Alexander disease (AxD) is a rare and severe neurodegenerative disorder caused by mutations in glial fibrillary acidic protein (GFAP). While the exact disease mechanism remains unknown, previous studies suggest that mutant GFAP influences many cellular processes, including cytoskeleton stability, mechanosensing, metabolism, and proteasome function. While most studies have primarily focused on GFAP-expressing astrocytes, GFAP is also expressed by radial glia and neural progenitor cells, prompting questions about the impact of GFAP mutations on central nervous system (CNS) development. In this study, we observed impaired differentiation of astrocytes and neurons in co-cultures of astrocytes and neurons, as well as in neural organoids, both generated from AxD patient-derived induced pluripotent stem (iPS) cells with a GFAPR239C mutation. Leveraging single-cell RNA sequencing (scRNA-seq), we identified distinct cell populations and transcriptomic differences between the mutant GFAP cultures and a corrected isogenic control. These findings were supported by results obtained with immunocytochemistry and proteomics. In co-cultures, the GFAPR239C mutation resulted in an increased abundance of immature cells, while in unguided neural organoids and cortical organoids, we observed altered lineage commitment and reduced abundance of astrocytes. Gene expression analysis revealed increased stress susceptibility, cytoskeletal abnormalities, and altered extracellular matrix and cell-cell communication patterns in the AxD cultures, which also exhibited higher cell death after stress. Overall, our results point to altered cell differentiation in AxD patient-derived iPS-cell models, opening new avenues for AxD research.
- MeSH
- Alexander Disease * genetics pathology metabolism MeSH
- Astrocytes * metabolism pathology MeSH
- Cell Differentiation * physiology MeSH
- Glial Fibrillary Acidic Protein * metabolism genetics MeSH
- Induced Pluripotent Stem Cells * metabolism MeSH
- Coculture Techniques MeSH
- Cells, Cultured MeSH
- Humans MeSH
- Mutation MeSH
- Neural Stem Cells metabolism MeSH
- Neurons metabolism pathology MeSH
- Organoids metabolism pathology MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
BACKGROUND: Lipopolysaccharide (LPS)-induced inflammation of lung tissues triggers irreversible alterations in the lung parenchyma, leading to fibrosis and pulmonary dysfunction. While the molecular and cellular responses of immune and connective tissue cells in the lungs are well characterized, the specific epithelial response remains unclear due to the lack of representative cell models. Recently, we introduced human embryonic stem cell-derived expandable lung epithelial (ELEP) cells as a novel model for studying lung injury and regeneration. METHODS: ELEPs were derived from the CCTL 14 human embryonic stem cell line through activin A-mediated endoderm specification, followed by further induction toward pulmonary epithelium using FGF2 and EGF. ELEPs exhibit a high proliferation rate and express key structural and molecular markers of alveolar progenitors, such as NKX2-1. The effects of Escherichia coli LPS serotype O55:B5 on the phenotype and molecular signaling of ELEPs were analyzed using viability and migration assays, mRNA and protein levels were determined by qRT-PCR, western blotting, and immunofluorescent microscopy. RESULTS: We demonstrated that purified LPS induces features of a hybrid epithelial-to-mesenchymal transition in pluripotent stem cell-derived ELEPs, triggers the unfolded protein response, and upregulates intracellular β-catenin level through retention of E-cadherin within the endoplasmic reticulum. CONCLUSIONS: Human embryonic stem cell-derived ELEPs provide a biologically relevant, non-cancerous lung cell model to investigate molecular responses to inflammatory stimuli and address epithelial plasticity. This approach offers novel insights into the fine molecular processes underlying lung injury and repair.
- MeSH
- Cell Line MeSH
- Antigens, CD metabolism MeSH
- Endoplasmic Reticulum * metabolism drug effects MeSH
- Epithelial-Mesenchymal Transition * drug effects MeSH
- Epithelial Cells * drug effects metabolism cytology MeSH
- Cadherins * metabolism MeSH
- Humans MeSH
- Human Embryonic Stem Cells * cytology MeSH
- Lipopolysaccharides * pharmacology MeSH
- Lung * cytology MeSH
- Thyroid Nuclear Factor 1 MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Background: The intermediate filament nestin was first described in stem and progenitor cells of neural and mesenchymal origin. Additionally, it is expressed in endothelial cells during wound healing and tumorigenesis. Thus, nestin is widely regarded as a marker for proliferative endothelium. However, little is known about its role in lymphatic endothelium. Methods: Here, we analyzed the expression of nestin in the endothelium of ten human haemangiomas and ten lymphangiomas in situ by immunohistochemistry. This study aimed to investigate the expression of nestin in haemangiomas and lymphangiomas to determine its potential role as a vascular marker. Specifically, we aimed to assess whether nestin expression is restricted to proliferating endothelial cells or also present in non-proliferative blood vessels. Results: Immunohistochemically, haemangiomas were positive for CD31 but negative for D2-40. The endothelial cells within these lesions showed a homogeneous expression of nestin. In contrast, the endothelium of lymphangiomas reacted positively for D2-40 and CD31 but did not show any nestin expression. Additionally, only a few endothelial cells of capillary haemangiomas showed a Ki-67 positivity. Conclusions: The differential expression of nestin in haemangiomas and lymphangiomas indicates a specificity of nestin for the endothelium of blood vessels. The Ki-67 negativity in the majority of the endothelial cells reveals the proliferative quiescence of these cells. These findings indicate that nestin could be used as a marker to differentiate between blood and lymphatic vessels.
- Publication type
- Journal Article MeSH
Ischemic stroke is a leading cause of mortality and long-term disability globally. One of its aspects is the breakdown of the blood-brain barrier (BBB). The disruption of BBB's integrity during stroke exacerbates neurological damage and hampers therapeutic intervention. Recent advances in regenerative medicine suggest that mesenchymal stem cells (MSCs) derived extracellular vesicles (EVs) show promise for restoring BBB integrity. This review explores the potential of MSC-derived EVs in mediating neuroprotective and reparative effects on the BBB after ischemic stroke. We highlight the molecular cargo of MSC-derived EVs, including miRNAs, and their role in enhancing angiogenesis, promoting the BBB and neural repair, and mitigating apoptosis. Furthermore, we discuss the challenges associated with the clinical translation of MSC-derived EV therapies and the possibilities of further enhancing EVs' innate protective qualities. Our findings underscore the need for further research to optimize the therapeutic potential of EVs and establish their efficacy and safety in clinical settings.
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- Journal Article MeSH
- Review MeSH
Medulloblastoma, the most prevalent brain tumor among children, requires a comprehensive understanding of its cellular characteristics for effective research and treatment. In this study, we focused on DAOY, a permanent cell line of medulloblastoma, and investigated the unique properties of DAOY cells when cultured as floating multicellular aggregates called spheres, as opposed to adherent monolayers. Through our comprehensive analysis, we identified distinct characteristics associated with DAOY spheres. Our findings demonstrate that DAOY spheres express markers for both neural stem cells, such as CD133 (PROM1), and differentiated neurons, exemplified by MAP2. Additionally, our investigation revealed that spheres-derived cells exhibit heightened resistance to ionizing radiation compared to adherent cells. Consequently, our results indicate that caution is advised when interpreting experimental results obtained from adherent cell cultures and extrapolating them to in vivo situations.
- Publication type
- Journal Article MeSH
Hypothalamic Adult Neurogenesis (hAN) has been implicated in regulating energy homeostasis. Adult-generated neurons and adult Neural Stem Cells (aNSCs) in the hypothalamus control food intake and body weight. Conversely, diet-induced obesity (DIO) by high fat diets (HFD) exerts adverse influence on hAN. However, the effects of anti-obesity compounds on hAN are not known. To address this, we administered a lipidized analogue of an anti-obesity neuropeptide, Prolactin Releasing Peptide (PrRP), so-called LiPR, to mice. In the HFD context, LiPR rescued the survival of adult-born hypothalamic neurons and increased the number of aNSCs by reducing their activation. LiPR also rescued the reduction of immature hippocampal neurons and modulated calcium dynamics in iPSC-derived human neurons. In addition, some of these neurogenic effects were exerted by another anti-obesity compound, Liraglutide. These results show for the first time that anti-obesity neuropeptides influence adult neurogenesis and suggest that the neurogenic process can serve as a target of anti-obesity pharmacotherapy.
- MeSH
- Prolactin-Releasing Hormone pharmacology therapeutic use MeSH
- Hypothalamus MeSH
- Humans MeSH
- Mice MeSH
- Neurogenesis MeSH
- Neuropeptides * MeSH
- Obesity * drug therapy MeSH
- Body Weight MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Glial cells expressing neuron-glial antigen 2 (NG2), also known as oligodendrocyte progenitor cells (OPCs), play a critical role in maintaining brain health. However, their ability to differentiate after ischemic injury is poorly understood. The aim of this study was to investigate the properties and functions of NG2 glia in the ischemic brain. Using transgenic mice, we selectively labeled NG2-expressing cells and their progeny in both healthy brain and after focal cerebral ischemia (FCI). Using single-cell RNA sequencing, we classified the labeled glial cells into five distinct subpopulations based on their gene expression patterns. Additionally, we examined the membrane properties of these cells using the patch-clamp technique. Of the identified subpopulations, three were identified as OPCs, whereas the fourth subpopulation had characteristics indicative of cells likely to develop into oligodendrocytes. The fifth subpopulation of NG2 glia showed astrocytic markers and had similarities to neural progenitor cells. Interestingly, this subpopulation was present in both healthy and post-ischemic tissue; however, its gene expression profile changed after ischemia, with increased numbers of genes related to neurogenesis. Immunohistochemical analysis confirmed the temporal expression of neurogenic genes and showed an increased presence of NG2 cells positive for Purkinje cell protein-4 at the periphery of the ischemic lesion 12 days after FCI, as well as NeuN-positive NG2 cells 28 and 60 days after injury. These results suggest the potential development of neuron-like cells arising from NG2 glia in the ischemic tissue. Our study provides insights into the plasticity of NG2 glia and their capacity for neurogenesis after stroke.
- MeSH
- Antigens metabolism MeSH
- Astrocytes metabolism MeSH
- Brain Ischemia * metabolism MeSH
- Brain metabolism MeSH
- Mice, Transgenic MeSH
- Mice MeSH
- Neural Stem Cells * metabolism MeSH
- Neuroglia metabolism MeSH
- Oligodendroglia metabolism MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Previously, a new biodegradable poly(ester urethane urea) was synthesized based on polycaprolactone-diol and fish gelatin (PU-Gel). In this work, the potential of this new material for neural tissue engineering is evaluated. Membranes with randomly oriented fibers and with aligned fibers are produced using electrospinning and characterized regarding their mechanical behavior under both dry and wet conditions. Wet samples exhibit a lower Young's modulus than dry ones and aligned membranes are stiffer and more brittle than those randomly oriented. Cyclic tensile tests are conducted and high values for recovery ratio and resilience are obtained. Both membranes exhibited a hydrophobic surface, measured by the water contact angle (WCA). Human mesenchymal stem cells from umbilical cord tissue (UC-MSCs) and human neural stem cells (NSCs) are seeded on both types of membranes, which support their adhesion and proliferation. Cells stained for the cytoskeleton and nucleus in membranes with aligned fibers display an elongated morphology following the alignment direction. As the culture time increased, higher cell viability is obtained on randomfibers for UC-MSCs while no differences are observed for NSCs. The membranes support neuronal differentiation of NSCs, as evidenced by markers for a neuronal filament protein (NF70) and for a microtubule-associated protein (MAP2).
- MeSH
- Biocompatible Materials chemistry pharmacology MeSH
- Cell Adhesion drug effects MeSH
- Cell Differentiation drug effects MeSH
- Cells, Cultured MeSH
- Humans MeSH
- Mesenchymal Stem Cells * cytology drug effects metabolism MeSH
- Neural Stem Cells * cytology drug effects metabolism MeSH
- Tensile Strength MeSH
- Polyesters * chemistry pharmacology MeSH
- Polyurethanes * chemistry pharmacology MeSH
- Cell Proliferation drug effects MeSH
- Materials Testing MeSH
- Tissue Engineering * methods MeSH
- Tissue Scaffolds chemistry MeSH
- Cell Survival drug effects MeSH
- Gelatin * chemistry MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
INTRODUCTION: Neural stem cells (NSCs) are essential for both embryonic development and adult neurogenesis, and their dysregulation causes a number of neurodevelopmental disorders, such as epilepsy and autism spectrum disorders. NSC proliferation and differentiation in the developing brain is a complex process controlled by various intrinsic and extrinsic stimuli. The mammalian target of rapamycin (mTOR) regulates proliferation and differentiation, among other cellular functions, and disruption in the mTOR pathway can lead to severe nervous system development deficits. In this study, we investigated the effect of inhibition of the mTOR pathway by rapamycin (Rapa) on NSC proliferation and differentiation. METHODS: The NSC cultures were treated with Rapa for 1, 2, 6, 24, and 48 h. The effect on cellular functions was assessed by immunofluorescence staining, western blotting, and proliferation/metabolic assays. RESULTS: mTOR inhibition suppressed NSC proliferation/metabolic activity as well as S-Phase entry by as early as 1 h of Rapa treatment and this effect persisted up to 48 h of Rapa treatment. In a separate experiment, NSCs were differentiated for 2 weeks after treatment with Rapa for 24 or 48 h. Regarding the effect on neuronal and glial differentiation (2 weeks post-treatment), this was suppressed in NSCs deficient in mTOR signaling, as evidenced by downregulated expression of NeuN, MAP2, and GFAP. We assume that the prolonged effect of mTOR inhibition is realized due to the effect on cytoskeletal proteins. DISCUSSION: Here, we demonstrate for the first time that the mTOR pathway not only regulates NSC proliferation but also plays an important role in NSC differentiation into both neuronal and glial lineages.
- Publication type
- Journal Article MeSH
BACKGROUND: Changes in the hippocampus after brain metastases radiotherapy can significantly impact neurocognitive functions. Numerous studies document hippocampal atrophy correlating with the radiation dose. This study aims to elucidate volumetric changes in patients undergoing whole-brain radiotherapy (WBRT) or targeted stereotactic radiotherapy (SRT) and to explore volumetric changes in the individual subregions of the hippocampus. METHOD: Ten patients indicated to WBRT and 18 to SRT underwent brain magnetic resonance before radiotherapy and after 4 months. A structural T1-weighted sequence was used for volumetric analysis, and the software FreeSurfer was employed as the tool for the volumetry evaluation of 19 individual hippocampal subregions. RESULTS: The volume of the whole hippocampus, segmented by the software, was larger than the volume outlined by the radiation oncologist. No significant differences in volume changes were observed in the right hippocampus. In the left hippocampus, the only subregion with a smaller volume after WBRT was the granular cells and molecular layers of the dentate gyrus (GC-ML-DG) region (median change -5 mm3, median volume 137 vs. 135 mm3; P = .027), the region of the presumed location of neuronal progenitors. CONCLUSIONS: Our study enriches the theory that the loss of neural stem cells is involved in cognitive decline after radiotherapy, contributes to the understanding of cognitive impairment, and advocates for the need for SRT whenever possible to preserve cognitive functions in patients undergoing brain radiotherapy.
- Publication type
- Journal Article MeSH
