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Survival and functionality of xeno-free human embryonic stem cell-derived retinal pigment epithelial cells on polyester substrate after transplantation in rabbits
摘要： Purpose: To study immunogenic properties of human embryonic stem cell–derived retinal pigment epithelium (hESC-RPE) and to evaluate subretinal xenotransplantation of hESC-RPE on porous polyethylene terephthalate (PET) in rabbits. Methods: Human ESC-RPE cells were characterized by morphology, transepithelial electrical resistance (TER), protein expression and photoreceptor outer segment phagocytosis in vitro. Expression of major histocompatibility complex (MHC) proteins was assessed in conventionally or xeno-free produced hESC-RPE with interferon-gamma (IFN-γ) stimulation (n = 1). Xeno-free hESC-RPE on PET with TER < 200 Ω·cm2 or PET alone were transplanted into 18 rabbits with short-term triamcinolone with extended tacrolimus immunosuppression. Rabbits were monitored by spectral domain optical coherence tomography. After 4 weeks, the eyes were processed for histology and transmission electron microscopy. Results: Upon in vitro IFN-γ stimulation, xeno-free hESC-RPE expressed lower level of MHC-II proteins compared to the conventional cells. Outer nuclear layer (ONL) atrophy was observed over the graft in most cases 4 weeks post-transplantation. In 3/4 animals with high TER hESC-RPE, but only in 1/3 animals with low TER hESC-RPE, ONL atrophy was observed already within 1 week. Retinal cell infiltrations were more frequent in animals with high TER hESC-RPE. However, the difference was not statistically significant. In three animals, preservation of ONL was observed. Weekly intravitreal tacrolimus did not affect ONL preservation. In all animals, hESC-RPE cells survived for 4 weeks, but without tacrolimus, enlarged vacuoles accumulated in hESC-RPE (n = 1). Conclusions: Xenografted xeno-free hESC-RPE monolayers can survive and retain some functionality for 4 weeks following short-term immunosuppression. The preliminary findings of this study suggest that further investigations to improve transplantation success of hESC-RPE xenografts in rabbits should be addressed especially toward the roles of hESC-RPE maturation stage and extended intravitreal immunosuppression.
关键词： xeno-free,age-related macular degeneration,cell therapy,RPE transplantation,pluripotent stem cell
更新于2025-09-23 15:23:52
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Scalable Measurements of Intrinsic Excitability in Human iPS Cell-Derived Excitatory Neurons Using All-Optical Electrophysiology
摘要： Induced pluripotent stem (iPS) cells offer the exciting opportunity for modeling neurological disorders in vitro in the context of a human genetic background. While significant progress has been made in advancing the use of iPS cell-based disease models, there remains an unmet need to characterize the electrophysiological profile of individual neurons with sufficient throughput to enable statistically robust assessment of disease phenotypes and pharmacological modulation. Here, we describe the Optopatch platform technology that utilizes optogenetics to both stimulate and record action potentials (APs) from human iPS cell-derived excitatory neurons with similar information content to manual patch clamp electrophysiology, but with ~ 3 orders of magnitude greater throughput. Cortical excitatory neurons were produced using the NGN2 transcriptional programming approach and cultured in the presence of rodent glial cells. Characterization of the neuronal preparations using immunocytochemistry and qRT-PCR assays reveals an enrichment of neuronal and glutamatergic markers as well as select ion channels. We demonstrate the scale of our intrinsic cellular excitability assay using pharmacological assessment with select ion channel modulators quinidine and retigabine, by measuring changes in both spike timing and waveform properties. The Optopatch platform in human iPS cell-derived cortical excitatory neurons has the potential for detailed phenotype and pharmacology evaluation, which can serve as the basis of cellular disease model exploration for drug discovery and phenotypic screening efforts.
关键词： Electrophysiology,Optogenetics,Induced pluripotent stem cells
更新于2025-09-23 15:23:52
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Nitrogen mapping from ADF imaging analysis in quaternary dilute nitride superlattices
摘要： A method for the determination of N distribution in dilute nitride GaAsSbN superlattices (SLs) by using different STEM imaging settings is proposed. The method combines the simultaneous acquisition under Low Angle (LA-) and High Angle (HA-) Annular Dark Field (ADF) conditions by exploiting two different dedicated angle intervals. On one hand, HAADF technique gives information that principally depends on the atomic number (Sb sensitive) and on the other hand, N atoms produce high static atomic displacements affecting the image intensity especially under LAADF conditions. However, the simultaneous presence of Sb and N supposes an important handicap to differentiate both elements. N distribution maps could be obtained from suitable normalization and the separation of the intensity ratios in regions with/without Sb. Semi-quantitative maps are also available by combination of the results from high-resolution X-ray diffraction and energy dispersive X-Ray spectroscopy techniques. Type-I (GaAsSbN/GaAs) and type-II (GaAsSb/GaAsN) SL structures are evaluated using the proposed methodology. Differences in the N distribution between both samples such as inhomogeneities and cluster formation are discussed. Specifically, we have found a greater number of N-rich regions in type-I structure as compared to their type-II counterparts, which could have an influence on the optical response of each design.
关键词： STEM,Dilute nitride,Compositional distribution,Superlattice structures
更新于2025-09-23 15:22:29
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Optical and Microstructural Investigation of Heavy B-Doping Effects in Sublimation-Grown 3C-SiC
摘要： In this work, a complementary microstructural and optical approach is used to define processing conditions favorable for the formation of deep boron-related acceptor centers that may provide a pathway for achieving an intermediate band behavior in highly B-doped 3C-SiC. The crystallinity, boron solubility and precipitation mechanisms in sublimation-grown 3C-SiC crystals implanted to 1-3 at.% B concentrations were investigated by STEM. The revealed defect formation and boron precipitation trends upon thermal treatment in the range 1100-2000oC have been cross-correlated with the optical characterization results provided by imaging PL spectroscopy. We discuss optical activity of the implanted B ions in terms of both shallow acceptors and deep D-centers, a complex formed by a boron atom and a carbon vacancy, and associate the observed spectral developments upon annealing with the strong temperature dependence of the D-center formation efficiency, which is further enhanced by the presence of implantation-induced defects.
关键词： photoluminescence,defects,3C-SiC,STEM,implantation,boron doping
更新于2025-09-23 15:22:29
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Intravitreal Administration of Human Bone Marrow CD34+ Stem Cells in a Murine Model of Retinal Degeneration
摘要： PURPOSE. Intravitreal murine lineage-negative bone marrow (BM) hematopoietic cells slow down retinal degeneration. Because human BM CD34t hematopoietic cells are not precisely comparable to murine cells, this study examined the effect of intravitreal human BM CD34t cells on the degenerating retina using a murine model. METHODS. C3H/HeJrd1/rd1 mice, immunosuppressed systemically with tacrolimus and rapamycin, were injected intravitreally with PBS (n ? 16) or CD34t cells (n ? 16) isolated from human BM using a magnetic cell sorter and labeled with enhanced green ?uorescent protein (EGFP). After 1 and 4 weeks, the injected eyes were imaged with scanning laser ophthalmoscopy (SLO)/optical coherence tomography (OCT) and tested with electroretinography (ERG). Eyes were harvested after euthanasia for immunohistochemical and microarray analysis of the retina. RESULTS. In vivo SLO fundus imaging visualized EGFP-labeled cells within the eyes following intravitreal injection. Simultaneous OCT analysis localized the EGFP-labeled cells on the retinal surface resulting in a saw-toothed appearance. Immunohistochemical analysis of the retina identi?ed EGFP-labeled cells on the retinal surface and adjacent to ganglion cells. Electroretinography testing showed a ?at signal both at 1 and 4 weeks following injection in all eyes. Microarray analysis of the retina following cell injection showed altered expression of more than 300 mouse genes, predominantly those regulating photoreceptor function and maintenance and apoptosis. CONCLUSIONS. Intravitreal human BM CD34t cells rapidly home to the degenerating retinal surface. Although a functional bene?t of this cell therapy was not seen on ERG in this rapidly progressive retinal degeneration model, molecular changes in the retina associated with CD34t cell therapy suggest potential trophic regenerative effects that warrant further exploration.
关键词： intravitreal,stem cells,CD34t,retinal degeneration
更新于2025-09-23 15:22:29
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Perforating Freestanding Molybdenum Disulfide Monolayers with Highly Charged Ions
摘要： Porous single layer molybdenum disulfide (MoS2) is a promising material for applications such as DNA sequencing and water desalination. In this work, we introduce irradiation with highly charged ions (HCIs) as a new technique to fabricate well-defined pores in MoS2. Surprisingly, we find a linear increase of the pore creation efficiency over a broad range of potential energies. Comparison to atomistic simulations reveals the critical role of energy deposition from the ion to the material through electronic excitation in the defect creation process, and suggests an enrichment in molybdenum in the vicinity of the pore edges at least for ions with low potential energies. Analysis of the irradiated samples with atomic resolution scanning transmission electron microscopy reveals a clear dependence of the pore size on the potential energy of the projectiles, establishing irradiation with highly charged ions as an effective method to create pores with narrow size distributions and radii between ca. 0.3 and 3 nm.
关键词： 2D material,perforation,ion irradiation,MD simulation,molybdenum disulfide,highly charged ions,STEM
更新于2025-09-23 15:22:29
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Comparative Analysis of Defects in Mg-Implanted and Mg-Doped GaN Layers on Freestanding GaN Substrates
摘要： Inefficient Mg-induced p-type doping has been remained a major obstacle in the development of GaN-based electronic devices for solid-state lighting and power applications. This study reports comparative structural analysis of defects in GaN layers on freestanding GaN substrates where Mg incorporation is carried out via two approaches: ion implantation and epitaxial doping. Scanning transmission electron microscopy revealed the existence of pyramidal and line defects only in Mg-implanted sample whereas Mg-doped sample did not show presence of these defects which suggests that nature of defects depends upon incorporation method. From secondary ion mass spectrometry, a direct correspondence is observed between Mg concentrations and location and type of these defects. Our investigations suggest that these pyramidal and line defects are Mg-rich species and their formation may lead to reduced free hole densities which is still a major concern for p-GaN-based material and devices. As freestanding GaN substrates offer a platform for realization of p-n junction-based vertical devices, comparative structural investigation of defects originated due to different Mg incorporation processes in GaN layers on such substrates is likely to give more insight towards understanding Mg self-compensation mechanisms and then optimizing Mg doping and/or implantation process for the advancement of GaN-based device technology.
关键词： Line defects,Pyramidal defects,SIMS,GaN,STEM
更新于2025-09-23 15:22:29
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Polybenzyl Glutamate Biocompatible Scaffold Promotes the Efficiency of Retinal Differentiation toward Retinal Ganglion Cell Lineage from Human-Induced Pluripotent Stem Cells
摘要： Optic neuropathy is one of the leading causes of irreversible blindness caused by retinal ganglion cell (RGC) degeneration. The development of induced pluripotent stem cell (iPSC)-based therapy opens a therapeutic window for RGC degeneration, and tissue engineering may further promote the efficiency of differentiation process of iPSCs. The present study was designed to evaluate the effects of a novel biomimetic polybenzyl glutamate (PBG) scaffold on culturing iPSC-derived RGC progenitors. The iPSC-derived neural spheres cultured on PBG scaffold increased the differentiated retinal neurons and promoted the neurite outgrowth in the RGC progenitor layer. Additionally, iPSCs cultured on PBG scaffold formed the organoid-like structures compared to that of iPSCs cultured on cover glass within the same culture period. With RNA-seq, we found that cells of the PBG group were differentiated toward retinal lineage and may be related to the glutamate signaling pathway. Further ontological analysis and the gene network analysis showed that the differentially expressed genes between cells of the PBG group and the control group were mainly associated with neuronal differentiation, neuronal maturation, and more specifically, retinal differentiation and maturation. The novel electrospinning PBG scaffold is beneficial for culturing iPSC-derived RGC progenitors as well as retinal organoids. Cells cultured on PBG scaffold differentiate effectively and shorten the process of RGC differentiation compared to that of cells cultured on coverslip. The new culture system may be helpful in future disease modeling, pharmacological screening, autologous transplantation, as well as narrowing the gap to clinical application.
关键词： induced pluripotent stem cells,retinal ganglion cells,tissue engineering,glaucoma,optic neuropathy,polybenzyl glutamate,electrospinning scaffold
更新于2025-09-23 15:22:29
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Prospective scintillation electron detectors for S(T)EM based on garnet film scintillators
摘要： The performance of a scintillation electron detector for a scanning electron microscope and/or a scanning transmission electron microscope (S(T)EM) based on new epitaxial garnet film scintillators was explored. The LuGAGG:Ce and LuGAGG:Ce,Mg film scintillators with chemical formula (Ce0.01Lu0.27Gd0.74)3–wMgw(Ga2.48Al2.46)O12 were prepared and their cathodoluminescence (CL) and optical properties were studied and compared with the properties of current standard bulk single crystal YAG:Ce and YAP:Ce scintillators. More specifically, CL decay characteristics, CL emission spectra, CL intensities, optical absorption coefficients, and the refractive indices of the mentioned scintillators were measured. Furthermore, electron interaction volumes with absorbed energy distributions, photomultiplier (PMT) photocathode matchings, modulation transfer functions (MTF), and the photon transport efficiencies of scintillation detectors with the mentioned scintillators were calculated. A CL decay time for the LuGAGG:Ce,Mg film scintillator as low as 28 ns with an afterglow of only 0.02% at 1 μs after the e-beam excitation was observed. As determined from calculated MTFs, the scintillation detectors with the new film scintillators lose contrast transfer ability above 0.6 lp/pixel, while the currently commonly used YAG:Ce single crystal scintillators already do so above 0.1 lp/pixel. It was also calculated that the new studied film scintillators have an 8% higher photon transfer efficiency, even for a simple disk shape compared with the standard bulk single crystal YAG:Ce scintillator. The studied LuGAGG:Ce,Mg epitaxial garnet film scintillators were evaluated as prospective fast scintillators for electron detectors, not only in S(T)EM but also in other e-beam devices.
关键词： electron detector,multicomponent garnet film,scintillator,STEM,SEM,LuGAGG:Ce,Mg,cathodoluminescence
更新于2025-09-23 15:22:29
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European Microscopy Congress 2016: Proceedings || Studying membrane proteins in intact cells using nanoparticle labels and liquid-phase electron microscopy
摘要： Cells have receptor proteins in their plasma membranes ‘listening’ to chemical signals from the outside world. These signals consist of ligands, small molecules that bind specifically to a receptor. But how those signals are interpreted and lead to decisions is incompletely understood mainly on account of limitations of present analytical methods. It is typically extremely difficult to directly see how endogenously expressed individual proteins respond to ligand binding in the intact cell, which can lead, for example, to the formation of protein complexes triggering signaling processes. Much knowledge about cellular function has been obtained via biochemical methods but these analyze pooled material from many thousands of cells and the knowledge is thus based on population averages. But we need to look at the individual cell in order to understand the fundamentals of how a cell interprets a signal. Studying membrane proteins at the nanoscale in intact eukaryotic cells is now possible using liquid-phase scanning transmission electron microscopy (STEM) [1, 2]. The key step is to specifically label the proteins of interest in a one-to-one ratio with small probes combined with nanoparticles, for example, gold nanoparticles or quantum dots. Cells in liquid are then placed in a microfluidic chamber enclosing the sample in the vacuum of the electron microscope, and are imaged with STEM. It is not always necessary to enclose the cells in the microfluidic chamber. For some studies, it is sufficient to obtain information from the thin outer regions of the cells, and those can be imaged with high resolution using environmental scanning electron microscopy (ESEM) with STEM detector [3]. Liquid-phase STEM was used to explore the formation of the epidermal growth factor HER2 at the single-molecule level in intact SKBR3 breast cancer cells in liquid state [4]. HER2 is a membrane protein and plays an important role in breast cancer aggressiveness and progression. Data analysis based on calculating the pair correlation function from individual HER2 positions revealed remarkable differences in functionality between different cellular regions, and between cells with possible relevance for studying cancer metastasis and drug response.
关键词： quantum dots,STEM,ESEM,whole cells,liquid-phase,EGFR,HER2
更新于2025-09-23 15:21:21