Isolation and characterization of rabbit limbal niche cells.

Limbal niche cells (LNCs) are one of the most important supporting cells for corneal epithelial stem cells (CES), however, research on LNCs has been mostly limited to humans and rats previously. To expand the research work into the rabbit animal model, one of the most often used animals in stem cell study, this study was carried out for the in vitro isolation and identification of rabbit LNCs. Rabbit LNCs were isolated by collagenase A digestion method and single cells were obtained, the cells were then seeded on 5% Matrigel-coated plastic surface and cultured in modified embryonic stem cell medium (MESCM). Three biological replicates of the isolating and characterization were recorded from New Zealand White rabbits aged from 2.5 months to 5 months. LNC markers (VIM/CD90/CD105/SCF/PDGFRß) were analyzed using tyramide signal amplification (TSA) staining, immunohistochemical staining (IHC), western blotting (WB), and real-time reverse transcription polymerase chain reaction (qPCR). TSA staining suggested that VIM was highly expressed in rabbit limbus stroma, which was confirmed by WB, and P63α was expressed in the basal limbus epithelium. Pan-CK and CK12 were highly expressed in the central corneal epithelium but lightly expressed in the limbal epithelium. The WB result indicated that PDGFRß and VIM expressions in rabbit-LNCs P4 were higher than in P1 and P7. In addition, rabbit corneal epithelium highly expressed Paired Box 6 (PAX6) and Epidermal growth factor-like domain 6(EGFL6). For the three repeat experiments, the cell expansion activity of rabbit-LNC was highest at P4. Rabbit-LNCs were passaged from P0 to P7, and the number of cell doublings (NCD) of P4 for the three repeat experiments was 2.816, 2.737, and 2.849. qPCR showed that high mRNA expression levels of VIM, CD90, CD105, SCF, and PDGFRß in rabbit-LNCs P4. In conclusion, rabbit-LNCs could be successfully isolated by the collagenase A digestion method as used in human tissue. There were similar characteristics between rabbit and human LNCs (VIM+/CD90+/CD105+/SCF+/PAX6+/PDGFRß+).

Isolation and Identification of Limbal Niche Cells.

Here we report a standard procedure for the isolation and identification of limbal niche cells (LNCs). Limbus tissue obtained from an eye bank was used for LNCs isolation. The tissue was divided into 12 pieces under aseptic conditions and digested for 18 h at 37 °C in the cell culture incubator using collagenase A to obtain cell clusters with LNCs and limbal epithelial progenitor cells. The cell clusters were further digested for 15 min at 37 °C using 0.25% trypsin-EDTA to obtain single cells and then cultured in modified embryonic stem cell medium (MESCM) on a plastic surface coated with 5% Matrigel. Cells were passaged upon 70% confluence, and LNCs were identified using immunofluorescence, real-time quantitative PCR (qPCR), and flow cytometry. Primary LNCs were isolated and passaged more than 12 times. The proliferation activity of LNCs from P4 to P6 was the highest. LNCs expressed higher stem cell markers than BMMSCs (SCF, Nestin, Rex1, SSEA4, CD73, CD90, MSX1, P75NTR, and PDGFRß). Furthermore, results showed that P4 LNCs uniformly expressed VIM, CD90, CD105, and PDGFRß, but not Pan-CK, which could be used as a marker for the identification of LNCs. Flow cytometric analysis showed that approximately 95%, 97%, 92%, and 11% of LNCs expressed CD73, CD90, CD105, and SCF respectively, while they were 68%, 99%, 20%, and 3% in BMMSCs. The standard process for LNC isolation and identification could provide a reliable laboratory basis for the widespread use of LNCs.

Visual Acuity and Visual Field Changes in Patients with End-Stage PACG with Tubular Visual Field after Cataract Surgery.

INTRODUCTION: To investigate the impact of cataract surgery on visual acuity and visual field (VF) in patients with end-stage glaucoma with tubular VF, and assess the risk of severe visual impairment. METHODS: Retrospective analysis of the case data of patients with end-stage glaucoma with tubular VF who underwent cataract surgery in our hospital in the past 7 years. RESULTS: A total of 59 patients with 63 eyes were enrolled, 62 eyes were primary angle-closure glaucoma (PACG) and 1 eye was primary open-angle glaucoma. The last follow-up time was an average of 9 months, and no cases of severe vision loss occurred. Best corrected visual acuity (BCVA) improved significantly after surgery (0.57 ± 0.46 vs. 0.45 ± 0.43 logarithm of the minimum angle of resolution, p < 0.01), and there was a significant drop in intraocular pressure (IOP; 22.85 ± 9.7 vs. 16.07 ± 3.38, p < 0.01), a reduced number of glaucoma medications (2 ± 1.32 vs. 0.5 ± 1, p < 0.01), statistical improvement in VF index (VFI) and mean defect (MD) (12.3% ± 7.65% vs. 16.1% ± 9.84%, p < 0.01; -29.09 ± 2.16 vs. -28.31 ± 3.01, p < 0.01) after surgery. The higher the preoperative VFI and MD were, the better the postoperative BCVA (r = -0.387, r = -0.347, respectively). The degree of postoperative VFI improvement was significantly correlated with preoperative MD (r = 0.372, p < 0.01). During the follow-up period, 5 eyes (8%) underwent anti-glaucoma surgery due to elevated IOP. CONCLUSION: Cataract surgery can significantly improve visual acuity and VF in patients with end-stage PACG with tubular VF, and no patients have severe visual impairment. The less preoperative VF damage there is, the greater the postoperative visual acuity and VF improvement. Poor IOP control is the main cause of further damage to postoperative visual acuity and VF.

Progress of EGFL6 in angiogenesis and tumor development.

The epidermal growth factor (EGF) superfamily includes the protein 6 with an epidermal growth factor-like protein (EGFL6). EGFL6 has a signal peptide domain with an amino terminus and a MAM domain with a carboxy terminus. There are four whole EGF-like repeat regions and one partial EGF-like repeat region. Three of these regions include calcium-binding structures and an arg-gly-asp (RGD) integrin interaction motif. The epidermal growth factor-like (EGFL) and EGF domains have identical amino acid residues. Cell division, differentiation, mortality, cell adhesion, and migration are all affected by EGFL6. EGFL proteins are involved in a broad range of biological activities, making it important in tumor development and angiogenesis. We highlighted the latest development of EGFL6 research on tumor proliferation, invasion, and migration in this review.

Limbal Niche Cells and Three-Dimensional Matrigel-Induced Dedifferentiation of Mature Corneal Epithelial Cells.

Purpose: To investigate the phenotypic changes of mature corneal epithelial cells (MCECs) that cocultured with limbal niche cells (LNCs) in three-dimensional Matrigel (3D Matrigel) in vitro. Methods: MCECs were isolated from central corneas, and limbal epithelial progenitor cells (LEPCs) were isolated from limbal segments with Dispase II. LNCs were isolated and cultured from limbal niche using the collagenase A digestion method and identified with PCK/VIM/CD90/CD105/SCF/PDGFRß. MCECs were cultured on 3D Matrigel (50%, v/v) with or without LNCs for 10 days. Expression of CK12 and p63α and clone formation test were used to compare the progenitor phenotypic changes for MCECs before and after induction using LEPCs as control. Results: Homogeneous LNCs were isolated and identified as spindle shape and adherent to a plastic surface coated with 5% Matrigel. Double immunostaining of the fourth-passage LNCs was uniformly PCK-/VIM+/CD90+/CD105+/SCF+/PDGFRß+. Reverse transcription and quantitative real-time polymerase chain reaction (RT-qPCR) revealed the decrease of PCK expression from the second passage and elevation of Vim, CD90, CD105, SCF, and PDGFRß transcripts from the third passage, and the transcription level of Vim, CD90, CD105, SCF, and PDGFRß was elevated statistically in the fourth passage compared to the first passage (P < 0.01). Both immunofluorescence (IF) staining for cross section and cytospin cells demonstrated that MCECs expressed higher CK12 while lower p63α than LEPCs (P < 0.01). Sphere growth formation was noticed as early as 24 hours in the MCEC + LNC group, 48 hours in the LEPC group, and 72 hours in the MCEC group. The diameters of the spheres were the biggest in the MCEC + LNC group (182.24 ± 57.91 µm), smaller in the LEPC group (125.71 ± 41.20 µm), and smallest in the MCEC group (109.39 ± 34.85 µm) by the end of the 10-day culture (P < 0.01). Double immunostaining with CK12/p63α showed that cells in the sphere formed from MCECs expressed CK12 but not p63α; in contrast, some cells in the MCEC + LNC group expressed CK12, but most of them expressed p63α. RT-qPCR revealed a significant reduction of CK12 transcript but elevation of p63α, Oct4, Nanog, Sox2, and SSEA4 (P < 0.05). Holoclone composed of cubic epithelial cells could be generated in the MCEC + LNC group but not in the other two groups. Conclusions: The data shows that human MCEC cell phenotype could be induced to the dedifferentiation stage when cocultured with LNCs in 3D Matrigel that simulated the microenvironment of limbal stem cells in vitro.

Differential Gene Expression between Limbal Niche Progenitors and Bone Marrow Derived Mesenchymal Stem Cells.

Purpose: To compare the difference in gene expression between human limbal niche cells (LNC) and bone marrow derived mesenchymal stem cells (BMMSC). Methods: LNC were isolated by collagenase and expanded in modified embryonic stem cell medium (MESCM) on a Matrigel coated plastic plate. Cell diameters were measured with Image J software. Relative gene expression levels between LNC and BMMSC were compared using Affymetrix Human Primer View Gene Expression Array. A subset of differentially expressed genes was verified by RT-qPCR. The protein level of LAMA1 and COL4A1 was confirmed by Western blot and immunostaining. Results: The average diameter of LNC was 10.2±2.4 µm, which was significantly smaller than that of BMMSC (14 ±3.4 µm) (p<0.0001). Expression of 20,432 genes was examined by Gene Expression Array, among which expression of 349 genes in LNC was 10-fold or higher than that of BMMSC and expression of 8 genes in LNC was 100-fold or higher than that of BMMSC, while expression of 3 genes in BMMSC was 100-fold higher than that of LNC. GO analysis and pathway analysis showed that the differentially expressed genes were mainly enriched in the extracellular matrix receptor interaction pathway and Wnt signaling pathway. In addition, RT-qPCR results demonstrated that the expression of CD73, CD90, CD105, PDGFRß, Vimentin, SCF, KIT (CD117), COL14A1, LAMA2, THBS2, FZD1, BMP2 and CXCL12 genes in LNC were at least 2 folds higher than BMMSC. The protein level of LAMA1 was higher but the protein level of COL4A1 was lower in LNC than that in BMMSC. Conclusion: LNC exhibit differential gene expression from BMMSC in the extracellular matrix (ECM) receptor interaction pathway and Wnt signaling pathway, suggesting that LNC have their unique signaling pathways to support limbal stem cell niches.

Lacrimal Duct Occlusion Is Associated with Infectious Keratitis.

Background: To explore the prevalence of lacrimal duct obstruction in patients with infectious keratitis, and the necessity of lacrimal duct dredge in the treatment of human infectious keratitis. Methodology/Principle Findings: The design is prospective, non-control case series. Thirty-one eyes from twenty-eight continuous patients with infectious keratitis were included in this study. The presence/absence of lacrimal duct obstruction was determined by the lacrimal duct irrigation test. The diagnosis of infectious keratitis was made based on clinical manifestations, cornea scraping microscopic examination and bacterial/fungus culture. Diagnosis of viral keratitis was set up based on the recurrent history, deep neovascularization and typical outlook of the cornea scar. The treatment of keratitis included drugs, eye drops or surgery, while treatment of chronic dacryocystitis was lacrimal duct dredging with supporting tube implantation surgery. In the thirty-one eyes with infectious keratitis, fifteen suffered from fungal keratitis (48%), two bacterial keratitis (6%), and fourteen viral keratitis (45%). Eleven eyes (35%) from ten patients with infectious keratitis also suffered from lacrimal duct obstruction. In those cases, six eyes also suffered from lower canalicular obstruction, three nasolacrimal duct obstruction and chronic dacryocystitis, one a combination of upper and lower canalicular obstruction, one upper canalicular obstruction. After local and systemic applications of anti-bacterial, anti-viral, anti-fungal and anti-inflammatory drugs, twenty-eight eyes (90%) recovered within three weeks, while the ulceration of three patients required the lacrimal duct dredging and supporting tube implantation surgery for the healing. Conclusions: Herein, we first report that the prevalence of infectious keratitis is closely correlated to the occurrence of lacrimal duct obstruction. When both confirmed, simultaneous treatment of keratitis and lacrimal duct obstruction promptly is required. Further evaluation of mechanism, prevention and control of the diseases are warranted.

Amniotic membrane covering promotes healing of cornea epithelium and improves visual acuity after debridement for fungal keratitis.

AIM: To investigate the effect of amniotic membrane covering (AMC) on the healing of cornea epithelium and visual acuity for fungal keratitis after debridement. METHODS: Twenty fungal keratitis patients were divided into two groups randomly, the AMC group and the control group, ten patients each group. Both debridement of the infected cornea tissue and standard anti-fungus drugs treatments were given to every patients, monolayer amniotic membrane were sutured to the surface of the entire cornea and bulbar conjunctiva with 10-0 nylon suture for patients in the AMC group. The diameter of the ulcer was determined with slit lamp microscope and the depth of the infiltration was determined with anterior segment optical coherence tomography. Uncorrected visual acuity (UCVA) was tested before surgery and three month after healing of the epithelial layer. The healing time of the cornea epithelium, visual acuity (VA) was compared between the two groups using t-test. RESULTS: There was no statistical difference of the diameter of the ulcer, depth of the infiltration, height of the hypopyon and VA between the two groups before surgery (P>0.05). The average healing time of the AMC group was 6.89±2.98d, which was statistically shorter than that of the control group (10.23±2.78d) (P<0.05). The average UCVA of the AMC group was 0.138±0.083, which was statistically better than that of the control group (0.053±0.068) (P<0.05). CONCLUSION: AMC surgery could promote healing of cornea epithelium after debridement for fungal keratitis and lead to better VA outcome.

Pediatric microbial keratitis: a tertiary hospital study.

PURPOSE: To study the predisposing factors, clinical and microbiologic characteristics, treatment, and outcomes of pediatric microbial keratitis. METHODS: The medical records of 80 eyes with (nonviral) microbial keratitis in 76 children aged 16 years or younger were retrospectively reviewed. Demographic features, predisposing factors, clinical features, etiologic microorganisms, and treatment outcomes were analyzed. RESULTS: Seventy-six patients met the inclusion criteria of this study, and the male to female ratio was 1.9:1. The average age of the children was 8.9 ± 5.7 years, and the mean duration of symptoms was 12.5 ± 9.8 days. The most common predisposing factor was trauma (58.8%). Thirty-nine (48.8%) of 80 cases were culture positive. Bacterial isolates were observed in 21 cases, being headed by coagulase-negative Staphylococcus, and fungi were found in 19 cases, with Fusarium sp. the predominant pathogen. Fifty-nine cases required surgery intervention. Fifty of the 58 examined eyes achieved best-corrected visual acuity of 20/200 or better at the final follow-up. CONCLUSIONS: The most common risk factor for childhood microbial keratitis was corneal trauma. The most frequent bacteria isolated were coagulase-negative Staphylococcus, whereas the predominant fungi isolated were Fusarium species. Early diagnosis, intensive drug therapy, and timely surgical intervention may effectively improve the prognosis of pediatric microbial keratitis.

Targeting therapy of choroidal neovascularization by use of polypeptide- and PEDF-loaded immunoliposomes under ultrasound exposure.

Pigment epithelium derived factor (PEDF) has been proven to be an effective drug for the treatment of choroidal neovascularization (CNV). However, the lack of ideal administration route is the biggest bottleneck preventing PEDF from wider clinical use. In this study, we developed a novel PEDF-carrying system which employed immuno-nano-liposomes (INLs) under ultrasound exposure. PEDF-loaded INLs were prepared by conjugating nanoliposomes to the peptide ATWLPPR specifically targeting the receptor-2 for vascular endothelial growth factor (VEGFR-2) and reversely encapsuling PEDF. RF/6A cells were incubated with PEDF-loaded INLs. CNV models of BN rats were injected with PEDF-loaded INLs. MTT assay was used to evaluate the cytotoxicity of the INLs on RF/6A cells. Flow cytometry was conducted to detect the apoptotic rate of cells. Laser scanning confocal microscopy was employed to observe the binding and transmitting process of PEDF-loaded INLs and to calculate the area of CNV in the rat model. The results showed that the PEDF-loaded INLs could exclusively bind to CNV but not to the normal choroidal vessels. The CNV area was significantly decreased in PEDF treatment groups in comparison with control group (P<0.05). Moreover, PEDF-loaded INLs exposed under ultrasound were more efficient in reducing the CNV area (P<0.05). It was concluded that INLs in combination with ultrasonic exposure can transmit PEDF into cytoplasma with high specificity and efficiency, which strengthens the inhibitory effects of PEDF on CNV and reduces its side effects. PEDF-loaded INLs possibly represent a new treatment paradigm for patients with ocular neovascularization.