Accurate and comprehensive evaluation of O6-methylguanine-DNA methyltransferase promoter methylation by nanopore sequencing.

AIMS: The methylation status of the O6-methylguanine-DNA methyltransferase (MGMT) promoter region is essential in evaluating the prognosis and predicting the drug response in patients with glioblastoma. In this study, we evaluated the utility of using nanopore long-read sequencing as a method for assessing methylation levels throughout the MGMT CpG-island, compared its performance to established techniques and demonstrated its clinical applicability. METHODS: We analysed 165 samples from CNS tumours, focusing on the MGMT CpG-island using nanopore sequencing. Oxford Nanopore Technologies (ONT) MinION and PromethION flow cells were employed for single sample or barcoded assays, guided by a CRISPR/Cas9 protocol, adaptive sampling or as part of a whole genome sequencing assay. Methylation data obtained through nanopore sequencing were compared to results obtained via pyrosequencing and methylation bead arrays. Hierarchical clustering was applied to nanopore sequencing data for patient stratification. RESULTS: Nanopore sequencing displayed a strong correlation (R2 = 0.91) with pyrosequencing results for the four CpGs of MGMT analysed by both methods. The MGMT-STP27 algorithm's classification was effectively reproduced using nanopore data. Unsupervised hierarchical clustering revealed distinct patterns in methylated and unmethylated samples, providing comparable survival prediction capabilities. Nanopore sequencing yielded high-confidence results in a rapid timeframe, typically within hours of sequencing, and extended the analysis to all 98 CpGs of the MGMT CpG-island. CONCLUSIONS: This study presents nanopore sequencing as a valid and efficient method for determining MGMT promotor methylation status. It offers a comprehensive view of the MGMT promoter methylation landscape, which enables the identification of potentially clinically relevant subgroups of patients. Further exploration of the clinical implications of patient stratification using nanopore sequencing of MGMT is warranted.

The human lens is capable of trilineage differentiation towards osteo-, chondro-, and adipogenesis-a model for studying cataract pathogenesis.

The potential for trilineage differentiation of cells in tissues represents a model for studying disease pathogenesis and regeneration pathways. Human lens trilineage differentiation has not yet been demonstrated, and so has calcification and osteogenic differentiation of human lens epithelial cells in the whole human lens. Such changes can pose a risk for complications during cataract surgery. Human lens capsules (n = 9) from cataract patients undergoing uneventful surgery were trilineage-differentiated toward osteogenesis, chondrogenesis, and adipogenesis. Furthermore, whole human healthy lenses (n = 3) collected from cadaveric eyes were differentiated into bone and characterized by immunohistochemistry. The cells in the human lens capsules were capable of undergoing trilineage differentiation, while the whole human healthy lenses could undergo osteogenesis differentiation, expressing osteocalcin, collagen I, and pigment epithelium-derived factor. We, hereby, show an ex vivo model for cataract formation through different stages of opacification, as well as provide in vivo evidence from patients undergoing calcified lens extraction with bone-like consistency.

Intracorneal Implantation of 3D Bioprinted Scaffolds Containing Mesenchymal Stromal Cells Using Femtosecond-Laser-Assisted Intrastromal Keratoplasty.

Injury of the cornea is a complex biological process. Regeneration of the corneal stroma can be facilitated by the presence of mesenchymal stromal cells (MSCs) and application of tissue equivalents. A new tissue-engineering strategy for corneal stroma regeneration is presented using cellularized 3D bioprinted hydrogel constructs implanted into organ cultured porcine corneas using femtosecond laser-assisted intrastromal keratoplasty. The ex vivo cultured, MSC-loaded 3D bioprinted structures remain intact, support cell survival, and contain de novo synthesized extracellular matrix components and migrating cells throughout the observation period. At day 14 postimplantation, the cellularized tissue equivalents contain few or no cells, as demonstrated by optical coherence tomography imaging and immunofluorescent staining. This study successfully combines a laboratory-based method with modern, patient-care practice to produce a cell-laden tissue equivalent for corneal implantation. Optimal bioink composition and cellularization of tissue equivalents are essential in fine-tuning a method to promote the current technique as a future treatment modality.

Subretinal Implantation of RPE on a Carrier in Minipigs: Guidelines for Preoperative Preparations, Surgical Techniques, and Postoperative Care.

Degenerative disorders of the retina (including age-related macular degeneration), which originate primarily at or within the retinal pigmented epithelial (RPE) layer, lead to a progressive disorganization of the retinal anatomy and the deterioration of visual function. The substitution of damaged RPE cells (RPEs) with in vitro cultured RPE cells using a subretinal cell carrier has shown potential for re-establishing the anatomical structure of the outer retinal layers and is, therefore, being further studied. Here, we present the principles of a surgical technique that allows for the effective subretinal transplantation of a cell carrier with cultivated RPEs into minipigs. The surgeries were performed under general anesthesia and included a standard lens-sparing three-port pars plana vitrectomy (PPV), subretinal application of a balanced salt solution (BSS), a 2.7 mm retinotomy, implantation of a nanofibrous cell carrier into the subretinal space through an additional 3.0 mm sclerotomy, fluid-air exchange (FAX), silicone oil tamponade, and closure of all the sclerotomies. This surgical approach was used in 29 surgeries (18 animals) over the past 8 years with a success rate of 93.1%. Anatomic verification of the surgical placement was carried out using in vivo fundus imaging (fundus photography and optical coherence tomography). The recommended surgical steps for the subretinal implantation of RPEs on a carrier in minipig eyes can be used in future preclinical studies using large-eye animal models.

Subretinal Implantation of Human Primary RPE Cells Cultured on Nanofibrous Membranes in Minipigs.

PURPOSE: The development of primary human retinal pigmented epithelium (hRPE) for clinical transplantation purposes on biodegradable scaffolds is indispensable. We hereby report the results of the subretinal implantation of hRPE cells on nanofibrous membranes in minipigs. METHODS: The hRPEs were collected from human cadaver donor eyes and cultivated on ultrathin nanofibrous carriers prepared via the electrospinning of poly(L-lactide-co-DL-lactide) (PDLLA). "Libechov" minipigs (12-36 months old) were used in the study, supported by preoperative tacrolimus immunosuppressive therapy. The subretinal implantation of the hRPE-nanofibrous carrier was conducted using general anesthesia via a custom-made injector during standard three-port 23-gauge vitrectomy, followed by silicone oil endotamponade. The observational period lasted 1, 2, 6 and 8 weeks, and included in vivo optical coherence tomography (OCT) of the retina, as well as post mortem immunohistochemistry using the following antibodies: HNAA and STEM121 (human cell markers); Bestrophin and CRALBP (hRPE cell markers); peanut agglutining (PNA) (cone photoreceptor marker); PKCα (rod bipolar marker); Vimentin, GFAP (macroglial markers); and Iba1 (microglial marker). RESULTS: The hRPEs assumed cobblestone morphology, persistent pigmentation and measurable trans-epithelial electrical resistance on the nanofibrous PDLLA carrier. The surgical delivery of the implants in the subretinal space of the immunosuppressed minipigs was successfully achieved and monitored by fundus imaging and OCT. The implanted hRPEs were positive for HNAA and STEM121 and were located between the minipig's neuroretina and RPE layers at week 2 post-implantation, which was gradually attenuated until week 8. The neuroretina over the implants showed rosette or hypertrophic reaction at week 6. The implanted cells expressed the typical RPE marker bestrophin throughout the whole observation period, and a gradual diminishing of the CRALBP expression in the area of implantation at week 8 post-implantation was observed. The transplanted hRPEs appeared not to form a confluent layer and were less capable of keeping the inner and outer retinal segments intact. The cone photoreceptors adjacent to the implant scaffold were unchanged initially, but underwent a gradual change in structure after hRPE implantation; the retina above and below the implant appeared relatively healthy. The glial reaction of the transplanted and host retina showed Vimentin and GFAP positivity from week 1 onward. Microglial activation appeared in the retinal area of the transplant early after the surgery, which seemed to move into the transplant area over time. CONCLUSIONS: The differentiated hRPEs can serve as an alternative cell source for RPE replacement in animal studies. These cells can be cultivated on nanofibrous PDLLA and implanted subretinally into minipigs using standard 23-gauge vitrectomy and implantation injector. The hRPE-laden scaffolds demonstrated relatively good incorporation into the host retina over an eight-week observation period, with some indication of a gliotic scar formation, and a likely neuroinflammatory response in the transplanted area despite the use of immunosuppression.

Impact of benzalkonium chloride-preserved and preservative-free latanoprost eye drops on cultured human conjunctival goblet cells upon acute exposure and differences in physicochemical properties of the eye drops.

OBJECTIVE: To investigate the short-term impact on human conjunctival goblet cell (GC) survival and mucin release of acute exposure to benzalkonium chloride (BAK) preserved and preservative-free (PF) 0.005% (w/v) latanoprost (LT) eye drops, and to compare the eye drops' physicochemical properties. METHODS AND ANALYSIS: Primary GC cultures were established from human conjunctival donor tissue. The impact of eye drops on GC survival was assessed using a lactate dehydrogenase assay. Mucin release was evaluated through mucin-specific immunostaining. pH value, osmolality, drop mass and surface tension for all LT eye drops were measured. RESULTS: After application with PF-LT for 30 min (min), the GC survival was maintained compared with control (p=0.9941), while all BAK-LT eye drops reduced survival with approximately 30% (p<0.02). Following application with PF-LT for 30 min, mucin was found around the GC nucleus, as seen in the vehicle control, indicating no secretion. In contrast, BAK-LT caused diffuse staining of mucin, similar to the secretagogue histamine, indicating stimulation of secretion. The pH value of the BAK-LT and PF-LT eye drops were 6.0-6.9 and 6.8, respectively. The osmolality was 258-288 mOsm/kg for the BAK-LT eye drops and 276 for PF-LT eye drops. The mean drop mass was 26-31 mg for the BAK-LT eye drops and 30 mg for PF-LT. The surface tension was lower for all BAK-LT eye drops (31.1-32.1 mN/m) compared with PF-LT (42 mN/m). CONCLUSION: PF-LT compared with various branded and generic LT preparations containing BAK are less cytotoxic when applied to cultured GCs.

Long-term myofibroblast persistence in the capsular bag contributes to the late spontaneous in-the-bag intraocular lens dislocation.

Late spontaneous in-the-bag intraocular lens (IOL) dislocation is a complication presenting 6 months or later after cataract surgery. We aimed to characterize the cells in the lens capsules (LCs) of 18 patients with spontaneous late in-the-bag IOL dislocation. Patients' average age was 82.6 ± 1.5 years (range 72-98), and most of them had pseudoexfoliation syndrome (PEX). Cells from the LCs were positive for myofibroblast (αSMA), proliferation (Ki-67, PCNA), early lens development/lens progenitor (SOX2, PAX6), chemokine receptor (CXCR4), and transmembrane (N-cadherin) markers, while negative for epithelial (E-cadherin) marker. Moreover, the cells produced abundant fibronectin, type I and type V collagen in the nearby extracellular matrix (ECM). During ex vivo cultivation of dislocated IOL-LCs in toto, the cells proliferated and likely migrated onto the IOL's anterior side. EdU proliferation assay confirmed the proliferation potential of the myofibroblasts (MFBs) in dislocated IOL-LCs. Primary cultured lens epithelial cells/MFBs isolated from the LC of dislocated IOLs could induce collagen matrix contraction and continuously proliferated, migrated, and induced ECM remodeling. Taken together, this indicates that long-lived MFBs of dislocated IOLs might contribute to the pathogenic mechanisms in late in-the-bag IOL dislocation.

Isolation and Culture of Corneal Stromal Stem Cells.

An increasing body of evidence authenticates the benefit of corneal stroma-derived stem cells (CSSCs) in tissue engineering and regeneration oriented research, and potentially in the development of clinically relevant cellular therapies. Postmortem corneal tissue obtained from otherwise discarded material after keratoplasties is oftentimes the source of the cells for ex vivo research. Relatively easy to isolate and cultivate as well as inexpensive to culture, CSSCs now represent a well-described cell type with attributes of mesenchymal stem cells (MSCs). These include differentiation- and immunosuppressive potential, as well as a favorable capacity to expand in vitro. Here, we in detail describe two straightforward methods to isolate and establish CSSC cultures ex vivo.

Resveratrol as Inducer of Autophagy, Pro-Survival, and Anti-Inflammatory Stimuli in Cultured Human RPE Cells.

Purpose: To investigate the mechanism by which resveratrol acts upon retinal pigment epithelial (RPE) cells and to characterize its effect upon autophagy, survival, and inflammation, with consequent implications to treatment for age-related macular degeneration (AMD). METHODS: Cultured ARPE-19 cells were exposed to 10 and 50 µM resveratrol. Cell survival/death was determined by annexin-FITC/propidium iodide using flow cytometry, while autophagy was studied by detecting autophagic vacuoles formation (acridine orange and transmission electron microscopy), as well as LC3II/I ratio and p62 expression by Western blot. In addition, time-lapse confocal microscopy of a pDENDRA-LC3 expression vector was performed to detect autophagy in transfected ARPE-19 cells under the different treatment conditions. Inhibition of proteasomal and autophagy-lysosomal fusion was carried out by MG-132 and chloroquine, respectively, while induction of autophagy was achieved by rapamycin treatment. Detection of secreted cytokines by ARPE-19 cells using Human XL Cytokine Array was performed under oxidative stress (H2O2) and resveratrol treatments, respectively. RESULTS: Resveratrol induced autophagy in ARPE-19 cells as determined by augmented presence of autophagic vacuoles, increased LC3II/I ratio and decreased p62 expression, as well as time-lapse confocal microscopy using pDENDRA-LC3 expression vector. Resveratrol acted similarly to proteasomal inhibition and downstream of mammalian target of rapamycin (mTOR), since upstream inhibition of autophagy by 3-methyladenine could not inhibit autophagy in ARPE-19 cells. Co-treatmeant by rapamycin and/or proteasome inhibition showed no additive effect upon autophagy induction. ARPE-19 cells treated by resveratrol showed lower cell death rate compared to untreated controls. Resveratrol induced a specific anti-inflammatory response in ARPE-19 cells. CONCLUSIONS: Resveratrol can induce autophagy, pro-survival, and anti-inflammatory stimuli in ARPE-19 cells, properties which could be plausible to formulate future treatment modalities for AMD.

Clinical and molecular markers in retinal detachment-From hyperreflective points to stem cells and inflammation.

PURPOSE: Retinal detachment (RD) is one of the most frequently diagnosed ophthalmologic conditions requiring prompt surgical intervention. Combination of proper surgical technique and new diagnostic markers, both clinical and molecular, can help improve the diagnosis and prognosis of RD treatment. METHODS: 12 patients with rhegmatogenous RD (rRD) were included into the study after obtaining patient consent and Regional Ethical Approval (average age: 58.1 ± 17.4 years). OCT was performed before and after 23G vitrectomy for RD. Pure subretinal fluid (SRF) was collected during surgery and analyzed by protein array profiling on a panel of 105 inflammatory cytokines (Human XL Cytokine Array), while the effect of SRF upon human macrophages-driven phagocytosis of apoptotic retinal pigment epithelial (RPE) cells ex vivo was quantified by flow cytometry. Immunohistochemistry (IHC) of retinectomized tissue due to PVR caused by RD was performed to determine presence of markers for microglial cells (CD34), macrophages and activated microglia (CD68), regulator of the immune response to infection (NFkB), progenitor and stem cell marker (Sox2), pluripotency marker (Oct4) and intermediate filament markers (GFAP and Nestin). RESULTS: OCT of fresh RD patients contained pre-operatively hyper reflective points (HRPs) at the detached neuroretina border and proximal to the RPE layer-their size and number decreased following successful reattachment surgery. IHC of the retinectomized tissue from detached retina due to severe PVR showed presence of cell conglomerates at the detached neuroretina border which were positive for CD68, NFkB, Sox2 and GFAP, less positive for CD47 and Nestin and negative for Oct4 and CD34. The SRF contained at least 37 cytokines with higher, and 4 cytokine with lower concentration compared to that in vitreous from non-RD pathology; when used as conditional medium to human macrophages ex vivo, the SRF doubled their capacity for engulfing dying RPEs. CONCLUSIONS: Fresh RD can be hallmarked by presence of HRPs at the detached neuroretina border on OCT; the HRPs decrease in size and number after successful reattachment surgery, and likely resemble the macrophage conglomerates seen by IHC. The neuroretina in RD contains progenitor/stem-like cells and signs of inflammatory reaction, while the SRF contains inflammatory cytokines and other factors which increase the ability of professional phagocytes to engulf dying RPE, or for that matter, other dying cells in the retina.

Ex vivo 3D human corneal stroma model for Schnyder corneal dystrophy - role of autophagy in its pathogenesis and resolution.

INTRODUCTION: Multilamellar bodies (MLBs) are concentric cytoplasmic membranes which form through an autophagy-dependent mechanism. In the cornea, the presence of MLBs is associated with Schnyder corneal dystrophy (SCD). Ex vivo 3D modelling of the corneal stroma and SCD can help study pathogenesis and resolution of the disorder. METHODS: Corneal stroma explants were isolated from cadavers and cultivated long-term for more than 3 months to achieve spontaneous 3D outgrowth of corneal stroma-derived mesenchymal stem-like cells (CSMSCs). The 3D tissues were then examined by transmission electron microscopy (TEM) for presence of MLBs, and by immunofluorescent labelling against markers for autophagy (p62, LC3). Autophagy was induced by classical serum starvation or rapamycin (RAP) treatment (50 nM), and inhibited by the autophagy inhibitor 3-methyladenine (3-MA, 10 mM) for 24 hours. RESULTS: CSMSCs can form spontaneously 3D outgrowths over a 3-4 weeks period, depositing their own extracellular matrix containing collagen I. TEM confirmed the presence of MLBs in the long-term (>3 months) 3D cultures, which became more abundant under starvation and RAP treatment, and decreased in number under autophagy inhibition with 3-MA. The presence of autophagy and its disappearance could be confirmed by an inversely related increase and decrease in the expression of LC3 and p62, respectively. CONCLUSIONS: MLB formation in long-standing CSMSC cultures could serve as a potential ex vivo model for studying corneal stroma diseases, including SCD. Inhibition of autophagy can decrease the formation of MLBs, which may lead to a novel treatment of the disease in the future.

Effect of Isolation Technique and Location on the Phenotype of Human Corneal Stroma-Derived Cells.

PURPOSE: To determine the effect of the isolation technique and location upon the phenotype of human corneal stroma-derived cells (CSCs). METHODS: CSCs were isolated from the corneal stroma center and periphery using the explant or enzymatic digestion technique. The native tissue was stained for functional markers, while cultured cells were analysed by FACS. PCR was used to determine gene expression in the cultured versus native cells. RESULTS: The native stroma was positive for α-actinin, ALDH1A1, CD31, CD34, Collagen I, and Vimentin. Cultured cells expressed CD73, CD90, CD105, CD51, Nestin, CD49a, CD49d, ABCG2, and CD47. PCR demonstrated a significant upregulation of ALDH1A1, AQP1, ITGB4, KLF4, CD31, CD34, and CXCR4 in the native tissue, while the expression of ABCG2, ITGAV, Nestin, CD73, CD90, CD105, and Vimentin were significantly higher in the cultured cells. GPC did not change. CONCLUSION: The study finds no significant difference between the phenotype of CSCs generated by the explant or enzymatic digestion technique from the center or periphery of the stroma. Isolation of the cells can be performed without regard to the location and isolation technique used for research. Cultivated CSCs undergo a complete surface marker and genotype profile change compared to the state in situ.

Cultivation and characterization of pterygium as an ex vivo study model for disease and therapy.

PURPOSE: Development of ex vivo model to study pathogenesis, inflammation and treatment modalities for pterygium. METHODS: Pterygium obtained from surgery was cultivated (3 months). Gravitational attachment method using viscoelastic facilitated adherence of graft and outgrowing cells. Medium contained serum as the only growth supplement with no use of scaffolds. Surface profiling of the multi-layered cells for hematopoietic- and mesenchymal stem cell markers was performed. Examination of cells by immunohistochemistry using pluripotency, oxidative stress, stemness, migration and proliferation, epithelial and secretory markers was performed. The effect of anti-proliferative agent Mitomycin C upon secretion of pro-inflammatory cytokines IL-6 and IL-8 was assessed. RESULTS: Cells showed high expression of migration- (CXCR4), secretory- (MUC1, MUC4) and oxidative damage- (8-OHdG) markers, and low expression of hypoxia- (HIF-1α) and proliferation- (Ki-67) markers. Moderate and low expression of the pluripotency markers (Vimentin and ΔNp63) was present, respectively, while the putative markers of stemness (Sox2, Oct4, ABCG-2) and epithelial cell markers- (CK19, CK8-18) were weak. The surface marker profile of the outgrowing cells revealed high expression of the hematopoietic marker CD47, mesenchymal markers CD90 and CD73, minor or less positivity for the hematopoietic marker CD34, mesenchymal marker CD105, progenitor marker CD117 and attachment protein markers while low levels of IL-6 and IL-8 secretion ex vivo, were inhibited upon Mitomycin C treatment. CONCLUSION: Ex vivo tissue engineered pterygium consists of a mixture of cells of different lineage origin, suitable for use as a disease model for studying pathogenesis ex vivo, while opening possibilities for new treatment and prevention modalities.

Cultivation and characterisation of the surface markers and carbohydrate profile of human corneal endothelial cells.

BACKGROUND: The study aims to characterise human corneal endothelial cell (HCEnC) cultures generated by the peel-and-digest method based on their surface protein/carbohydrate expression pattern. METHODS: Quantitative polymerase chain reaction was used to compare expression of vimentin, CD90, Cytokeratin-19, ZO-1 and Claudin 14 in cultured HCEnC and cell line B4G12 versus stromal cells. Fluorescence-activated cell sorting was used to assess surface protein distribution of cultured and uncultured HCEnC. Distribution of surface proteins/carbohydrates was visualised by immunofluorescent and lectin staining. RESULTS: Human corneal endothelial cell and B4G12 showed lower expression level for vimentin, CD90, Cytokeratin-19 compared with stromal cells; while ZO-1 was expressed in endothelial cells, Claudin 14 was detected in B4G12 only. Fluorescence-activated cell sorting analyses revealed CD166, CD47, CD44, CD54, CD73, CD90, CD105, CD106, CD112, CD146 and CD325 to be present, with CD34 to be absent from cultured HCEnC. Freshly isolated, non-cultivated HCEnCs were CD90, CD73, CD146 and CD325 positive. Carbohydrates were detected by lectins LCA, PHA E, PHA L, PSA, sWGA, Con A, RCA 120 and WGA, but cultured HCEnC showed negative for GSL I, SBA, DBA, PNA and UEA I. CONCLUSION: Cultures established by the peel-and-digest method are probably not prone to stromal contamination, but the cells are likely to undergo endothelial-to mesenchymal transition as suggested by apparent morphological changes.

Long-Term Cultures of Human Cornea Limbal Explants Form 3D Structures Ex Vivo - Implications for Tissue Engineering and Clinical Applications.

Long-term cultures of cornea limbal epithelial stem cells (LESCs) were developed and characterized for future tissue engineering and clinical applications. The limbal tissue explants were cultivated and expanded for more than 3 months in medium containing serum as the only growth supplement and without use of scaffolds. Viable 3D cell outgrowth from the explants was observed within 4 weeks of cultivation. The outgrowing cells were examined by immunofluorescent staining for putative markers of stemness (ABCG2, CK15, CK19 and Vimentin), proliferation (p63α, Ki-67), limbal basal epithelial cells (CK8/18) and differentiated cornea epithelial cells (CK3 and CK12). Morphological and immunostaining analyses revealed that long-term culturing can form stratified 3D tissue layers with a clear extracellular matrix deposition and organization (collagen I, IV and V). The LESCs showed robust expression of p63α, ABCG2, and their surface marker fingerprint (CD117/c-kit, CXCR4, CD146/MCAM, CD166/ALCAM) changed over time compared to short-term LESC cultures. Overall, we provide a model for generating stem cell-rich, long-standing 3D cultures from LESCs which can be used for further research purposes and clinical transplantation.