In vitro generation of pancreatic endocrine cells from human adult fibroblast-like limbal stem cells.

Stem cells might provide unlimited supply of transplantable cells for ß-cell replacement therapy in diabetes. The human limbus is a highly specialized region hosting a well-recognized population of epithelial stem cells, which sustain the continuous renewal of the cornea, and the recently identified stromal fibroblast-like stem cells (f-LSCs), with apparent broader plasticity. However, the lack of specific molecular markers for the identification of the multipotent limbal subpopulation has so far limited the investigation of their differentiation potential. In this study we show that the human limbus contains uncommitted cells that could be potentially harnessed for the treatment of diabetes. Fourteen limbal biopsies were obtained from patients undergoing surgery for ocular diseases not involving the conjunctiva or corneal surface. We identified a subpopulation of f-LSCs characterized by robust proliferative capacity, expressing several pluripotent stem cell markers and exhibiting self-renewal ability. We then demonstrated the potential of f-LSCs to differentiate in vitro into functional insulin-secreting cells by developing a four-step differentiation protocol that efficiently directed f-LSCs towards the pancreatic endocrine cell fate. The expression of specific endodermal, pancreatic, islet, and ß-cell markers, as well as functional properties of f-LSC-derived insulin-producing cells, were evaluated during differentiation. With our stage-specific approach, up to 77% of f-LSCs eventually differentiated into cells expressing insulin (also assessed as C-peptide) and exhibited phenotypic features of mature ß-cells, such as expression of critical transcription factors and presence of secretory granules. Although insulin content was about 160-fold lower than what observed in adult islets, differentiated cells processed ∼98% of their proinsulin content, similar to mature ß-cells. Moreover, they responded in vitro in a regulated manner to multiple secretory stimuli, including glucose. In conclusion, f-LSCs represent a possible relevant source of autologous, transplantable, insulin-producing cells that could be tested for the reversal of diabetes.

OCT is not useful for detection of minimal diabetic retinopathy in type 1 diabetes.

Optical coherence tomography (OCT) has been proven useful in measuring retinal thickness (RT) in patients with diabetes, although with discordant results in different studies. We examined RT in patients with type 1 diabetes (T1D) with or without minimal diabetic retinopathy (MDR) to test whether OCT is able to identify early retinal changes and potential correlations with metabolic parameters. RT of 102 patients with T1D (53 females, 49 males, aged 27.03 +/- 7.4 years) and of 42 healthy controls was examined, with analysis of nine different sectors (fovea, four pericentral and four peripheral sectors). According to the results of basal fundus photography, patients were divided into two groups, without MDR (48 cases) and with MDR (54 cases). Patients with proliferative DR or macular edema were excluded. No difference was found between patients with or without MDR and the control group for all OCT parameters investigated. Mean HbA1c of the last 5 years (P < 0.001), microalbuminuria (P = 0.002), total (P = 0.046) and LDL cholesterol (P = 0.007) and triglyceride (P < 0.001) levels were higher in patients with MDR, along with higher prevalence of hypertension (P = 0.013), younger age at diagnosis (P = 0.018) and longer diabetes duration (P < 0.001) with regard to the patients without MDR and controls, although no significant correlation between these parameters and RT was found. Our study suggests that MDR without macular edema in patients with T1D cannot be detected with OCT. Therefore, the conventional diagnostic methods are mandatory to detect early DR.

E-PTFE (Gore-Tex) implant with or without low-dosage mitomycin-C as an adjuvant in penetrating glaucoma surgery: 2 year randomized clinical trial.

PURPOSE: To test the expanded polytetrafluoroethylene (ePTFE) as a new adjuvant in trabeculectomy. METHODS: Consecutive glaucoma surgical inpatients were observed at the Department of Ophthalmology of Palermo University. Sixty patients (60 eyes) were randomly assigned to undergo trabeculectomy (T), trabeculectomy with mitomycin-C (TMMC), with ePTFE (TG) or with mitomycin-C and ePTFE (TGMMC). Postoperative visits were scheduled at 24 hr, 7 days, 1, 3, 6, 12, 18 and 24 months. Complete success and qualified success were assessed at two target intraocular pressure (IOP) levels -< or =21 and < or =17 mmHg - by Kaplan-Meier curves. RESULTS: The postoperative IOP reduction was significant (P < 0.01) at the endpoint in all groups, with a mean IOP of 16.9 (+/-2.9), 16.2 (+/-2.7), 15.3 (+/-3.4) and 15.2 (+/-4.3) mmHg in T, TMMC, TG and TGMMC eyes, respectively. No intergroup difference was found at either IOP targets. The Kaplan-Meier curves relating to either the < or =21 mmHg or the < or =17 mmHg target IOP did not show significant intergroup differences for complete and qualified success rate. When ePTFE was used, a trend favouring the medium-term survival rate was noted. No adverse reaction to the ePTFE was present, and no membrane extrusion or conjuctival erosion were noted in any cases. Hypotony was significantly more frequent (P = 0.035) in groups without ePTFE. Moreover, the late MMC-related complications were more frequent when MMC was applied. CONCLUSION: Expanded polytetrafluoroethylene implant in trabeculectomy is well tolerated and could act as a filtration modulating device. Therefore, it is useful in reducing early hypotony-related complications and contributes to attaining medium-term IOP control that is comparable to the low-dosage MMC.

Deep sclerectomy versus punch trabeculectomy with or without phacoemulsification: a randomized clinical trial.

PURPOSE: To compare the efficacy of non-penetrating deep sclerectomy without implant with Crozafon-De Laage punch trabeculectomy, and to evaluate the effect of simultaneous temporal approach phacoemulsification on both techniques. SETTING: Department of Ophthalmology of the University of Palermo. DESIGN: Prospective randomized clinical trial. Patients and intervention procedures: Sixty-five patients (65 eyes) with primary open angle glaucoma (POAG) or pseudoexfoliative glaucoma (PEXG): 32 eyes underwent non-penetrating deep sclerectomy (NPDS), 17 as single procedure and 15 combined with phacoemulsification (phaco-NPDS), and 33 eyes underwent punch trabeculectomy (PT), 18 single and 15 with phaco (phaco-PT). The patients were randomly assigned to each procedure. No adjuvants, such as Nd: YAG laser goniopuncture, laser suturelysis, and antimetabolites were used. MAIN OUTCOME MEASURES: Postoperative complications, number of antiglaucoma medications, and IOP level were checked at each control. Complete success indicated the achievement of the target IOP without antiglaucoma medications, while qualified success indicated the same goal with or without medications. These categories were assessed at two target IOP levels, namely < or =21 mm Hg and < or =17 mm Hg in all four groups. RESULTS: The mean follow-up period was 22.5 +/- 2.5 months. The mean preoperative IOP was 30.2 mm Hg in NPDS eyes, 26.8 in phaco-NPDS eyes, 32.1 in PT eyes, and 27.0 in phaco-PT ones, without significant intergroup difference. At the end point the mean IOP was 17.7 +/- 0.8, 15.7 +/- 0.9, 14.2 +/- 1.1, and 13.8 +/- 1.1 mm Hg respectively with postoperative IOP significantly lower (P = 0.005) than preoperative IOP in all groups. No difference was observed among groups at any observation time when simple and combined surgery were compared. Significant difference at the end point was found between NPDS and PT (P = 0.030). As for complete and qualified success with a < or =21 and < or =17 mm Hg target IOP no significant differences were noticed in all groups. Among postoperative complications, hypotony was significantly more frequent in both PT groups when compared with the NPDS groups. The same was true, but relating only to the single procedures, for shallow anterior chamber and choroidal detachment. The Kaplan-Meier cumulative survival curves relating to the qualified success rate in the four surgical groups for a < or =21 mm Hg target IOP (log rank, P = 0.564) and for a < or =17 mm Hg target IOP (log rank, P = 0.591) showed no significant intergroup differences. When the < or =21 mm Hg target IOP was considered, a mild positive trend in combined procedures (both phaco-NPDS and phaco-PT) was found in comparison with simple procedures. At lower IOP target (ie, < or =17 mm Hg) a better trend was found in favor of simple or combined PT procedure. CONCLUSIONS: Both techniques, NPDS and PT, without enhancements (ie, implants or antimetabolites) control IOP efficaciously at our end point. Phacoemulsification combined with penetrating and non-penetrating procedures does not seem to interfere with final results. When a lower target IOP and probability of success over time are considered, PT, single or combined, exhibits a better trend. PT, therefore, could be more suitable for higher IOP levels or longer life expectancies.