Ex vivo cultivated retinal pigment epithelial cell transplantation for the treatment of rabbit corneal endothelial dysfunction

Abstract Objective Stem cell therapy is a promising strategy for the treatment of corneal endothelial dysfunction, and the need to find functional alternative seed cells of corneal endothelial cells (CECs) is urgent. Here, we determined the feasibility of using the retinal pigment epithelium (RPE) as an equivalent substitute for the treatment of corneal endothelial dysfunction. Methods RPE cells and CECs in situ were obtained from healthy New Zealand male rabbits, and the similarities and differences between them were analyzed by electron microscopy, immunofluorescent staining, and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Rabbit primary RPE cells and CECs were isolated and cultivated ex vivo, and Na+/K+-ATPase activity and cellular permeability were detected at passage 2. The injection of cultivated rabbit primary RPE cells, CECs and human embryonic stem cell (hESC)-derived RPE cells was performed on rabbits with corneal endothelial dysfunction. Then, the therapeutic effects were evaluated by corneal transparency, central corneal thickness, enzyme linked immunosorbent assay (ELISA), qRT-PCR and immunofluorescent staining. Results The rabbit RPE cells were similar in form to CECs in situ and ex vivo, showing a larger regular hexagonal shape and a lower cell density, with numerous tightly formed cell junctions and hemidesmosomes. Moreover, RPE cells presented a stronger barrier and ionic pumping capacity than CECs. When intracamerally injected into the rabbits, the transplanted primary RPE cells could dissolve corneal edema and decrease corneal thickness, with effects similar to those of CECs. In addition, the transplantation of hESC-derived RPE cells exhibited a similar therapeutic effect and restored corneal transparency and thickness within seven days. qRT-PCR results showed that the expressions of CEC markers, like CD200 and S100A4, increased, and the RPE markers OTX2, BEST1 and MITF significantly decreased in the transplanted RPE cells. Furthermore, we have demonstrated that rabbits transplanted with hESC-derived RPE cells maintained normal corneal thickness and exhibited slight pigmentation in the central cornea one month after surgery. Immunostaining results showed that the HuNu-positive transplanted cells survived and expressed ZO1, ATP1A1 and MITF. Conclusion RPE cells and CECs showed high structural and functional similarities in barrier and pump characteristics. Intracameral injection of primary RPE cells and hESC-derived RPE cells can effectively restore rabbit corneal clarity and thickness and maintain normal corneal function. This study is the first to report the effectiveness of RPE cells for corneal endothelial dysfunction, suggesting the feasibility of hESC-derived RPE cells as an equivalent substitute for CECs

Characteristics of bacterial community in eyelashes of patients with Demodex blepharitis

Abstract Background Demodex blepharitis (DB) is a common disease of the ocular surface. The characteristics of the bacterial community in eyelash roots after Demodex infestation are still unknown. Knowledge of the characteristics of the bacterial community of eyelash follicles in patients with DB can provide valuable insights for guiding the diagnosis and treatment of DB. Methods Twenty-five patients with DB (DB group) and 21 non-DB volunteers (control group) were enrolled in the study. Eyelashes from the upper eyelid of the right eye were sampled, and 16S ribosomal DNA (rDNA) sequencing was performed to determine the V3-V4 regions of the microbial 16S rDNA gene within 1 month of infestation. The sequencing data of the two groups were analyzed and compared. The effect of the bacterium Burkholderia on the survival of Demodex mites was evaluated using Demodex obtained from 12 patients with DB other that the patients in the DB group. Results A total of 31 phyla and 862 genera were identified in the DB and control groups. The five most abundant phyla in the two groups were Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes and Cyanobacteria. The abundance of Actinomycetes was significantly higher in the DB group than in the control group. At the genus level, the five most abundant genera in the two groups were Pseudomonas, Burkholderia-Caballeronia-Paraburkholderia, Rolstonia and Acinetobacter; Clostridium sensu stricto 1 was abundant in the control group and Corynebacterium_1 was abundant in the DB group. Compared with the control group, the abundance of Burkholderia-Caballeronia-Paraburkholderia was 2.36-fold lower in the DB group. Linear discriminant analysis Effect Size (LEfSe) analysis revealed Burkholderia-Caballeronia-Paraburkholderia, SC_I_84_unclassified, Nonmyxobacteria and Succinvibrio to be the major biomarkers in the control group and Catenibacterium and Lachnospiraceae NK4A136 group to be the major biomarkers in the DB group. To explore the performance of these optimal marker models, receiver operational characteristic curve analysis was performed, and the average area under the curve value of Burkholderia-Caballeronia-Paraburkholderia was 0.7448. Burkholderia cepacia isolated from normal human eyelashes was fermented, and the Demodex mites isolated from patient eyelashes were cultured together with its fermented supernatant. The results showed that the fermentation supernatant could significantly reduce the survival time of the Demodex mites, suggesting the potential therapeutic value of this bacterium against Demodex. Conclusions The composition of the bacterial community in the eyelashes of DB patients differed from that in eyelashes of healthy volunteers, revealing a decrease in bacterial diversity in infested eyelashes. This decrease may be related to the occurrence and development of DB. The supernatant of Burkholderia cepacia culture medium was found to inhibit the growth of Demodex in eyelash hair follicles, providing a new insight with potential applications for the clinical treatment of Demodex infestation. Graphical Abstrac

Additional file 1 of Characteristics of bacterial community in eyelashes of patients with Demodex blepharitis

Additional file 1. Supplementary materials for identification of Burkholderia and survival time of each Demodex