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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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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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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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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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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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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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Generating iPSC-Derived Choroidal Endothelial Cells to Study Age-Related Macular Degeneration
摘要： PURPOSE. Age-related macular degeneration (AMD), the most common cause of incurable blindness in the western world, is characterized by the dysfunction and eventual death of choroidal endothelial (CECs), RPE, and photoreceptor cells. Stem cell–based treatment strategies designed to replace photoreceptor and RPE cells currently are a major scienti?c focus. However, the success of these approaches likely also will require replacement of the underlying, supportive choroidal vasculature. The purpose of this study was to generate stem cell–derived CECs to develop ef?cient differentiation and transplantation protocols. METHODS. Dermal ?broblasts from the Tie2-GFP mouse were isolated and reprogrammed into two independent induced pluripotent stem cell (iPSC) lines via viral transduction of the transcription factors Oct4, Sox2, Klf4, and c-Myc. Tie2-GFP iPSCs were differentiated into CECs using a coculture method with either the RF6A CEC line or primary mouse CECs. Induced pluripotent stem cell–derived CECs were characterized via RT-PCR and immunocytochemistry for EC- and CEC-speci?c markers. RESULTS. Induced pluripotent stem cells generated from mice expressing green ?uorescent protein (GFP) under control of the endothelial Tie2 promoter display classic pluripotency markers and stem cell morphology. Induced pluripotent stem cell–derived CECs express carbonic anhydrase IV, eNOS, FOXA2, PLVAP, CD31, CD34, ICAM-1, Tie2, TTR, VE-cadherin, and vWF. CONCLUSIONS. Induced pluripotent stem cell–derived CECs will be a valuable tool for modeling of choriocapillaris-speci?c insults in AMD and for use in future choroidal endothelial cell replacement approaches.
关键词： choroidal endothelial cells,induced pluripotent stem cells,macular degeneration
更新于2025-09-23 15:21:01
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Bone quality assessment of osteogenic cell cultures by Raman microscopy
摘要： The use of autologous stem/progenitor cells represents a promising approach to the repair of craniofacial bone defects. The calvarium is recognized as a viable source of stem/progenitor cells that can be transplanted in vitro to form bone. However, it is unclear if bone formed in cell culture is similar in quality to that found in native bone. In this study, the quality of bone mineral formed in osteogenic cell cultures were compared against calvarial bone from postnatal mice. Given the spectroscopic resemblance that exists between cell and collagen spectra, the feasibility of extracting information on cell activity and bone matrix quality were also examined. Stem/progenitor cells isolated from fetal mouse calvaria were cultured onto fused‐quartz slides under osteogenic differentiation conditions for 28 days. At specific time intervals, slides were removed and analyzed by Raman microscopy and mineral staining techniques. We show that bone formed in culture at Day 28 resembled calvarial bone from 1‐day‐old postnatal mice with comparable mineralization, mineral crystallinity, and collagen crosslinks ratios. In contrast, bone formed at Day 28 contained a lower degree of ordered collagen fibrils compared with 1‐day‐old postnatal bone. Taken together, bone formed in osteogenic cell culture exhibited progressive matrix maturation and mineralization but could not fully replicate the high degree of collagen fibril order found in native bone.
关键词： Raman microscopy,osteogenic differentiation,tissue engineering,stem cells,bone quality
更新于2025-09-23 15:21:01
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Lung Cancer - Strategies for Diagnosis and Treatment || Targeted Photodynamic Therapy for Improved Lung Cancer Treatment
摘要： Cancer develops from the outgrowth of a clonal population of cells with a genetic pathology to evade cell death and exponential proliferation. It has become a global burden with increasing mortality rates. Lung cancer is a major contributor to cancer fatalities. Conventional therapies have shown advances in treating lung cancer, but the successful eradication of cancer lies in targeting both cancer and cancer stem cells. Cancer stem cells (CSCs) are a ration of cells found within the tumour bulk, capable of cancer initiation, therapy resistance, metastasis and cancer relapse. Photodynamic therapy (PDT) has proven effective in treating lung cancer. PDT exerts selective cell death mechanisms toward cancerous cells. With the use of a photosensitizer (PS) which becomes excited upon irradiation with laser light at a specific wavelength, the PS forms reactive oxygen species (ROS) in turn killing neoplastic cells. Leading therapeutic sequel can be obtained by transcending PDT though combination therapies such as immunotherapy and nanotechnology which will enable PDT to target lung CSCs preventing lung cancer recurrence.
关键词： lung cancer stem cells,targeted PDT,lung cancer,PDT
更新于2025-09-23 15:21:01