Enrichment, Characterization, and Proteomic Profiling of Small Extracellular Vesicles Derived from Human Limbal Mesenchymal Stromal Cells and Melanocytes.

Limbal epithelial progenitor cells (LEPC) rely on their niche environment for proper functionality and self-renewal. While extracellular vesicles (EV), specifically small EVs (sEV), have been proposed to support LEPC homeostasis, data on sEV derived from limbal niche cells like limbal mesenchymal stromal cells (LMSC) remain limited, and there are no studies on sEVs from limbal melanocytes (LM). In this study, we isolated sEV from conditioned media of LMSC and LM using a combination of tangential flow filtration and size exclusion chromatography and characterized them by nanoparticle tracking analysis, transmission electron microscopy, Western blot, multiplex bead arrays, and quantitative mass spectrometry. The internalization of sEV by LEPC was studied using flow cytometry and confocal microscopy. The isolated sEVs exhibited typical EV characteristics, including cell-specific markers such as CD90 for LMSC-sEV and Melan-A for LM-sEV. Bioinformatics analysis of the proteomic data suggested a significant role of sEVs in extracellular matrix deposition, with LMSC-derived sEV containing proteins involved in collagen remodeling and cell matrix adhesion, whereas LM-sEV proteins were implicated in other cellular bioprocesses such as cellular pigmentation and development. Moreover, fluorescently labeled LMSC-sEV and LM-sEV were taken up by LEPC and localized to their perinuclear compartment. These findings provide valuable insights into the complex role of sEV from niche cells in regulating the human limbal stem cell niche.

The impact of substrate stiffness on morphological, transcriptional and functional aspects in RPE.

Alterations in the structure and composition of Bruch's membrane (BrM) and loss of retinal pigment epithelial (RPE) cells are associated with various ocular diseases, notably age-related macular degeneration (AMD) as well as several inherited retinal diseases (IRDs). We explored the influence of stiffness as a major BrM characteristic on the RPE transcriptome and morphology. ARPE-19 cells were plated on soft ( E = 30 kPa ) or stiff ( E = 80 kPa ) polyacrylamide gels (PA gels) or standard tissue culture plastic (TCP). Next-generation sequencing (NGS) data on differentially expressed small RNAs (sRNAs) and messenger RNAs (mRNAs) were validated by qPCR, immunofluorescence or western blotting. The microRNA (miRNA) fraction of sRNAs grew with substrate stiffness and distinct miRNAs such as miR-204 or miR-222 were differentially expressed. mRNA targets of differentially expressed miRNAs were stably expressed, suggesting a homeostatic effect of miRNAs. mRNA transcription patterns were substrate stiffness-dependent, including components of Wnt/beta-catenin signaling, Microphthalmia-Associated Transcription Factor (MITF) and Dicer. These findings highlight the relevance of mechanical properties of the extracellular matrix (ECM) in cell culture experiments, especially those focusing on ECM-related diseases, such as AMD.

Oncostatin M Reduces Pathological Neovascularization in the Retina Through Müller Cell Activation.

Purpose: Continuous vision loss due to vasoproliferative eye disease still represents an unsolved issue despite anti-vascular endothelial growth factor (VEGF) therapy. The impact of signal transducer and activator of transcription 3 (STAT3) signaling on retinal angiogenesis and its potential use as a therapeutic target remain controversial. In vitro, oncostatin M (OSM), as a strong STAT3 activator, possesses robust proangiogenic activity. This study investigated to what extent the proangiogenic effects of OSM translate to the in vivo setting of vasoproliferative eye disease. Methods: The in vitro effect of OSM on endothelial cells was investigated in the spheroid sprouting assay and through RNA sequencing. The mouse model for oxygen-induced retinopathy (OIR) was used to evaluate the impact of OSM in vivo. Signaling patterns were measured by western blot and retinal cryosections. Primary Müller cell cultures were used to evaluate the effect of OSM on the Müller cell secretome. Murine retinal vascular endothelial cells were isolated from OIR retinas using fluorescence-activated cell sorting (FACS) and were used for RNA sequencing. Results: Although OSM induced pro-angiogenic responses in vitro, in the OIR model intravitreal injection of OSM reduced retinal neovascularization by 65.2% and vaso-obliteration by 45.5% in Müller cells. Injecting OSM into the vitreous activated the STAT3 signaling pathway in multiple retinal cell types, including Müller cells. In vitro, OSM treatment increased CXCL10 secretion. RNA sequencing of sorted vascular endothelial cells at OIR P17 following OSM treatment indicated downregulation of angiogenesis- and mitosis-associated genes. Conclusions: In vivo, OSM reveals a beneficial angiomodulatory effect by activating Müller cells and changing their secretome. The data highlight contradictions between cytokine-induced effects in vitro and in vivo depending on the cell types mediating the effect.

Corneal tissue induces transcription of metallothioneins in monocyte-derived human macrophages.

PURPOSE: Immune reactions following corneal transplantation are the most common cause of transplant failure. However, the underlying mechanisms of corneal graft rejection are not yet fully understood but increasing evidence points to a crucial role of the innate immune system in this context. Using a human in vitro model, we aimed to assess the response of human macrophages to stimulation with human corneal tissue and whether corneal endothelial cells (CEC) have immune-modulating properties. METHODS: Human monocytes were isolated from peripheral blood mononuclear cells and differentiated into monocyte-derived macrophages (MDM). A standardized protocol was used for disaggregation of human corneas into fragments of defined sizes. MDMs were stimulated using processed corneal material with or without CEC. Lipopolysaccharide (LPS) or interferon-gamma (IFNγ) served as controls. RNA sequencing was applied to analyze the impact of differential stimulation of MDMs on their transcriptional profile. RNA sequencing results were validated using digital PCR. RESULTS: The transcriptional profile of MDMs was significantly modulated by the type of stimulus used for MDM activation as well as by the individual MDM donor. LPS- or IFNγ-stimulation resulted in distinct transcriptional alterations compared to unstimulated MDMs including an upregulation of various cytokines such as CCL3, 4, 5, 19 or CXCL9. Corneal tissue induced the differential expression of 45 genes when compared to unstimulated MDMs, with several metallothioneins (MTs) among the upregulated factors (MT1A, MT1E, MT1F, MT1G, MT1H, MT1L, MT1M, MT1X, MT2A). This effect was independent of the presence or absence of CEC. PCR validation confirmed induction of 3 different metallothioneins (MT1G, MT1H and MT2A) in MDMs stimulated by corneal tissue. CONCLUSIONS: The MDM in vitro model proved to be a robust tool to study the effects of LPS, IFNγ and corneal tissue homogenates on the transcriptional activity of MDM. Human macrophages showed a distinct upregulation of various MTs when challenged with human corneal allogen with or without corneal endothelium, which might have an immune-modulatory effect. As a general observation, it appears that in MDM-based studies a significant donor-dependent effect on the transcriptional profile of MDMs needs to be considered and adjusted before downstream analysis.