RESUMEN
AIMS/HYPOTHESIS: Diabetes is associated with epigenetic modifications including DNA methylation and miRNA changes. Diabetic complications in the cornea can cause persistent epithelial defects and impaired wound healing due to limbal epithelial stem cell (LESC) dysfunction. In this study, we aimed to uncover epigenetic alterations in diabetic vs non-diabetic human limbal epithelial cells (LEC) enriched in LESC and identify new diabetic markers that can be targeted for therapy to normalise corneal epithelial wound healing and stem cell expression. METHODS: Human LEC were isolated, or organ-cultured corneas were obtained, from autopsy eyes from non-diabetic (59.87±20.89 years) and diabetic (71.93±9.29 years) donors. The groups were not statistically different in age. DNA was extracted from LEC for methylation analysis using Illumina Infinium 850K MethylationEPIC BeadChip and protein was extracted for Wnt phospho array analysis. Wound healing was studied using a scratch assay in LEC or 1-heptanol wounds in organ-cultured corneas. Organ-cultured corneas and LEC were transfected with WNT5A siRNA, miR-203a mimic or miR-203a inhibitor or were treated with recombinant Wnt-5a (200 ng/ml), DNA methylation inhibitor zebularine (1-20 µmol/l) or biodegradable nanobioconjugates (NBCs) based on polymalic acid scaffold containing antisense oligonucleotide (AON) to miR-203a or a control scrambled AON (15-20 µmol/l). RESULTS: There was significant differential DNA methylation between diabetic and non-diabetic LEC. WNT5A promoter was hypermethylated in diabetic LEC accompanied with markedly decreased Wnt-5a protein. Treatment of diabetic LEC and organ-cultured corneas with exogenous Wnt-5a accelerated wound healing by 1.4-fold (p<0.05) and 37% (p<0.05), respectively, and increased LESC and diabetic marker expression. Wnt-5a treatment in diabetic LEC increased the phosphorylation of members of the Ca2+-dependent non-canonical pathway (phospholipase Cγ1 and protein kinase Cß; by 1.15-fold [p<0.05] and 1.36-fold [p<0.05], respectively). In diabetic LEC, zebularine treatment increased the levels of Wnt-5a by 1.37-fold (p<0.01)and stimulated wound healing in a dose-dependent manner with a 1.6-fold (p<0.01) increase by 24 h. Moreover, zebularine also improved wound healing by 30% (p<0.01) in diabetic organ-cultured corneas and increased LESC and diabetic marker expression. Transfection of these cells with WNT5A siRNA abrogated wound healing stimulation by zebularine, suggesting that its effect was primarily due to inhibition of WNT5A hypermethylation. Treatment of diabetic LEC and organ-cultured corneas with NBC enhanced wound healing by 1.4-fold (p<0.01) and 23.3% (p<0.05), respectively, with increased expression of LESC and diabetic markers. CONCLUSIONS/INTERPRETATION: We provide the first account of epigenetic changes in diabetic corneas including dual inhibition of WNT5A by DNA methylation and miRNA action. Overall, Wnt-5a is a new corneal epithelial wound healing stimulator that can be targeted to improve wound healing and stem cells in the diabetic cornea. DATA AVAILABILITY: The DNA methylation dataset is available from the public GEO repository under accession no. GSE229328 ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE229328 ).
Asunto(s)
Diabetes Mellitus , MicroARNs , Humanos , Represión Epigenética , Proteína Wnt-5a/genética , Proteína Wnt-5a/metabolismo , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Células Madre/metabolismo , ARN Interferente Pequeño/metabolismo , Cicatrización de Heridas/genética , Células Epiteliales/metabolismoRESUMEN
Human diabetic corneas develop delayed wound healing, epithelial stem cell dysfunction, recurrent erosions, and keratitis. Adenoviral gene therapy modulating c-Met, cathepsin F and MMP-10 normalized wound healing and epithelial stem cells in organ-cultured diabetic corneas but showed toxicity in stem cell-enriched cultured limbal epithelial cells (LECs). For a safer treatment, we engineered a novel nanobiopolymer (NBC) that carried antisense oligonucleotide (AON) RNA therapeutics suppressing cathepsin F or MMP-10, and miR-409-3p that inhibits c-Met. NBC was internalized by LECs through transferrin receptor (TfR)-mediated endocytosis, inhibited cathepsin F or MMP-10 and upregulated c-Met. Non-toxic NBC modulating c-Met and cathepsin F accelerated wound healing in diabetic LECs and organ-cultured corneas vs. control NBC. NBC treatment normalized levels of stem cell markers (keratins 15 and 17, ABCG2, and ΔNp63), and signaling mediators (p-EGFR, p-Akt and p-p38). Non-toxic nano RNA therapeutics thus present a safe alternative to viral gene therapy for normalizing diabetic corneal cells.
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Córnea/patología , Diabetes Mellitus/patología , Células Epiteliales/patología , Nanopartículas/química , Polímeros/química , ARN/uso terapéutico , Células Madre/patología , Cicatrización de Heridas , Adenoviridae/fisiología , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/metabolismo , Supervivencia Celular , Células Cultivadas , Córnea/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/virología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Nanopartículas/ultraestructura , Oligonucleótidos Antisentido/farmacología , ARN/farmacología , Receptores de Superficie Celular/metabolismo , Transducción de Señal/efectos de los fármacos , Células Madre/efectos de los fármacos , Cicatrización de Heridas/efectos de los fármacosRESUMEN
PURPOSE: To examine the expression of putative limbal epithelial stem cell (LESC) markers and wound healing rates in primary healthy and diabetic human limbal epithelial cells (LECs) cultured on different substrata. METHODS: Primary limbal epithelial cells were isolated from human autopsy corneas and discarded corneoscleral rims with dispase II treatment. LECs were cultured in EpiLife medium on human amniotic membrane (AM) denuded with mild alkali treatment, on plastic dishes and on glass slides coated with a mixture of human fibronectin, collagen type IV, and laminin (FCL). Cultured LECs were fixed in p-formaldehyde or methanol, and the expression of the putative LESC markers ΔNp63α, PAX6, and ABCG2 and keratins K12, K15, and K17 was examined with immunostaining. Wound healing was evaluated in scratch wound assay in LECs cultured on FCL-coated plates 20 h after wounding. RESULTS: LECs cultured on denuded AM expressed ΔNp63α, PAX6 (both showed nuclear staining), K15, K17 (cytoskeleton staining), and ABCG2 (cytoplasmic and/or plasma membrane staining). LECs cultured on FCL-coated slides also expressed these markers, whereas no expression was detected for differentiated corneal epithelial cell marker K12. Decreased expression of LESC markers was observed in diabetic LECs compared to healthy LECs cultured on the FCL-coated slides. This reduction was most prominent for K15 and K17. Diabetic LECs were found to heal scratch wounds slower than healthy cells in accordance with previous results in corneal organ cultures. CONCLUSIONS: Healthy human LECs cultured either on AM or FCL-coated slides preserved LESC marker expression. The observed reduction in LESC marker expression and slower wound healing in cultured diabetic LECs are in line with our earlier reports and may account for diabetic LESC dysfunction and clinically observed impaired corneal epithelial wound healing.
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Complicaciones de la Diabetes/metabolismo , Limbo de la Córnea/metabolismo , Células Madre/metabolismo , Cicatrización de Heridas/fisiología , Anciano , Anciano de 80 o más Años , Biomarcadores/metabolismo , Células Cultivadas , Enfermedades de la Córnea/etiología , Enfermedades de la Córnea/metabolismo , Enfermedades de la Córnea/patología , Medios de Cultivo , Complicaciones de la Diabetes/patología , Células Epiteliales/metabolismo , Células Epiteliales/patología , Epitelio Corneal/metabolismo , Epitelio Corneal/patología , Femenino , Humanos , Limbo de la Córnea/patología , Masculino , Persona de Mediana Edad , Células Madre/patologíaRESUMEN
Wide availability and uptake of contact lenses came with the development of the first polymethyl methacrylate corneal lenses during the late 1940s. It is less well known that colored contact lenses were developed simultaneously. These innovations allowed both a degree of spectacle independence for ametropes and an ability to vary eye color. The impact on facial and ocular cosmesis was substantial, particularly for public figures such as actors. We have obtained contact lenses and matching casts manufactured by their inventor, Kevin M. Tuohy. Measurements of these suggest they were made for a myope, and we provide indirect evidence that the lenses were made for Marilyn Monroe. We also provide evidence that Monroe is likely to have been myopic, used colored contact lenses to change her eye color and may have been an early sufferer of contact lens overwear syndrome. The importance of ocular cosmesis can be overlooked in ophthalmic practice, yet it is of great interest to and importance for patients. It appears that discomfort, and even risk, will be tolerated to achieve a particular appearance. This is reflected in current techniques aimed at achieving improved cosmesis.
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Lentes de Contacto Hidrofílicos , Lentes de Contacto , Miopía , Humanos , Femenino , Miopía/terapia , CórneaRESUMEN
PURPOSE: To assess the relative impact of antibodies specific for HSV-1 glycoproteins on eye disease in response to HSV-1 infection, the composition of antibodies specific for 10 of the viral glycoproteins, and the effect of anti-glycoprotein (g)D and anti-gK antibodies on antibody-dependent enhancement (ADE). METHODS: In a prospective case-control study, sera from patients with a history of herpes stromal keratitis (HSK) were compared with sera from nonocular HSV-1-seropositive and HSV-seronegative control subjects. HSV-1 neutralizing antibody titer and type-specific IgG and IgM were measured. In addition, the presence of anti-HSV-1 gD and gK antibodies in the sera of all patients also was determined by ELISA using gD and gK antigens. Finally, the role of anti-gD- and gK-specific antibodies to ADE was investigated. RESULTS: Average neutralizing antibody titers and levels of HSV-1 IgG were similar between HSK- and non-HSK-seropositive patients. However, the contribution of gD to the neutralizing antibody titer in HSK sera was significantly lower than that in non-HSK-seropositive patients, despite higher anti-gD ELISA titers. Overall, sera from patients with HSK had higher anti-gK antibody titers and induced ADE in vitro compared with non-HSK or seronegative sera. The ADE response in HSK sera was attributed to anti-gK antibody. CONCLUSIONS: These results suggest that sera from HSK patients had higher anti-gD and -gK antibody titers than sera from seropositive patients who had no history of HSK despite similar levels of neutralizing antibody titers and HSV-1 IgG, that HSK sera induced ADE whereas sera from non-HSK patients did not induce ADE, and that anti-gD antibody in sera of HSK patients contributed less to the HSV-1 neutralization antibody titer than did sera from non-HSK patients.
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Anticuerpos Antivirales/sangre , Herpesvirus Humano 1/inmunología , Inmunoglobulina G/sangre , Queratitis Herpética/inmunología , Proteínas del Envoltorio Viral/inmunología , Proteínas Virales/inmunología , Adolescente , Adulto , Animales , Estudios de Casos y Controles , Sustancia Propia/virología , Ensayo de Inmunoadsorción Enzimática , Femenino , Herpesvirus Humano 2/inmunología , Humanos , Inmunoglobulina M/sangre , Queratitis Herpética/virología , Masculino , Estudios Prospectivos , ConejosRESUMEN
PURPOSE: To evaluate the thickness and diameter accuracy of the IntraLase femtosecond laser in harvesting corneal discs for lamellar keratoplasty. The stromal bed quality of resected corneas was evaluated by scanning electron microscopy. METHODS: Two IntraLase units were used to create corneal discs at 225-, 300-, 400-, and 500-microm thickness settings and 7-, 8-, 8.5-, and 9-mm diameter settings (n = 28). Achieved thickness was measured using a digital caliper and achieved diameter was measured using imaging software. Samples were preserved and processed for scanning electron microscopy to observe stromal bed quality. RESULTS: On both units, the mean deviation from attempted thickness was -9.5 +/- 8.6 microm (range: +6 to -28 microm). All of the obtained corneal discs were circular (horizontal versus vertical diameters, P > .05). The raster pattern produced a smoother stromal bed compared to the spiral pattern. CONCLUSIONS: The IntraLase femtosecond laser keratome provides depth and diameter accuracy even at increased depth settings. Stromal bed was smooth with the raster pattern and can further be smoothed by excimer phototherapeutic keratectomy treatment. Femtosecond laser lamellar keratoplasty is a promising procedure and clinical data are needed to determine effectiveness.
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Córnea/citología , Trasplante de Córnea/métodos , Colgajos Quirúrgicos/normas , Córnea/cirugía , Sustancia Propia/ultraestructura , Trasplante de Córnea/instrumentación , Diseño de Equipo , Humanos , Microscopía Electrónica de Rastreo , Donantes de TejidosRESUMEN
PURPOSE: To describe what is, to our knowledge, the first documented case of vancomycin-resistant enterococcus (VRE) conjunctivitis and its successful topical treatment. METHODS: A 77-year-old white man with end-stage multiple myeloma was hospitalized for congestive heart failure and pneumonia. During hospitalization, the patient developed conjunctivitis. Cultures of the eye were directly plated into several media. The bacterium was tested for antibiotic minimum inhibitory concentration (MIC) with the Clinical and Laboratory Standards Institute (CLSI) method. RESULTS: Culture of the affected eye grew Enterococcus faecalis resistant to vancomycin. Topical treatment with moxifloxacin 0.5% (Vigamox; Alcon, Ft. Worth, TX) resulted in clinical resolution despite a MIC showing resistance. CONCLUSION: Clinical resolution of VRE conjunctivitis was shown with topical moxifloxacin therapy in this case. At the same time, we suggest the use of combined topical and systemic therapy for treatment of VRE in immunocompromised patients.
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Antituberculosos/administración & dosificación , Compuestos Aza/administración & dosificación , Conjuntivitis Bacteriana/tratamiento farmacológico , Enterococcus faecalis/aislamiento & purificación , Infecciones por Bacterias Grampositivas/tratamiento farmacológico , Quinolinas/administración & dosificación , Resistencia a la Vancomicina , Anciano , Antituberculosos/uso terapéutico , Compuestos Aza/uso terapéutico , Conjuntivitis Bacteriana/microbiología , Enterococcus faecalis/efectos de los fármacos , Fluoroquinolonas , Estudios de Seguimiento , Infecciones por Bacterias Grampositivas/microbiología , Humanos , Masculino , Moxifloxacino , Soluciones Oftálmicas , Quinolinas/uso terapéuticoRESUMEN
PURPOSE: To examine immunohistochemically 2 human corneal buttons after corneal transplantation for post-laser in situ keratomileusis (LASIK) keratectasia. METHODS: Two ectatic corneas after penetrating keratoplasty and 2 postmortem control corneas from a patient after uncomplicated LASIK were used. Cryostat sections were stained by immunofluorescence for >30 extracellular matrix (ECM) components and proteinases. RESULTS: The ratios of distance between LASIK flap interface and the upper epithelial layer to total corneal thickness were 0.27-0.34 in all cases. The whole flap interface was positive only for total and cellular fibronectin. Stromal types VI and XIV collagen, fibrillin-1, tenascin-C, and vitronectin were unchanged with no evidence of fibrosis. In ectasia cases, keratocytes adjacent to the flap did not express nidogens. Staining for type IV collagen alpha5 chain, nidogen-2, chains of laminin-8, and laminin-10 was weak and discontinuous in the epithelial basement membrane (EBM). Type IV collagen alpha1/alpha2 chains were found in the EBM of ectasia cases only. Matrix metalloproteinase (MMP)-10 showed increase in the epithelium, and MMP-3, in some keratocytes near the flap interface of ectatic corneas. Also, cathepsin F was seen at the flap margin only. Staining for limbal basal epithelial marker, alpha-enolase, was mostly absent in the ectatic cases, suggesting largely normal epithelial differentiation. CONCLUSIONS: Abnormal EBM structure similar to that previously observed in keratoconus and bullous keratopathy and an increase in certain proteinases suggest ongoing EBM lysis and remodeling. Immunohistochemical staining for fibronectin may be used to reveal the position of flap interface.
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Biomarcadores/metabolismo , Córnea/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Queratomileusis por Láser In Situ/efectos adversos , Queratoplastia Penetrante , Complicaciones Posoperatorias , Adulto , Membrana Basal/patología , Córnea/patología , Topografía de la Córnea , Dilatación Patológica/etiología , Dilatación Patológica/metabolismo , Dilatación Patológica/cirugía , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Masculino , Persona de Mediana Edad , Colgajos QuirúrgicosRESUMEN
PURPOSE: To determine the efficacy of INTACS insertion using a femtosecond laser in the treatment of keratoconus and to compare it to the technique using a mechanical spreader. METHODS: INTACS were inserted in 10 eyes using the mechanical spreader to create the channels and subsequently on another 20 eyes using the femtosecond laser. Uncorrected (UCVA) and best spectacle-corrected visual acuity (BSCVA), manifest refraction, and corneal topography were measured prior to surgery, at 6 months (femtosecond group), and 1 year (mechanical group). Pre- and postoperative data were analyzed to determine changes in the above parameters. RESULTS: Both groups showed significant reduction in average keratometry (K), spherical equivalent refraction, BSCVA, UCVA, surface regularity index (SRI), and surface asymmetry index (SAI). The laser group performed better in all parameters except change in SRI. Results of the laser versus the mechanical spreader were as follows: reduction in spherical equivalent refraction (3.98 vs 2.96), change in average K (2.91 vs 2.52), improvement in UCVA (4.13 vs 3.63), improvement in BSCVA (3.92 vs 1.63), change in SRI (0.37 vs 0.64), and change in SAI (1.00 vs 0.70). Statistical analysis, however, did not reveal any statistically significant differences between the two groups for any single parameter studied. The biggest improvement in the laser group versus the mechanical group was BSCVA (P=.09). Overall success, defined as contact lens or spectacles tolerance, was 85% in the laser group and 70% in the mechanical group. CONCLUSIONS: Inserting INTACS using the femtosecond laser to create the channels is as effective as using the mechanical spreader.
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Sustancia Propia/cirugía , Queratocono/cirugía , Terapia por Láser/instrumentación , Prótesis e Implantes , Implantación de Prótesis/métodos , Adulto , Sustancia Propia/patología , Topografía de la Córnea , Femenino , Estudios de Seguimiento , Humanos , Queratocono/patología , Queratocono/fisiopatología , Masculino , Persona de Mediana Edad , Diseño de Prótesis , Agudeza VisualRESUMEN
PURPOSE: To examine the distribution of extracellular matrix (ECM) and basement membrane (BM) components and of Na+,K+-ATPase in postcataract surgery (PCS) corneas. These corneas were from patients who never developed pseudophakic or aphakic bullous keratopathy (PBK/ABK) after cataract surgery. PCS corneas were compared with PBK/ABK and Fuchs' dystrophy corneas. METHODS: The distribution of PBKIABK ECM and BM markers and of all three Na+,K+-ATPase alpha subunits was studied by immunofluorescence in 10 healthy, 11 PCS, 16 PBK/ABK, and 12 Fuchs' dystrophy corneas. RESULTS: Fibrotic ECM proteins, tenascin-C and fibrillin-1, were found in only 1 of 10 healthy and in 2 of 11 PCS corneas. In contrast, these proteins were expressed in all PBK/ABK and more than half of the Fuchs' dystrophy corneas. BM components in PCS corneas were altered to a greater extent (40-60%), especially fibronectin and laminin-10. A decreased epithelial immunostaining for Na+,K+-ATPase alpha subunits was seen in approximately 40% of PCS corneas and in approximately two thirds of PBK/ABK and Fuchs' dystrophy corneas. However, the endothelial staining was normal in all groups. CONCLUSIONS: Because tenascin-C and fibrillin-1 were mostly found in diseased but not in PCS corneas, their expression may be related to later, clinical stages of corneal edema development. However, BM components abnormal in PBK/ABK and Fuchs' dystrophy corneas were also altered in PCS corneas without clinical evidence of ocular disease. This may result from subclinical corneal changes resulting from cataract surgery, lens removal, exposure to the intraocular lens, or endothelial cell damage. Alterations of epithelial Na+,K+-ATPase point to the importance of epithelial changes in the development of corneal edematous diseases.
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Edema Corneal/metabolismo , Epitelio Corneal/metabolismo , Matriz Extracelular/metabolismo , Distrofia Endotelial de Fuchs/metabolismo , Queratoplastia Penetrante/efectos adversos , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Membrana Basal/metabolismo , Western Blotting , Edema Corneal/etiología , Epitelio Corneal/patología , Proteínas de la Matriz Extracelular/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Distrofia Endotelial de Fuchs/etiología , HumanosRESUMEN
PURPOSE: To characterize extracellular matrix (ECM) and nine matrix metalloproteinase (MMP) changes in two corneas that underwent a complicated laser-assisted in situ keratomileusis (LASIK) procedure. METHODS: The first patient underwent bilateral LASIK. The flap on the left eye was transected in several locations and placed back. This cornea later developed edema, and the removed flap was analyzed after lamellar keratoplasty. The second patient had a LASIK flap lifted, replaced twice, and then completely removed. The epithelium grew over the stroma, but haze and severe ectasia occurred. After penetrating keratoplasty, the recipient cornea was analyzed. An autopsy cornea from a person who underwent uneventful LASIK and ten normal autopsy corneas served as controls. Corneas were analyzed by immunohistochemistry. RESULTS: Both flap regions in the treated corneas had marked alterations of ECM components and MMPs. Stromal deposits of various ECM proteins, including those normally absent in the central cornea (tenascin-C, fibrillin-1, type XIV collagen), were found. Rare myofibroblasts and inflammatory cells were present. The epithelial basement membrane (BM) was altered in both cases. The most dramatic change was poor or no staining for alpha3-alpha6 type IV collagen chains and thrombospondin. The limbal alpha1-alpha2 type IV collagen and laminin-2 (alpha2beta1gamma1) appeared in the central epithelial BM. Other components were altered to a lesser extent. The anterior stroma was positive for MMP-1 and MMP-2, and some MMP-7 was seen in the epithelium. These ECM and MMP patterns were not seen in uneventful LASIK or normal corneas. CONCLUSIONS: In the flaps of LASIK-treated corneas, fibrosed areas of anterior stroma had increased levels of MMP-1 and MMP-2 that may have caused loss of specific type IV collagen isoforms in the epithelial BM. These changes may reflect an ongoing wound healing process and contribute to the development of ectasia.
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Córnea/metabolismo , Enfermedades de la Córnea/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Matriz Extracelular/metabolismo , Queratomileusis por Láser In Situ/efectos adversos , Metaloproteinasas de la Matriz/metabolismo , Adulto , Anciano , Córnea/patología , Enfermedades de la Córnea/etiología , Enfermedades de la Córnea/cirugía , Matriz Extracelular/patología , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Queratoplastia Penetrante , Masculino , Persona de Mediana Edad , Colgajos Quirúrgicos , Cicatrización de HeridasRESUMEN
Limbal epithelial stem cell (LESC) deficiency (LSCD) leads to corneal abnormalities resulting in compromised vision and blindness. LSCD can be potentially treated by transplantation of appropriate cells, which should be easily expandable and bankable. Induced pluripotent stem cells (iPSCs) are a promising source of transplantable LESCs. The purpose of this study was to generate human iPSCs and direct them to limbal differentiation by maintaining them on natural substrata mimicking the native LESC niche, including feederless denuded human amniotic membrane (HAM) and de-epithelialized corneas. These iPSCs were generated with nonintegrating vectors from human primary limbal epithelial cells. This choice of parent cells was supposed to enhance limbal cell differentiation from iPSCs by partial retention of parental epigenetic signatures in iPSCs. When the gene methylation patterns were compared in iPSCs to parental LESCs using Illumina global methylation arrays, limbal-derived iPSCs had fewer unique methylation changes than fibroblast-derived iPSCs, suggesting retention of epigenetic memory during reprogramming. Limbal iPSCs cultured for 2 weeks on HAM developed markedly higher expression of putative LESC markers ABCG2, ΔNp63α, keratins 14, 15, and 17, N-cadherin, and TrkA than did fibroblast iPSCs. On HAM culture, the methylation profiles of select limbal iPSC genes (including NTRK1, coding for TrkA protein) became closer to the parental cells, but fibroblast iPSCs remained closer to parental fibroblasts. On denuded air-lifted corneas, limbal iPSCs even upregulated differentiated corneal keratins 3 and 12. These data emphasize the importance of the natural niche and limbal tissue of origin in generating iPSCs as a LESC source with translational potential for LSCD treatment.
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Técnicas de Cultivo de Célula/métodos , Epitelio Corneal/citología , Células Madre Pluripotentes Inducidas/citología , Limbo de la Córnea/citología , Diferenciación Celular/fisiología , Humanos , Inmunohistoquímica , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Human amniotic membrane is a standard substratum used to culture limbal epithelial stem cells for transplantation to patients with limbal stem cell deficiency. Various methods were developed to decellularize amniotic membrane, because denuded membrane is poorly immunogenic and better supports repopulation by dissociated limbal epithelial cells. Amniotic membrane denuding usually involves treatment with EDTA and/or proteolytic enzymes; in many cases additional mechanical scraping is required. Although ensuring limbal cell proliferation, these methods are not standardized, require relatively long treatment times and can result in membrane damage. We propose to use 0.5 M NaOH to reliably remove amniotic cells from the membrane. This method was used before to lyse cells for DNA isolation and radioactivity counting. Gently rubbing a cotton swab soaked in NaOH over the epithelial side of amniotic membrane leads to nearly complete and easy removal of adherent cells in less than a minute. The denuded membrane is subsequently washed in a neutral buffer. Cell removal was more thorough and uniform than with EDTA, or EDTA plus mechanical scraping with an electric toothbrush, or n-heptanol plus EDTA treatment. NaOH-denuded amniotic membrane did not show any perforations compared with mechanical or thermolysin denuding, and showed excellent preservation of immunoreactivity for major basement membrane components including laminin α2, γ1-γ3 chains, α1/α2 and α6 type IV collagen chains, fibronectin, nidogen-2, and perlecan. Sodium hydroxide treatment was efficient with fresh or cryopreserved (10% dimethyl sulfoxide or 50% glycerol) amniotic membrane. The latter method is a common way of membrane storage for subsequent grafting in the European Union. NaOH-denuded amniotic membrane supported growth of human limbal epithelial cells, immortalized corneal epithelial cells, and induced pluripotent stem cells. This simple, fast and reliable method can be used to standardize decellularized amniotic membrane preparations for expansion of limbal stem cells in vitro before transplantation to patients.
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Amnios/citología , Técnicas de Cultivo de Célula , Separación Celular/métodos , Amnios/metabolismo , Biomarcadores , Células Epiteliales/citología , Células Epiteliales/metabolismo , Femenino , HumanosRESUMEN
PURPOSE: To perform an immunohistochemical evaluation of corneas with INTACS for post-laser in situ keratomileusis (LASIK) keratectasia and keratoconus, obtained after corneal transplantation. METHODS: Corneas from 1 patient with INTACS for post-LASIK keratectasia and 2 patients with INTACS for keratoconus were obtained within 3 hours after penetrating keratoplasty, and cryostat sections were analyzed by immunostaining for 35 extracellular matrix (ECM) components and proteinases. RESULTS: In the stroma of all corneas next to an INTACS implant, ECM components typically associated with fibrosis were observed. These included tenascin-C, fibrillin-1, and types III, IV (alpha1/alpha2 chains), and XIV collagen. Also, significant deposition of perlecan, nidogen-2, and cellular fibronectin was revealed in the same locations. The keratoconus cases displayed typical Bowman layer breaks and subepithelial fibrosis with deposition of various ECM components. In all cases, some keratocytes around INTACS were positive for specific proteinases associated with stromal remodeling, including cathepsins F and H, matrix metalloproteinase (MMP)-1, MMP-3, and MMP-10. Staining for MMP-7 was variable; MMP-2 and MMP-9 were mostly negative. Patterns of type IV collagen alpha 3, alpha 4, and alpha 6 chains; types VI and VIII collagen; laminin-332, alpha 4, alpha 5, beta1, beta2, and gamma 1 laminin chains; vitronectin; thrombospondin-1; urokinase; EMMPRIN; and cathepsins B and L were unchanged around INTACS in all 3 cases compared with normal. CONCLUSIONS: Abnormal accumulation of fibrotic ECM components and proteinases near INTACS suggests ongoing lysis and remodeling of corneal stroma. Specific changes observed in each case may be related to underlying pathology.