Proliferation Of Neural Progenitor Cells Research Articles

Low-intensity ultrasound ameliorates brain organoid integration and rescues microcephaly deficits.

Human brain organoids represent a remarkable platform for modeling neurological disorders and a promising brain repair approach. However, the effects of physical stimulation on their development and integration remain unclear. Here, we report that low-intensity ultrasound significantly increases neural progenitor cell proliferation and neuronal maturation in cortical organoids. Histological assays and single-cell gene expression analyses reveal that low-intensity ultrasound improves the neural development in cortical organoids. Following organoid grafts transplantation into the injured somatosensory cortices of adult mice, longitudinal electrophysiological recordings and histological assays reveal that ultrasound-treated organoid grafts undergo advanced maturation. They also exhibit enhanced pain-related gamma-band activity and more disseminated projections into the host brain than the untreated groups. Finally, low-intensity ultrasound ameliorates neuropathological deficits in a microcephaly brain organoid model. Hence, low-intensity ultrasound stimulation advances the development and integration of brain organoids, providing a strategy for treating neurodevelopmental disorders and repairing cortical damage.

Impact of metformin on neocortical development during pregnancy: Involvement of ERK and p35/CDK5 pathways

Metformin, the most commonly prescribed drug for the treatment of diabetes, is increasingly used during pregnancy to address various disorders such as diabetes, obesity, preeclampsia, and metabolic diseases. However, its impact on neocortex development remains unclear. Here, we investigated the direct effects of metformin on neocortex development, focusing on ERK and p35/CDK5 regulation. Using a pregnant rat model, we found that metformin treatment during pregnancy induces small for gestational age (SGA) and reduces relative cortical thickness in embryos and neonates. Additionally, we discovered that metformin inhibits neural progenitor cell proliferation in the sub-ventricular zone (SVZ)/ventricular zone (VZ) of the developing neocortex, a process possibly mediated by ERK inactivation. Furthermore, metformin induces neuronal apoptosis in the SVZ/VZ area of the developing neocortex. Moreover, metformin retards neuronal migration, cortical lamination, and differentiation, potentially through p35/CDK5 inhibition in the developing neocortex. Remarkably, compensating for p35 through in utero electroporation partially rescues metformin-impaired neuronal migration and development. In summary, our study reveals that metformin disrupts neocortex development by inhibiting neuronal progenitor proliferation, neuronal migration, cortical layering, and cortical neuron maturation, likely via ERK and p35/CDK5 inhibition. Consequently, our findings advocate for caution in metformin usage during pregnancy, given its potential adverse effects on fetal brain development.

Harnessing developmental dynamics of spinal cord extracellular matrix improves regenerative potential of spinal cord organoids

Neonatal spinal cord tissues exhibit remarkable regenerative capabilities as compared to adult spinal cord tissues after injury, but the role of extracellular matrix (ECM) in this process has remained elusive. Here, we found that early developmental spinal cord had higher levels of ECM proteins associated with neural development and axon growth, but fewer inhibitory proteoglycans, compared to those of adult spinal cord. Decellularized spinal cord ECM from neonatal (DNSCM) and adult (DASCM) rabbits preserved these differences. DNSCM promoted proliferation, migration, and neuronal differentiation of neural progenitor cells (NPCs) and facilitated axonal outgrowth and regeneration of spinal cord organoids more effectively than DASCM. Pleiotrophin (PTN) and Tenascin (TNC) in DNSCM were identified as contributors to these abilities. Furthermore, DNSCM demonstrated superior performance as a delivery vehicle for NPCs and organoids in spinal cord injury (SCI) models. This suggests that ECM cues from early development stages might significantly contribute to the prominent regeneration ability in spinal cord.

Abstract 2776: Exploring the differences between brain tumor initiating cell extracellular vesicles and secreted factors that influence the tumor microenvironment

Abstract Glioblastoma (GBM) is the most common and lethal primary brain tumor in adults. Within the tumor, there is a subtype of cells deemed brain tumor initiating cells (BTICs), which contribute to tumor progression, therapy resistance, and tumoral heterogeneity. The tumor microenvironment, surrounding tumors, contains an ecosystem of cells in which BTICs can interact with and further promote tumor progression. In the lateral wall of the lateral ventricles, the neurogenic niche of the subventricular zone (SVZ), contains neural progenitor cells (NPCs) that self-renew and generate highly migratory neuroblasts. Interestingly, GBM tumors proximal to the SVZ are more proliferative, migratory, and negatively impact the survival of patients. We have previously observed that the presence of GBM induces reciprocal changes to the SVZ altering its neurogenic capacity and resulting in a more proliferative tumor. BTICs share similar self-renewal capacity to NPCs and may exert intercellular communication resulting in increased malignancy features. The mechanisms of intercellular communication, present in the SVZ include paracrine, autocrine, direct cell contact, gap junctions, nanotubes, and more recently extracellular vesicles (EVs). However, the exact mechanisms of how BTICs communicate with NPCs is not well understood. EVs are a heterogenous group of cell-derived membranous structures that serve as means of intercellular communication, allowing for cells to exchange proteins, lipids, RNA, and other genetic material. Cancer cells release higher amounts of EVs than non-malignant cells, and these EVs help communicate with other nearby cells, leading to promotion of tumorigenesis. We have observed that BTICs-derived EVs increase the proliferation of human NPCs, while altering their morphology and expression of stem cell markers. These effects are not observed when NPCs are treated with vesicle-free BTIC-derived secreted factors. To investigate the distinctions between BTIC secreted factors and protein cargo contained in BTIC-EVs, we employ the proximity protein labelling system, TurboID, and express this system in BTIC-EVs. The overarching goal of this project is to characterize BTIC-derived EV cargo to identify proteins that modify the GBM tumor microenvironment. We hypothesize that the phenotype of non-cancer NPCs is altered by distinct protein cargo contained in BTIC-derived EVs. The results of these experiments will help us to elucidate what BTIC-specific EV cargo impacts the tumor microenvironment, with the potential of revealing therapeutic targets for GBM. Citation Format: Marissa Russo, Maria Jose Ulloa Navas, Emily Norton-Ramos, Alfredo Quiñones Hinojosa, Hugo Guerrero Cazares. Exploring the differences between brain tumor initiating cell extracellular vesicles and secreted factors that influence the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2776.

The Role of Bcl11 Transcription Factors in Neurodevelopmental Disorders.

Neurodevelopmental disorders (NDDs) comprise a diverse group of diseases, including developmental delay, autism spectrum disorder (ASD), intellectual disability (ID), and attention-deficit/hyperactivity disorder (ADHD). NDDs are caused by aberrant brain development due to genetic and environmental factors. To establish specific and curative therapeutic approaches, it is indispensable to gain precise mechanistic insight into the cellular and molecular pathogenesis of NDDs. Mutations of BCL11A and BCL11B, two closely related, ultra-conserved zinc-finger transcription factors, were recently reported to be associated with NDDs, including developmental delay, ASD, and ID, as well as morphogenic defects such as cerebellar hypoplasia. In mice, Bcl11 transcription factors are well known to orchestrate various cellular processes during brain development, for example, neural progenitor cell proliferation, neuronal migration, and the differentiation as well as integration of neurons into functional circuits. Developmental defects observed in both, mice and humans display striking similarities, suggesting Bcl11 knockout mice provide excellent models for analyzing human disease. This review offers a comprehensive overview of the cellular and molecular functions of Bcl11a and b and links experimental research to the corresponding NDDs observed in humans. Moreover, it outlines trajectories for future translational research that may help to better understand the molecular basis of Bcl11-dependent NDDs as well as to conceive disease-specific therapeutic approaches.

Substrate-bound and soluble domains of tenascin-C regulate differentiation, proliferation and migration of neural stem and progenitor cells.

The lack of regenerative capacity of the central nervous system is one of the major challenges nowadays. The knowledge of guidance cues that trigger differentiation, proliferation, and migration of neural stem and progenitor cells is one key element in regenerative medicine. The extracellular matrix protein tenascin-C (Tnc) is a promising candidate to regulate cell fate due to its expression in the developing central nervous system and in the adult neural stem cell niches. Of special interest are the alternatively spliced fibronectin type III (FnIII) domains of Tnc whose combinatorial diversity could theoretically generate up to 64 isoforms in the mouse. A total of 27 isoforms have already been discovered in the developing brain, among others the domain combinations A1D, CD, and A124BCD. In the present study, these domains as well as the combination of the constitutively expressed FnIII domains 7 and 8 (78) were expressed in Chinese hamster ovary cells as pseudo-antibodies fused to the Fc-fragment of a human immunoglobulin G antibody. The fusion proteins were presented to primary mouse neural stem/progenitor cells (NSPCs) grown as neurospheres, either as coated culture substrates or as soluble additives in vitro. The influence of the domains on the differentiation, proliferation and migration of NSPCs was analyzed. We observed that the domain combination A124BCD promoted the differentiation of neurons and oligodendrocytes, whereas the domain A1D supported astrocyte differentiation. The constitutively expressed domain 78 had a proliferation and migration stimulating impact. Moreover, most effects were seen only in one of the presentation modes but not in both, suggesting different effects of the Tnc domains in two- and three-dimensional cultures. This knowledge about the different effect of the Tnc domains might be used to create artificial three-dimensional environments for cell transplantation. Hydrogels spiked with Tnc-domains might represent a promising tool in regenerative medicine.

Abstract WP14: Photobiomodulation Treatment Enhancesand Sustains Neurogenesis Following Acute Ischemic Stroke

Rationale: Previous work has indicated that photobiomodulation (PBM), may provide neuroprotection and improve neurological function after acute ischemic stroke. However, the underlying mechanism remains unclear. The present study was performed to examine whether PBM, a noninvasive neurostimulation technique, promotes neurogenesis in a middle cerebral artery occlusion (MCAO) stroke rat model. Methods: Beginning 6 h after MCAO, once-daily 2-min PBM treatments(808 nm) were applied to the infarcted hemisphere for 7 consecutive days. Samples were collected 7 days and 14 days after MCAO stroke. Fluorescent labeling, Western blotting, and behavioral tests were performed accordingly. Results: We found that PBM-treated animals displayed improved motor and sensory functions. PBM treatment significantly expanded the pool of neural progenitor cell (NPC) and newly generated immature neurons in the cortical peri-infarct region after acute ischemic stroke. Application of PBM strikingly diminished the MCAO-induced loss of NPCs and newly-formed neurons. In addition, the amount of newly generated mature neurons in the peri-infarct zone was significantly promoted by PBM treatment. Intriguingly, PBM reduced reactive gliogenesis significantly while promoting oligodendrogenesis and preserving myelination. Mechanistic studies uncovered that PBM enhances the expression of brain-derived neurotrophic factor (BDNF) and initiates the activation of the BDNF/TrkB signaling pathway. Conclusions: Our findings indicate that PBM promotes neurogenesis and functional recovery through stimulating NPC proliferation, reducing NPC and immature neuron cell death, and tamping excessive gliogenesis. These effects were due, in part, to the upregulated release of trophic factors. These findings support PBM as an effective neuroregenerative treatment for acute ischemic stroke. Keywords: Ischemic stroke, Photobiomodulation (PBM), Neurogenesis, Functional recovery, Neurotrophic factors

Ischemic and hemorrhagic stroke lesion environments differentially alter the glia repair potential of neural progenitor cell and immature astrocyte grafts

Using cell grafting to direct glia-based repair mechanisms in adult CNS injuries represents a potential therapeutic strategy for supporting functional neural parenchymal repair. However, glia repair directed by neural progenitor cell (NPC) grafts is dramatically altered by increasing lesion size, severity, and mode of injury. To address this, we studied the interplay between astrocyte differentiation and cell proliferation of NPC in vitro to generate proliferating immature astrocytes (ImA) using hysteretic conditioning. ImA maintain proliferation rates at comparable levels to NPC but showed robust immature astrocyte marker expression including Gfap and Vimentin. ImA demonstrated enhanced resistance to myofibroblast-like phenotypic transformations upon exposure to serum enriched environments in vitro compared to NPC and were more effective at scratch wound closure in vitro compared to quiescent astrocytes. Glia repair directed by ImA at acute ischemic striatal stroke lesions was equivalent to NPC but better than quiescent astrocyte grafts. While ischemic injury environments supported enhanced survival of grafts compared to healthy striatum, hemorrhagic lesions were hostile towards both NPC and ImA grafts leading to poor survival and ineffective modulation of natural wound repair processes. Our findings demonstrate that lesion environments, rather than transcriptional pre-graft states, determine the survival, cell-fate, and glia repair competency of cell grafts applied to acute CNS injuries.

Cationic Hydrogels Modulate Neural Stem and Progenitor Cell Proliferation and Differentiation Behavior in Dependence of Cationic Moiety Concentration in 2D Cell Culture.

The central nervous system (CNS) has a limited regenerative capacity because a hostile environment prevents tissue regeneration after damage or injury. Neural stem/progenitor cells (NSPCs) are considered a potential resource for CNS repair, which raises the issue of adequate cultivation and expansion procedures. Cationic charge supports the survival and adhesion of NSPCs. Typically, tissue culture plates with cationic coatings, such as poly-l-ornithine (PLO), have been used to culture these cell types (NSPCs). Yet presently, little is known about the impact of cationic charge concentration on the viability, proliferation, and differentiation capacity of NSPCs. Therefore, we have recently developed well-defined, fully synthetic hydrogel systems G1 (gel 1) to G6 (gel 6) that allow for the precise control of the concentration of the cationic trimethylaminoethyl acrylate (TMAEA) molecule associated with the polymer in a range from 0.06 to 0.91 μmol/mg. When murine NSPCs were cultured on these gels under differentiation conditions, we observed a strong correlation of cationic charge concentration with NSPC survival. In particular, neurons were preferentially formed on gels with a higher cationic charge concentration, whereas astrocytes and oligodendrocytes favored weakly charged or even neutral gel surfaces. To test the properties of the gels under proliferative conditions, the NSPCs were cultivated in the presence of fibroblast growth factor 2 (FGF2). The cytokine significantly increased the number of NSPCs but delayed the differentiation toward neurons and glia cells. Thus, the hydrogels are compatible with the survival, expansion, and differentiation of NSPCs and may be useful to create supportive environments in transplantation approaches.

The Neural Progenitor Cell-Associated Transcription Factor FoxG1 Regulates Cardiac Epicardial Cell Proliferation.

The epicardium is a layer of mesothelial cells that covers the surface of the heart. During development, epicardial cells undergo epithelial-to-mesenchymal transition (EMT) to form multipotent precursors that migrate into the heart and contribute to the coronary vasculature by differentiating into adventitial fibroblasts, smooth muscle cells, and endothelial cells. Epicardial cells also provide paracrine signals to cardiac myocytes that are required for appropriate heart growth. In adult hearts, a similar process of epicardial cell EMT, migration, and differentiation occurs after myocardial infarction (MI, heart attack). Pathological cardiac hypertrophy is associated with fibrosis, negative remodeling, and reduced cardiac function. In contrast, aerobic exercises such as swimming and running promote physiological (i.e., beneficial) hypertrophy, which is associated with angiogenesis and improved cardiac function. As epicardial cell function(s) during physiological hypertrophy are poorly understood, we analyzed and compared the native epicardial cells isolated directly from the hearts of running-exercised mice and age-matched, nonrunning littermates. To obtain epicardial cells, we enzymatically digested the surfaces of whole hearts and performed magnetic-activated cell sorting (MACS) with antibodies against CD104 (integrin β4). By cDNA microarray assays, we identified genes with increased transcription in epicardial cells after running exercise; these included FoxG1, a transcription factor that controls neural progenitor cell proliferation during brain development and Snord116, a small noncoding RNA that coordinates expression of genes with epigenetic, circadian, and metabolic functions. In cultured epicardial cells, shRNA-mediated FoxG1 knockdown significantly decreased cell proliferation, as well as Snord116 expression. Our results demonstrate that FoxG1 regulates epicardial proliferation, and suggest it may affect cardiac remodeling.

JADE1 is dispensable for the brain development in mice

In mammalian brain development, WNT signaling balances proliferation and differentiation of neural progenitor cells, and is essential for the maintenance of regular brain development. JADE1 is a candidate transcription co-factor essential for DNA replication, cell division, and cell cycle regulation. In 293T cells, JADE1 is stabilized by von Hippel–Lindau protein pVHL, promotes the β-catenin ubiquitination and thus blunts canonical WNT signaling. Furthermore, JADE1 inhibits β-catenin-induced ectopic axis formation in Xenopus embryos. However, JADE1's role in mammalian brain development remains unknown. Here, we generated a new Jade1 knockout mouse line using CRISPR-Cas9 technology. We found that JADE1 null resulted in decreased survival rate, reduced body weight and brain weight in mice. However, histological analysis revealed a normal brain development. Furthermore, Jade1 null neural progenitor cells proliferated normally in vivo and in vitro. RNA-seq analysis further showed that JADE1 loss did not affect the cerebral cortex gene expression. Our findings indicate that JADE1 is dispensable for developing the cerebral cortex in mice.

The Role of KDM2A and H3K36me2 Demethylation in Modulating MAPK Signaling During Neurodevelopment.

Intellectual disability (ID) is a condition characterized by cognitive impairment and difficulties in adaptive functioning. In our research, we identified two de novo mutations (c.955C>T and c.732C>A) at the KDM2A locus in individuals with varying degrees of ID. In addition, by using the Gene4Denovo database, we discovered five additional cases of de novo mutations in KDM2A. The mutations we identified significantly decreased the expression of the KDM2A protein. To investigate the role of KDM2A in neural development, we used both 2D neural stem cell models and 3D cerebral organoids. Our findings demonstrated that the reduced expression of KDM2A impairs the proliferation of neural progenitor cells (NPCs), increases apoptosis, induces premature neuronal differentiation, and affects synapse maturation. Through ChIP-Seq analysis, we found that KDM2A exhibited binding to the transcription start site regions of genes involved in neurogenesis. In addition, the knockdown of KDM2A hindered H3K36me2 binding to the downstream regulatory elements of genes. By integrating ChIP-Seq and RNA-Seq data, we made a significant discovery of the core genes' remarkable enrichment in the MAPK signaling pathway. Importantly, this enrichment was specifically linked to the p38 MAPK pathway. Furthermore, disease enrichment analysis linked the differentially-expressed genes identified from RNA-Seq of NPCs and cerebral organoids to neurodevelopmental disorders such as ID, autism spectrum disorder, and schizophrenia. Overall, our findings suggest that KDM2A plays a crucial role in regulating the H3K36me2 modification of downstream genes, thereby modulating the MAPK signaling pathway and potentially impacting early brain development.

Loss of Aspm causes increased apoptosis of developing neural cells during mouse cerebral corticogenesis.

Abnormal spindle-like microcephaly associated (ASPM) is a causative gene of primary autosomal recessive microcephaly. Microcephaly is considered to be a consequence of a small brain, but the associated molecular mechanisms are not fully understood. In this study, we generated brain-specific Aspm knockout mice to evaluate the fetal brain phenotype and observed cortical reduction in the late stage of murine cortical development. It has been reported that the total number of neurons is regulated by the number of neural stem and progenitor cells. In the Aspm knockout mice, no apparent change was shown in the neural progenitor cell proliferation and there was no obvious effect on the number of newly generated neurons in the developing cortex. On the other hand, the knockout mice showed a constant increase in apoptosis in the cerebral cortex from the early through the late stages of cortical development. Furthermore, apoptosis occurred in the neural progenitor cells associated with DNA damage. Overall, these results suggest that apoptosis of the neural progenitor cells is involved in the thinning of the mouse cerebral cortex, due to the loss of the Aspm gene in neocortical development.

Telomere shortening induces aging-associated phenotypes in hiPSC-derived neurons and astrocytes

Telomere shortening is a well-established hallmark of cellular aging. Telomerase reverse transcriptase (TERT) plays a crucial role in maintaining the length of telomeres, which are specialised protective caps at the end of chromosomes. The lack of in vitro aging models, particularly for the central nervous system (CNS), has impeded progress in understanding aging and age-associated neurodegenerative diseases. In this study, we aimed to explore the possibility of inducing aging-associated features in cell types of the CNS using hiPSC (human induced pluripotent stem cell) technology. To achieve this, we utilised CRISPR/Cas9 to generate hiPSCs with a loss of telomerase function and shortened telomeres. Through directed differentiation, we generated motor neurons and astrocytes to investigate whether telomere shortening could lead to age-associated phenotypes. Our findings revealed that shortened telomeres induced age-associated characteristics in both motor neurons and astrocytes including increased cellular senescence, heightened inflammation, and elevated DNA damage. We also observed cell-type specific age-related morphology changes. Additionally, our study highlighted the fundamental role of TERT and telomere shortening in neural progenitor cell (NPC) proliferation and neuronal differentiation. This study serves as a proof of concept that telomere shortening can effectively induce aging-associated phenotypes, thereby providing a valuable tool to investigate age-related decline and neurodegenerative diseases.Graphical abstract

A “toxic window” study on the hippocampal development of mice offspring exposed to azithromycin at different doses, courses, and time during pregnancy

BackgroundAzithromycin, one of the new-generation macrolides, is an effective medicine for the treatment of mycoplasma infection during pregnancy. Epidemiological studies have reported adverse pregnancy outcomes with prenatal azithromycin exposure (PAzE). However, the effect of PAzE on fetal hippocampal development is unclear. This study aimed to explore the effects and potential mechanism of PAzE-induced fetal hippocampal development at different doses, courses, and time. MethodPregnant mice were administered azithromycin by gavage at different doses (50, 100 or 200 mg/kg.d), different courses (gestational day (GD)15–17 for three consecutive days, or GD17 once a day) and different time (GD10-12, GD15-17). ResultsCompared with the control group, morphological development damage of the fetal hippocampus was observed in the PAzE group, with a dysbalance in neuronal proliferation and apoptosis, decreased expression of the neuronal-specific marker Snap25, NeuN, PSD95 and Map2, increased expression of the glial-specific marker Iba1, GFAP, and S-100β, and decreased expression of P2ry12. The PAzE-induced hippocampal developmental deficiency varied based on different doses, courses, and time, and the developmental toxicity was most significant in the late pregnancy, high dose, multi-course group (AZHT). The significant reduction of SOX2 and Wnt, which were related to regulation of neural progenitor cells (NPCs) proliferation in PAzE fetus compared with the control group indicated that the SOX2/Wnt signaling may be involved in PAzE-induced hippocampal developmental toxicity. ConclusionIn this study, PAzE was associated with hippocampal developmental toxicity in a variety of nerve cells. Hippocampal developmental toxicity due to azithromycin was most significant in the late pregnancy, high-dose (equivalent to maximum clinical dose) and multi-course group (AZHT). The findings provide an experimental and theoretical foundation for guiding the sensible use of medications during pregnancy and effectively assessing the risk of fetal hippocampal developmental toxicity.

Glucocorticoid Receptor Down-Regulation Affects Neural Stem Cell Proliferation and Hippocampal Neurogenesis.

Dysregulation of the hypothalamic-pituitary-adrenal axis and abnormalities in the glucocorticoid receptor (GR) have been linked to major depressive disorder. Given the critical role of GR in stress response regulation, we investigated the impact of GR changes on neural stem cells (NSCs) proliferation and hippocampal neurogenesis. Stress response was induced using dexamethasone (DEX), a GR agonist, which led to reduced proliferation of neural stem cells and neural progenitor cells, as well as decreased expression of GR. Additionally, a reduction of serum concentration within the culture media resulted in suppressed cell proliferation, accompanied by decreased GR expression. The association between GR expression and cell proliferation was further confirmed through GR siRNA knockdown and overexpression experiments. Furthermore, in vivo studies utilizing young male C57BL/6 mice demonstrated that corticosterone (CORT) (35μg/ml) administered through drinking water for four weeks induced depression-like behavior, as indicated by increased immobility times in forced swimming and tail suspension tests. CORT exposure led to reduced GR and nestin expression levels, along with diminished numbers of BrdU-positive cells in the hippocampi, indicating impaired hippocampal neurogenesis. Taken together, our findings provide the first evidence that stress-induced downregulation of GR negatively affects neurogenesis by inhibiting NSCs proliferation.

Development of synthetic modulator enabling long-term propagation and neurogenesis of human embryonic stem cell-derived neural progenitor cells

Neural progenitor cells (NPCs) are essential for in vitro drug screening and cell-based therapies for brain-related disorders, necessitating well-defined and reproducible culture systems. Current strategies employing protein growth factors pose challenges in terms of both reproducibility and cost. In this study, we developed a novel DNA-based modulator to regulate FGFR signaling in NPCs, thereby facilitating the long-term maintenance of stemness and promoting neurogenesis. This DNA-based FGFR-agonist effectively stimulated FGFR1 phosphorylation and activated the downstream ERK signaling pathway in human embryonic stem cell (HESC)-derived NPCs. We replaced the basic fibroblast growth factor (bFGF) in the culture medium with our DNA-based FGFR-agonist to artificially modulate FGFR signaling in NPCs. Utilizing a combination of cell experiments and bioinformatics analyses, we showed that our FGFR-agonist could enhance NPC proliferation, direct migration, and promote neurosphere formation, thus mimicking the functions of bFGF. Notably, transcriptomic analysis indicated that the FGFR-agonist could specifically influence the transcriptional program associated with stemness while maintaining the neuronal differentiation program, closely resembling the effects of bFGF. Furthermore, our culture conditions allowed for the successful propagation of NPCs through over 50 passages while retaining their ability to efficiently differentiate into neurons. Collectively, our approach offers a highly effective method for expanding NPCs, thereby providing new avenues for disease-in-dish research and drug screening aimed at combating neural degeneration.

Single-cell transcriptomics analysis of zebrafish brain reveals adverse effects of manganese on neurogenesis

Manganese (Mn) is considered as an important environmental risk factor for Parkinson's disease. Excessive exposure to Mn can damage various neural cells and affect the neurogenesis, resulting in neurological dysfunction. However, the specific mechanisms of Mn exposure affecting neurogenesis have not been well understood, including compositional changes and heterogeneity of various neural cells. Zebrafish have been successfully used as a neurotoxicity model due to its homology with mammals in several key regions of the brain, as well as its advantages such as small size. We performed single-cell RNA sequencing of zebrafish brains from normal and Mn-exposed groups. Our results suggested that low levels of Mn exposure activated neurogenesis in the zebrafish brain, including promoting the proliferation of neural progenitor cells and differentiation to newborn neurons and oligodendrocytes, while high levels of Mn exposure inhibited neurogenesis and neural function. Mn could affect neurogenesis through specific molecular pathways. In addition, Mn regulated intercellular communication and affected cellular communication in neural cells through specific signaling pathways. Taken together, our study elucidates the cellular composition of the zebrafish brain and adds to the understanding of the mechanisms involved in Mn-induced neurogenesis damage.

Identification of novel NRF2-dependent genes as regulators of lead and arsenic toxicity in neural progenitor cells

Lead (Pb) and arsenic (As) are prevalent metal contaminants in the environment. Exposures to these metals are associated with impaired neuronal functions and adverse effects on neurodevelopment in children. However, the molecular mechanisms by which Pb and As impair neuronal functions remain poorly understood. Here, we identified F2RL2, TRIM16L, and PANX2 as novel targets of Nuclear factor erythroid 2-related factor 2 (NRF2)—the master transcriptional factor for the oxidative stress response—that are commonly upregulated with both Pb and As in human neural progenitor cells (NPCs). Using a ChIP (Chromatin immunoprecipitation)-qPCR assay, we showed that NRF2 directly binds to the promoter region of F2RL2, TRIM16L, and PANX2 to regulate expression of these genes. We demonstrated that F2RL2, PANX2, and TRIM16L have differential effects on cell death, proliferation, and differentiation of NPCs in both the presence and absence of metal exposures, highlighting their roles in regulating NPC function. Furthermore, the analyses of the transcriptomic data on NPCs derived from autism spectrum disorder (ASD) patients revealed that dysregulation of F2RL2, TRIM16L, and PANX2 was associated with ASD genetic backgrounds and ASD risk genes. Our findings revealed that Pb and As induce a shared NRF2-dependent transcriptional response in NPCs and identified novel genes regulating NPC function. While further in vivo studies are warranted, this study provides a novel mechanism linking metal exposures to NPC function and identifies potential genes of interest in the context of neurodevelopment.

Pluripotent stem cell-derived neural progenitor cells can be used to model effects of IL-6 on human neurodevelopment

ABSTRACT Maternal immune activation (MIA) increases the risks for neurodevelopmental disorders in offspring through inflammatory cytokines, including interleukin-6 (IL-6). We therefore aimed to establish a human two-dimensional (2D) in vitro neural model to investigate the effects of IL-6 exposure on neurodevelopment. IL-6 signal transduction requires two receptors: interleukin-6 signal transducer (IL6ST) and interleukin-6 receptor (IL6R). Prenatally, neural cells lack IL6R, and hence cannot elicit cis IL-6 signaling, but IL6R can be provided by microglia in trans. We demonstrate here that an immortalized human neural progenitor cell (NPC) line, ReNCell CX, expresses IL6ST and elicits both cis and trans IL-6 signaling, limiting its use as a model of MIA. In contrast, induced pluripotent stem cell (iPSC)-derived NPCs only activate the IL-6 cascade in trans. Activation of the trans IL-6 cascade did not result in increased proliferation of iPSC-derived NPCs or ReNCell CX, as has been demonstrated in animal models. iPSC-derived NPCs upregulated NR2F1 expression in response to IL-6 signaling in line with analogous experiments in organoids. Thus, iPSC-derived NPCs can be used to model gene expression changes in response to MIA in 2D cultures.