Henning Thomasen

The Effect of Long-Term Storage on the Biological and Histological Properties of Cryopreserved Amniotic Membrane

Purpose: Cryopreserved amniotic membrane (AM) is widely used in ophthalmology because of its anti-angiogenic, anti-inflammatory, and wound healing promoting capabilities. A common method to conserve the tissue is the storage in cryo-medium containing 50% glycerol at −80°C. The aim of this study was to examine the influence of storage time on the sterility as well as the histological and biological properties of cryopreserved AM. Methods: Amniotic membrane from different donors was stored in cell culture media containing 50% glycerol for different time periods, on average 4 months (group 1), 15 months (group 2), and 24 months (group 3), at −80°C. Samples of the tissue and cryo-medium were examined for bacterial and fungal contamination. Tissue samples were incubated in 0.5 ml/cm2 serum-free medium at 37°C. The medium was changed after 1, 2, and 3 days. The proteins released by AM were TCA-precipitated and the presence of the proteins TIMP-1 and IL-1ra was analyzed using Western blotting and semi quantified by means of image analysis. Integrity of the amniotic epithelium and the basement membrane components collagen IV, collagen VII, laminin, laminin 5, and fibronectin were examined by haematoxylin eosin stain and immunohistochemistry in cryosections of AM. Results: None of the examined samples showed bacterial or fungal contamination. The soluble proteins TIMP-1 and IL-1ra were found in all samples of medium incubated for all time periods. The examined proteins were detectable after one-day incubation but the staining signal diminished significantly in the second and third wash after 48 hr and 72 hr. Differences in the intensity of the Western blot signal between the three particular groups were statistically not significant. The epithelia of all samples were intact. The basement membranes of all samples showed a similar distribution of collagen IV, collagen VII, laminin, laminin 5, and fibronectin. Conclusions: Long-term storage of amniotic membrane in cell culture media with 50% glycerol does not significantly impair sterility, histology, or biological properties of AM.

Expression of pluripotency and multipotency factors in human ocular surface tissues.

Aim: Mechanisms that control ocular surface stem cells (SCs) are unclear. Recent studies have shown that several adult SCs express pluripotency markers. Our objective was to analyze the expression of key molecules of pluripotency in human ocular surface tissues as well as in cultivated limbal epithelium. Methods: Four samples of human corneal, limbal and on amniotic membrane cultivated limbal epithelium (HLEC-AM), as well as bulbar and fornical conjunctiva were analyzed. Human embryonic stem (ES) cells and human umbilical vein endothelial cells served as controls. Expression of corneal epithelial differentiation markers (K3, K12, Cx43), putative limbal SC markers (ABCG2, p63, K15), and molecules associated with pluripotency/multipotency (NANOG, OCT4, SOX2, KLF4, KIT, NESTIN, PAX6, NOTCH1) was examined using real-time polymerase chain reaction (PCR) and immunohistochemical staining. Results: Limbal epithelium showed a significantly (p < 0.05) higher expression of K15, ABCG2, OCT4, SOX2, NESTIN and NOTCH1, but a lower expression of K3 than corneal epithelium. Besides a higher expression of ABCG2 in fornix, the expression of pluripotency markers was similar in both conjunctival regions, although lower than in limbal epithelium. Expression of pluripotency factors in ES cells was significantly higher than in ocular surface SCs, whereas the expression in limbal epithelium was the closest to ES cells. HLEC-AM in comparison to limbal epithelium showed a lower expression of differentiation markers, a similar expression of ABCG2 but a significantly lower expression of pluripotency factors. Conclusion: Human ocular surface epithelial cells and especially limbal epithelial cell express genes are important for pluripotency and may have preserved some common mechanisms with pluripotent SCs.

Amnionmembrantransplantation bei herpetischen Hornhautinfektionen

Severe infectious corneal ulceration such as herpetic stromal keratitis commonly causes loss of vision and may lead to blindness. Treatment depending on the underlying disease includes antimicrobial medication and the development of surgical strategies to restore the integrity of the corneal ocular surface. Ulcerative herpetic stromal keratitis and/or neurotrophic keratopathy with the risk of corneal perforation are still clinically challenging conditions in ophthalmic surgery of the ocular surface. Since the introduction of newly developed preservation methods, amniotic membrane (AM) functioning as a basement membrane substitute has gained widespread popularity in ocular surface reconstruction. Various ways of clinical application such as the use of AM as a graft, patch or culture substrate and carrier system to expand ocular surface epithelia have been recently reported. In this article, the basis and clinical application of amniotic membrane transplantation for the management of corneal infections with Herpes simplex and Herpes zoster virus are reviewed.

Frequent TERT Promoter Mutations in Ocular Surface Squamous Neoplasia

PURPOSE Ocular surface squamous neoplasia, including intraepithelial neoplasia (CIN) and invasive squamous cell carcinoma (SCC), are one of the most common malignant tumors of the conjunctiva. Little is known of the genetic alterations involved in their pathogenesis. Promoter mutations in telomerase reverse transcriptase (TERT) have been identified in various cancers, including many associated with ultraviolet (UV) exposure. Our study analyzes the mutation rate and clinicopathological associations of TERT promoter mutations in ocular surface squamous neoplasia. METHODS DNA was isolated and the region of the TERT promoter where hotspot mutations can occur analyzed by Sanger-sequencing in 48 ocular surface squamous neoplasia tumor samples (6 CIN and 42 SCC). An analysis of associations between TERT promoter mutation status and various clinicopathological parameters was performed. RESULTS We identified TERT promoter mutations in 21 of 48 ocular surface squamous neoplasia samples (43.8%), including 4 in CIN and 17 in SCC. The mutations consisted of 8 Chr.5:1295228C>T, 1 Chr.5:1295228_1295229CC>TT, 5 Chr.5:1295242_1295243CC>TT, and 12 Chr.5:1295250C>T mutations. All mutations were C>T or CC>TT alterations, demonstrating a UV-signature. TERT promoter mutations showed no statistically significant associations with clinicopathological parameters. CONCLUSIONS Telomerase reverse transcriptase promoter mutations are found in almost half of ocular surface squamous neoplasias and have a mutation profile supporting UV induction as the major source of mutagenesis. We conclude that UV induced TERT promoter mutations leading to aberrant overexpression of telomerase is a major pathogenetic factor in ocular surface squamous neoplasia.

Establishment of a Cell Line From Conjunctival Squamous Cell Carcinoma: PeCa-UkHb-01

PURPOSE Until now, no epithelial cell line from conjunctival squamous cell carcinoma (SCC), to our knowledge, has existed; therefore, the establishment of a model cell line would be a useful tool for further studies. In particular, the phenotypic and molecular characterization in comparison to other SCC cells is of high interest because this would enable the development of new treatment options for clinical application in ophthalmic oncology. METHODS Epithelial cells were isolated from a bulbar conjunctiva SCC obtained from a 74-year-old male, harvested by stepwise trypsinization and named PeCa-UkHb-01. Cell doubling and the number of passages were determined. Short tandem repeats (STR) and karyotype analyses were performed. Semiquantitative real-time PCR and immunocytochemical fluorescence staining were carried out to detect tumor and epithelial cell markers. RESULTS The cells had an epithelial and conjunctival phenotype. They grew above passage number 50 in a doubling time at approximately 34.5 hours. Short tandem repeat analyses confirmed the cell origin, although loss of heterozygosity occurred. Karyotype analyses revealed a heterogeneous composition of the cell culture and the karyogram itself showed aberrations and changes in the chromosome numbers. Real-time PCR and immunocytochemical fluorescence staining revealed the expression of the stem cell markers such as ABCG2, p63, OCT4, c-MYC, and SOX2 as well as the conjunctival cytokeratin K19. CONCLUSIONS PeCa-UkHb-01 cells fulfill the criteria of a cell line. They display characteristics of ocular carcinoma cells and therefore the presented cell line might serve for further basic research in ophthalmic oncology.