this process remains limited. Therefore, extensive and focused investigation is highly demanding to overcome this challenging task. Here, we have designed an efficient peptide-based EphA4 receptor-targeted ligand through an in silico approach. Further, this strategy involves chemical conjugation of the peptide with adipose tissue stem cell-derived EV (Exo-pep-11). Interestingly, our newly designed engineered EV, Exo-pep-11, targets NSC through EphA4 receptors, which offers promising therapeutic advantages by stimulating NSC proliferation and subsequent differentiation. Our result demonstrates that NSC successfully internalized Exo-pep-11 in both in vitro culture conditions as well as in the in vivo aging rats. We found that the uptake of Exo-pep-11 decreased by ∼2.3-fold when NSC was treated with EphA4 antibody before Exo-pep-11 incubation, which confirms the receptor-specific uptake of Exo-pep-11. Exo-pep-11 treatment also increases NSC proliferation by ∼1.9-fold and also shows ∼1.6- and ∼2.4-fold increase in expressions of Nestin and ID1, respectively. Exo-pep-11 also has the potential to increase neurogenesis in aging rats, which is confirmed by ∼1.6- and ∼1.5-fold increases in expressions of TH and Tuj1, respectively, in rat olfactory bulb. Overall, our findings highlight the potential role of Exo-pep-11 for prospective applications in combating age-related declines in NSC activity and neurogenesis.

An injectable decellularized extracellular matrix hydrogel with cortical neuron-derived exosomes enhances tissue repair following traumatic spinal cord injury

Traumatic spinal cord injury (SCI), known for its limited intrinsic regeneration capacity, often results in considerable neurological impairment. Studies suggest that therapeutic techniques utilizing exosomes (Exo) to promote tissue regeneration and modulate immune responses are promising for SCI treatment. However, combining exosome therapy with biomaterials for SCI treatment is not very effective. This study developed an adhesive hydrogel using exosomes secreted by cortical neurons derived from human induced pluripotent stem cells (iPSCs) and decellularized extracellular matrix (dECM) from human umbilical cord mesenchymal stem cells (hUCMSCs) to enhance motor function recovery post-SCI. In vitro assessments demonstrated the excellent cytocompatibility of the dECM hydrogel. Additionally, the Exo-dECM hydrogel facilitated the polarization of early M2 macrophages, reduced neuronal apoptosis, and established a pro-regenerative microenvironment in a rodent SCI model. Subsequent analyses revealed significant activation of endogenous neural stem cells and promotion of axon regeneration and remyelination at eight weeks post-surgery. The Exo-dECM hydrogel also promoted the functional recovery and preservation of urinary tissue in SCI-afflicted rats. These findings highlighted that the Exo-dECM hydrogel is a promising therapeutic strategy for treating SCI.

Mapping the cellular expression patterns of vascular endothelial growth factor aa and bb genes and their receptors in the adult zebrafish brain during constitutive and regenerative neurogenesis

The complex interplay between vascular signaling and neurogenesis in the adult brain remains a subject of intense research. By exploiting the unique advantages of the zebrafish model, in particular the persistent activity of neural stem cells (NSCs) and the remarkable ability to repair brain lesions, we investigated the links between NSCs and cerebral blood vessels. In this study, we first examined the gene expression profiles of vascular endothelial growth factors aa and bb (vegfaa and vegfbb), under physiological and regenerative conditions. Employing fluorescence in situ hybridization combined with immunostaining and histology techniques, we demonstrated the widespread expression of vegfaa and vegfbb across the brain, and showed their presence in neurons, microglia/immune cells, endothelial cells and NSCs. At 1 day post-lesion (dpl), both vegfaa and vegfbb were up-regulated in neurons and microglia/peripheral immune cells (macrophages). Analysis of vegf receptors (vegfr) revealed high expression throughout the brain under homeostatic conditions, with vegfr predominantly expressed in neurons and NSCs and to a lower extent in microglia/immune cells and endothelial cells. These findings were further validated by Vegfr3 and Vegfr4 immunostainings, which showed significant expression in neurogenic radial glial cells.Following brain lesion (1 dpl), while vegfr gene expression remained stable, vegfr transcripts were detected in proliferative cells within the injured parenchyma. Collectively, our results provide a first overview of Vegf/Vegfr signaling in the brain and suggest important roles for Vegf in neurogenesis and regenerative processes.

Metformin enhances endogenous neural stem cells proliferation, neuronal differentiation, and inhibits ferroptosis through activating AMPK pathway after spinal cord injury

BackgroundInadequate nerve regeneration and an inhibitory local microenvironment are major obstacles to the repair of spinal cord injury (SCI). The activation and differentiation fate regulation of endogenous neural stem cells (NSCs) represent one of the most promising repair approaches. Metformin has been extensively studied for its antioxidative, anti-inflammatory, anti-aging, and autophagy-regulating properties in central nervous system diseases. However, the effects of metformin on endogenous NSCs remains to be elucidated.MethodsThe proliferation and differentiation abilities of NSCs were evaluated using CCK-8 assay, EdU/Ki67 staining and immunofluorescence staining. Changes in the expression of key proteins related to ferroptosis in NSCs were detected using Western Blot and immunofluorescence staining. The levels of reactive oxygen species, glutathione and tissue iron were measured using corresponding assay kits. Changes in mitochondrial morphology and membrane potential were observed using transmission electron microscopy and JC-1 fluorescence probe. Locomotor function recovery after SCI in rats was assessed through BBB score, LSS score, CatWalk gait analysis, and electrophysiological testing. The expression of the AMPK pathway was examined using Western Blot.ResultsMetformin promoted the proliferation and neuronal differentiation of NSCs both in vitro and in vivo. Furthermore, a ferroptosis model of NSCs using erastin treatment was established in vitro, and metformin treatment could reverse the changes in the expression of key ferroptosis-related proteins, increase glutathione synthesis, reduce reactive oxygen species production and improve mitochondrial membrane potential and morphology. Moreover, metformin administration improved locomotor function recovery and histological outcomes following SCI in rats. Notably, all the above beneficial effects of metformin were completely abolished upon addition of compound C, a specific inhibitor of AMP-activated protein kinase (AMPK).ConclusionMetformin, driven by canonical AMPK-dependent regulation, promotes proliferation and neuronal differentiation of endogenous NSCs while inhibiting ferroptosis, thereby facilitating recovery of locomotor function following SCI. Our study further elucidates the protective mechanism of metformin in SCI, providing new mechanistic insights for its candidacy as a therapeutic agent for SCI.

Epigenetic maintenance of adult neural stem cell quiescence in the mouse hippocampus via Setd1a

Quiescence, a hallmark of adult neural stem cells (NSCs), is required for maintaining the NSC pool to support life-long continuous neurogenesis in the adult dentate gyrus (DG). Whether long-lasting epigenetic modifications maintain NSC quiescence over the long term in the adult DG is not well-understood. Here we show that mice with haploinsufficiency of Setd1a, a schizophrenia risk gene encoding a histone H3K4 methyltransferase, develop an enlarged DG with more dentate granule cells after young adulthood. Deletion of Setd1a specifically in quiescent NSCs in the adult DG promotes their activation and neurogenesis, which is countered by inhibition of the histone demethylase LSD1. Mechanistically, RNA-sequencing and CUT & RUN analyses of cultured quiescent adult NSCs reveal Setd1a deletion-induced transcriptional changes and many Setd1a targets, among which down-regulation of Bhlhe40 promotes quiescent NSC activation in the adult DG in vivo. Together, our study reveals a Setd1a-dependent epigenetic mechanism that sustains NSC quiescence in the adult DG.

Primitive and Definitive Neural Precursor Cells Are Present in Human Cerebral Organoids.

Activation of neural stem cells (NSCs) correlates with improved functional outcomes in mouse models of injury. In the murine brain, NSCs have been extensively characterized and comprise (1) primitive NSCs (pNSCs) and (2) definitive NSCs (dNSCs). pNSCs are the earliest cells in the NSC lineage giving rise to dNSCs in the embryonic and adult mouse brain. pNSCs are quiescent under baseline conditions and can be activated upon injury. Herein, we asked whether human pNSCs and dNSCs can be isolated during the maturation of human cerebral organoids (COs) and activated by drugs known to regulate mouse NSC behavior. We demonstrate that self-renewing, multipotent pNSC and dNSC populations are present in human COs and express genes previously characterized in mouse NSCs. The drug NWL283, an inhibitor of apoptosis, reduced cell death in COs but did not improve NSC survival. Metformin, a drug used to treat type II diabetes that is known to promote NSC activation in mice, was found to expand human NSC pools. Together, these findings are the first to identify and characterize human pNSCs, advancing our understanding of the human NSC lineage and highlighting drugs that enhance their activity.

Classification of Neural Stem Cell Activation State In Vitro using Autofluorescence.

Neural stem cells (NSCs) divide and produce newborn neurons in the adult brain through a process called adult neurogenesis. Adult NSCs are primarily quiescent, a reversible cell state where they have exited the cell cycle (G0) yet remain responsive to the environment. In the first step of adult neurogenesis, quiescent NSCs (qNSCs) receive a signal and activate, exiting quiescence and re-entering the cell cycle. Thus, understanding the regulators of NSC quiescence and quiescence exit is critical for future strategies targeting adult neurogenesis. However, our understanding of NSC quiescence is limited by technical constraints in identifying quiescent NSCs (qNSCs) and activated NSCs (aNSCs). This protocol describes a new approach to identify and enrich qNSCs and aNSCs generated in in vitro cultures by imaging NSC autofluorescence. First, this protocol describes how to use a confocal microscope to identify autofluorescent markers of qNSCs and aNSCs to classify NSC activation state using autofluorescence intensity. Second, this protocol describes how to use a fluorescent activated cell sorter (FACS) to classify NSC activation state and enrich samples for qNSCs or aNSCs using autofluorescence intensity. Third, this protocol describes how to use a multiphoton microscope to perform fluorescence lifetime imaging (FLIM) at single-cell resolution, classify NSC activation state, and track the dynamics of quiescent exit using both autofluorescence intensitiesand fluorescence lifetimes. Thus, this protocol provides a live-cell, label-free, single-cell resolution toolkit for studying NSC quiescence and quiescence exit.

A core scientific problem in the treatment of central nervous system diseases: newborn neurons.

It has long been asserted that failure to recover from central nervous system diseases is due to the system's intricate structure and the regenerative incapacity of adult neurons. Yet over recent decades, numerous studies have established that endogenous neurogenesis occurs in the adult central nervous system, including humans'. This has challenged the long-held scientific consensus that the number of adult neurons remains constant, and that new central nervous system neurons cannot be created or renewed. Herein, we present a comprehensive overview of the alterations and regulatory mechanisms of endogenous neurogenesis following central nervous system injury, and describe novel treatment strategies that target endogenous neurogenesis and newborn neurons in the treatment of central nervous system injury. Central nervous system injury frequently results in alterations of endogenous neurogenesis, encompassing the activation, proliferation, ectopic migration, differentiation, and functional integration of endogenous neural stem cells. Because of the unfavorable local microenvironment, most activated neural stem cells differentiate into glial cells rather than neurons. Consequently, the injury-induced endogenous neurogenesis response is inadequate for repairing impaired neural function. Scientists have attempted to enhance endogenous neurogenesis using various strategies, including using neurotrophic factors, bioactive materials, and cell reprogramming techniques. Used alone or in combination, these therapeutic strategies can promote targeted migration of neural stem cells to an injured area, ensure their survival and differentiation into mature functional neurons, and facilitate their integration into the neural circuit. Thus can integration replenish lost neurons after central nervous system injury, by improving the local microenvironment. By regulating each phase of endogenous neurogenesis, endogenous neural stem cells can be harnessed to promote effective regeneration of newborn neurons. This offers a novel approach for treating central nervous system injury.

Autofluorescence is a biomarker of neural stem cell activation state

Neural stem cells (NSCs) must exit quiescence to produce neurons; however, our understanding of this process remains constrained by the technical limitations of current technologies. Fluorescence lifetime imaging (FLIM) of autofluorescent metabolic cofactors has been used in other cell types to study shifts in cell states driven by metabolic remodeling that change the optical properties of these endogenous fluorophores. Using this non-destructive, live-cell, and label-free strategy, we found that quiescent NSCs (qNSCs) and activated NSCs (aNSCs) have unique autofluorescence profiles. Specifically, qNSCs display an enrichment of autofluorescence localizing to a subset of lysosomes, which can be used as a graded marker of NSC quiescence to predict cell behavior at single-cell resolution. Coupling autofluorescence imaging with single-cell RNA sequencing, we provide resources revealing transcriptional features linked to deep quiescence and rapid NSC activation. Together, we describe an approach for tracking mouse NSC activation state and expand our understanding of adult neurogenesis.

Hippocampal neurodegeneration induces transient endogenous regeneration and long-term exhaustion of the neurogenic niche.

The hippocampal dentate gyrus, responds to diverse pathological stimuli through neurogenesis. This phenomenon, observed following brain injury or neurodegeneration, is postulated to contribute to neuronal repair and functional recovery, thereby presenting an avenue for endogenous neuronal restoration. This study investigated the extent of regenerative response in hippocampal neurogenesis by leveraging the well-established kainic acid-induced status epilepticus model in vivo. In our study, we observed the activation and proliferation of neuronal progenitors or neural stem cell (NSC) and their subsequent migration to the injury sites following the seizure. At the injury sites, new neurons (Tuj1+BrdU+ and NeuN+BrdU+) have been generated indicating regenerative and reparative roles of the progenitor cells. We further detected whether this transient neurogenic burst, which might be a response towards an attempt to repair the brain, is associated with persistent long-term exhaustion of the dentate progenitor cells and impairment of adult neurogenesis marked by downregulation of Ki67, HoPX, and Sox2 with BrdU+ cell in the later part of life. Our studies suggest that the adult brain has the constitutive endogenous regenerative potential for brain repair to restore the damaged neurons, meanwhile, in the long term, it accelerates the depletion of the finite NSC pool in the hippocampal neurogenic niche by changing its proliferative and neurogenic capacity. A thorough understanding of the impact of modulating adult neurogenesis will eventually be required to design novel therapeutics to stimulate or assist brain repair while simultaneously preventing the adverse effects of early robust neurogenesis on the proliferative potential of endogenous neuronal progenitors.

Transcriptional control of neural stem cell activity.

In the adult brain, neural stem cells (NSCs) are under the control of various molecular mechanisms to produce an appropriate number of neurons that are essential for specific brain functions. Usually, the majority of adult NSCs stay in a non-proliferative and undifferentiated state known as quiescence, occasionally transitioning to an active state to produce newborn neurons. This transition between the quiescent and active states is crucial for the activity of NSCs. Another significant state of adult NSCs is senescence, in which quiescent cells become more dormant and less reactive, ceasing the production of newborn neurons. Although many genes involved in the regulation of NSCs have been identified using genetic manipulation and omics analyses, the entire regulatory network is complicated and ambiguous. In this review, we focus on transcription factors, whose importance has been elucidated in NSCs by knockout or overexpression studies. We mainly discuss the transcription factors with roles in the active, quiescent, and rejuvenation states of adult NSCs.

The m6A methylation and expression profiles of mouse neural stem cells after hypoxia/reoxygenation

BackgroundIschemia–reperfusion injury to the central nervous system often causes severe complications. The activation of endogenous neural stem cells (NSCs) is considered a promising therapeutic strategy for nerve repair. However, the specific biological processes and molecular mechanisms of NSC activation remain unclear, and the role of N6-methyladenosine (m6A) methylation modification in this process has not been explored.MethodsNSCs were subjected to hypoxia/reoxygenation (H/R) to simulate ischemia–reperfusion in vivo. m6A RNA methylation quantitative kit was used to measure the total RNA m6A methylation level. Quantitative real-time PCR was used to detect methyltransferase and demethylase mRNA expression levels. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) were conducted for NSCs in control and H/R groups, and the sequencing results were analyzed using bioinformatics. Finally, the migration ability of NSCs was identified by wound healing assays, and the proliferative capacity of NSCs was assessed using the cell counting kit-8, EdU assays and cell spheroidization assays.ResultsOverall of m6A modification level and Mettl14 mRNA expression increased in NSCs after H/R treatment. The m6A methylation and expression profiles of mRNAs in NSCs after H/R are described for the first time. Through the joint analysis of MeRIP-seq and RNA-seq results, we verified the proliferation of NSCs after H/R, which was regulated by m6A methylation modification. Seven hub genes were identified to play key roles in the regulatory process. Knockdown of Mettl14 significantly inhibited the proliferation of NSCs. In addition, separate analysis of the MeRIP-seq results suggested that m6A methylation regulates cell migration and differentiation in ways other than affecting mRNA expression. Subsequent experiments confirmed the migration ability of NSCs was suppressed by knockdown of Mettl14.ConclusionThe biological behaviors of NSCs after H/R are closely related to m6A methylation of mRNAs, and Mettl14 was confirmed to be involved in cell proliferation and migration.

Iron/ROS/Itga3 mediated accelerated depletion of hippocampal neural stem cell pool contributes to cognitive impairment after hemorrhagic stroke

Hemorrhagic stroke, specifically intracerebral hemorrhage (ICH), has been implicated in the development of persistent cognitive impairment, significantly compromising the quality of life for affected individuals. Nevertheless, the precise underlying mechanism remains elusive. Here, we report for the first time that the accumulation of iron within the hippocampus, distal to the site of ICH in the striatum, is causally linked to the observed cognitive impairment with both clinical patient data and animal model. Both susceptibility-weighted imaging (SWI) and quantitative susceptibility mapping (QSM) demonstrated significant iron accumulation in the hippocampus of ICH patients, which is far from the actual hematoma. Logistical regression analysis and multiple linear regression analysis identified iron level as an independent risk factor with a negative correlation with post-ICH cognitive impairment. Using a mouse model of ICH, we demonstrated that iron accumulation triggers an excessive activation of neural stem cells (NSCs). This overactivation subsequently leads to the depletion of the NSC pool, diminished neurogenesis, and the onset of progressive cognitive dysfunction. Mechanistically, iron accumulation elevated the levels of reactive oxygen species (ROS), which downregulated the expression of Itga3. Notably, pharmacological chelation of iron accumulation or scavenger of aberrant ROS levels, as well as conditionally overexpressed Itga3 in NSCs, remarkably attenuated the exhaustion of NSC pool, abnormal neurogenesis and cognitive decline in the mouse model of ICH. Together, these results provide molecular insights into ICH-induced cognitive impairment, shedding light on the value of maintaining NSC pool in preventing cognitive dysfunction in patients with hemorrhagic stroke or related conditions.

Coordination between circadian neural circuit and intracellular molecular clock ensures rhythmic activation of adult neural stem cells

The circadian clock throughout the day organizes the activity of neural stem cells (NSCs) in the dentate gyrus (DG) of adult hippocampus temporally. However, it is still unclear whether and how circadian signals from the niches contribute to daily rhythmic variation of NSC activation. Here, we show that norepinephrinergic (NEergic) projections from the locus coeruleus (LC), a brain arousal system, innervate into adult DG, where daily rhythmic release of norepinephrine (NE) from the LC NEergic neurons controlled circadian variation of NSC activation through β3-adrenoceptors. Disrupted circadian rhythmicity by acute sleep deprivation leads to transient NSC overactivation and NSC pool exhaustion over time, which is effectively ameliorated by the inhibition of the LC NEergic neuronal activity or β3-adrenoceptors-mediated signaling. Finally, we demonstrate that NE/β3-adrenoceptors-mediated signaling regulates NSC activation through molecular clock BMAL1. Therefore, our study unravels that adult NSCs precisely coordinate circadian neural circuit and intrinsic molecular circadian clock to adapt their cellular behavior across the day.

A Reversible Neural Stem Cell Quiescence and Activation Culture System for Metabolic Study.

Stem cells in vivo can transit between quiescence and activation, two metabolically distinct states. It is increasingly appreciated that cell metabolism assumes profound roles in stem cell maintenance and tissue homeostasis. However, the lack of suitable models greatly hinders our understanding of the metabolic control of stem cell quiescence and activation. In the present study, we have utilized classical signaling pathways and developed a cell culture system to model reversible NSC quiescence and activation. Unlike activated ones, quiescent NSCs manifested distinct morphology characteristics, cell proliferation, and cell cycle properties but retained the same cell proliferation and differentiation potentials once reactivated. Further transcriptomic analysis revealed that extensive metabolic differences existed between quiescent and activated NSCs. Subsequent experimentations confirmed that NSC quiescence and activation transition was accompanied by a dramatic yet coordinated and dynamic shift in RNA metabolism, protein synthesis, and mitochondrial and autophagy activity. The present work not only showcases the broad utilities of this powerful in vitro NSC quiescence and activation culture system but also provides timely insights for the field and warrants further investigations.

Serine and Arginine-Rich Splicing Factor 3 Promotes the Activation of Quiescent Mouse Neural Stem Cells.

The quiescence and activation of adult stem cells are regulated by many kinds of molecular mechanisms, and RNA alternative splicing participates in regulating many cellular processes. However, the relationship between stem cell quiescence and activation regulation and gene alternative splicing has yet to be studied. In this study, we aimed to elucidate the regulation of stem cell quiescence and activation by RNA alternative splicing. The upregulated genes in activated mouse neural stem cells (NSCs), muscle stem cells, and hematopoietic stem cells were collected for Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis. The genes from three tissue stem cells underwent Venn analysis. The mouse NSCs were used for quiescence and reactivation induction. The immunostaining of cell-specific markers was performed to identify cell properties. The reverse transcription-polymerase chain reaction and western blotting were used to detect the gene expression and protein expression, respectively. We found that the upregulated genes in activated stem cells from three tissues were all enriched in RNA splicing-related biological processes; the upregulated RNA splicing-related genes in activated stem cells displayed tissue differences; mouse NSCs were successfully induced into quiescence and reactivation in vitro without losing differentiation potential; serine and arginine-rich splicing factor 3 (Srsf3) was highly expressed in the activated mouse NSCs, and the overexpression of SRSF3 protein promoted the activation of quiescent mouse NSCs and increased the neural cell production. Our data indicate that the alternative splicing change may underline the transition of quiescence and activation of stem cells. The manipulation of the splicing factor may benefit tissue repair by promoting the activation of quiescent stem cells.

3D Bioprinted differentiated murine neurospheres as a neurogenesis model

AbstractBackgroundAdult neurogenesis, the generation of neurons by neural stem cells (NSCs), occurs mainly in neurogenic niches, located in the dentate gyrus of the hippocampus and in the subventricular zone (SVZ) of the lateral ventricles. However, in Alzheimer’s disease (AD), proliferative activity of NSCs and the generation of new neurons is deregulated. Although there are animal models of AD, developing an in vitro three‐dimensional (3D) model of the disease may certainly enhance the approach of a multidinamic microenvironment able to evaluate specific areas of pathogenesis. The aim of this study was to produce 3D constructs targeting their application as an in vitro model of neurogenesis.MethodNeurosphere (clusters of NSCs and neural progenitor cells) cultures were developed from isolated cells from the SVZ of 6 weeks old C57bl/6 mice. Cells were cultivated in poly‐HEMA coated flasks for 7 days, then, formed neurospheres (∼100µm) were dissociated and bioprinted in an alginate:gelatin solution (6%:4%, respectively). The bioink was 3D printed, crosslinked with 2% CaCl2, and the constructs were maintained for 2 days in culture medium to allow neurosphere expansion. From days 3‐7, the constructs were cultivated in growth or differentiation medium, to stimulate neuronal differentiation. Morphological organization of cells within the neurospheres was evaluated.ResultThe produced 3D constructs are cytocompatible and allow neurosphere formation and growth under growth condition (Fig. 1A). As the growth medium was substituted by the neuronal differentiation medium, processes outgrowth was observed spreading outside the spheres (Fig. 1B).ConclusionA biocompatible construct loaded with neurospheres was produced by 3D bioprinting. The outgrowth of cellular processes outside the spheres suggest that neuronal differentiation occurs, showing that the biomaterial consists in an excellent model to investigate neurogenesis and neurogenic niches dynamics, two impaired elements in AD pathogenesis. Further steps of this study include the improvement of the 3D constructs, creating a microenvironment suitable to better simulate the potential of cellular differentiation under AD pathological conditions. Acknowledgments: This study was supported by São Paulo Research Foundation (FAPESP, grants 2022/08664‐4 and 2018/45605‐8), and by the Brazilian National Council for Scientific and Technological Development (CNPq, grant 406258/2022‐8).