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.

2D and 3D in vitro angiogenesis assays highlight different aspects of angiogenesis.

In angiogenesis research, scientists need to carefully select appropriate in vitro models to test their hypotheses to minimize the risk for false negative or false positive study results. In this study, we investigate molecular differences between simple two-dimensional and more complex three-dimensional angiogenesis assays and compare them to in vivo data from cancer-associated angiogenesis using an unbiased transcriptomic analysis. Human umbilical vein endothelial cells were treated with VEGF in 2D wound healing and proliferation assays and the 3D spheroid sprouting assay. VEGF-induced transcriptomic shifts were assessed in both settings by bulk RNA sequencing. Immunocytochemistry was used for protein detection. The data was linked to the transcriptomic profile of vascular endothelial cells from a single cell RNA sequencing dataset of various cancer tissue compared to adjacent healthy tissue control. VEGF induced a more diverse transcriptomic shift in vascular endothelial cells in a 3D experimental setting (767 differentially expressed genes) compared to the 2D settings (167 differentially expressed genes). Particularly, VEGF-induced changes in cell-matrix interaction, tip cell formation, and glycolysis were pronounced in the 3D spheroid sprouting experiments. Immunocytochemistry for VCAM1 and CD34 confirmed enhanced expression in response to VEGF-treatment in 3D settings. In vivo, vascular endothelial cells within various cancer tissue were characterized by strong transcriptomic changes in cell-matrix interaction and glycolysis similar to the 3D setting. Consequently, 3D assays may better address certain key aspects of angiogenesis in comparison to fast and scalable 2D assays. This should be taken into consideration within the context of each research question.

Influence of Organ Culture on the Characteristics of the Human Limbal Stem Cell Niche.

Organ culture storage techniques for corneoscleral limbal (CSL) tissue have improved the quality of corneas for transplantation and allow for longer storage times. Cultured limbal tissue has been used for stem cell transplantation to treat limbal stem cell deficiency (LSCD) as well as for research purposes to assess homeostasis mechanisms in the limbal stem cell niche. However, the effects of organ culture storage conditions on the quality of limbal niche components are less well described. Therefore, in this study, the morphological and immunohistochemical characteristics of organ-cultured limbal tissue are investigated and compared to fresh limbal tissues by means of light and electron microscopy. Organ-cultured limbal tissues showed signs of deterioration, such as edema, less pronounced basement membranes, and loss of the most superficial layers of the epithelium. In comparison to the fresh limbal epithelium, organ-cultured limbal epithelium showed signs of ongoing proliferative activity (more Ki-67+ cells) and exhibited an altered limbal epithelial phenotype with a loss of N-cadherin and desmoglein expression as well as a lack of precise staining patterns for cytokeratin ((CK)14, CK17/19, CK15). The analyzed extracellular matrix composition was mainly intact (collagen IV, fibronectin, laminin chains) except for Tenascin-C, whose expression was increased in organ-cultured limbal tissue. Nonetheless, the expression patterns of cell-matrix adhesion proteins varied in organ-cultured limbal tissue compared to fresh limbal tissue. A decrease in the number of melanocytes (Melan-A+ cells) and Langerhans cells (HLA-DR+, CD1a+, CD18+) was observed in the organ-cultured limbal tissue. The organ culture-induced alterations of the limbal epithelial stem cell niche might hamper its use in the treatment of LSCD as well as in research studies. In contrast, reduced numbers of donor-derived Langerhans cells seem associated with better clinical outcomes. However, there is a need to consider the preferential use of fresh CSL for limbal transplants and to look at ways of improving the limbal stem cell properties of stored CSL tissue.

Transcriptional Comparison of Human and Murine Retinal Neovascularization.

Purpose: Retinal neovascularization (RNV) is the leading cause of vision loss in diseases like proliferative diabetic retinopathy (PDR). A significant failure rate of current treatments indicates the need for novel treatment targets. Animal models are crucial in this process, but current diabetic retinopathy models do not develop RNV. Although the nondiabetic oxygen-induced retinopathy (OIR) mouse model is used to study RNV development, it is largely unknown how closely it resembles human PDR. Methods: We therefore performed RNA sequencing on murine (C57BL/6J) OIR retinas (n = 14) and human PDR RNV membranes (n = 7) extracted during vitrectomy, each with reference to control tissue (n=13/10). Differentially expressed genes (DEG) and associated biological processes were analyzed and compared between human and murine RNV to assess molecular overlap and identify phylogenetically conserved factors. Results: In total, 213 murine- and 1223 human-specific factors were upregulated with a small overlap of 94 DEG (7% of human DEG), although similar biological processes such as angiogenesis, regulation of immune response, and extracellular matrix organization were activated in both species. Phylogenetically conserved mediators included ANGPT2, S100A8, MCAM, EDNRA, and CCR7. Conclusions: Even though few individual genes were upregulated simultaneously in both species, similar biological processes appeared to be activated. These findings demonstrate the potential and limitations of the OIR model to study human PDR and identify phylogenetically conserved potential treatment targets for PDR.

Slow Interstitial Fluid Flow Activates TGF-ß Signaling and Drives Fibrotic Responses in Human Tenon Fibroblasts.

BACKGROUND: Fibrosis limits the success of filtering glaucoma surgery. We employed 2D and 3D in vitro models to assess the effects of fluid flow on human tenon fibroblasts (HTF). METHODS: HTF were exposed to continuous or pulsatile fluid flow for 48 or 72 h, at rates expected at the transscleral outflow site after filtering surgery. In the 2D model, the F-actin cytoskeleton and fibronectin 1 (FN1) were visualized by confocal immunofluorescence microscopy. In the 3D model, mRNA and whole cell lysates were extracted to analyze the expression of fibrosis-associated genes by qPCR and Western blot. The effects of a small-molecule inhibitor of the TGF-ß receptor ALK5 were studied. RESULTS: Slow, continuous fluid flow induced fibrotic responses in the 2D and 3D models. It elicited changes in cell shape, the F-actin cytoskeleton, the deposition of FN1 and activated the intracellular TGF-ß signaling pathway to induce expression of fibrosis-related genes, such as CTGF, FN1 and COL1A1. ALK5-inhibition reduced this effect. Intermittent fluid flow also induced fibrotic changes, which decreased with increasing pause duration. CONCLUSIONS: Slow interstitial fluid flow is sufficient to induce fibrosis, could underlie the intractable nature of fibrosis following filtering glaucoma surgery and might be a target for antifibrotic therapy.

Characterization of the Immediate and Delayed Biomechanical Response to UV-A Crosslinking of Human Corneas.

PURPOSE: Keratoconus leads to visual deterioration due to irregular astigmatism and corneal thinning. Riboflavin-based corneal UV-A crosslinking (CXL) induces novel intramolecular and intermolecular links resulting in corneal tissue stiffening, thereby halting disease progression. The purpose of this study was to analyze the immediate and delayed biomechanical responses of human donor corneas to CXL. METHODS: CXL was performed according to the Dresden protocol to corneas not suitable for transplantation. Biomechanical properties were subsequently monitored by measuring the Young modulus using nanoindentation. The immediate tissue response was determined after 0, 1, 15, and 30 minutes of irradiation. Delayed biomechanical effects were investigated with follow-up measurements immediately and 1, 3, and 7 days after CXL. RESULTS: Young's modulus indicated a linear trend in direct response to increasing irradiation times (mean values: total 61.31 kPa [SD 25.53], 0 minutes 48.82 kPa [SD 19.73], 1 minute 53.44 kPa [SD 25.95], 15 minutes 63.56 kPa [SD 20.99], and 30 minutes 76.76 kPa [SD 24.92]). The linear mixed model for the elastic response of corneal tissue was 49.82 kPa + (0.91 kPa/min × time [minutes]); P < 0.001. The follow-up measurements showed no significant delayed changes in the Young modulus (mean values: total 55,28 kPa [SD 15.95], immediately after CXL 56,83 kPa [SD 18.74], day 1 50.28 kPa [SD 14.15], day 3 57.08 kPa [SD 14.98], and day 7 56.83 kPa [SD 15.07]). CONCLUSIONS: This study suggests a linear increase of corneal Young modulus as a function of CXL timing. No significant short-term delayed biomechanical changes posttreatment were observed.

Efficient Isolation and Expansion of Limbal Melanocytes for Tissue Engineering.

Limbal melanocytes (LMs) are found in the corneoscleral limbus basal epithelial layer and interact with neighboring limbal epithelial progenitor cells. The difficulty of isolating and cultivating LMs is due to the small fraction of LMs in the overall limbal population and the frequent contamination of primary cultures by other cell types. This has limited the research on freshly isolated LMs and the investigation of their biological significance in the maintenance of the limbal stem cell niche. Here, we describe an optimized protocol for the efficient isolation and expansion of LMs from cadaveric corneal limbal tissue using CD90 and CD117 as selective markers in fluorescence-activated cell sorting to obtain a pure population of LMs (CD90- CD117+) with self-renewal capacity and sustained melanin production. The isolation of pure LMs from a single preparation enables direct transcriptomic and proteomic analyses, as well as functional studies on freshly isolated LMs, which can be considered the proper counterparts of LMs in vivo and have potential applications in tissue engineering.

[Keratoconus: biomechanics ex vivo]. / Keratokonus: Biomechanik ex vivo.

BACKGROUND: Keratoconus is associated with an impairment in corneal biomechanics. Using nanoindentation, spatially resolved measurement of biomechanical properties can be performed on corneal tissue. The aim of this study is to assess the biomechanical properties of corneas with keratoconus in comparison to healthy controls. METHODS: 17 corneas with keratoconus and 10 healthy corneas unsuitable for transplantation were included in the study. After explantation, corneas were kept in culture medium containing 15% dextran for at least 24 h. Nanoindentation was then performed to a depth of 25 µm at a force increase of 300 µN/min. RESULTS: A total of 2328 individual indentations were performed for this study. In the keratoconus group; the mean modulus of elasticity was 23.2 kPa (± 15.0 kPa) for a total of 1802 indentations. In the control group, the mean modulus of elasticity was 48.7 kPa (± 20.5 kPa) with a total of 526 indentations. The Wilcoxon test showed that the differences were statistically significant. CONCLUSION: Using nanoindentation, a significantly lower elastic modulus was found in corneas with keratoconus compared to corneas without keratoconus. Further studies are needed to gain a better understanding of how keratoconus affects corneal biomechanics.

Characterization of Porcine Ocular Surface Epithelial Microenvironment.

The porcine ocular surface is used as a model of the human ocular surface; however, a detailed characterization of the porcine ocular surface has not been documented. This is due, in part, to the scarcity of antibodies produced specifically against the porcine ocular surface cell types or structures. We performed a histological and immunohistochemical investigation on frozen and formalin-fixed, paraffin-embedded ocular surface tissue from domestic pigs using a panel of 41 different antibodies related to epithelial progenitor/differentiation phenotypes, extracellular matrix and associated molecules, and various niche cell types. Our observations suggested that the Bowman's layer is not evident in the cornea; the deep invaginations of the limbal epithelium in the limbal zone are analogous to the limbal interpalisade crypts of human limbal tissue; and the presence of goblet cells in the bulbar conjunctiva. Immunohistochemistry analysis revealed that the epithelial progenitor markers cytokeratin (CK)15, CK14, p63α, and P-cadherin were expressed in both the limbal and conjunctival basal epithelium, whereas the basal cells of the limbal and conjunctival epithelium did not stain for CK3, CK12, E-cadherin, and CK13. Antibodies detecting marker proteins related to the extracellular matrix (collagen IV, Tenascin-C), cell-matrix adhesion (ß-dystroglycan, integrin α3 and α6), mesenchymal cells (vimentin, CD90, CD44), neurons (neurofilament), immune cells (HLA-ABC; HLA-DR, CD1, CD4, CD14), vasculature (von Willebrand factor), and melanocytes (SRY-homeobox-10, human melanoma black-45, Tyrosinase) on the normal human ocular surface demonstrated similar immunoreactivity on the normal porcine ocular surface. Only a few antibodies (directed against N-cadherin, fibronectin, agrin, laminin α3 and α5, melan-A) appeared unreactive on porcine tissues. Our findings characterize the main immunohistochemical properties of the porcine ocular surface and provide a morphological and immunohistochemical basis useful to research using porcine models. Furthermore, the analyzed porcine ocular structures are similar to those of humans, confirming the potential usefulness of pig eyes to study ocular surface physiology and pathophysiology.

PAX6 Expression Patterns in the Adult Human Limbal Stem Cell Niche.

Paired box 6 (PAX6), a nuclear transcription factor, determines the fate of limbal epithelial progenitor cells (LEPC) and maintains epithelial cell identity. However, the expression of PAX6 in limbal niche cells, primarily mesenchymal stromal cells (LMSC), and melanocytes is scarce and not entirely clear. To distinctly assess the PAX6 expression in limbal niche cells, fresh and organ-cultured human corneoscleral tissues were stained immunohistochemically. Furthermore, the expression of PAX6 in cultured limbal cells was investigated. Immunostaining revealed the presence of PAX6-negative cells which were positive for vimentin and the melanocyte markers Melan-A and human melanoma black-45 in the basal layer of the limbal epithelium. PAX6 staining was not observed in the limbal stroma. Moreover, the expression of PAX6 was observed by Western blot in cultured LEPC but not in cultured LMSC or LM. These data indicate a restriction of PAX6 expression to limbal epithelial cells at the limbal stem cell niche. These observations warrant further studies for the presence of other PAX isoforms in the limbal stem cell niche.

Transcriptional Profiling Identifies Prognostic Gene Signatures for Conjunctival Extranodal Marginal Zone Lymphoma.

This study characterizes the transcriptional profile and the cellular tumor microenvironment of conjunctival extranodal marginal zone lymphoma (EMZL) and identifies prognostically relevant biomarkers. Ten formalin-fixed and paraffin-embedded conjunctival EMZL and eight healthy conjunctival specimens were analyzed by Massive Analysis of cDNA Ends (MACE) RNA sequencing. The 3417 upregulated genes in conjunctival EMZL were involved in processes such as B cell proliferation and Rac protein signaling, whereas the 1188 downregulated genes contributed most significantly to oxidative phosphorylation and UV protection. The tumor microenvironment, as determined by deconvolution analysis, was mainly composed of multiple B cell subtypes which reflects the tumor's B cell lineage. However, several T cell types, including T helper 2 cells and regulatory T cells, as well as innate immune cell types, such as anti-inflammatory macrophages and plasmacytoid dendritic cells, were also strongly enriched in conjunctival EMZL. A 13-biomarker prognostic panel, including S100A8 and S100A9, classified ocular and extraocular tumor recurrence, exceeded prognostic accuracy of Ann Arbor and American Joint Committee on Cancer (AJCC) staging, and demonstrated prognostic value for patient survival in 21 different cancer types in a database of 12,332 tumor patients. These findings may lead to new options of targeted therapy and may improve prognostic prediction for conjunctival EMZL.

[The economic importance of individual health services (IGeL) in ophthalmology over time: survey results from 2010 and 2020]. / Die wirtschaftliche Bedeutung von Individuellen Gesundheitsleistungen (IGeL) in der Augenheilkunde über die Zeit: Umfrageergebnisse aus 2010 und 2020.

BACKGROUND: Individual health services (IGeL) enable patients to receive medical services not covered by social health care; however, there is no central data collection on IGeL in Germany. OBJECTIVE: This study illustrates the spectrum of IGeL provided in the field of ophthalmology as an example of the importance of IGeL in Germany based on survey results. MATERIAL AND METHODS: Nationwide, 10% of ophthalmologists in private practice were randomly selected in this anonymous survey in 2010 and 2020 while in 2020 in addition to the randomized 10% of ophthalmologists the same ophthalmologists from 2010 were contacted. By means of a written questionnaire, ophthalmologists were asked about their practice structure, total revenue from IGeL as well as the frequency and price of specific IGeL. RESULTS: Income from IGeL was estimated at an average of 21% of the regular service volume in 2010 and 23% in 2020. Glaucoma IGeL and medical report IGeL were offered by almost all ophthalmologists and glaucoma screening being performed most frequently with an average frequency of over 150 examinations/month. IGeL, such as HRT IGeL were offered by significantly fewer ophthalmologists in 2020 than in 2010, while IGeL based on other technological procedures such as glaucoma OCT were offered more frequently in 2020. CONCLUSION: The volume of IGeL provided in established ophthalmological practices was stable between 2010 and 2020. The range of services offered in 2020 compared to 2010 reflects a dynamic change associated with the entry of new technologies into routine care.

STAT3 signaling induced by the IL-6 family of cytokines modulates angiogenesis.

Aberrant angiogenesis is a hallmark of cardiovascular and retinal neovascular disease. The STAT3 signaling pathway represents a potential pharmacological target for these diseases due to its impact on angiogenesis. Surprisingly, some STAT3 activators, such as the IL-6 cytokine family member oncostatin M (OSM), enhance angiogenesis, whereas others, such as ciliary neurotropic factor (CNTF), reduce it. This study aimed to clarify these conflicting effects. In contrast to the anti-angiogenic cytokine CNTF, the pro-angiogenic cytokine OSM was able to activate intracellular signaling pathways beyond the STAT3 pathway, including the ERK and AKT pathways. These differences translated into transcriptomic and metabolic shifts. siRNA-mediated STAT3 knockdown experiments showed a decrease in VEGF-induced endothelial migration and sprouting, enhancing the pro-angiogenic drive of OSM and switching the CNTF response from anti-angiogenic to pro-angiogenic. These effects correlated with a transcriptomic shift representing enhanced STAT1 and ERK activity following STAT3 knockdown, including a compensatory prolonged phosphorylated STAT1 activity. In conclusion, the angiogenic effect of STAT3 appears to be determined by cytokine-induced STAT3 specificity and simultaneous activity of other intracellular signaling pathways, whereas the STAT3 pathway, predominantly recognized for its pro-angiogenic phenotypes, reveals novel anti-angiogenic potential.

Web-based gene expression analysis-paving the way to decode healthy and diseased ocular tissue.

BACKGROUND: Gene expression analysis using RNA sequencing has helped to improve the understanding of many diseases. Databases, such as the Gene Expression Omnibus database of the National Center for Biotechnology Information provide RNA sequencing raw data from various diseased tissue types but their analysis requires advanced bioinformatics skills. Therefore, specific ocular databases provide the transcriptional profiles of different ocular tissues and in addition enable intuitive web-based data analysis. OBJECTIVE: The aim of this narrative review is to provide an overview of ocular transcriptome databases and to compare them with the Human Eye Transcriptome Atlas newly established in Freiburg. METHODS: PubMed literature search. RESULTS: A total of nine ocular transcriptome databases focusing on different aspects were identified. The iSyTE and Express platforms specialize in gene expression during lens and retinal development in mice, whereas retina.tigem.it, Eye in a Disk, and Spectacle focus on selected ocular tissues such as the retina. Spectacle, UCSC Cell Browser and Single Cell Portal allow intuitive exploration of single cell RNA sequencing data derived from retinal, choroid, cornea, iris, trabecular meshwork and sclera specimens. The microarray profiles of a variety of healthy ocular tissues are included in the Ocular Tissue Database. The Human Eye Transcriptome Atlas provides the largest collection of different ocular tissue types, contains the highest number of ocular diseases and is characterized by a high level of quality achieved by methodological consistency. CONCLUSION: Ocular transcriptome databases provide comprehensive and intuitive insights into the transcriptional profiles of a variety of healthy and diseased ocular tissues. Thus, they improve our understanding of the underlying molecular mediators, support hypothesis generation and help in the search for new diagnostic and therapeutic targets for various ocular diseases.

Transcriptional profiling specifies the pathogen-specific human host response to infectious keratitis.

Purpose: Corneal infections are a leading cause of visual impairment and blindness worldwide. Here we applied high-resolution transcriptomic profiling to assess the general and pathogen-specific molecular and cellular mechanisms during human corneal infection. Methods: Clinical diagnoses of herpes simplex virus (HSV) (n=5) and bacterial/fungal (n=5) keratitis were confirmed by histology. Healthy corneas (n=7) and keratoconus (n=4) samples served as controls. Formalin-fixed, paraffin-embedded (FFPE) human corneal specimens were analyzed using the 3' RNA sequencing method Massive Analysis of cDNA Ends (MACE RNA-seq). The cellular host response was investigated using comprehensive bioinformatic deconvolution (xCell and CYBERSORTx) analyses and by integration with published single cell RNA-seq data of the human cornea. Results: Our analysis identified 216 and 561 genes, that were specifically overexpressed in viral or bacterial/fungal keratitis, respectively, and allowed to distinguish the two etiologies. The virus-specific host response was driven by adaptive immunity and associated molecular signaling pathways, whereas the bacterial/fungal-specific host response mainly involved innate immunity signaling pathways and cell types. We identified several genes and pathways involved in the host response to infectious keratitis, including CXCL9, CXCR3, and MMP9 for viral, and S100A8/A9, MMP9, and the IL17 pathway for bacterial/fungal keratitis. Conclusions: High-resolution molecular profiling provides new insights into the human corneal host response to viral and bacterial/fungal infection. Pathogen-specific molecular profiles may provide the foundation for novel diagnostic biomarker and therapeutic approaches that target inflammation-induced damage to corneal host cells with the goal to improve the outcome of infectious keratitis.

Addressing bias in manual segmentation of spheroid sprouting assays with U-Net.

Purpose: Angiogenesis research faces the issue of false-positive findings due to the manual analysis pipelines involved in many assays. For example, the spheroid sprouting assay, one of the most prominent in vitro angiogenesis models, is commonly based on manual segmentation of sprouts. In this study, we propose a method for mitigating subconscious or fraudulent bias caused by manual segmentation. This approach involves training a U-Net model on manual segmentations and using the readout of this U-Net model instead of the potentially biased original segmentations. Our hypothesis is that U-Net will mitigate any bias in the manual segmentations because this will impose only random noise during model training. We assessed this idea using a simulation study. Methods: The training data comprised 1531 phase contrast images and manual segmentations from various spheroid sprouting assays. We randomly divided the images 1:1 into two groups: a fictitious intervention group and a control group. Bias was simulated exclusively in the intervention group. We simulated two adversarial scenarios: 1) removal of a single randomly selected sprout and 2) systematic shortening of all sprouts. For both scenarios, we compared the original segmentation, adversarial segmentation, and respective U-Net readouts. In the second step, we assessed the sensitivity of this approach to detect a true positive effect. We sampled multiple treatment and control groups with decreasing treatment effects based on unbiased ground truth segmentation. Results: This approach was able to mitigate bias in both adversarial scenarios. However, in both scenarios, U-Net detected the real treatment effects based on a comparison to the ground truth. Conclusions: This method may prove useful for verifying positive findings in angiogenesis experiments with a manual analysis pipeline when full investigator masking has been neglected or is not feasible.

Transcriptomic Landscape and Functional Characterization of Human Induced Pluripotent Stem Cell-Derived Limbal Epithelial Progenitor Cells.

Limbal stem cell deficiency (LSCD) is a complex, multifactorial disease affecting limbal epithelial progenitor cells (LEPC), which are essential for maintaining corneal stability and transparency. Human induced pluripotent stem cell-derived (hiPSC-) LEPC are a promising cell source for the treatment of LSCD. However, their similarity to native tissue-derived (T-) LEPC and their functional characterization has not been studied in detail. Here, we show that hiPSC-LEPC and T-LEPC have rather similar gene expression patterns, colony-forming ability, wound-healing capacity, and melanosome uptake. In addition, hiPSC-LEPC exhibited lower immunogenicity and reduced the proliferation of peripheral blood mononuclear cells compared with T-LEPC. Similarly, the hiPSC-LEPC secretome reduced the proliferation of vascular endothelial cells more than the T-LEPC secretome. Moreover, hiPSC-LEPC successfully repopulated decellularized human corneolimbal (DHC/L) scaffolds with multilayered epithelium, while basal deposition of fibrillary material was observed. These findings suggest that hiPSC-LEPC exhibited functional properties close to native LEPC and that hiPSC-LEPC-DHC/L scaffolds might be feasible for transplantation in patients suffering from LSCD in the future. Although hiPSC-LEPC-based stem cell therapy is promising, the current study also revealed new challenges, such as abnormal extracellular matrix deposition, that need to be overcome before hiPSC-LEPC-based stem cell therapies are viable.

Transcriptional Profiling Provides New Insights into Organ Culture-Induced Changes in Human Donor Corneas.

Corneal transplantation is one of the most common forms of tissue transplantation worldwide. Donor corneal tissue used in transplantation is provided by eye banks, which store the tissue in culture medium after procurement. To date, the effects of cell culture on human corneal tissue have not been fully elucidated. Using the 3' RNA sequencing method for massive analysis of cDNA ends (MACE), we show that cultivation of corneal tissue leads to significant changes in a variety of molecular processes in human corneal tissue that go well beyond aspects of previously known culture effects. Functionally grouped network analysis revealed nine major groups of biological processes that were affected by corneal organ culture, among them keratinization, hypoxia, and angiogenesis, with genes from each group being affected by culture time. A cell type deconvolution analysis revealed significant modulations of the corneal immune cell profile in a time dependent manner. The results suggest that current culture conditions should be further refined and that prolonged cultivation may be detrimental. Recently, we showed that MACE enables transcriptional profiling of formalin-fixed and paraffin-embedded (FFPE) conjunctival tissue with high accuracy even after more than 10 years of storage. Here we demonstrate that MACE provides comparable results for native and FFPE corneal tissue, confirming that the technology is suitable for transcriptome analysis of a wide range of archived diseased corneal samples stored in histological archives. Finally, our data underscore the feasibility of bioinformatics cell-type enrichment analysis in bulk RNA-seq data to profile immune cell composition in fixed and archived corneal tissue samples, for which RNA-seq analysis of individual cells is often not possible.