neural stem/progenitor cell
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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
- Alexanderova nemoc * genetika patologie metabolismus MeSH
- astrocyty * metabolismus patologie MeSH
- buněčná diferenciace * fyziologie MeSH
- gliový fibrilární kyselý protein * metabolismus genetika MeSH
- indukované pluripotentní kmenové buňky * metabolismus MeSH
- kokultivační techniky MeSH
- kultivované buňky MeSH
- lidé MeSH
- mutace MeSH
- nervové kmenové buňky metabolismus MeSH
- neurony metabolismus patologie MeSH
- organoidy metabolismus patologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články 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
- buněčné linie MeSH
- CD antigeny metabolismus MeSH
- endoplazmatické retikulum * metabolismus účinky léků MeSH
- epitelo-mezenchymální tranzice * účinky léků MeSH
- epitelové buňky * účinky léků metabolismus cytologie MeSH
- kadheriny * metabolismus MeSH
- lidé MeSH
- lidské embryonální kmenové buňky * cytologie MeSH
- lipopolysacharidy * farmakologie MeSH
- plíce * cytologie MeSH
- tyreoidální jaderný faktor 1 MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články 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.
- Publikační typ
- časopisecké články 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
- biokompatibilní materiály chemie farmakologie MeSH
- buněčná adheze účinky léků MeSH
- buněčná diferenciace účinky léků MeSH
- kultivované buňky MeSH
- lidé MeSH
- mezenchymální kmenové buňky * cytologie účinky léků metabolismus MeSH
- nervové kmenové buňky * cytologie účinky léků metabolismus MeSH
- pevnost v tahu MeSH
- polyestery * chemie farmakologie MeSH
- polyurethany * chemie farmakologie MeSH
- proliferace buněk účinky léků MeSH
- testování materiálů MeSH
- tkáňové inženýrství * metody MeSH
- tkáňové podpůrné struktury chemie MeSH
- viabilita buněk účinky léků MeSH
- želatina * chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články 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.
- Publikační typ
- časopisecké články MeSH
Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) exhibit multilineage differentiation potential, adhere to plastic, and express a specific set of surface markers-CD105, CD73, CD90. Although there are relatively well-established differentiation protocols for WJ-MSCs, the exact molecular mechanisms involved in their in vitro long-term culture and differentiation remain to be elucidated. In this study, the cells were isolated from Wharton's jelly of umbilical cords obtained from healthy full-term deliveries, cultivated in vitro, and differentiated towards osteogenic, chondrogenic, adipogenic and neurogenic lineages. RNA samples were isolated after the differentiation regimen and analyzed using an RNA sequencing (RNAseq) assay, which led to the identification of differentially expressed genes belonging to apoptosis-related ontological groups. ZBTB16 and FOXO1 were upregulated in all differentiated groups as compared to controls, while TGFA was downregulated in all groups. In addition, several possible novel marker genes associated with the differentiation of WJ-MSCs were identified (e.g., SEPTIN4, ITPR1, CNR1, BEX2, CD14, EDNRB). The results of this study provide an insight into the molecular mechanisms involved in the long-term culture in vitro and four-lineage differentiation of WJ-MSCs, which is crucial to utilize WJ-MSCs in regenerative medicine.
- MeSH
- apoptóza genetika MeSH
- buněčná diferenciace genetika MeSH
- chondrocyty MeSH
- kultivované buňky MeSH
- lidé MeSH
- mezenchymální kmenové buňky * MeSH
- osteoblasty MeSH
- proteiny nervové tkáně MeSH
- transkriptom MeSH
- tukové buňky MeSH
- Whartonův rosol * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Cyanobacterial blooms are known sources of environmentally-occurring retinoid compounds, including all-trans and 9-cis retinoic acids (RAs). The developmental hazard for aquatic organisms has been described, while the implications for human health hazard assessment are not yet sufficiently characterized. Here, we employ a human neural stem cell model that can differentiate in vitro into a mixed culture of neurons and glia. Cells were exposed to non-cytotoxic 8-1000 nM all-trans or 9-cis RA for 9-18 days (DIV13 and DIV22, respectively). Impact on biomarkers was analyzed on gene expression (RT-qPCR) and protein level (western blot and proteomics) at both time points; network patterning (immunofluorescence) on DIV22. RA exposure significantly concentration-dependently increased gene expression of retinoic acid receptors and the metabolizing enzyme CYP26A1, confirming the chemical-specific response of the model. Expression of thyroid hormone signaling-related genes remained mostly unchanged. Markers of neural progenitors/stem cells (PAX6, SOX1, SOX2, NESTIN) were decreased with increasing RA concentrations, though a basal population remained. Neural markers (DCX, TUJ1, MAP2, NeuN, SYP) remained unchanged or were decreased at high concentrations (200-1000 nM). Conversely, (astro-)glial marker S100β was increased concentration-dependently on DIV22. Together, the biomarker analysis indicates an RA-dependent promotion of glial cell fates over neural differentiation, despite the increased abundance of neural protein biomarkers during differentiation. Interestingly, RA exposure induced substantial changes to the cell culture morphology: while low concentrations resulted in a network-like differentiation pattern, high concentrations (200-1000 nM RA) almost completely prevented such network patterning. After functional confirmation for implications in network function, such morphological features could present a proxy for network formation assessment, an apical key event in (neuro-)developmental Adverse Outcome Pathways. The described application of a human in vitro model for (developmental) neurotoxicity to emerging environmentally-relevant retinoids contributes to the evidence-base for the use of differentiating human in vitro models for human health hazard and risk assessment.
- MeSH
- alitretinoin * toxicita MeSH
- buněčná diferenciace MeSH
- lidé MeSH
- nervové kmenové buňky * účinky léků MeSH
- receptory kyseliny retinové genetika metabolismus MeSH
- retinoidy farmakologie MeSH
- tretinoin * toxicita MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Závěrečná zpráva o řešení grantu Agentury pro zdravotnický výzkum MZ ČR
nestr.
Kmenové buňky představují unikátní buněčný typ schopný sebeobnovy a diferenciace do funkčních somatických typů. Jakákoli klinicky zaměřená aplikace kmenových buněk či z nich odvozených progenitorů, derivátů a produktů vyžaduje přísnou kontrolu kvality celého procesu přípravy. V současné době existuje potřeba robustních, snadno proveditelných a přitom dostatečně citlivých metod, které by určily nebo potvrdily status kultivovaných buněk, a odhalily možné skryté odchylky fenotypu od optimálního stavu. Změny které se neprojeví na morfologické úrovni, v karyotypu nebo které nejsou cíleně sledovány pomocí specifických molekulárních markerů mohou nicméně zůstat neodhaleny a představovat tak značné riziko pro pacienty. V tomto projektu navrhujeme inovativní postup kombinující hmotnostní spektrometrii a metody umělé inteligence, které umožní překonat limity stávajících metod a poskytnout tak nový nástroj pro identifikaci, autentizaci a klasifikaci pluripotentních kmenových buněk pro klinické aplikace.; Stem cells represent a unique cell type capable of self-renewal and differentiation to functional somatic cell types. Any patient-oriented application of stem cells or their differentiated progenitors, derivatives and products, requires a stringent quality control of the whole manufacturing process before entering a clinical trial. Therefore, there is an ongoing need for robust, feasible, and sensitive methods of determining or confirming status of otherwise identical cells, and for revealing hidden divergences from the optimal states. However, changes in cell phenotype that are not pronounced as alterations in cell morphology, abnormal karyotype, genome re-arrangements or simply are not covered by particular molecular markers may stay unrecognized, posing a potential risk to patients. In this project, we propose an innovative approach combining mass spectrometry and artificial intelligence that bypasses limits of standard biological methods and provides a novel tool to identify, authenticate and classify pluripotent stem cells in clinical applications.
The inhibition of glycogen synthase kinase-3 (GSK-3) can induce neurogenesis, and the associated activation of Wnt/β-catenin signaling via GSK-3 inhibition may represent a means to promote motor function recovery following spinal cord injury (SCI) via increased astrocyte migration, reduced astrocyte apoptosis, and enhanced axonal growth. Herein, we assessed the effects of GSK-3 inhibition in vitro on the neurogenesis of ependymal stem/progenitor cells (epSPCs) resident in the mouse spinal cord and of human embryonic stem cell-derived neural progenitors (hESC-NPs) and human-induced pluripotent stem cell-derived neural progenitors (hiPSC-NPs) and in vivo on spinal cord tissue regeneration and motor activity after SCI. We report that the treatment of epSPCs and human pluripotent stem cell-derived neural progenitors (hPSC-NPs) with the GSK-3 inhibitor Ro3303544 activates β-catenin signaling and increases the expression of the bIII-tubulin neuronal marker; furthermore, the differentiation of Ro3303544-treated cells prompted an increase in the number of terminally differentiated neurons. Administration of a water-soluble, bioavailable form of this GSK-3 inhibitor (Ro3303544-Cl) in a severe SCI mouse model revealed the increased expression of bIII-tubulin in the injury epicenter. Treatment with Ro3303544-Cl increased survival of mature neuron types from the propriospinal tract (vGlut1, Parv) and raphe tract (5-HT), protein kinase C gamma-positive neurons, and GABAergic interneurons (GAD65/67) above the injury epicenter. Moreover, we observed higher numbers of newly born BrdU/DCX-positive neurons in Ro3303544-Cl-treated animal tissues, a reduced area delimited by astrocyte scar borders, and improved motor function. Based on this study, we believe that treating animals with epSPCs or hPSC-NPs in combination with Ro3303544-Cl deserves further investigation towards the development of a possible therapeutic strategy for SCI.
- MeSH
- kinasa 3 glykogensynthasy antagonisté a inhibitory MeSH
- lidé MeSH
- modely nemocí na zvířatech MeSH
- multipotentní kmenové buňky účinky léků MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- neurogeneze účinky léků MeSH
- poranění míchy farmakoterapie enzymologie MeSH
- transplantace kmenových buněk MeSH
- western blotting MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
