[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: 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.
Cancer Immunology and Immunotherapy 07/2013; · 3.64 Impact Factor
[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. METHODS:: 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. RESULTS:: 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. CONCLUSION:: 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.
[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.
BioMed research international. 01/2013; 2013:531579.
[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. Methods: 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. Results: 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. Conclusion: 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.
[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. Methods: 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. Results: 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). Conclusions: 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.
[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.
[show abstract][hide abstract] ABSTRACT: A case of perforating ocular injury with a retrobulbar foreign body and a large full-thickness posterior pole defect near the optic disc was scheduled for vitrectomy after primary corneal suturing. Because it was difficult to remove the retrobulbar foreign body by orbitotomy and perform the outside suture, the retrobulbar foreign body was removed through the posterior hole by a transocular approach, and an autologous Tenon capsule flap was used to internally patch the large full-thickness posterior pole defect, thus enabling silicon tamponade. After 3 months of follow-up, there was no immune response around the patch. The retina remained mostly attached with a maintained peripheral visual field, normal intraocular pressure, and good cosmetic appearance. This surgical technique may be valuable in patients with a perforating retrobulbar foreign body and a large full-thickness posterior pole defect.
Archives of ophthalmology 04/2012; 130(4):493-6. · 3.86 Impact Factor
[show abstract][hide abstract] ABSTRACT: In addition to the ability for self-renewal and functional differentiation, neural stem/progenitor cells (NSCs) can respond to CNS injuries by targeted migration. In lower vertebrates, retinal injury is known to activate NSCs in the ciliary marginal zone (CMZ). Cells expressing markers of NSCs are also present in the ciliary body epithelium (CE) and in Müller glia in the peripheral retina (PR) of the adult human eye. However, these cells seem to be quiescent in the adult human eye and recent reports have shown that CE cells have limited properties of NSCs. In order to further clarify whether NSCs exist in the adult human eye, we tested whether NSC-like cells could be activated in eyes with proliferative vitreoretinopathy (PVR). The PR and CE were studied for NSC-associated markers in human enucleated control eyes and eyes with confirmed PVR, as well as in a mouse model of PVR. Furthermore, cells isolated from vitreous samples obtained during vitrectomies for retinal detachment were directly fixed or cultured in a stem cell-promoting medium and compared to cells cultured from the post-mortem retina and CE. In situ characterization of the normal eyes revealed robust expression of markers present in NSCs (Nestin, Sox2, Pax6) only around peripheral cysts of the proximal pars plana region and the PR, the latter population also staining for the glial marker GFAP. Although there were higher numbers of dividing cells in the CE of PVR eyes than in controls, we did not detect NSC-associated markers in the CE except around the proximal pars plana cysts. In the mice PVR eyes, Nestin activation was also found in the CE. In human PVR eyes, proliferation of both non-glial and glial cells co-staining NSC-associated markers was evident around the ora serrata region. Spheres formed in 7/10 vitreous samples from patients with PVR compared to 2/15 samples from patients with no known PVR, and expressed glial - and NSC-associated markers both after direct fixation and repetitive passages. In conclusion, the adult human eye may harbor two different populations of neuroepithelial stem/progenitor cells; a non-glial population located in the proximal pars plana around peripheral cysts in addition to a population with Müller glia characteristics. Yet, we only found that the glial population was able to respond to retinal injury by targeted migration into the vitreous.
Experimental Eye Research 03/2012; 98:28-36. · 3.03 Impact Factor
[show abstract][hide abstract] ABSTRACT: Purpose: Storage time for donor corneas in Optisol GS is limited compared to Eye Bank Organ Culture (EBOC). We here examine the epithelium on donor corneoscleral rims after primary storage in Optisol GS and subsequent incubation in EBOC. Methods: Morphology was monitored by light and electron microscopy, expression of phenotypic and genotypic markers by immunohistochemistry and RT-PCR and changes in oxidative lipid and DNA damage by ELISA and COMET assay. Results: A prominent loss of cells was observed after storage in Optisol GS. After maintenance in EBOC, spreading apical cells were Occludin(+) , while the staining for E-cadherin and Connexin-43 was less intense. There were an upregulation of Occludin and a downregulation of E-cadherin and Connexin-43. Eye Bank Organ Culture was associated with an ongoing proliferative activity and a downregulation of putative progenitor/stem cell marker ABCG2 and p63. Staining for 8-OHdG and Caspase-3 did not increase, while levels of malondialdehyde and number of DNA strand breaks and oxidized bases increased. Conclusions: This dual procedure should be pursued as an option to increase the storage time and the pool of available donor corneas. The observed downregulation of markers associated with stemness during EBOC is relevant considering the potential use of donor epithelium in the treatment of ocular surface disorders.
[show abstract][hide abstract] ABSTRACT: In patients with limbal stem cell deficiency (LSCD), transplantation of ex vivo expanded human limbal epithelial cells (HLECs) can restore the structural and functional integrity of the corneal surface. However, the protocol for cultivation and transplantation of HLECs differ significantly, and in most protocols growth additives such as cholera toxins, exogenous growth factors, hormones and fetal calf serum are used. In the present article, we compare for the first time human limbal epithelial cells (HLECs) cultivated on human amniotic membrane (HAM) in a complex medium (COM) including fetal bovine serum to a medium with human serum as single growth supplement (HSM), and report on our first examinations of HLECs expanded in autologous HSM and used for transplant procedures in patients with LSCD. Expanded HLECs were examined by genome-wide microarray, RT-PCR, Western blotting, and for cell viability, morphology, expression of immunohistochemical markers and colony forming efficiency. Cultivation of HLECs in HSM produced a multilayered epithelium where cells with markers associated with LESCs were detected in the basal layers. There were few transcriptional differences and comparable cell viability between cells cultivated in HSM and COM. The p63 gene associated with LESCs were expressed 3.5 fold more in HSM compared to COM, and Western blotting confirmed a stronger p63α band in HSM cultures. The cornea-specific keratin CK12 was equally found in both culture conditions, while there were significantly more CK3 positive cells in HSM. Cells in epithelial sheets on HAM remaining after transplant surgery of patients with LSCD expressed central epithelial characteristics, and dissociated cells cultured at low density on growth-arrested fibroblasts produced clones containing 21 ± 12% cells positive for p63α (n = 3). In conclusion, a culture medium without growth additives derived from animals or from animal cell cultures and with human serum as single growth supplement may serve as an equivalent replacement for the commonly used complex medium for ex vivo expansion of HLECs on HAM.
Experimental Eye Research 02/2012; 97(1):1-9. · 3.03 Impact Factor
[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: 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.
PLoS ONE 01/2012; 7(10):e47187. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Age-related macular degeneration (AMD) is attributed to a complex interaction of genetic and environmental factors. It is characterized by degeneration involving the retinal photoreceptors, retinal pigment epithelium (RPE) and Bruch's membrane, as well as alterations in choroidal capillaries. AMD pathogenesis is strongly associated with chronic oxidative stress and inflammation that ultimately lead to protein damage, aggregation and degeneration of RPE. Specific degenerative findings for AMD are accumulation of intracellular lysosomal lipofuscin and extracellular drusens. In this review, we discuss thoroughly RPE-derived mechanisms in AMD pathology.
[show abstract][hide abstract] ABSTRACT: The identification of cells with properties of retinal progenitor cells (RPCs) in the adult human ciliary margin (CM) prompted a number of studies of their proliferative and differentiation potential. One of the remaining challenges is to find a feasible method of isolating RPCs from the patient's eye. In the human CM, only the iris pigment epithelium (IPE) is easily obtained by a minimally invasive procedure. In the light of recent studies questioning the existence of RPCs in the adult mammalian CM, we wanted to assess the potential of the adult human IPE as source of RPCs.
The IPE were isolated from peripheral iridectomies during glaucoma surgery, and IPE and ciliary body (CB) epithelium were also isolated from post-mortem tissue. Cells were cultivated in sphere-promoting conditions or as monolayers. Whole-tissue samples, undifferentiated and differentiated cells were studied by immunocytochemistry, RT-PCR and transmission electron microscopy.
The adult human IPE, like the CB, expressed markers of RPCs such as Pax6, Sox2 and Nestin in vivo. Both sphere-promoting and monolayer cultures preserved this phenotype. However, both IPE/CB cultures expressed markers of differentiated epithelial cells such as Claudin, microphtalmia-associated transcription factor (MITF) and Cytokeratin-19. Ultrastructurally, IPE spheres displayed epithelial-like junctions and contained mature melanosomes. After induced differentiation, IPE-derived cells showed only partial neuronal differentiation expressing β-III-tubulin, Map-2 and Rhodopsin, whereas no mature glial markers were found.
Proliferative cells with some properties of RPCs can be isolated from the adult human IPE by peripheral iridectomies. Yet, many cells retain properties of differentiated epithelial cells and lack central properties of somatic stem cells.
[show abstract][hide abstract] ABSTRACT: To explore the potential role of vascular endothelial growth factor compared with transforming growth factor-β2 in the regulation of human retinal pigment epithelium cell-mediated collagen gel contraction.
The retinal pigment epithelium cell mediated type I collagen gel contraction assay was performed to evaluate and compare the effect of vascular endothelial growth factor and transforming growth factor-β2. The number of viable retinal pigment epithelium cells in the gel and the expression of α-smooth muscle actin were analysed.
Both vascular endothelial growth factor and transforming growth factor-β2 caused a time dependent gel contraction, associated with over expression of α-smooth muscle actin in retinal pigment epithelium cells undergoing a fibroblast like transformation. The decrease in volume of the collagen gel and increase in α-smooth muscle actin expression were more significant in the transforming growth factor-β2-treated group than in vascular endothelial growth factor-treated group beginning at day 2, and the growth of retinal pigment epithelium cells was significantly more inhibited in the transforming growth factor-β2-treated group compared with the vascular endothelial growth factor-treated group after day 1 (P < 0.05). Transforming growth factor-β2 stimulation increased both vascular endothelial growth factor mRNA expression and secretion. The α-smooth muscle actin expression and the change in volume of collagen gel were significantly positively correlated in both experimental groups.
Both vascular endothelial growth factor and transforming growth factor-β2 can cause induction of retinal pigment epithelium cell-mediated collagen gel contraction in vitro via partial upregulation of α-smooth muscle actin expression.
Clinical and Experimental Ophthalmology 06/2011; 40(1):e76-86. · 1.96 Impact Factor