[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: To provide data on plasma vascular endothelial growth factor (VEGF) concentration during three consecutive monthly intravitreal aflibercept injections and after transition to bimonthly treatment in patients with neovascular age-related macular degeneration (nAMD).
Sixteen consecutive treatment-naïve Caucasian patients with nAMD were included in the study. The treatment consisted of one intravitreal aflibercept (2 mg) injection every 28 days for three consecutive months followed by a fourth injection 8 weeks later. VEGF plasma concentrations were measured with Luminex on day 0 (baseline, prior to first injection); days 1, 6 and 27 (prior to second injection); day 55 (prior to third injection) and days 97 and 111 (after third injection).
Baseline plasma VEGF concentration was 59.6±13.3 pg/mL. Aflibercept decreased plasma VEGF concentration to 32.5±3 pg/mL on day 1 (p<0.0001) and 34.7±6.3 pg/mL on day 6 (p<0.0001). On day 27, the VEGF plasma level increased to 50.6±6.5 pg/mL (p=0.009) and on day 55 to 52.8±8.8 pg/mL (p=0.027). There was no statistically significant difference between mean plasma VEGF concentrations on days 27 and 55 (p=0.139). Plasma VEGF concentration recovered completely 6 weeks after the third injection, reaching 57.9±9.6 pg/mL on day 97 (p=0.600) and 59.5±11.6 pg/mL on day 111 (p=0.987).
Intravitreal aflibercept decreases plasma VEGF concentration mostly in the first week after treatment. Despite repeated monthly intravitreal injections, there was a monthly increase in plasma VEGF values to near baseline levels, with complete recovery 6 weeks after the third injection.
Identifier no. NCT02125864.
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No preview · Article · May 2015 · The British journal of ophthalmology
[Show abstract][Hide abstract] ABSTRACT: Autophagy and apoptosis function as important early cellular defense mechanisms in infections and other diseases. The outcome of an infection is determined by a complex interplay between the pathogenic microorganism and these intracellular pathways. To better understand the cytopathogenicity of Herpes simplex virus types 1 and 2 (HSV-1 and -2), we studied the effect of these viruses on the autophagic and apoptotic processes in the SIRC corneal cell line. Infection with the KOS strain of HSV-1 and a wild-type strain of HSV-2 enhanced autophagosome formation, triggered cytoplasmic acidification, increased LC3B lipidation and elevated the ratio of apoptotic cells. The autophagy inhibitor bafilomycin A1 triggered a significant increase in the apoptotic responses of HSV-1- and HSV-2-infected cells. Thus, both HSV types affect autophagy and apoptosis in a coordinated fashion, and autophagy plays cytoprotective role in HSV-infected cells via antagonizing apoptosis. Together these data implicate autophagy in the pathogenic mechanism of herpetic keratitis.
Full-text · Article · Jul 2014 · Journal of Biosciences
[Show abstract][Hide abstract] ABSTRACT: The surface of the human eye is covered by corneal epithelial cells (CECs) which regenerate from a small population of limbal epithelial stem cells (LESCs). Cell therapy with LESCs is a non-penetrating treatment for preventing blindness due to LESC deficiency or dysfunction. Our aim was to identify new putative molecular markers and upstream regulators in the LESCs and associated molecular pathways.
Genome-wide microarray transcriptional profiling was used to compare LESCs to differentiated human CECs. Ingenuity-based pathway analysis was applied to identify upstream regulators and pathways specific to LESCs. ELISA and flow cytometry were used to measure secreted and surface expressed proteins, respectively. More than 2 fold increase and decrease in expression could be found in 1830 genes between the two cell types. A number of molecules functioning in cellular movement (381), proliferation (567), development (552), death and survival (520), and cell-to-cell signaling (290) were detected having top biological functions in LESCs and several of these were confirmed by flow cytometric surface protein analysis. Custom-selected gene groups related to stemness, differentiation, cell adhesion, cytokines and growth factors as well as angiogenesis could be analyzed. The results show that LESCs play a key role not only in epithelial differentiation and tissue repair, but also in controlling angiogenesis and extracellular matrix integrity. Some pro-inflammatory cytokines were found to be important in stemness-, differentiation- and angiogenesis-related biological functions: IL-6 and IL-8 participated in most of these biological pathways as validated by their secretion from LESC cultures.
The gene and molecular pathways may provide a more specific understanding of the signaling molecules associated with LESCs, therefore, help better identify and use these cells in the treatment of ocular surface diseases.
[Show abstract][Hide abstract] ABSTRACT: Aim: Cultures for engineering of transplantable limbal epithelial grafts for treatment of ocular surface disorders may be initiated using dissociation of limbal epithelial cells by trypsin-EDTA or dispase or by a sequential incubation with these enzymes. The safety of such procedures is debated, and in the present study we examined levels of DNA damage in cells dissociated by a commonly used concentration of trypsin. Limbal samples subjected to the dissociation procedure were subsequently cultivated and monitored for outgrowth of cells.
Methods: Corneo-limbal rings were retrieved after transplant surgery, divided into samples measuring approx. 2x2 mm (n=32), and incubated in 0.05% trypsin-EDTA for one or three hours in either 250 μl or in 3 ml of the enzyme solution at 37 °C. DNA damage (strand breaks plus alkali-labile sites) was assessed using single cell gel electrophoresis (Comet assay) and evaluation of tail intensity (TI). Outgrowths from the cultivated samples were monitored by phase contrast microscopy and cells were subjected to Hoechst.
Results: Noticeable levels of DNA damage were seen regardless of incubation time and volume of enzyme solution. There was a trend towards increased levels of damage in cells when using 3 ml compared to values recorded in cells dissociated in 250 μl of the enzyme solution. Outgrowth of cells was observed from all of the 32 cultivated samples.
Conclusion: Dissociation of human limbal epithelial cells by a commonly used concentration of trypsin-EDTA may induce evident DNA damage in the cell population destined for graft production. The current methods for cell dissociation should be examined more closely for induction of damage to essential molecular constituents of the cells including to the stem cell population. Procedural steps and components of the ex vivo system that may reduce such damage and/or facilitate repair should be identified.
[Show abstract][Hide abstract] ABSTRACT: Recent reports of retinal stem cells being present in several locations of the adult eye have sparked great hopes that they may be used to treat the millions of people worldwide who suffer from blindness as a result of retinal disease or injury. A population of proliferative cells derived from the ciliary body epithelium (CE) has been considered one of the prime stem cell candidates, and as such they have received much attention in recent years. However, the true nature of these cells in the adult human eye has still not been fully elucidated, and the stem cell claim has become increasingly controversial in light of new and conflicting reports. In this paper, we will try to answer the question of whether the available evidence is strong enough for the research community to conclude that the adult human CE indeed harbors stem cells.
[Show abstract][Hide abstract] ABSTRACT: 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<0.001). Conclusion Our data show clear distinction between the studied stem cells based upon their gene expression patterns and strengthen the hypothesis that CSMSCs are derived from bmMSCs and not from LESCs.
No preview · Article · Aug 2013 · Acta ophthalmologica
[Show abstract][Hide abstract] ABSTRACT: The growth and recurrence of several cancers appear to be driven by a population of cancer stem cells (CSCs). Glioblastoma, the most common primary brain tumor, is invariably fatal, with a median survival of approximately 1 year. Although experimental data have suggested the importance of CSCs, few data exist regarding the potential relevance and importance of these cells in a clinical setting.
We here present the first seven patients treated with a dendritic cell (DC)-based vaccine targeting CSCs in a solid tumor. Brain tumor biopsies were dissociated into single-cell suspensions, and autologous CSCs were expanded in vitro as tumorspheres. From these, CSC-mRNA was amplified and transfected into monocyte-derived autologous DCs. The DCs were aliquoted to 9-18 vaccines containing 10(7) cells each. These vaccines were injected intradermally at specified intervals after the patients had received a standard 6-week course of post-operative radio-chemotherapy. The study was registered with the ClinicalTrials.gov identifier NCT00846456.
Autologous CSC cultures were established from ten out of eleven tumors. High-quality RNA was isolated, and mRNA was amplified in all cases. Seven patients were able to be weaned from corticosteroids to receive DC immunotherapy. An immune response induced by vaccination was identified in all seven patients. No patients developed adverse autoimmune events or other side effects. Compared to matched controls, progression-free survival was 2.9 times longer in vaccinated patients (median 694 vs. 236 days, p = 0.0018, log-rank test).
These findings suggest that vaccination against glioblastoma stem cells is safe, well-tolerated, and may prolong progression-free survival.
Full-text · Article · Jul 2013 · Cancer Immunology and Immunotherapy
[Show abstract][Hide abstract] ABSTRACT: Purpose:
To investigate the use of ultra-widefield fundus autofluorescence imaging in the early postoperative evaluation of scleral buckling surgery for retinal detachment.
Forty-five eyes from 44 patients with rhegmatogenous retinal detachment were included. Ultra-widefield fundus autofluorescence imaging (Optomap P200Tx) was performed from both eyes preoperatively and early (1-2 days) postoperatively. All patients were operated with 2.5-mm encircling band, 6-mm to 9-mm segmental buckle, transscleral cryopexy, and the choice of drainage and air/gas endotamponade.
The mean age of the patients was 58 ± 12 years, and the ratio of macula on-off detachments was 19/26. Light cryopexy induced hyperfluorescence of the treated area (in 11% of cases). Moderate cryopexy resulted in central hypofluorescence with a hyperfluorescent halo (in 51% of cases), whereas extensive cryopexy and disruption of the retinal pigment epithelium resulted in a broad hypofluorescent area (in 36% of cases). Tightening of the indenting elements induced peripheral hyperfluorescent radial streaks in 47% of cases and distinct areas of hyperfluorescence in 58% of cases. Demarcation lines and residual subretinal fluid were observed as hyperfluorescent areas. Central autofluorescence changes were observed in 96% of macula-off surgeries, whereas only 27% of these cases showed distinct hyper- and hypo-autofluorescent streaks.
Ultra-widefield fundus autofluorescence imaging is a useful adjuvant tool for evaluating early outcome and retinal pigment epithelium function after scleral buckling surgery for retinal detachment.
No preview · Article · Mar 2013 · Retina (Philadelphia, Pa.)
[Show abstract][Hide abstract] ABSTRACT: Purpose:
DNA damage has been described in the human cataractous lens epithelium, and oxidative stress generated by UV radiation and endogenous metabolic processes has been suggested to play a significant role in the pathogenesis of cataract. In this study, the aim was to explore the quality and relative quantity of DNA damage in lens epithelium of cataract patients in vivo and after incubation in a cell culture system.
Capsulotomy specimens were analysed, before and after 1 week of ex vivo cultivation, using the comet assay to measure DNA strand breaks, oxidized purine and pyrimidine bases and UV-induced cyclobutane pyrimidine dimers.
DNA strand breaks were barely detectable, oxidized pyrimidines and pyrimidine dimers were present at low levels, whereas there was a relatively high level of oxidized purines, which further increased after cultivation.
The observed levels of oxidized purines in cataractous lens epithelium may support a theory consistent with light damage and oxidative stress as mediators of molecular damage to the human lens epithelium. Damage commonly associated with UV-B irradiation was relatively low. The levels of oxidized purines increased further in a commonly used culture system. This is of interest considering the importance and versatility of ex vivo systems in studies exploring the pathogenesis of cataract.
No preview · Article · Sep 2012 · Acta ophthalmologica
[Show abstract][Hide abstract] ABSTRACT: A simple, reproducible, animal-material free method for cultivating and characterizing cornea limbal epithelial stem cells (LESCs) on human lens capsule (LC) was developed for future clinical transplantation. The limbal tissue explants (2×2×0.25 mm) were harvested from 77 cadavers and expanded ex vivo on either cell culture plates or LC in medium containing human serum as the only growth supplement. Cell outgrowth at the edge of the explants was observed within 24 hours of cultivation and achieved viable outgrowth (>97% viability as measured by MTT assay and flow cytometry) within two weeks. The outgrowing cells were examined by genome-wide microarray including markers of stemness (p63α, ABCG2, CK19, Vimentin and Integrin α9), proliferation (Ki-67), limbal epithelial cells (CK 8/18 and 14) and differentiated cornea epithelial cells (CK 3 and 12). Immunostaining revealed the non-hematopoietic, -endothelial and -mesenchymal stem cell phenotype of the LESCs and the localization of specific markers in situ. Cell adhesion molecules, integrins and lectin-based surface carbohydrate profiling showed a specific pattern on these cells, while colony-formation assay confirmed their clonal potency. The LESCs expressed a specific surface marker fingerprint (CD117/c-kit, CXCR4, CD144/VE-Cadherin, CD146/MCAM, CD166/ALCAM, and surface carbohydrates: WGA, ConA, RCA, PNA and AIL) which can be used for better localization of the limbal stem cell niche. In summary, we report a novel method combining the use of a medium with human serum as the only growth supplement with LC for cultivating, characterizing and expanding cornea LESCs from cadavers or alternatively from autologous donors for possible treatment of LESC deficiency.
[Show abstract][Hide abstract] ABSTRACT: Purpose:
Presently, our clinic is the only centre in Scandinavia that offers patients with corneal surface pathology including limbal stem cell deficiency (LSCD) transplantation of ex vivo expanded limbal epithelial cells (LECs). We here present clinical data of the first nine patients with LSCD who were transplanted with autologous LECs expanded in medium completely free of any animal-derived products and non-human/recombinant growth factors (including Cholera Toxin), and with autologous human serum as the only growth supplement.
We conducted a noncomparative retrospective study of patients with LSCD at our centre between 2009 and 2011. The diagnosis was based on history and clinical signs. A biopsy was taken from healthy limbus, and the epithelium was expanded on amniotic membrane (AM) in medium containing autologous serum and subsequently transplanted to the affected eye.
Successful outcome was defined as relief of pain and photophobia and/or improved best corrected visual acuity (BCVA) and/or reestablishment of a stable corneal epithelium and regression of corneal vascularization. Five of the nine transplanted patients (55.6%) had an improvement in either subjective symptoms or objective findings (11- to 28-month follow-up).
Our clinical study shows that patients with LSCD can be treated successfully with transplantation of LECs expanded ex vivo in a medium with autologous serum as the only growth supplement. The use of this novel culture system, which is devoid of animal-derived products and non-human/recombinant growth factors (including Cholera Toxin), reduces the risks of inter-species disease transmission and host immune responses to xenogenic proteins, both obvious advantages for the patient.
No preview · Article · Aug 2012 · Acta ophthalmologica
[Show abstract][Hide abstract] ABSTRACT: Lipid peroxidation content was measured in an organ culture medium after one-week storage of human donor corneas. Moreover, the effects of the medium on oxidative stress, antioxidant capacity, and the proliferation of cultured human corneal cells were studied.
The medium was sampled from the upper and lower halves of storage vials and from controls (n=42). Malondialdehyde (MDA) was measured by high pressure liquid chromatography (HPLC). Cultured human corneal epithelium (CRL-11515) was exposed to different medium samples and monitored for changes in MDA (enzyme-linked immunosorbent assay [ELISA]), total antioxidant capacity (antioxidant assay kit), and proliferation (Ki-67).
A significant increase in MDA was observed in the organ culture medium in the lower level of storage vials. The addition of this fraction to cultured cells increased MDA significantly after 3 days, and the medium from both levels significantly increased MDA after 7 days. The medium from both levels significantly decreased the total antioxidant capacity of the cells but did not affect proliferative activity.
An oxidative gradient with an evident biologic effect is established in the medium in vials during organ culture of human donor corneas. Donor tissue stored at the bottom or in lower levels of such vials is exposed to a significant amount of oxidative stress.
[Show abstract][Hide abstract] ABSTRACT: Opticin, a small leucine rich repeat protein (SLRP) contributes to vitreoretinal adhesion. This study was conducted to investigate the effects of hypoxia and vascular endothelial growth factor (VEGF) on matrix metalloproteinase (MMP) mediated opticin production in retinal pigment epithelium (RPE) cells. Primary cultured human RPE cells were treated with hypoxia (low oxygen and cobalt chloride) or VEGF (0-100 ng/mL). The mRNA levels of opticin and the protein levels of intra and extracellular opticin in RPE cells were examined by RT-PCR and Western blot assay, respectively. Furthermore, the MMP activity was analyzed by zymography, and EDTA was used as an MMP inhibitor. Analysis of the effect of MMP-2 on opticin was performed by recombinant human (rh) MMP-2 stimulation in RPE cultures and by human vitreous sample digestion with activated rhMMP-2. Our results showed that opticin was expressed by primary cultured human RPE cells. Hypoxia and VEGF stimulation did not alter opticin mRNA and protein expression in RPE cells, but markedly decreased the protein levels of extracellular opticin following increased latent MMP-2 activity. The VEGF- and hypoxia induced opticin degradation in the culture medium was blocked by EDTA. Together, opticin levels in the culture medium were also reduced after rhMMP-2 treatment. In addition, opticin in human vitreous samples could be cleaved by rhMMP-2. These results reveal that VEGF and hypoxia could decrease opticin protein levels in the human RPE secretome, and that opticin may be an enzymatic substrate for MMP-2.