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Effects of Cytokine Activation and Oxidative Stress on the Function of the Human Embryonic Stem Cell–Derived Retinal Pigment Epithelial Cells
摘要： PURPOSE. In several retinal complications, such as age-dependent macular degeneration (AMD), oxidative stress is increased and cytokine level is elevated. These are shown to alter the activation and expression of matrix metalloproteinase (MMP) both in human primary and immortalized retinal pigment epithelial (RPE) cells. However, the effects on human embryonic stem cell (hESC)–derived RPE cells remain to be elucidated. METHODS. The mature hESC-RPE cells were exposed to inflammatory cytokines (IFN-γ or TNF-α) for 24 hours or oxidative stress (H2O2) for 1 hour. Effects on barrier properties were analyzed with transepithelial electrical resistance (TEER), the expression of MMP-1, MMP-2, MMP-3, MMP-9, collagen I, and collagen IV genes with quantitative RT-PCR, and the expression of MMP-1 and MMP-3 proteins with Western blot or ELISA, respectively. Also, activation and secretion of MMP-2 and -9 proteins were analyzed with zymography. RESULTS. In normal state, mature hESC-RPE cells expressed MMP-1, -2, -3, and -9 genes in low levels, respectively. Tumor necrosis factor-α increased MMP-1 and -2 gene expression, and H2O2 increased MMP-3 and -9 gene expression. Zymography revealed IFN-γ– and TNF-α–induced secretion of MMP-2 and high-molecular-weight species of MMP (HMW MMP), but H2O2 decreased their secretion. Furthermore, TNF-α and H2O2 significantly decreased barrier properties. CONCLUSIONS. Here, cytokines induced the MMP-1 and -2 gene and protein expression. Also, H2O2 induced MMP-3 and -9 gene expression, but not their protein secretion. These data propose that under oxidative stress and cytokine stimuli, mature hESC-RPE cells resemble their native counterpart in the human eye in regard to MMP secretion and expression and could be used to model retinal disorders involving alterations in MMP activity such as AMD, diabetic retinopathy, or proliferative vitreoretinopathy in vitro.
关键词： matrix metalloproteinase,MMP,stem cells,retinal pigment epithelium
更新于2025-11-21 11:08:12
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A novel near-infrared light responsive 4D printed nanoarchitecture with dynamically and remotely controllable transformation
摘要： Four-dimensional (4D) printing is an emerging and highly innovative additive manufacturing process by which to fabricate pre-designed, self-assembly structures with the ability to transform over time. However, one of the critical challenges of 4D printing is the lack of advanced 4D printing systems that not only meet all the essential requirements of shape change but also possess smart, dynamic capabilities to spatiotemporally and instantly control the shape-transformation process. Here, we present a facile 4D printing platform which incorporates nanomaterials into the conventional stimuli-responsive polymer, allowing the 4D printed object to achieve a dynamic and remote controlled, on-time and position shape transformation. A proof-of-concept 4D printed brain model was created using near-infrared light (NIR) responsive nanocomposite to evaluate the capacity for controllable 4D transformation, and the feasibility of photothermal stimulation for modulating neural stem cell behaviors. This novel 4D printing strategy can not only be used to create dynamic 3D patterned biological structures that can spatiotemporally control their shapes or behaviors of transformation under a human benign stimulus (NIR), but can also provide a potential method for building complex self-morphing objects for widespread applications.
关键词： brain,4D printing,dynamically and remotely controllable,neural stem cell,near-infrared light responsive,graphene
更新于2025-11-21 11:08:12
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Fabrication of conductive fibrous scaffold for photoreceptor differentiation of mesenchymal stem cell
摘要： Conductive nanofibrous scaffolds with that can conduct electrical current have a great potential in neural tissue engineering. The purpose of this study was to survey effects of electrical stimulation and polycaprolactone/polypyrrole/multiwall carbon nanotube (PCL/PPY/MWCNTs) fibrous scaffold on photoreceptor differentiation of trabecular meshwork mesenchymal stem cells (TM‐MSCs). PCL/PPY/MWCNTs scaffold was made by electrospinning method. TM‐MSCs were seeded on PCL/PPY/MWCNTs scaffold and stimulated with a potential of 115 V/m. Scanning electron microscopy, transmission electron microscopy, and FT‐IR were used to evaluate the fabricated scaffold. Immunofluorescence and quantitative real‐time polymerase chain reaction were used to examine differentiated cells. Scanning electron microscopy, transmitting electron microscopy, and FT‐IR confirmed the creation of the composite structure of fibers. RT‐qPCR analysis showed that the expression of rhodopsin and peripherin genes in electrically stimulated cells were significantly higher (5.7‐ and 6.23‐fold, respectively; p ≤ 0.05) than those with no electrical stimulation. Collectively, it seems that the combination of PCL/PPY/MWCNTs scaffold, as a suitable conductive scaffold, and electrical stimulation could be an effective approach in the differentiation of stem cells in retinal tissue engineering.
关键词： electrical conductive,trabecular meshwork mesenchymal stem cells,photoreceptor‐like cells,nanostructure
更新于2025-11-14 15:18:02
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[Methods in Molecular Biology] Astrocytes Volume 1938 (Methods and Protocols) || Fluorescence-Activated Cell Sorting-Based Isolation and Characterization of Neural Stem Cells from the Adult Zebrafish Telencephalon
摘要： Adult mammalian brain, including humans, has rather limited addition of new neurons and poor regenerative capacity. In contrast, neural stem cells (NSC) with glial identity and neurogenesis are highly abundant throughout the adult zebrafish brain. Importantly, the activation of NSC and production of new neurons in response to injuries lead to the brain regeneration in zebrafish brain. Therefore, understanding of the molecular pathways regulating NSC behavior in response to injury is crucial in order to set the basis for experimental modification of these pathways in glial cells after injury in the mammalian brain and to elicit neuronal regeneration. Here, we describe the procedure that we successfully used to prospectively isolate NSCs from adult zebrafish telencephalon, extract RNA, and prepare cDNA libraries for next generation sequencing (NGS) and full transcriptome analysis as the first step toward understanding regulatory mechanisms leading to restorative neurogenesis in zebrafish. Moreover, we describe an alternative approach to analyze antigenic properties of NSC in the adult zebrafish brain using intracellular fluorescence activated cell sorting (FACS). We employ this method to analyze the number of proliferating NSCs positive for proliferating cell nuclear antigen (PCNA) in the prospectively isolated population of stem cells.
关键词： Neural stem cells,Neural stem cell purification,Intracellular FACS,Zebrafish
更新于2025-09-23 15:23:52
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Utilization of Multiphoton Imaging For Real-Time Fate Determination of Mesenchymal Stem Cells in an Immunocompetent Mouse Model
摘要： The clinical application of Mesenchymal Stem Cells (MSCs) for the treatment of a variety of diseases is the focus of intense research. Despite large research efforts many questions regarding MSC biology in vivo remain unanswered. For instance, we do not know for certain whether MSCs exert their therapeutic effects directly within the target tissue or indirectly by influencing the polarization of other cell types, such as macrophages, which can then home to the target tissue microenvironment. To help address this issue, the application of intravital multiphoton microscopy allows for the determination of the dynamic action of intact MSCs versus endogenous host cells at the target tissue site in real time.
关键词： Mesenchymal Stem Cell,Granulocyte,MSC,Intravital,Mouse Model,Multiphoton
更新于2025-09-23 15:23:52
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Functional Assessment of Patient-Derived Retinal Pigment Epithelial Cells Edited by CRISPR/Cas9
摘要： Retinitis pigmentosa is the most common form of inherited blindness and can be caused by a multitude of different genetic mutations that lead to similar phenotypes. Specifically, mutations in ubiquitously expressed splicing factor proteins are known to cause an autosomal dominant form of the disease, but the retina-specific pathology of these mutations is not well understood. Fibroblasts from a patient with splicing factor retinitis pigmentosa caused by a missense mutation in the PRPF8 splicing factor were used to produce three diseased and three CRISPR/Cas9-corrected induced pluripotent stem cell (iPSC) clones. We differentiated each of these clones into retinal pigment epithelial (RPE) cells via directed differentiation and analyzed the RPE cells in terms of gene and protein expression, apicobasal polarity, and phagocytic ability. We demonstrate that RPE cells can be produced from patient-derived and corrected cells and they exhibit morphology and functionality similar but not identical to wild-type RPE cells in vitro. Functionally, the RPE cells were able to establish apicobasal polarity and phagocytose photoreceptor outer segments at the same capacity as wild-type cells. These data suggest that patient-derived iPSCs, both diseased and corrected, are able to differentiate into RPE cells with a near normal phenotype and without differences in phagocytosis, a result that differs from previous mouse models. These RPE cells can now be studied to establish a disease-in-a-dish system relevant to retinitis pigmentosa.
关键词： retinal pigment epithelial cells,CRISPR/Cas9,retinitis pigmentosa,PRPF8,induced pluripotent stem cells
更新于2025-09-23 15:23:52
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Structural characterization of bulk and nanoparticle lead halide perovskite thin films by (S)TEM techniques
摘要： Lead halide (APbX3) perovskites, in polycrystalline thin films but also perovskite nanoparticles (NPs) have demonstrated excellent performances to implement a new generation of photovoltaic and photonic devices. The structural characterization of APbX3 thin films using (scanning) transmission electron microscopy ((S)TEM) techniques can provide valuable information that can be used to understand and model their optoelectronic performance and device properties. However, since APbX3 perovskites are soft materials, their characterization using (S)TEM is challenging. Here, we study and compare the structural properties of two different metal halide APbX3 perovskite thin films: bulk CH3NH3PbI3 prepared by spin-coating of the precursors in solution and CsPbBr3 colloidal NPs synthetized and deposited by doctor blading. Both specimen preparation methods and working conditions for analysis by (S)TEM are properly optimized. We show that CH3NH3PbI3 thin films grown by a one-step method results in the formation of tetragonal perovskite thin films with good adherence to an underlying TiO2 layer, which is characterized by a photoluminescence (PL) emission band centered at 775 nm. The perovskite thin films based on CsPbBr3 colloidal NPs, which are used as the building blocks of the film, they are preserved by the deposition process, even if small gaps are observed between adjacent NPs. The crystal structure of CsPbBr3 NPs is cubic, which is beneficial for optical properties due to its optimal band gap. The absorption and PL spectra measured in both the thin film and the colloidal solution of CsPbBr3 NPs are very similar, indicating a good homogeneity of the thin films and the absence of aggregation of NPs. However, a particular care was required to avoid long electron irradiation times during our structural studies, even at a low voltage of 80 kV, as the material was observed to decompose through Pb segregation.
关键词： structural characterization,TEM,thin films,optoelectronics,STEM,nanoparticles,perovskite
更新于2025-09-23 15:23:52
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Progrès dans les méthodes diagnostiques du déficit en cellules souches limbiques. Apport de la microscopie confocale et de la tomographie en cohérence optique
摘要： The limbus is the anatomical and functional barrier between corneal and conjunctival epithelia. It is characterized by presence of the limbal stem cell niche which allows corneal homeostasis to be maintained. Limbal stem cell deficiency is characterized by a dual process: insufficient regeneration of corneal epithelium, which cannot therefore assure its function of physiological support, associated with corneal invasion by conjunctival proliferation. Diagnosis is currently made via routine clinical examination, corneal impression cytology and in vivo confocal microscopy (IVCM). Slit lamp examination shows abnormal limbal anatomy, thin and irregular epithelium with late fluorescein staining, and superficial vascularization. With its high resolution, IVCM allows identification of limbal and corneal epithelial changes at a cellular level in en face views, parallel to the corneal surface, but with a restricted viewing field of the corneal surface. It shows a poor transition between the corneal and conjunctival epithelia, associated with a loss of the normal corneal epithelial stratification, low basal cell and sub-basal nerve plexus densities, even with sub-epithelial fibrosis. Optical coherence tomography in central cornea and at the limbus, with scans in different orientations, allows a quick, global and non-invasive analysis of normal eyes and those with limbal stem cell deficiency. It shows a thin limbal epithelium, lacking normal thickening, featuring absence of stromal undulations and limbal crypts in cross-sections and sections parallel to the limbus, lack of visible limbal crypts in en face sections, loss of clear transition between the hyporeflective corneal epithelium and the hyperreflective conjunctival epithelium, and hyperreflective sub-epithelial fibrosis.
关键词： Limbal stem cell deficiency,Limbus,Optical coherence tomography,In vivo confocal microscopy
更新于2025-09-23 15:23:52
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Electron energy-loss spectroscopy (EELS) with a cold-field emission scanning electron microscope at low accelerating voltage in transmission mode
摘要： A commercial electron energy-loss spectrometer (EELS) attached to a high-resolution cold-field emission scanning electron microscope in transmission mode (STEM) is evaluated and its potential for characterizing materials science thin specimens at low accelerating voltage is reviewed. Despite the increased beam radiation damage at SEM voltages on sensitive compounds, we describe some potential applications which benefit from lowering the primary electrons voltage on less-sensitive specimens. We report bandgap measurements on several dielectrics which were facilitated by the lack of Cherenkov radiation losses at 30 kV. The possibility of volume plasmon imaging to probe local composition changes in complex materials was demonstrated using energy-filtered STEM, either via spectrum imaging or elemental mapping using the 'three-windows' method. As plasmonic materials are increasing used for energy, electronics or biomedical applications, the ability of reliably evaluate their properties at low accelerating voltage in a SEM is very appealing and is demonstrated. The energy resolution of the spectrometer, taken as the full width at half maximum of the zero-loss peak, was routinely measured at around 0.55 eV and it is demonstrated that t/λ ratios up to 1.5 allowed practical EEL spectroscopy at 30 kV.
关键词： Damage,Plasmon resonance,Silver,STEM,Lithium,Zero-loss,Bandgap
更新于2025-09-23 15:23:52
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Inverse size-dependence of piezoelectricity in single BaTiO3 nanoparticles
摘要： The piezoelectric charge coefficients d33 of single BaTiO3 (BT) nanoparticles (NPs) were characterized using a transmission electron microscope (TEM) that is equipped with a precise charge meter and an in-situ TEM indentation holder that enables controlled compression experiments. An exceptionally high d33 of 1775 pC/N was obtained in NPs that are smaller than the critical diameter (D; typically known as < 100 nm) that has been regarded as the lower limit to permit for ferroelectricity in BT. The mechanical conversion efficiency of piezoelectric BT nanogenerators enhanced as D of BT NPs was decreased; this result corresponds with the single-NP compression measurements of d33. This quantification of the effect of D in ferroelectric materials may guide development of efficient and high-powered nanostructured piezoelectric energy devices such as piezoelectric nanogenerators.
关键词： in-situ TEM,size effect,STEM,Ferroelectric,piezoelectric,nanogenerator
更新于2025-09-23 15:23:52