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Summary Human embryonic stem cell-derived retinal pigment epithelial cells (hESC-RPE) provide a promising cell source for studying retinal development, for disease modelling, and retinal cell replacement therapies. Several research groups, including ours, have demonstrated that hESC-RPE structure, function, and physiology resembles that of native RPE regarding cellular fine structure and expression of many RPE signature genes and proteins. To characterize the hESC-RPE proteome in larger scale, we have compared hESC-RPE protein expression to primary human RPE using isobaric tags for relative quantitation (iTRAQ) technology. The hESC-RPE proteome reflected that of native RPE with a large number of metabolic, mitochondrial, cytoskeletal, and transport proteins expressed. No adverse signs such as increased stress, proliferation, or retinal degeneration-related changes were seen in hESC-RPE, while proteins involved in key RPE functions such as visual cycle and phagocytosis were detected. Proteomics provides valuable tools for validation of hESC-RPE, studying disease mechanisms, and discovery of new biomarkers and therapeutic targets.

Purpose Inefficient removal of dying retinal pigment epithelial (RPE) cells by professional and non-professional phagocytes can result in debris formation and development of age-related macular degeneration (AMD). We aimed to study the clearance of autophagic dying RPE cells serving as a model for dry and wet type of AMD, respectively. Methods Autophagic cell death was induced by serum deprivation and H2O2 co-treatment in ARPE-19 and primary human RPE (hRPE) cells. Annexin-V FITC/PI labelling was used to determine the cell death rate, while autophagy detection was achieved by Western blot quantification of LC3 II/LC3 I ratio. The clearance of autophagic dying cells by non-professional and professional phagocytes was quantified using flow cytometry. Results An increasing percentage of dying RPE cells was observed in a time- and concentration dependent manner upon H2O2 treatment. Paralelly, an induction of autophagy could be detected within 2hrs of H2O2 treatment. Phagocytosis studies found autophagic dying cells to be efficiently and increasingly engulfed by both professional and non-professional phagocytes over time. Conclusion The clearance of autophagic dying RPE cells can be used as a model for studying both dry and wet type of AMD in vitro, as well as for testing future pharmacological agents for treating this disease.

Purpose Purpose Stem cells in the central part of the cornea serve an important regenerative and homeostatic function. We aimed to describe the genetic fingerprint of these cells and compare that to limbal epithelial stem cells (LESC) and bone marrow-derived MSCs (bmMSCs). Methods Corneal tissue and bmMSCs were harvested from cadavers and healthy donors, respectively (according to the Guidelines of the Helsinki Declaration) and cultured ex vivo. MSC-specific cell surface markers- and genome-wide microarray analysis were performed using FACS and Affymetrix GeneChip Human Gene 1.0 ST Array (~23,000 gene transcripts). Results Genes related to stemness (228), differentiation and lineage (220), cell cycle (108) and HOX, SOCS, Notch signaling (218) were collected into functional groups and clustered hierarchically: 45 genes were found to be specific for corneal stroma (CS)-MSCs, 62 for LESCs and 9 for bmMSCs. The hierarchical clustering clearly separated the CS-MSCs from the LESCs and bmMSCs, but formed a higher cluster with the later. The top 10 genes related to the differences were VCAM1, FNDC1, MFAP5, SFRP2, IGF2, MMP, ITGA2, COLEC12, SEMA3A and MGARP. Number of molecules functioning in cellular movement (381), cellular growth and proliferation (408), development (370) and cellular development (360) were found with top biological functions in CSMSCs compared to LESCs or bmMSCs (p

Purpose To characterize the cell surface marker phenotype and function of ex vivo cultured cells growing out of human epiretinal membranes (ERMs) from proliferative diabetic retinopathy (PDR). Methods All tissue collection complied with the Guidelines of the Helsinki Declaration. ERMs were obtained from vitrectomies due to intravitreal hemorrhage in PDR. Ex vivo cultivation under adherent conditions was performed in DMEM supplemented with FBS and the cell surface marker phenotype determined. Release of IL-6, IL-8 and TNFalpha was measured upon activation of the cells with TLR lingands and TNFalpha. The dynamics of the intracellular calcium was measured using fluorescent dye Fura-2 and imaged in response to mechano-stimulation. Results The cultivated ERMs formed proliferating cell monolayers when cultivated ex vivo. These cells were negative for endothelial markers (CD31, VEGFR2), partially positive for hematopoietic (CD34, CD47) and mesenchymal markers (CD90, PDGFRb, CD73) and negative for CD105. IL-6, IL-8 and TNFalpha secretion could be measured upon activation of the cells by LPS, Poly I:C and TNFalpha. Mechano-stimulation of the outgrowing cells induced intracellular calcium propagation representing their functional viability. Conclusion ERMs from PDR contain cells of hematopoietic and mesenchimal origin which have proliferative potential, release pro-inflammatory cytokines upon selective inflammatory stimulation and show functionality reflected through calcium dynamics upon mechano-stimulation.

Purpose Mesenchymal stem cells (MSC) are the stromal cells of bone marrow, but they can be also found in other tissues including the cornea. Our goal was to isolate and cultivate human corneal stroma MSC-like cells (CSMSCs) and study their role in immunity and wound healing. Methods Corneal buttons were harvested from cadavers (according to the Guidelines of the Helsinki Declaration). The isolated stromal cells were cultured ex vivo in human serum containing medium. Fluorescent microscopy, FACS and gene array analysis, as well as standardized in vitro differentiation assays were performed to investigate the stemness and phenotype of the CSMSCs. To investigate the immunosuppressive function of these cells, mitogen activated lymphocyte reaction and activation by pro-inflammatory cytokines were used. Results According to the definition of the ISCT, the most important MSC markers (CD73, CD90 and CD105) were highly expressed on the surface of the CSMSCs with the absence of endothelial or hematopoietic cell markers. The CSMSCs were able to differentiate into fat, bone, cartilage tissues and close wounds within 24 hrs in vitro. They could suppress the proliferation of activated peripheral blood lymphocytes and secrete suppressive cytokines upon pro-inflammatory activation. Conclusion We demonstrate a method for isolating and cultivating MSC-like cells from human corneal stroma. The ex vivo data suggest that these cells may have a role in wound healing and immunological processes in the eye that can possibly be used in future treatments of ocular diseases and corneal stroma injuries.

Purpose Besides bone marrow, mesenchymal stem cells (MSCs) can be separated from various tissues including the cornea. We investigated whether human corneal stroma-derived cells resemble MSCs and if they can be grown in animal-materials free medium containing human serum as the only supplement. Methods Human cornea stromal cells were isolated from cadavers after removal of the epithelial and endothelial layers (approved by the Hungarian Regional Ethical Committee). The cells were grown in DMEM containing human serum as the only supplement. Immunophenotyping with MSC markers, integrins/cell-adhesion-, endothelial- and hematopoietic markers was carried out by FACS analysis. Standard manufacturer protocols were used for differentiating the cells into fat, cartilage or bone. Results Cells isolated from human corneal stroma grew as monolayers in vitro and could be maintained in culture for more than 10 passages (n=6). They expressed the most important markers for MSCs (CD73, CD90, CD105, CD44, CD147, PDGFRb) and were negative for the hematopoietic markers CD34, CD45, HLA-DR, CD69 and CD133. High per cent of the cells expressed the pluripotency markers CD117, C-kit and CD47, but not the endothelial cell markers CD31, CD105/V-CAM, VEGFR2. One of the hallmarks of human MSCs being capable to differentiate towards adipocytes, chondrocytes and osteocytes could also be demonstrated. Conclusion Our results indicate the presence of MSC-like cells in the human corneal stroma, which can be grown in human serum-containing medium. This opens the door for studying human keratopathies, as well as corneal tissue engineering and cell-based therapies.