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Altered esophageal ion transport mechanisms play a key role in inflammatory and cancerous diseases of the esophagus, but epithelial ion processes have been less studied in the esophagus because of the lack of a suitable experimental model. In this study, we generated three-dimensional (3D) esophageal organoids (EOs) from two different mouse strains and characterized the ion transport processes of the EOs. EOs form a cell-filled structure with a diameter of 250–300 µm and were generated from epithelial stem cells as shown by FACS analysis. Using conventional PCR and immunostaining, the presence of Slc26a6 Cl−/HCO3− anion exchanger (AE), Na+/H+ exchanger (NHE), Na+/HCO3− cotransporter (NBC), cystic fibrosis transmembrane conductance regulator (CFTR), and anoctamin 1 Cl− channels was detected in EOs. Microfluorimetric techniques revealed high NHE, AE, and NBC activities, whereas that of CFTR was relatively low. In addition, inhibition of CFTR led to functional interactions between the major acid-base transporters and CFTR. We conclude that EOs provide a relevant and suitable model system for studying the ion transport mechanisms of esophageal epithelial cells, and they can be also used as preclinical tools to assess the effectiveness of novel therapeutic compounds in esophageal diseases associated with altered ion transport processes.

We previously described a novel in vitro culture technique for dedifferentiated human adult skin melanocytes. Melanocytes cultured in a defined, cholera toxin and PMA free medium became bipolar, unpigmented, and highly proliferative. Furthermore, TRP-1 and c-Kit expression disappeared and EGFR receptor and nestin expression were induced in the cells. Here, we further characterized the phenotype of these dedifferentiated cells and by comparing them to mature pigmented melanocytes we detected crucial steps in their phenotype change. Our data suggest that normal adult melanocytes easily dedifferentiate into pluripotent stem cells given the right environment. This dedifferentiation process described here for normal melanocyte is very similar to what has been described for melanoma cells, indicating that phenotype switching driven by environmental factors is a general characteristic of melanocytes that can occur independent of malignant transformation.

Recent animal studies, as well as quantitative sodium MRI observations on humans demonstrated that remarkable amounts of sodium can be stored in the skin. It is also known that excess sodium in the tissues leads to inflammation in various organs, but its role in dermal pathophysiology has not been elucidated. Therefore, our aim was to study the effect of dietary salt loading on inflammatory process and related extracellular matrix (ECM) remodeling in the skin. To investigate the effect of high salt consumption on inflammation and ECM production in the skin mice were kept on normal (NSD) or high salt (HSD) diet and then dermatitis was induced with imiquimod (IMQ) treatment. The effect of high salt concentration on dermal fibroblasts (DF) and peripheral blood mononuclear cells (PBMC) was also investigated in vitro. The HSD resulted in increased sodium content in the skin of mice. Inflammatory cytokine Il17 expression was elevated in the skin of HSD mice. Expression of anti-inflammatory Il10 and Il13 decreased in the skin of HSD or HSD IMQ mice. The fibroblast marker Acta2 and ECM component Fn and Col1a1 decreased in HSD IMQ mice. Expression of ECM remodeling related Pdgfb and activation phosphorylated (p)-SMAD2/3 was lower in HSD IMQ mice. In PBMCs, production of IL10, IL13 and PDGFB was reduced due to high salt loading. In cultured DFs high salt concentration resulted in decreased cell motility and ECM production, as well. Our results demonstrate that high dietary salt intake is associated with increased dermal pro-inflammatory status. Interestingly, although inflammation induces the synthesis of ECM in most organs, the expression of ECM decreased in the inflamed skin of mice on high salt diet. Our data suggest that salt intake may alter the process of skin remodeling.

Purpose. This study is aimed at investigating the phenotype, differentiation potential, immunomodulatory properties, and responsiveness of saphenous vein vessel wall-derived mesenchymal stromal cells (SV-MSCs) to various TLR ligands and proinflammatory cytokines, as well as comparing their features to those of their bone marrow-derived counterparts (BM-MSCs). Methods. SV-MSCs were isolated by enzymatic digestion of the saphenous vein vessel wall. Phenotype analysis was carried out by flow cytometry and microscopy, whereas adipogenic, chondrogenic, and osteogenic differentiation potentials were tested in in vitro assays. For comparative analysis, the expression of different stemness, proliferation, and differentiation-related genes was determined by Affymetrix gene array. To compare the immunomodulatory properties of SV-MSCs and BM-MSCs, mixed lymphocyte reaction was applied. To investigate their responses to various activating stimuli, MSCs were treated with TLR ligands (LPS, PolyI:C) or proinflammatory cytokines (TNFα, IL-1β, IFNγ), and the expression of various early innate immune response-related genes was assessed by qPCR, while secretion of selected cytokines and chemokines was measured by ELISA. Results. The isolated SV-MSCs were able to differentiate into bone, fat, and cartilage cells/direction in vitro. SV-MSCs expressed the most important MSC markers (CD29, CD44, CD73, CD90, and CD105) and shared almost identical phenotypic characteristics with BM-MSCs. Their gene expression pattern and activation pathways were close to those of BM-MSCs. SV-MSCs showed better immunosuppressive activity inhibiting phytohemagglutinin-induced T lymphocyte proliferation in vitro than BM-MSCs. Cellular responses to treatments mimicking inflammatory conditions were comparable in the bone marrow- and saphenous vein-derived MSCs. Namely, similar to BM-MSCs, SV-MSCs secreted increased amount of IL-6 and IL-8 after 12- or 24-hour treatment with LPS, PolyI:C, TNFα, or IL-1β, compared to untreated controls. Interestingly, a different CXCL-10/IP-10 secretion pattern could be observed under inflammatory conditions in the two types of MSCs. Conclusion. Based on our results, cells isolated from saphenous vein vessel wall fulfilled the ISCT’s (International Society for Cellular Therapy) criteria for multipotent mesenchymal stromal cells, and no significant differences in the phenotype, gene expression pattern, and responsiveness to inflammatory stimuli could be observed between BM-MSCs and SV-MSCs, while the latter cells have more potent immunosuppressive activity in vitro. Further functional assays have to be performed to reveal whether SV-MSCs could be useful for certain regenerative therapeutic applications or tissue engineering purposes.

Inefficient removal of dying retinal pigment epithelial (RPE) cells by professional phagocytes can result in debris formation and development of age-related macular degeneration (AMD). Chronic oxidative stress and inflammation play an important role in AMD pathogenesis. Only a few well-established in vitro phagocytosis assay models exist. We propose human embryonic stem cell-derived-RPE cells as a new model for studying RPE cell removal by professional phagocytes. The characteristics of human embryonic stem cells-derived RPE (hESC-RPE) are similar to native RPEs based on their gene and protein expression profile, integrity, and barrier properties or regarding drug transport. However, no data exist about RPE death modalities and how efficiently dying hESC-RPEs are taken upby macrophages, and whether this process triggers an inflammatory responses. This study demonstrates hESC-RPEs can be induced to undergo anoikis or autophagy-associated cell death due to extracellular matrix detachment or serum deprivation and hydrogen-peroxide co-treatment, respectively, similar to primary human RPEs. Dying hESC-RPEs are efficiently engulfed by macrophages which results in high amounts of IL-6 and IL-8 cytokine release. These findings suggest that the clearance of anoikic and autophagy-associated dying hESC-RPEs can be used as a new model for investigating AMD pathogenesis or for testing the in vivo potential of these cells in stem cell therapy.

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.

Human embryonic stem cell-derived retinal pigment epithelial cells (hESC-RPE) provide an unlimited cell source for retinal cell replacement therapies. Clinical trials using hESC-RPE to treat diseases such as age-related macular degeneration (AMD) are currently underway. Human ESC-RPE cells have been thoroughly characterized at the gene level but their protein expression profile has not been studied at larger scale. In this study, proteomic analysis was used to compare hESC-RPE cells differentiated from two independent hESC lines, to primary human RPE (hRPE) using Isobaric tags for relative quantitation (iTRAQ). 1041 common proteins were present in both hESC-RPE cells and native hRPE with majority of the proteins similarly regulated. The hESC-RPE proteome reflected that of normal hRPE with a large number of metabolic, mitochondrial, cytoskeletal, and transport proteins expressed. No signs of increased stress, apoptosis, immune response, proliferation, or retinal degeneration related changes were noted in hESC-RPE, while important RPE specific proteins involved in key RPE functions such as visual cycle and phagocytosis, could be detected in the hESC-RPE. Overall, the results indicated that the proteome of the hESC-RPE cells closely resembled that of their native counterparts.

Retinal pigment epithelial (RPE) cells can undergo different forms of cell death, including autophagy-associated cell death during age-related macular degeneration (AMD). Failure of macrophages or dendritic cells (DCs) to engulf the different dying cells in the retina may result in the accumulation of debris and progression of AMD. ARPE-19 and primary human RPE cells undergo autophagy-associated cell death upon serum depletion and oxidative stress induced by hydrogen peroxide (H2O2). Autophagy was revealed by elevated light-chain-3 II (LC3-II) expression and electron microscopy, while autophagic flux was confirmed by blocking the autophago-lysosomal fusion using chloroquine (CQ) in these cells. The autophagy-associated dying RPE cells were engulfed by human macrophages, DCs and living RPE cells in an increasing and time-dependent manner. Inhibition of autophagy by 3-methyladenine (3-MA) decreased the engulfment of the autophagy-associated dying cells by macrophages, whereas sorting out the GFP-LC3-positive/autophagic cell population or treatment by the glucocorticoid triamcinolone (TC) enhanced it. Increased amounts of IL-6 and IL-8 were released when autophagy-associated dying RPEs were engulfed by macrophages. Our data suggest that cells undergoing autophagy-associated cell death engage in clearance mechanisms guided by professional and non-professional phagocytes, which is accompanied by inflammation as part of an in vitro modeling of AMD pathogenesis.

Limbal epithelial stem cells (LESCs) are tissue-specific stem cells responsible for renewing the corneal epithelium. Acute trauma or chronic disease affecting LESCs may disrupt corneal epithelial renewal, causing vision threatening and painful ocular surface disorders, collectively referred to as LESC deficiency (LESCD). These disorders cannot be treated with traditional corneal transplantation and therefore alternative cell sources for successful cell-based therapy are needed. LESCs derived from human pluripotent stem cells (hPSCs) are a prospective source for ocular surface reconstruction, yet critical evaluation of these cells is crucial before considering clinical applications. In order to quantitatively evaluate hPSC-derived LESCs, we compared protein expression in native human corneal cells to that in hPSC-derived LESCs using isobaric tag for relative and absolute quantitation (iTRAQ) technology. We identified 860 unique proteins present in all samples, including proteins involved in cell cycling, proliferation, differentiation and apoptosis, various LESC niche components and limbal and corneal epithelial markers. Protein expression profiles were nearly identical in LESCs derived from two different hPSC lines, indicating that the differentiation protocol is reproducible, yielding homogeneous cell populations. Their protein expression profile suggests that hPSC-derived LESCs are similar to the human ocular surface epithelial cells and possess LESC-like characteristics.

Background The apopto-phagocytic gene expression patterns during clearance of dying cells in the retina and the effect of triamcinolone (TC) upon these processes have relevance to development of age-related macular degeneration (AMD). Methods ARPE-19 cells and primary human retinal pigment epithelium (hRPE) were induced to undergo cell death by anoikis and the clearance of these cells by living hRPE/ARPE-19 or human monocyte-derived macrophages (HMDMs) in the presence or absence of TC was quantified by flow cytometry. TaqMan low-density gene expression array determining known markers of phagocytosis and loss-of-function studies on selected apopto-phagocytic genes was carried out in HMDM engulfing anoikic cells. Results The glucocorticoid TC had a profound phagocytosis-enhancing effect on HMDM engulfing anoikic ARPE-19 or hRPE cells, causing a selective upregulation of the Mer tyrosine kinase (MERTK) receptor, while decreasing the expression of the AXL receptor tyrosine kinase and thrombospondin-1 (THSB-1). The key role of the MERTK could be demonstrated in HMDM engulfing dying cells using gene silencing as well as blocking antibodies. Similar pathways were found upregulated in living ARPE-19 engulfing anoikic ARPE-19 cells. Gas6 treatment enhanced phagocytosis in TC-treated HMDMs. Conclusions Specific agonists of the Mertk receptor may have a potential role as phagocytosis enhancers in the retina and serve as future targets for AMD therapy. General significance The use of Gas6 as enhancer of retinal phagocytosis via the MerTK receptor, alone or in combination with other specific ligands of the tyrosine kinase receptors' family may have a potential role in AMD therapy.

Severe shortage of donor corneas for transplantation, particularly in developing countries, has prompted the advancement of bioengineered tissue alternatives. Bioengineered corneas that can withstand transplantation while maintaining transparency and compatibility with host cells, and that are additionally amenable to standardized low-cost mass production are sought. In this study, a bioengineered porcine construct (BPC) was developed to function as a biodegradable scaffold to promote corneal stromal regeneration by host cells. Using high-purity medical-grade type I collagen, high 18% collagen content and optimized EDC-NHS cross-linker ratio, BPCs were fabricated into hydrogel corneal implants with over 90% transparency and four-fold increase in strength and stiffness compared with previous versions. Remarkably, optical transparency was achieved despite the absence of collagen fibril organization at the nanoscale. In vitro testing indicated that BPC supported confluent human epithelial and stromal-derived mesenchymal stem cell populations. With a novel femtosecond laser-assisted corneal surgical model in rabbits, cell-free BPCs were implanted in vivo in the corneal stroma of 10 rabbits over an 8-week period. In vivo, transparency of implanted corneas was maintained throughout the postoperative period, while healing occurred rapidly without inflammation and without the use of postoperative steroids. BPC implants had a 100% retention rate at 8 weeks, when host stromal cells began to migrate into implants. Direct histochemical evidence of stromal tissue regeneration was observed by means of migrated host cells producing new collagen from within the implants. This study indicates that a cost-effective BPC extracellular matrix equivalent can incorporate cells passively to initiate regenerative healing of the corneal stroma, and is compatible with human stem or organ-specific cells for future therapeutic applications as a stromal replacement for treating blinding disorders of the cornea.

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.

Recent studies have indicated that nitric oxide (NO)-induced cGMP synthesis is involved in different steps of neurogenesis in invertebrates. The development of putative NO synthetising elements was described earlier in the embryonic and juvenile pond snail, Lymnaea stagnalis, applying NADPH-diaphorase histochemistry (Serfozo et al., 1998). In the present study, we examined the distribution of NO synthase (NOS) during Lymnaea development by in situ hybridization for Lymnaea-NOS mRNA, histochemical, and immunohistochemical techniques for the NOS and NO-stimulated cGMP. Peripheral fibers projecting to the CNS and terminating in the ganglionic neuropils showed NOS immunoreactivity from 85% of embryonic development. At the same time, a fine dot-like, immunostaining indicated the presence of cGMP in the neuropil area. In the CNS, Lymnaea-NOS mRNA positive, as well as NOS and cGMP immunoreactive perikarya were detected first during postembryonic development; their number significantly increased from P3 juvenile stage. Some of the cell groups in the CNS containing NOS immunoreactive material also displayed Lymnaea-NOS mRNA hybridization signal and were cGMP-positive. However, in the subesophageal ganglia, the distribution of Lymnaea-NOS mRNA positive cell groups did not correspond to that of the NOS immunoreactive cells. Neurons revealing transient NOS and cGMP immunoreactivity, respectively, could also be detected in this part of the CNS. In most of the ganglia the number of Lymnaea-NOS mRNA containing and cGMP immunopositive neurons, respectively, exceeded that of the NOS immunoreactive cells from P4 juvenile stage. The localization of NADPH-diaphorase reaction also correlated well with that of the NOS immunoreactivity in the developing CNS. At the periphery, colocalization of Lymnaea-NOS mRNA signal, NOS and cGMP immunoreactivities were observed in the epithelial cells of the esophagus and mantle after hatching. The findings suggest the functional maturity of the NO/cGMP signal transduction pathway at both central and peripheral levels during the development of the snail, Lymnaea stagnalis. The differences in the localization of Lymnaea-NOS mRNA labeling and NOS immunoreactivity in the CNS and PNS can be explained by the existence of different NOS isoforms, posttranslational regulation of NOS, and/or some non-specific antibody labeling.

Age-related macular degeneration (AMD) is a multifactorial eye disease that is associated with aging, family history, smoking, obesity, cataract surgery, arteriosclerosis, hypertension, hypercholesterolemia and unhealthy diet. Gender has commonly been classified as a weak or inconsistent risk factor for AMD. This disease is characterized by degeneration of retinal pigment epithelial (RPE) cells, Bruch’s membrane, and choriocapillaris, which secondarily lead to damage and death of photoreceptor cells and central visual loss. Pathogenesis of AMD involves constant oxidative stress, chronic inflammation, and increased accumulation of lipofuscin and drusen. Estrogen has both anti-oxidative and anti-inflammatory capacity and it regulates signaling pathways that are involved in the pathogenesis of AMD. In this review, we discuss potential cellular signaling targets of estrogen in retinal cells and AMD pathology.

Age-related macular degeneration (AMD) is the leading cause of severe and irreversible loss of vision in the elderly in developed countries. AMD is a complex chronic neurodegenerative disease associated with many environmental, lifestyle, and genetic factors. Oxidative stress and the production of reactive oxygen species (ROS) seem to play a pivotal role in AMD pathogenesis. It is known that the macula receives the highest blood flow of any tissue in the body when related to size, and anything that can reduce the rich blood supply can cause hypoxia, malfunction, or disease. Oxidative stress can affect both the lipid rich retinal outer segment structure and the light processing in the macula. The response to oxidative stress involves several cellular defense reactions, for example, increases in antioxidant production and proteolysis of damaged proteins. The imbalance between production of damaged cellular components and degradation leads to the accumulation of detrimental products, for example, intracellular lipofuscin and extracellular drusen. Autophagy is a central lysosomal clearance system that may play an important role in AMD development. There are many anatomical changes in retinal pigment epithelium (RPE), Bruch’s membrane, and choriocapillaris in response to chronic oxidative stress, hypoxia, and disturbed autophagy and these are estimated to be crucial components in the pathology of neovascular processes in AMD.

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.

Age-related macular degeneration (AMD) is a complex, degenerative and progressive eye disease that usually does not lead to complete blindness, but can result in severe loss of central vision. Risk factors for AMD include age, genetics, diet, smoking, oxidative stress and many cardiovascular-associated risk factors. Autophagy is a cellular housekeeping process that removes damaged organelles and protein aggregates, whereas heterophagy, in the case of the retinal pigment epithelium (RPE), is the phagocytosis of exogenous photoreceptor outer segments. Numerous studies have demonstrated that both autophagy and heterophagy are highly active in the RPE. To date, there is increasing evidence that constant oxidative stress impairs autophagy and heterophagy, as well as increases protein aggregation and causes inflammasome activation leading to the pathological phenotype of AMD. This review ties together these crucial pathological topics and reflects upon autophagy as a potential therapeutic target in AMD.

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.

Background and objectives Mesenchymal stem cells (MSC) are the stromal cells of bone marrow, but they can also be found in other tissues including the synovia. Our goal was to isolate and cultivate human synovial fluid-derived MSC-like cells (SYF-MSC) and study their role in immunity and angiogenesis with relevance to rheumatoid arthritis (RA). Materials and methods Synovial fluid was harvested from patients with active RA (according to the Guidelines of the Helsinki Declaration). The isolated fluid/cells were cultured in vitro , and the expression of well-known MSC, hematopoietic, endothelial markers as well as high-end glycosylation products were measured by flow cytometry. In vitro differentiation assays were used to determine the stemness of SYF-MSCs. The immunosuppressive function of these cells was studied by mitogen-activated lymphocyte reaction, and their immunophenotype was investigated by cell activation with TLR ligands and pro-inflammatory cytokines; the secreted cytokines were measured by ELISA. Results The cells isolated from synovial fluid of patients with RA grew as monolayers in vitro and could be maintained in culture for more than 10 passages. The cells expressed the most important MSC markers (CD44, CD73, CD90 and CD105) with absence of endothelial (CD31, VEGFR2) or hematopoietic cell markers (CD34, CD45, CD69, CD133), respectively. SYF-MSCs were able to differentiate into bone, fat and cartilage tissue in vitro fulfilling the ISCT criteria, and could suppress the proliferation of mitogen activated peripheral blood lymphocytes. Furthermore, SYF-MSCs secreted increased amount of IL-6 after 12 and 24 h treatment by LPS (15423.61 + 9348.61 pg/ml at 12 h; 17479.17 + 3848.63 pg/ml at 24 h time points), Poly:IC (15215.28 + 10834.72 and 24090.28 + 3626.40 pg/ml), TNFα (7437.5 + 1168.06 and 11562.5 + 845.83 pg/ml) and IL-1β (19465.28 + 2306.94 and 21229.17 + 12.5 pg/ml) compared to untreated controls (1590.28 + 1015.28 and 2187.50 + 640.28 pg/ml), respectively. IL-8 could be detected in the supernatants of SYF-MSC only upon LPS (10876.76 + 7553.24 and 12117.94 + 2912.06 pg/ml), Poly:IC (8972.35 + 8707.65 and 30567.94 + 3637.94 pg/ml) TNFα (5516.47 + 1336.47 and 110325.29 + 5645.29 pg/ml) and IL-1β (18063.53 + 3083.53 and 24970.88 + 9940.88 pg/ml) treatment, but not in the untreated controls. Conclusions The in vitro data suggest that SYF-MSC may play a role in the inflammatory processes of RA, and their activation could lead to the release of pro-angiogenic cytokines as well.

The role of nitric oxide (NO) in the pathophysiology of inflammatory bowel disease (IBD) is controversial. The aim of this study was to investigate the expression and localization of nitric oxide synthase isoforms (iNOS, eNOS) in IBD colonic mucosa.Forty-four patients with IBD (24 ulcerative colitis (UC), 20 Crohn's disease (CD) and 16 controls) were investigated by colonoscopy. iNOS and eNOS in tissue sections was demonstrated by histochemistry (NADPH-diaphorase reaction) and immunohistochemistry. Cell type analysis and quantitative assessment of the iNOS immunoreactive (IR) cells and densitometry of iNOS in immunoblots were also performed.iNOS-IR cells were significantly numerous in inflamed mucosa of UC (64+/-4 cells/mm2) than in CD (4+/-2 cells/mm2). iNOS-IR/CD15-IR cells showed significant elevation in inflamed (i) versus uninflamed (u) UC mucosa (UCu 8+/-3%, UCi 85+/-10%) In CD, the percentage of iNOS-IR/CD68-IR cells was lower in inflamed sites (CDu 23+/-8%, CDi 4+/-3%). Immunoblot of biopsies revealed significant elevation of iNOS in active UC compared with uninflamed sites, whereas in CD no significant changes were detected. Differences were observed in eNOS and endothelial marker CD31 immunoreactivity. In patients with UC and in controls the ratios of eNOS/CD31-IR vessels were 82.3% and 92.0% respectively, whereas in CD the ratio was 8.3% with a concomitantly significant increase of CD31-IR vessels. The distribution and morphological characteristics of the NOS-IR inflammatory cells and endothelia were similar to those showing NADPH-diaphorase reactivity.Differences observed in the expression and distribution of NOS isoforms in immune and endothelial cells may contribute to better understanding of the structural and physiological changes in UC and CD.

Introduction. Amputation is the only current option for relief of rest pain or gangrene in patients with severe peripheral arterial disease (SPAD). Up to now, no effective blood-flow enhancement therapies are available. Autologous bone marrow-derived stem cell transplantation (ABMSCT) is an arising therapy modality with an option of building new blood vessels through endothelial stem and/or progenitor cells. Methods. Five patients with SPAD (4 with Buerger’s disease and 1 with arteriosclerosis obliterans) were treated by ABMSCT. CD34+/CD133+ cell counts were determined in aspirated bone marrow, CD34+ cells were isolated by magnetic separation and collected into a 10 ml sample. The stem cell suspension was administered by local intramuscular injections (0.5–1.0 ml injections in the musculus gastrocnemius). The follow-up (before; 1−, 3− and 6 months after ABMSCT) based on clinical (rest pain, walking distance without pain, changes of non-healing ulcers, ABI) and laboratory (DS-angiography, Color- and Laser-Doppler scan, TcPO2 measurement and endothel function test) parameters was documented and analyzed. Our goal was to treat the worse limb of all the 5 patients. Results. Therapeutic benefit was shown by complete regression of rest pain in all 5 patients, and by the significant increase of pain-free walking distance (36 m vs. 440 m). Six months after ABMSCT the ischemic ulcers disappeared in 2 patients, and in another 2 patients the large and deep ulcers became smaller and thinner (from 8 cm2 to 2.6 cm2 in one case, and from 30 cm2 to 8 cm2 in another case), and in 1 case no change was realized, where osteomyelitis of the affected toe was diagnosed. The average of ABI improved significantly on the treated lower limb (before: 0.41, 3 and 6 months after: 0.64/0.82). ABI values of the contra-lateral legs did not change. The clinical improvement started 1 months after ABMSCT, it became more prominent after 3 months, and the best clinical results were observed after 6 months of transplantation. Confirmed by post-trial observations obtained at 9 months the clinical improvement was evaluated as stable and long lasting. Using angiography we realized improvement in 3 cases, but only in 1 case using Color-Doppler scan. Before and 6/20 weeks after transplantation the TcPO2 changed on the foot from 18.80/16.78/23.83 mmHg, and on the calf from 36.66/31.25/45.00 mmHg. The laboratory parameters did not show improvement after 1 month, however, after 3 and 6 months improved parameters were recorded. No severe complications, adverse events were detected during the 6 months follow-up period. Conclusions. ABMSCT with isolated CD34+ cells was proved to be safe, effective and resulted in significant and sustained improvement of clinical parameters and quality of life for patients with SPAD. Multicentric, controlled clinical trials are required to confirm these preliminary clinical results.

To study the expression of endothelial and inducible nitric oxide synthases (eNOS and iNOS) and their role in inflammatory bowel disease (IBD).We examined the effect of sera obtained from patients with active Crohn's disease (CD) and ulcerative colitis (UC) on the function and viability of human umbilical vein endothelial cells (HUVEC). HUVECs were cultured for 0-48 h in the presence of a medium containing pooled serum of healthy controls, or serum from patients with active CD or UC. Expression of eNOS and iNOS was visualized by immunofluorescence, and quantified by the densitometry of Western blots. Proliferation activity was assessed by computerized image analyses of Ki-67 immunoreactive cells, and also tested in the presence of the NOS inhibitor, 10(-4) mol/L L-NAME. Apoptosis and necrosis was examined by the annexin-V-biotin method and by propidium iodide staining, respectively.In HUVEC immediately after exposure to UC, serum eNOS was markedly induced, reaching a peak at 12 h. In contrast, a decrease in eNOS was observed after incubation with CD sera and the eNOS level was minimal at 20 h compared to control (18%+/-16% vs 23%+/-15% P0.01). UC or CD serum caused a significant increase in iNOS compared to control (UC: 300%+/-21%; CD: 275%+/-27% vs 108%+/-14%, P0.01). Apoptosis/necrosis characteristics did not differ significantly in either experiment. Increased proliferation activity was detected in the presence of CD serum or after treatment with L-NAME. Cultures showed tube-like formations after 24 h treatment with CD serum.IBD sera evoked changes in the ratio of eNOS/iNOS, whereas did not influence the viability of HUVEC. These involved down-regulation of eNOS and up-regulation of iNOS simultaneously, leading to increased proliferation activity and possibly a reduced anti-inflammatory protection of endothelial cells.