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.

Amniotic membrane transplantation in the human eye.

BACKGROUND: Amniotic membrane transplantation (AMT) has a long tradition in ophthalmic surgery and has become very popular recently because of newly developed methods of tissue preservation. METHODS: We selectively review the literature on recent developments, mechanisms of action, and established indications of AMT in the treatment of various diseases of the ocular surface. We searched the PubMed database for articles that appeared from 1994 to 2009 with the key words "amniotic membrane," "cornea," and/or "conjunctiva." RESULTS: Amniotic membrane (AM) can function in the eye as a basement membrane substitute or as a temporary graft. It has anti-inflammatory and anti-scarring effects and contains growth factors that promote epithelial wound healing on the surface of the eye. AMT has been found to be a good alternative for corneal and conjunctival reconstruction in many clinical situations, including acute burns, persistent epithelial defects of the cornea, and diseases that cause conjunctival scarring. Nonetheless, there have been no more than a few randomized and controlled trials of AMT to date. Other studies have shown that AM can serve as a culture substrate to expand epithelial progenitor cells for use in ocular surface reconstruction. CONCLUSION: AMT is an established technique in the treatment of various diseases of the external eye. In the last few years, AMT has brought about major advances in the reconstructive surgery of the ocular surface.

Midterm results of cultivated autologous and allogeneic limbal epithelial transplantation in limbal stem cell deficiency.

BACKGROUND: Limbal stem cell deficiency (LSCD) leads to growth of abnormal fibro-vascular pannus tissue onto the corneal surface as well as chronic inflammation and impaired vision. Our aim was to investigate the clinical outcome of ocular surface reconstruction in LSCD using limbal epithelial cells expanded on amniotic membrane (AM). METHODS: Forty-four eyes of 38 patients (27 male, 11 female) with total (n = 32) or partial (n = 12) LSCD were treated by transplantation of autologous (n = 30) or allogeneic (n = 14) limbal epithelial cells expanded on intact AM. LSCD was caused by chemical and thermal burns (n = 22), pterygium (n = 9), congenital aniridia (n = 6), tumor excision (n = 2), perforating eye injury, mitomycin C, epidermolysis bullosa, bilateral graft-versus-host disease and chlamydial conjunctivitis (each n = 1). RESULTS: Mean follow-up time was 28.5 +/- 14.9 months. The corneal surface could be reconstructed to full stability in 30 (68%), and clear central cornea was achieved in 37 (84%) eyes. Grafting was significantly more successful in eyes treated by autologous than by allogeneic transplantation (76.7 vs. 50%, p < 0.05). The corneal surface could be successfully restored in 10 (83.3%) eyes with partial LSCD and in 20 (63.3%) eyes with total LSCD. Visual acuity (VA) increased significantly in 32 (73%) eyes, was stable in 10 (23%) eyes and decreased in 2 (4%) eyes. Mean VA increased significantly (p < 0.0001), from preoperative 1.7 +/- 0.9 log MAR (20/1,000) to 0.9 +/- 0.7 log-MAR (20/160). VA increased significantly after both autologous (p < 0.0001) and allogeneic transplantation (p < 0.005). CONCLUSIONS: In most patients with LSCD, transplantation of limbal epithelium cultivated on intact AM restores the corneal surface and results in significantly increased VA.

Ocular surface reconstruction with cultivated limbal epithelium in a patient with unilateral stem cell deficiency caused by Epidermolysis bullosa dystrophica hallopeau-Siemens.

PURPOSE: To report a case of partial limbal stem cell deficiency (LSCD) caused by epidermolysis bullosa dystrophica mutilans Hallopeau-Siemens treated by transplantation of autologous ex vivo expanded limbal epithelium. METHODS: Review of the clinical findings of an 11.5-year-old boy with unilateral LSCD and epidermolysis bullosa dystrophica who underwent ocular surface reconstruction in the right eye with autologous on intact human amniotic membrane cultivated limbal epithelial cells. RESULTS: Twenty-eight months after reconstruction, the corneal surface is clear, smooth, and stable showing no signs of LSCD recurrence. Three subconjunctival bevacizumab (Avastin) injections reduced the recurrent growth of symblepharon and corneal vascularization. The visual acuity has increased from hand motion to 20/50. CONCLUSION: Autologous transplantation of cultivated human limbal epithelial cells on intact human amniotic membrane can be a safe and effective method for corneal surface reconstruction in LSCD caused by recessive epidermolysis bullosa dystrophica.

Comparison of cryopreserved and air-dried human amniotic membrane for ophthalmologic applications.

BACKGROUND: Cryopreserved amniotic membrane (Cryo-AM) is widely used in ocular surface surgery because of its positive effect on wound healing and its anti-inflammatory properties. A new peracetic acid/ethanol sterilized air-dried amniotic membrane (AD-AM) recently became available which might be an alternative to Cryo-AM. Our aim was to compare AM preserved with both methods with regard to the release of wound-healing modulating proteins, the preservation of basement membrane components, and the ability to serve as a substrate for the cultivation of human limbal epithelial cells (HLECs). METHODS: Pieces of Cryo-AM and AD-AM from three different donors were incubated in DMEM for five days. The culture supernatant was collected after an incubation period of 0.1, 24, 48, 72 and 120 h; in the case of AD-AM, this period was extended up to 14 days. TIMP-1, IL-1ra, CTGF and TGF-beta1 were detected in the culture supernatant using Western blotting. Twenty human limbal epithelial cultures were initiated on both AD- and Cryo-AM. The cultures were analyzed morphologically, and the outgrowth area was measured in 3-day intervals. Cryosections of Cryo- and AD-AM from three different donors were analyzed histochemically to detect the basement membrane components collagen IV, collagen VII, laminin, laminin 5 and fibronectin. RESULTS: The release of TIMP-1, IL-1ra and TGF-beta1 from Cryo-AM was constant for the studied period. CTGF showed a stronger signal after 120 h. None of the analyzed proteins, except for a small amount of IL-1ra, could be detected in the supernatant of AD-AM. An outgrowth of HLEC was observed in all cultures on Cryo-AM, but in only 30% of cultures on AD-AM. The outgrowth area on Cryo-AM was at all time points significantly higher than on AD-AM (p < 0.0001). Collagen IV, -VII, laminins and fibronectin were detectable in the basement membrane of Cryo-AM, but only collagen IV and fibronectin in AD-AM. CONCLUSIONS: Cryo-AM is a more suitable substrate for the cultivation of HLECs than AD-AM. The higher outgrowth rate of cultured limbal epithelium, release of intact soluble wound-healing modulating factors and a better preservation of basement membrane components suggest the superiority of Cryo-AM for use in ophthalmology in comparison to AD-AM.

Characterization of the corneal surface in limbal stem cell deficiency and after transplantation of cultivated limbal epithelium.

PURPOSE: Transplantation of in vitro-cultivated limbal epithelium (TCLE) recently was developed to treat limbal stem cell deficiency (LSCD). The objective of this study was to characterize changes in the cornea during LSCD and on the corneal surface after TCLE. DESIGN: Experimental study. PARTICIPANTS AND CONTROLS: The pannus tissue excised from the corneas of 17 LSCD patients was analyzed to characterize the changes in the cornea during LSCD. Five corneal buttons obtained during perforating keratoplasty (pKP) from patients who had undergone TCLE at least 6 months before pKP were examined to assess the effect of TCLE. Six samples of healthy central cornea and 6 of bulbar conjunctiva served as control tissue. METHODS: The expression of epithelial lineage markers (keratin [K] 3, K12, K19, and mucin 5AC) and inflammatory markers (interleukin-1alpha [IL-1alpha], IL-1beta, intercellular adhesion molecule 1 [ICAM-1], vascular cell adhesion molecule 1 [VCAM-1], and vascular endothelial growth factor [VEGF]) were analyzed using real-time polymerase chain reaction, Western blotting, and immunofluorescence in the tissue samples. MAIN OUTCOME MEASURES: Comparison of the markers' expression patterns. RESULTS: The expression of all markers differed in healthy cornea and conjunctiva. Expression of lineage markers was similar in pannus to conjunctiva, but not to cornea. Interleukin-1beta, ICAM-1, VCAM-1, and VEGF were increased significantly in pannus compared with the levels in healthy cornea. Interleukin-1alpha, IL-1beta, and ICAM-1 were increased compared with healthy conjunctiva. The TCLE improved vision and reduced inflammation, vascularization, and discomfort. After TCLE, the lineage markers in the excised corneal buttons showed a corneal phenotype and a significant reduction in inflammatory markers in 4 of 5 cases. CONCLUSIONS: Limbal stem cell deficiency is characterized by ingrowth of abnormal inflamed tissue with a conjunctival phenotype. Transplantation of limbal epithelium cultivated in vitro on intact amniotic membrane restored a noninflamed ocular surface and a corneal phenotype. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Expression of membrane-associated mucins in limbal stem cell deficiency and after transplantation of cultivated limbal epithelium.

PURPOSE: Membrane-associated mucins are important components of the ocular tear film. Our objective was to characterize the expression of membrane-associated mucins MUC1, MUC4, and MUC16 in healthy cornea, healthy conjunctiva, fibrovascular tissue (pannus) covering the corneal surface in limbal stem cell deficiency (LSCD), human limbal epithelial cells cultivated on intact amniotic membrane (HLEC-AM), and corneal epithelium after transplantation of cultivated limbal epithelium (TCLE). METHODS: Total RNA was isolated from six samples of healthy human cornea, healthy conjunctiva, pannus, limbal epithelial cell cultures, and four corneal samples after TCLE. The expression of MUC1, MUC4, and MUC16 was evaluated by real-time polymerase chain reaction (PCR), Western blotting, and immunofluorescence. RESULTS: Conjunctiva showed a significantly higher expression of MUC1 and MUC4 than cornea, but the expression of MUC16 was similar in both tissues. The expression of MUC1 in HLEC-AM increased, MUC16 decreased, and MUC4 showed no significant difference when compared to cornea. The expression of all studied mucins in conjunctiva was significantly higher than in HLEC-AM. Pannus revealed a similar expression pattern of membrane-associated mucins to conjunctiva. The expression of all studied mucins in pannus was significantly higher than in cornea or HLEC-AM. A corneal expression pattern of membrane-associated mucins was found in all four samples of cornea after TCLE. CONCLUSION: A tissue-specific expression of MUC1 and MUC4 but not MUC16 was found in human cornea and conjunctiva. LSCD led to a conjunctival expression pattern of mucins on the corneal surface. A corneal phenotype could be restored after TCLE, confirming the potential of this method to reconstruct the corneal surface.

Correlation of cell cycle kinetics with p63 expression in human limbal epithelial cells expanded on intact human amniotic membrane.

AIM: To analyse the correlation of p63 expression and cell cycle kinetics of human limbal epithelial cells (HLECs) expanded on amniotic membrane (AM) or plastic. METHODS: Primary HLECs were cultured either on cryopreserved intact AM or plastic dishes for 2 weeks. Cells were labelled with 5 microM 5-bromo-2'-deoxyuridine (BrdU) for 3 days, followed by an interval of either 7 or 14 days in BrdU-free medium. The expression of p63 and BrdU labelling was detected by immunocytochemistry. RESULTS: More cells on AM (56%) than on plastic (24%) retained their BrdU label after the 7-day interval (p < 0.001). The difference was even more pronounced after 14 days (20 and 2%, p < 0.001). All BrdU-labelled cells were also p63 positive. 2.5-fold more cells on AM (54%) than on plastic (21%) were BrdU positive/p63 positive after 7 days (p < 0.001). It increased to a 9-fold difference after 14 days (p < 0.001). The BrdU label was lost more quickly than the p63 expression during the observation period, indicating that p63 expression was not confined to stem cells but existed also in transient amplifying cells. CONCLUSIONS: The combination of p63 expression and BrdU label retention is a better criterion to characterize stemness than either marker alone. AM as a culture substrate preserves stemness better than plastic.

Ocular surface reconstruction in graft-versus-host disease with HLA-identical living-related allogeneic cultivated limbal epithelium after hematopoietic stem cell transplantation from the same donor.

PURPOSE: To report a case of HLA-identical allogeneic living-related ex vivo expanded limbal epithelium in ocular surface reconstruction for chronic graft-versus-host disease (cGVHD). METHODS: Review the clinical findings in a 58-year-old woman with bilateral limbal stem cell deficiency caused by cGVHD who underwent ocular surface reconstruction on the left eye with cultivated limbal epithelial cells (LECs) on intact human amniotic membrane combined with extracapsular cataract extraction and intraocular lens implantation. LECs were harvested from a small biopsy of the same HLA-identical living-related donor who already donated peripheral blood cells for hematopoietic stem cell transplantation. RESULTS: At the present state, after a follow-up of 31 months, the patient shows a successful ocular surface reconstruction with a clear, smooth, and stable corneal ocular surface without recurrence of limbal stem cell deficiency. Nine months postoperatively, patients' corneal thinning progressed to a small perforation, which was successfully treated with a tectonic perforating keratoplasty combined with the removal of irritating lid lashes. CONCLUSION: The technique of HLA-typed allogeneic ex vivo expansion of LECs harvested from the same living-related donors who already donated hematopoietic stem cells offers a possibility to reconstruct ocular surface in cGVHD.

Reciprocal translocation t(7;16)(q21.2;p13.3) in an infertile man.

OBJECTIVE: To report the first case of reciprocal translocation t(7;16)(q21.2;p13.3) associated with male factor infertility. DESIGN: Case report. SETTING: University genetics laboratory and university andrology unit. PATIENT(S): A 38-year-old man with infertility and oligoasthenoteratozoospermia, but otherwise healthy. INTERVENTION(S): Chromosome analyses from peripheral blood lymphocyte cultures using Giemsa (G)-banding (GTG) and C-banding (CBG) and fluorescent in situ hybridization (FISH) were performed. MAIN OUTCOME MEASURE(S): Sperm count, motility, morphology, GTG and CBG banding, and FISH. RESULT(S): We report an apparently unique reciprocal translocation t(7;16)(q21.2;p13.3) confirmed by FISH in an infertile man. Semen analyses showed oligoasthenoteratozoospermia, with sperm count ranging from 2 x 10(6)/mL to 5 x 10(6)/mL and head defects (98%) in sperm morphology. CONCLUSION(S): In the present patient the breakpoint at 16p13.3 could have disrupted or harbored the PRM1, PRM2, or TNP2 genes responsible for the replacement of the histones involved in packaging the DNA into the sperm head. Resulting haploinsufficiency of these genes is likely to be the cause of sperm head defects and infertility in the patient. This case supports the opinion that alterations in the expression of protamine genes may be one of the causes of male factor infertility.