Neuronal Cell Differentiation Research Articles

Optimal Production of 3D Neuronal Lineage Population by Morphological Classification.

The increasing prevalence of neurodegenerative diseases and toxic substance exposure highlights the need for neuronal cell models that closely mimic human neurons in vivo. Compared to traditional models, human pluripotent stem cell (hPSC)-derived three-dimensional models mimic human physiological characteristics and complex nervous system interactions. These models enable patient-specific treatments and improve the predictive accuracy of drug toxicity evaluations. However, differentiation efficiency varies based on organoid size, structure, and cell line characteristics, necessitating standardized protocols for consistent outcome. The morphological characteristics of hPSC-derived embryonic bodies (EBs) formed by concave microwells were analyzed at the early stage of neuronal differentiation. Criteria were established to identify cells with high differentiation efficiency, enabling the optimization of differentiation methods applicable across various cell lines. Neuronal organoids were generated using a microfluidic-concave chip, and their suitability for drug toxicity testing was assessed. EBs, formed in 500µm concave microwells, exhibited the highest efficiency for neuronal cell differentiation. Cavity-like EBs were more suitable for neuronal differentiation and maturation than cystic-like forms. The optimal neuronal lineage differentiation method was established, and the drug toxicity sensitivity of organoids generated from this method was validated. This study identified EB structures suitable for neuronal lineage differentiation based on morphological classification. Furthermore, this study suggested an optimal method for generating neuronal organoids. This method can be applied to various cell lines, enabling its precise use in patient-specific treatments and drug toxicity tests.

Identifying Environmental Chemicals Impacting Neurodevelopment using a random Mixture-Based Screening Approach.

Identifying Environmental Chemicals Impacting Neurodevelopment using a random Mixture-Based Screening Approach.

Novel miRNAs, miR-937-3p, miR-4536-3p, and miR-4650-5p, can Modulate Neuronal Differentiation via the Wnt/MAPK Pathway in SH-SY5Y Cells.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate various biological processes, including cell differentiation. Despite their potential, their role in promoting neuronal differentiation by targeting neuronal genes and modulating signaling pathways is poorly understood. In this study, we aimed to elucidate the functions of miR-937-3p-, miR-4536-3p-, and miR-4650-5p-inhibitors in the neuronal differentiation of SH-SY5Y cells. We also aimed to determine the underlying mechanisms via qPCR, luciferase assay, immunocytochemistry, and western blotting analysis. Our findings confirmed that miRNAs participated in neuronal differentiation and regulated the Wnt/MAPK signaling pathway. Specifically, we identified Netrin1 (NTN1), Drebrin1 (DBN1), and Netrin-G1 (NTNG1) as target genes of miR-937-3p, miR-4536-3p, and miR-4650-5p, respectively. The treatment with the miRNA inhibitors increased the expression levels of neuronal markers such as TUBB3, NEFH, NEFM, NEFL, and MAP2. It also enhanced the protein expression levels of Wnt and MAPK signaling. Therefore, the inhibitors of miR-937-3p, miR-4536-3p, and miR-4650-5p could promote neuronal differentiation by targeting neuronal genes and activating the Wnt/MAPK signaling pathway.

Effects of selected heavy metals on neuronal differentiation of Asian seabass spinal cord cell line.

Effects of selected heavy metals on neuronal differentiation of Asian seabass spinal cord cell line.

The differentiation of glial precursors into neuronal-like cells through the Wnt and Neurotrophin signaling pathways via Ctnnβ1

ABSTRACT Glial precursor cells are among the major types of glia in the dorsal root ganglias (DRGs) of the peripheral nervous system. Previous studies have shown that the transdifferentiation of DRGs-derived glial precursor cells contributes to peripheral neurogenesis. In the present study, we investigated the mRNA expression profiles and examined the effects of differential expression mRNAs (DEMs) during the differentiation of glial precursor cells derived from the rat DRGs. We characterized glial precursor cells derived from rat DRGs explants using immunofluorescence. Sequencing was subsequently conducted, followed by enrichment analysis utilizing gene ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The identified genes were subsequently subjected to protein–protein interaction (PPI) network analysis during the differentiation process of glial precursor cells derived from the rat DRGs. The establishment of a sciatic nerve injury (SNI) model was followed by the detection of the expression of key genes in the Wnt and Neurotrophin pathways in the DRGs of SNI rats via qRT–PCR. Additionally, the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay was employed to assess apoptosis in the DRGs. We detected the mRNA expression profiles during the neuronal differentiation of rat DRGs-derived glial precursor cells. More DEMs and GO terms were detected on the third day of DRGs-derived glial precursor cells transdifferentiation, accompanied by morphological alterations in the cells; that is, some cells presented neuronal-like phenotypic characteristics (the early neuronal marker Tuj1 was positive). KEGG enrichment and PPI network analyses revealed that Wnt and Neurotrophin pathways play crucial roles in the process of glial precursor cell differentiation into neuronal-like cells. After knocking down cadherin-associated protein beta 1 (Ctnnβ1) in the SNI model, the number of apoptotic cells was significantly reduced, and the expression of Wnt4 and Ntrk3 was significantly increased. The Ctnnβ1 gene may be a crosstalk factor between the Wnt and Neurotrophin pathways that negatively regulates the differentiation of glial precursor cells.

Benzeneboronic acid-modified hyaluronic acid hydrogel enhances the differentiation of dorsal root ganglion stem cells in a three-dimensional environment.

Benzeneboronic acid-modified hyaluronic acid hydrogel enhances the differentiation of dorsal root ganglion stem cells in a three-dimensional environment.

Potential of hyaluronic acid and collagen-based scaffolds in promoting stem cell neuronal differentiation for neuroregenerative therapies: A review.

Potential of hyaluronic acid and collagen-based scaffolds in promoting stem cell neuronal differentiation for neuroregenerative therapies: A review.

Magnesium-L-threonate Ameliorates Cognitive Deficit by Attenuating Adult Hippocampal Neurogenesis Impairment in a Mouse Model of Alzheimer's Disease.

Impaired adult hippocampal neurogenesis is a key pathological mechanism contributing to memory deficits in Alzheimer's disease (AD). Recent studies have shown that elevating magnesium levels promotes neurogenesis by enhancing the neuronal differentiation of adult neural progenitor cells in vitro. Therefore, this in vivo study aims to determine if magnesium-L-threonate (MgT) can ameliorate cognitive deficit of AD mice by attenuating adult hippocampal neurogenesis impairment and to reveal the underlying mechanisms. APPswe/PS1dE9 mice were treated with different doses of MgT and ERK inhibitor PD0325901. The memory ability of each mouse was recorded by Morris Water Maze test. After cognitive test, hippocampus tissues were collected to measure the proportion of BrdU/doublecortin double-labeled cells using the flow cytometry test and assess the expression of doublecortin using PCR and Western blot. Furthermore, the activations of CREB, ERK, P38 and JNK were measured by Western blot to identify the involved mechanisms. The cognitive test confirmed that MgT treatment attenuated the memory impairment of APPswe/PS1dE9 mice. Flow cytometry test showed that Brdu/doublecortin labeled newborn neurons gradually increased following MgT administration. In line with the flow cytometry results, Western blot and PCR confirmed that MgT administration significantly increased doublecortin expression levels. Furthermore, the ratios of p-ERK/ERK and p-CREB/CREB increased with MgT elevation. In addition, these effects of MgT treatment were markedly reversed by PD0325901 supplementation. In conclusion, MgT treatment improved cognitive decline by ameliorating adult hippocampal neurogenesis impairment in this AD model, possibly via ERK/CREB activation.

Nervous System Repair and Immunity: Chiral Effects of Biomimetic Self‐Assembled Neuropeptide Hydrogels

AbstractFunction‐encoding peptides have emerged as promising biomaterials capable of replicating the robust biological functions of the extracellular matrix (ECM). Nevertheless, the full potential of their sequence designability remains to be explored to develop highly bioactive peptide‐based biomaterials with minimal immunogenicity. In this study, chiral peptides are self‐assembled into supramolecular hydrogels (FFFKTTKS/fffkttks) incorporating an active sequence derived from collagen hydrolysis, a key ECM factor. While FFFKTTKS (L‐type) and fffkttks (D‐type) peptide‐based hydrogels exhibit comparable viscoelasticity, porosity, and supramolecular architecture, they differ in their nanofiber composition, particularly in helical orientation. In a model of spinal cord injury, the FFFKTTKS hydrogel demonstrates superior neuronal regeneration and motor function recovery compared to its fffkttks counterpart. Further investigations reveal that both FFFKTTKS and fffkttks hydrogels equally promote the expression of ECM‐related genes, subsequently regulating nerve cell adhesion, neuronal differentiation, and synaptic regeneration. Notably, the FFFKTTKS hydrogel elicits a mild immune response and exhibits moderate anti‐inflammatory properties. In contrast, the fffkttks hydrogel triggers a robust immune response, activating the TNF pathway in microglia in vivo. These findings underscore that nanoscale chiral superstructures of specific peptide sequences can effectively modulate biocapability and neuroregeneration, providing critical insights for the rational design of peptide‐based synthetic ECM.

Human neuron chimeric mice reveal impairment of DVL-1-mediated neuronal migration by sevoflurane and potential treatment by rTMS

,Whether early exposure to general anesthetics hurts human brain development is still under discussion. Animal studies have documented multiple neurotoxicities of repeated/prolonged exposure to sevoflurane (Sev, a commonly used pediatric anesthetic) at the neonatal stage. Its effects on human neural development remain elusive. Here, by investigating neural progenitor cells derived from two human embryonic stem cell lines, human cerebral organoids and human neuronal chimeric mice, we found that, although Sev inhibits neuronal differentiation and synaptogenesis of human neural progenitor cells in vitro, it only inhibits human neuronal migration in vivo. Chemogenetic activation of human neurons rescued the defects of cell migration and social dysfunction of Sev-pretreated human neuronal chimeric mice. Mechanistically, Sev inhibits DVL-1/Ca2+ signaling and multiple cell migration-related genes. Overexpressing DVL-1 enhanced the Ca2+ response, neuronal migration and social function of Sev-pretreated chimeric mice. Furthermore, specific modulation of human neurons by high-frequency transcranial magnetic stimulation not only activated DVL-1/Ca2+ signaling but also improved human neuronal migration and social function in chimeric mice. Our data demonstrate that early Sev exposure is toxic to human neuronal migration via inhibiting DVL-1 signaling and that transcranial magnetic stimulation could be potentially therapeutic.

Quantitative phosphoproteomics reveals that nestin is a downstream target of dual leucine zipper kinase during retinoic acid-induced neuronal differentiation of Neuro-2a cells

BackgroundDual leucine zipper kinase (DLK) is critical for neurite outgrowth in the developing nervous system and during nerve regeneration, but the underlying mechanisms remain largely unknown. To address this issue, we generated stable shRNA-mediated DLK-depleted Neuro-2a cell lines and analyzed their phosphoproteome after induction of neuronal differentiation by retinoic acid (RA).ResultsHere, we report the identification of 32 phosphopeptides that exhibited significant differences in relative abundance between control and DLK-depleted cells. Two of the most downregulated phosphopeptides identified after DLK depletion were derived from nestin, a type VI intermediate filament (IF) protein typically expressed in neural progenitor cells. The reduced abundance of these phosphopeptides in response to DLK knockdown was validated using parallel reaction monitoring (PRM)-based quantitative proteomics and paired with a concomitant reduction in nestin mRNA and protein expression, indicating that the decrease in nestin phosphorylation was due to a decrease in total nestin in DLK-depleted cells compared to control cells. This DLK-mediated regulation of nestin expression had no apparent effect on neurite formation because nestin knockdown alone was not sufficient to impair RA-induced neurite extension in parental Neuro-2a cells, and nestin overexpression failed to rescue the neurite outgrowth defect observed in DLK-depleted Neuro-2a cells.ConclusionsTogether, these results demonstrate that nestin is a novel downstream target of DLK signaling but not a mediator of its ability to promote neurite outgrowth during neuronal differentiation.

Activin A enhances neurofunctional recovery following traumatic spinal cord injury by inhibiting autophagy.

In the early stages of traumatic spinal cord injury, extensive accumulation of autophagosomes creates a neurotoxic microenvironment, exacerbating neuronal cell death and worsening tissue damage, ultimately hindering neurofunctional recovery. Activin A is a critical growth factor necessary for the development of the embryonic nervous system and for maintaining neuronal function in the adult cerebral cortex. It can inhibit excessive autophagy in ischemic stroke to reduce neuronal damage. However, the specific mechanism through which Activin A functions in the spinal cord remains poorly understood. In this study, we administered different concentrations of Activin A to neural stem cells from the spinal cord and found that Activin A stimulated the proliferation and neuronal differentiation of neural stem cells. Then, we established an in vitro oxidative stress model by using hydrogen peroxide to stimulate the neural stem cells-induced neurons. We found that Activin A could reduce apoptosis caused by oxidative stress. Subsequently, we treated a mouse model of spinal cord contusion with intrathecal injection of Activin A. Behavioral and electrophysiological results showed that Activin A promoted recovery of motor function and reconstruction of neural circuits in the model mice. Finally, RNA sequencing indicated that Activin A inhibited autophagy by activating the PI3K/AKT/mTOR pathway and upregulating the expression of synaptogenesis-related factor Sema3A in the spinal cord. These results suggest that Activin A may mediate the excessive autophagic response after spinal cord injury, promote the reconstruction of damaged neural circuits, and restore neurological function in the injured spinal cord.

Metformin Protects Human Induced Pluripotent Stem Cell (hiPSC)-Derived Neurons from Oxidative Damage Through Antioxidant Mechanisms.

The antidiabetic drug metformin possesses antioxidant and cell protective effects including in neuronal cells, suggesting its potential use for treating neurodegenerative diseases. This study aimed to assess metformin's effects on viability and antioxidant activity in human-induced pluripotent stem cell (hiPSC)-derived neurons under varying concentrations and stress conditions. Six lines of hiPSC-derived neuronal progenitors derived from healthy human iPSCs were treated with metformin (1-500 µM) on day 18 of differentiation. For mature neurons (day 30), three concentrations (10 µM, 50 µM, and 100 µM) were used to assess cytotoxicity. MG132 proteasomal inhibitor and sodium arsenite (NaArs) were used to investigate oxidative stress, and 50 µM of metformin was tested for its protective effects against oxidative stress in hiPSC-derived neurons. Metformin treatment did not affect cell viability, neuronal differentiation, or trigger reactive oxygen species (ROS) generation in healthy hiPSC-derived motor neurons. Additionally, mitochondrial membrane potential (MMP) loss was not observed at 50 µM metformin. Metformin effectively protected neurons from stress agents and elevated the expression of antioxidant genes when treated with MG132. However, an interplay between MG132 and metformin resulted in lower expression of Nrf2 and NQO1 compared to the MG132 group alone, indicating reduced JC-1 aggregate levels due to MG132 proteasomal inhibition. Metformin upregulated antioxidant genes in hiPSC-derived neurons under stress conditions and protected the cells from oxidative damage.

Rapid and Effective Neuronal Conversion of Human Glioblastoma InVitro and InVivo Using Potent Small Molecules.

Exploring effective, prompt and universally applicable approaches for inducing the differentiation of glioblastoma (GBM) into terminally differentiated cells, such as astrocytes or neurons that cease cell division, is pivotal for the success of GBM differentiation therapy. In this study, a neuronal-specific promoter-reporter system was employed to screen small molecules that promote neural differentiation. The cocktail YFSS, consisting of Y27632, Forskolin, SB431542 and SP600125, which selectively targets the ROCK, cAMP, TGF-β and JNK signalling pathways, respectively, was found to effectively trigger differentiation in human GBM cells. This process yielded neuron-like cells within 7 days, inhibited GBM cell proliferation and reduced malignancy traits, such as stemness, migratory and invasive capabilities. Transcriptome sequencing revealed the pathways altered by YFSS, shedding light on its dual role in halting cell proliferation and initiating neuronal differentiation. A notable increase in CEND1 expression, a key molecule in cell cycle and neuronal differentiation regulation, was observed during differentiation. However, CEND1 alone could not replicate YFSS's high conversion efficiency and its depletion reduced the differentiation and restored proliferation of the GBM cells. Invivo, prolonged and localised YFSS application significantly curtailed tumour growth and extended survival in patient-derived xenograft mice models. In summary, our findings reveal that the small-molecule cocktail YFSS is an effective means for inducing neuronal differentiation in GBM cells, representing a novel and promising pathway for the advancement of GBM treatment.

SAP30, a novel autophagy regulatory gene in neuroblastoma.

SAP30, a novel autophagy regulatory gene in neuroblastoma.

Mesenchymal stem cells overexpressing neuropeptide S promote the recovery of rats with spinal cord injury by activating the PI3K/AKT/GSK3β signaling pathway

BackgroundTransplantation of nasal mucosa-derived mesenchymal stem cells (EMSCs) overexpressing neuropeptide S (NPS) is a promising approach for treating spinal cord injury (SCI). Despite the potential of stem cell therapy, challenges remain regarding cell survival and differentiation control. We aimed to conduct orthotopic transplantation of transected spinal cord to treat rats with complete SCI.MethodsIn this study, we loaded NPS-overexpressing EMSCs onto hydrogels to enhance cell survival in vivo and promote neuronal differentiation both in vitro and in vivo. However, in vitro co-culture promoted greater neuronal differentiation of neural stem cells (P < 0.01). When transplanted in vivo, NPS-overexpressing EMSCs showed greater cell survival in the transplanted area compared with stem cells without gene modification within 4 weeks after spinal cord implantation in rats (P < 0.01).ResultsCompared with those in the other groups, stable overexpression of NPS-EMSCs in a rat model with SCI significantly improved the treatment effect, reduced glial scar formation, promoted neural regeneration and endogenous neural stem cell proliferation and differentiation into neurons, and improved motor function.ConclusionsThese results indicate that this effect may be achieved by the overexpression of NPS-EMSCs through the activation of the PI3K/Akt/GSK3β signaling pathway. Overall, the overexpression of EMSCs significantly improved the therapeutic effect of SCI in rats, strongly supporting the potential for gene modification of mesenchymal stem cells in clinical applications.

Comparative characterization of human accelerated regions in neurons.

Human accelerated regions (HARs) are conserved genomic loci that have experienced rapid nucleotide substitutions following the divergence from chimpanzees1,2. HARs are enriched in candidate regulatory regions near neurodevelopmental genes, suggesting their roles in gene regulation3. However, their target genes and functional contributions to human brain development remain largely uncharacterized. Here we elucidate the cis-regulatory functions of HARs in human and chimpanzee induced pluripotent stem (iPS) cell-induced excitatory neurons. Using genomic4 and chromatin looping information, we prioritized 20 HARs and their chimpanzee orthologues for functional characterization via single-cell CRISPR interference, and demonstrated their species-specific gene regulatory functions. Our findings reveal diverse functional outcomes of HAR-mediated cis-regulation in human neurons, including attenuated NPAS3 expression by altering the binding affinities of multiple transcription factors in HAR202 and maintaining iPS cell pluripotency and neuronal differentiation capacities through the upregulation of PUM2 by 2xHAR.319. Finally, we used prime editing to demonstrate differential enhancer activity caused by several HAR26;2xHAR.178 variants. In particular, we link one variant in HAR26;2xHAR.178 to elevated SOCS2 expression and increased neurite outgrowth in human neurons. Thus, our study sheds new light on the endogenous gene regulatory functions of HARs and their potential contribution to human brain evolution.

Transcriptional enhancers in human neuronal differentiation provide clues to neuronal disorders

Genome-wide association studies (GWASs) have identified thousands of variants associated with complex phenotypes, including neuropsychiatric disorders. To better understand their pathogenesis, it is necessary to identify the functional roles of these variants, which are largely located in non-coding DNA regions. Here, we employ a human mesencephalic neuronal cell differentiation model, LUHMES, with sensitive and high-resolution methods to discover enhancers (NET-CAGE), perform DNA conformation analysis (Capture Hi-C) to link enhancers to their target genes, and finally validate selected interactions. We expand the number of known enhancers active in differentiating human LUHMES neurons to 47,350, and find overlap with GWAS variants for Parkinson’s disease and schizophrenia. Our findings reveal a fine-tuned regulation of human neuronal differentiation, even between adjacent developmental stages; provide a valuable resource for further studies on neuronal development, regulation, and disorders; and emphasize the importance of exploring the vast regulatory potential of non-coding DNA and enhancers.

Cerebellar liponeurocytoma: An updated comprehensive review of clinicopathologic, immunohistochemical, and molecular features of an unusual but distinct tumor.

Cerebellar liponeurocytoma (CL) is a rare WHO grade 2 tumor characterized by advanced neuronal differentiation and variable lipomatous features. Initially classified as a subtype of medulloblastoma, CL was later considered as a distinct entity owing to its peculiar morphological and molecular features and significant better outcome. Typically affecting adults, CL often presents with symptoms related to cerebellar dysfunction, including headaches, ataxia, and gait disturbances. On magnetic resonance imaging, this tumor presents as a well-defined, heterogeneous mass with lipomatous components, which may be less or more apparent depending on their extent. Histologically, CL is composed of neurocytic cells and lipidized tumor cells; the immunohistochemical positivity for synaptophysin and NeuN confirms the neuronal differentiation of neoplastic cells. In spite of its morphological similarity to medulloblastoma, CL lacks the genetic alterations commonly found in this tumor, but some cases display TP53 mutations. Complete surgical resection is the gold standard treatment, whereas the benefit of adjuvant radiotherapy is controversial. CL generally harbors a favorable prognosis, with low recurrence rates in cases with incomplete resection or high proliferative index. The present paper comprehensively reviews the literature about CL, emphasizing the clinicopathologic and molecular features of this unusual but distinct neuropathological entity.

Bioelectric stimulation outperforms brain derived neurotrophic factor in promoting neuronal maturation

Neuronal differentiation and maturation are crucial for developing research models and therapeutic applications. Brain-derived neurotrophic factor (BDNF) is a widely used biochemical stimulus for promoting neuronal maturation. However, the broad effects of biochemical stimuli on multiple cellular functions limit their applicability in both in vitro models and clinical settings. Electrical stimulation (ES) offers a promising physical method to control cell fate and function, but it is hampered by lack of standard and optimised protocols. In this study, we demonstrate that ES outperforms BDNF in promoting neuronal maturation in human neuroblastoma SH-SY5Y. Additionally, we address the question regarding which ES parameters regulate biological responses. The neuronal differentiation and maturation of SH-SY5Y cells were tested under several pulsed ES regimes. We identified accumulated charge and effective electric field time as novel criteria for determining optimal ES regimes. ES parameters were obtained using electrochemical characterisation and equivalent circuit modelling. Our findings show that neuronal maturation in SH-SY5Y cells correlates with the amount of accumulated charge during ES. Higher charge accumulation (~ 50 mC/h) significantly promotes extensive neurite outgrowth and ramification, and enhances the expression of synaptophysin, yielding effects exceeding those of BDNF. In contrast, fewer charge injection to the culture (~ 0.1 mC/h) minimally induces maturation but significantly increases cell proliferation. Moreover, ES altered the concentration and protein cargo of secreted extracellular vesicles (EV). ES with large enough accumulated charge significantly enriched EV proteome associated with neural development and function. These results demonstrate that each ES regime induces distinct cellular responses. Increased accumulated charge facilitates the development of complex neuronal morphologies and axonal ramification, outperforming exogenous neurotrophic factors. Controlled ES methods are immediately applicable in creating mature neuronal cultures in vitro with minimal chemical intervention.