Immunopanning purification and long-term culture of human retinal ganglion cells

Purpose: To establish a robust method to isolate primary retinal ganglion cells (RGCs) from human fetal retina for long-term culture while maintaining neuronal morphology and marker protein expression. Methods: A total of six human retinas were obtained from aborted fetuses at 10 to 12 weeks of gestation with informed consent from mothers. RGCs were isolated and purified by a modified two-step immunopanning procedure. The cells were maintained in a serum-free defined medium supplemented with brain-derived neurotrophic factor, ciliary neutrophic factor, and forskolin. The viable RGCs and the extent of neurite outgrowth were examined by calcein-acetoxymethylester assay. Expression of RGC markers was studied by immunocytochemistry. Results: Primary RGCs from human fetal retinas were isolated and maintained in vitro for one month with substantial neurite elongation. In cell culture, almost 70% of the isolated cells attached, spread, and displayed numerous dendrites. They were immunoreactive to RGC-specific markers (Thy-1, TUJ-1, and Brn3a) and negative for glial fibrillary acidic protein and amacrine cells marker HPC-1. Conclusions: Human RGCs were successfully isolated and maintained in long-term culture. This can serve as an ideal model for biologic, toxicological, and genomic assays of human RGCs in vitr

AC and AG Dinucleotide Repeats in the PAX6 P1 Promoter are Associated with High Myopia

Purpose: The PAX6 gene, located at the reported myopia locus MYP7 on chromosome 11p13, was postulated to be associated with myopia development. This study investigated the association of PAX6 with high myopia in 379 high myopia patients and 349 controls. Methods: High myopia patients had refractive errors of –6.00 diopters or greater and axial length longer than 26 mm. Control subjects had refractive errors less than –1.00 diopter and axial length shorter than 24 mm. The P1 promoter, all coding sequences, and adjacent splice-site regions of the PAX6 gene were screened in all study subjects by polymerase chain reaction and direct sequencing. PAX6 P1 promoter-luciferase constructs with variable AC and AG repeat lengths were prepared and transfected into human ARPE-19 cells prior to assaying for their transcriptional activities. Results: No sequence alterations in the coding or splicing regions showed an association with high myopia. Two dinucleotide repeats, (AC)m and (AG)n, in the P1 promoter region were found to be highly polymorphic and significantly associated with high myopia. Higher repeat numbers were observed in high myopia patients for both (AC)m (empirical p = 0.013) and (AG)n (empirical p = 0.012) dinucleotide polymorphisms, with a 1.327-fold increased risk associated with the (AG)n repeat (empirical p = 0.016; 95% confidence interval: 1.059–1.663). Luciferase-reporter analysis showed elevated transcription activity with increasing individual (AC)m and (AG)n and combined (AC)m(AG)n repeat lengths. Conclusions: Our results revealed an association between high myopia and AC and AG dinucleotide repeat lengths in the PAX6 P1 promoter, indicating the involvement of PAX6 in the pathogenesis of high myopia

MicroRNA-145 Regulates Human Corneal Epithelial Differentiation

Epigenetic factors, such as microRNAs, are important regulators in the self-renewal and differentiation of stem cells and progenies. Here we investigated the microRNAs expressed in human limbal-peripheral corneal (LPC) epithelia containing corneal epithelial progenitor cells (CEPCs) and early transit amplifying cells, and their role in corneal epithelium.Human LPC epithelia was extracted for small RNAs or dissociated for CEPC culture. By Agilent Human microRNA Microarray V2 platform and GeneSpring GX11.0 analysis, we found differential expression of 18 microRNAs against central corneal (CC) epithelia, which were devoid of CEPCs. Among them, miR-184 was up-regulated in CC epithelia, similar to reported finding. Cluster miR-143/145 was expressed strongly in LPC but weakly in CC epithelia (P = 0.0004, Mann-Whitney U-test). This was validated by quantitative polymerase chain reaction (qPCR). Locked nucleic acid-based in situ hybridization on corneal rim cryosections showed miR-143/145 presence localized to the parabasal cells of limbal epithelium but negligible in basal and superficial epithelia. With holoclone forming ability, CEPCs transfected with lentiviral plasmid containing mature miR-145 sequence gave rise to defective epithelium in organotypic culture and had increased cytokeratin-3/12 and connexin-43 expressions and decreased ABCG2 and p63 compared with cells transfected with scrambled sequences. Global gene expression was analyzed using Agilent Whole Human Genome Oligo Microarray and GeneSpring GX11.0. With a 5-fold difference compared to cells with scrambled sequences, miR-145 up-regulated 324 genes (containing genes for immune response) and down-regulated 277 genes (containing genes for epithelial development and stem cell maintenance). As validated by qPCR and luciferase reporter assay, our results showed miR-145 suppressed integrin β8 (ITGB8) expression in both human corneal epithelial cells and primary CEPCs.We found expression of miR-143/145 cluster in human corneal epithelium. Our results also showed that miR-145 regulated the corneal epithelium formation and maintenance of epithelial integrity, via ITGB8 targeting

Measurement of Photoreceptor Layer in Glaucoma: A Spectral-Domain Optical Coherence Tomography Study

Objective. To measure and compare photoreceptor layer thickness between normal and glaucomatous eyes using spectral-domain optical coherence tomography (OCT). Methods. Thirty-eight healthy normal volunteers and 47 glaucoma patients were included in the analysis. One eye from each participant was randomly selected for macula imaging by a spectral-domain OCT (3D OCT-1000, Topcon, Tokyo, Japan). The foveal and parafoveal (1.5 mm from the fovea) outer nuclear layer (ONL) and inner and outer segments (IS+OS) layer thicknesses were measured by a single masked observer. The measurements were repeated 3 times in a random sample of 30 normal eyes to determine the repeatability coefficient and intraclass correlation coefficient. Results. The measurement variabilities of photoreceptor thickness were low. The respective intraclass correlation coefficients of ONL and IS+OS thicknesses were 0.96 (95% confidence interval: 0.94–0.98) and 0.82 (95% confidence interval 0.70–0.90). While there were no differences in parafoveal ONL and IS+OS thicknesses between normal and glaucoma groups (≤.410), the foveal ONL thickness was greater in glaucomatous eyes (=.011) than in normal eyes. Conclusions. Glaucomatous damage may involve structural change in the photoreceptor layer

The transfer of ocular cells using collagen

The present study was designed to evaluate the use of collagen gel loaded with human retinal pigment epithelium (ARPE19) in cellular transfer and to assess its viability within the gel. Collagen solution was prepared by dissolving calfskin in hydrochloric acid to make a final concentration of 2.0 mg/ml and this was mixed with 10,000 ARPE19 cells/ml. The cell viability in gel was determined using MTT assay. van Gieson stain and proliferating cell nuclear antigen (PCNA) were used to identify the location of collagen and to localize the site of cell proliferation, respectively. The ARPE19 cells in gel appeared to be healthy with a rounded morphology. The optimal collagen concentration was 1.9 mg/ml. When this concentration was used to hold cells for over 12 days, it could be seen that the growth rate was the same between day 2 and day 8 in get and on plastic. When the cell-loaded gels were transferred onto standard tissue culture plastics, progressive cell migrations over time resembling cell migrations in organotypic explant cultures were observed. Upon intravitreal injection of cell-containing collagen suspension into a rabbit's eye, the gel became suspended within the vitreous a few hours after injection (day 0). However, it became obvious that the gel dispersed and spread around the vitreous even after just 24 h. These cells inside the vitreous were PCNA positive, indicating that the human ARPE19 cells have the capacity to proliferate even after I I days. The present study demonstrated the potential use of collagen gel as a tool in the transfer of cellular matrix onto other substrates. The results show that the cell seeding number must be critically balanced with the concentration of gel for it to be used as transplant material