PurposeTo evaluate the performance of reconstructed tissue-engineered human corneal endothelium (TE-HCE) by corneal transplantation in cat models.MethodsTE-HCE reconstruction was performed by culturing 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-labeled monoclonal HCE cells on denuded amniotic membranes (dAMs) in 20% fetal bovine serum-containing Dulbecco’s Modified Eagle’s Medium/Ham’s Nutrient Mixture F12 (1:1) medium and 5% CO2 at 37 °C on a 24-well culture plate. The reconstructed TE-HCE was transplanted into cat corneas via lamellar keratoplasty with all of the endothelium and part of Descemet’s membrane stripped. Postsurgical corneas were monitored daily with their histological properties examined during a period of 104 days after transplantation.ResultsThe reconstructed TE-HCE at a density of 3,413.33±111.23 cells/mm2 in average established intense cell-cell and cell-dAM junctions. After lamellar keratoplasty surgery, no obvious edema was found in TE-HCE-transplanted cat corneas, which were transparent throughout the monitoring period. In contrast, intense corneal edema developed in dAM-transplanted cat corneas, which were turbid. The corneal thickness gradually decreased to 751.33±11.37 μm on day 104 after TE-HCE transplantation, while that of dAM eye was over 1,000 μm in thickness during the monitoring period. A monolayer of endothelium consisting of TE-HCE-originated cells at a density of 2,573.33±0.59 cells/mm2 attached tightly to the surface of remnant Descemet’s membrane over 104 days; this was similar to the normal eye control in cell density.ConclusionsThe reconstructed TE-HCE was able to function as a corneal endothelium equivalent and restore corneal function in cat models.

Abstract:To evaluate the therapeutic efficiency of tissue-engineered human corneal endothelia (TE-HCEs) on rabbit primary corneal endotheliopathy (PCEP), TE-HCEs reconstructed with monoclonal human corneal endothelial cells (mcHCECs) and modified denuded amniotic membranes (mdAMs) were transplanted into PCEP models of New Zealand white rabbits using penetrating keratoplasty. The TE-HCEs were examined using diverse techniques including slit-lamp biomicroscopy observation and pachymeter and tonometer measurements in vivo, and fluorescent microscopy, alizarin red staining, paraffin sectioning, scanning and transmission electron microscopy observations in vitro. The corneas of transplanted eyes maintained transparency for as long as 200 d without obvious edema or immune rejection. The corneal thickness of transplanted eyes decreased gradually after transplanting, reaching almost the thickness of normal eyes after 156 d, while the TE-HCE non-transplanted eyes were turbid and showed obvious corneal edema. The polygonal corneal endothelial cells in the transplanted area originated from the TE-HCE transplant. An intact monolayer corneal endothelium had been reconstructed with the morphology, cell density and structure similar to those of normal rabbit corneal endothelium. In conclusion, the transplanted TE-HCE can reconstruct the integrality of corneal endothelium and restore corneal transparency and thickness in PCEP rabbits. The TE-HCE functions normally as an endothelial barrier and pump and promises to be an equivalent of HCE for clinical therapy of human PCEP.

Human corneal endothelium (HCE) is vital in maintaining corneal transparency and thickness, and damages to it may result in endotheliopathy and even corneal blindness, which can only be cured by keratoplasty. Due to the shortage of donor corneas, tissueengineered HCE (TE-HCE) has become an equivalent to HCE. This study constructs a high endothelial cell density (ECD) TE-HCE and evaluates its functions by corneal transplantation in rabbits. A TE-HCE was constructed by culturing DiI-labeled nontransfected HCE cells on modified denuded amniotic membrane (mdAM) using collagen IV, fibronectin, and laminin, and rabbit corneas were characterized both in vivo and ex vivo after TE-HCE transplantation. Our results indicated that the constructed TE-HCE with a high ECD of 3611 ± 56.66 cells/mm2 had similar morphology and structure to a native HCE. In vivo postsurgery detections showed that the TE-HCE-transplanted cornea gained transparency and recovered its thickness gradually during a monitoring period of 358 days, while the mdAM-transplanted cornea remained opaque and edematous. Ex vivo examinations revealed that a native-like corneal endothelium with an ECD of 2703 ± 70.37 cells/mm2 was reconstructed by the transplanted TE-HCE. Our findings suggested that the TE-HCE might be used as a HCE equivalent and has promising applications in therapy of corneal endotheliopathy.

Background Recently, many patients with corneal blindness caused by endothelial dysfunction have no opportunity to receive keratoplasty therapy because of the extremely limited number of donor corneas. Corneal tissue engineering opens a new path for in vitro reconstruction of tissue‐engineered HCE which will cure the corneal endotheliopathy by clinical corneal transplantation. In this study, we construct a human corneal endothelium (HCE) equivalent with non‐transfected monoclonal HCE (mcHCE) cells and modified denuded amniotic membrane (mdAM), and evaluate its functions in monkey models. Methods Tissue‐engineered HCE (TE‐HCE) was constructed by culturing DiI‐labeled mcHCE cells on mdAMs in 20% fetal bovine serum‐containing DMEM/Ham’s Nutrient Mixture F12 (1:1) medium and 5% CO2 at 37°C on a 24‐well culture plate. The constructed TE‐HCE was transplanted into monkey corneas via penetrating keratoplasty with Descemet’s membrane and endothelium stripped. The corneal transparency, thickness, and intraocular pressure were monitored in vivo, and the corneal morphology and histological structure were examined ex vivo 181 days after surgery. Results The constructed TE‐HCE, with an average density of 3602.22 ± 45.22 cells/mm2, mimicked its natural counterpart both in morphology and histological structure. In vivo, corneal transparency was maintained, and the corneal thickness gradually decreased to 567.33 ± 72.77 μm at day 181 after TE‐HCE transplanted into monkey eyes, while intense corneal edema and turbid were found in mdAM‐transplanted eyes with their corneal thicknesses maintained over 1000 μm during the monitoring period. Ex vivo, a monolayer of corneal endothelium, consisting of mcHCE cells at a density of 2795.65 ± 156.83 cells/mm2, was reconstructed in transplanted monkey eyes. The cells in the transplanted area had the hexagonal or polygonal morphology and normal ultrastructure, and established plenty of cell‐cell and cell‐stromal matrix junctions. Besides, huge membrane‐bounded flat stacks with electric dense inclusions were found in mcHCE cells beneath the plasma membrane at the stromal side. Conclusions The constructed TE‐HCE has normal histological property and functions well in monkey models. The TE‐HCE could be used as a promising HCE equivalent in therapy of corneal endothelium dysfunction and corneal regenerative medicine.

Skin epidermal stem cells (SESCs), which share a single origin with corneal epithelial cells (CECs), are considered to be one of the most ideal seed cells for the construction of tissue engineered corneas. However, the mechanism underlying the transdifferentiation of SESCs to CECs has not been fully elucidated. In the present study, to identify critical microRNAs (miRNAs/miRs) and genes that regulate the transdifferentiation of SESCs to CECs, SESCs and CECs were collected from sheep and used for small RNA sequencing and mRNA microarray analyses. Among the differentially expressed miRNAs and genes, 36 miRNAs were downregulated and 123 genes were upregulated in the CECs compared with those in the SESCs. miR-10b exhibited the largest change in expression between the cell types. Target genes of the 36 downregulated miRNAs were predicted and a computational approach demonstrated that these target genes may be involved in several signaling pathways, including the 'PI3K signaling pathway', the 'Wnt signaling pathway' and the 'MAPK signaling pathway', as well as in 'focal adhesion'. Comparison of these target genes to the 123 upregulated genes identified 43 intersection genes. A regulatory network of these 43 intersection genes and its correlative miRNAs were constructed, and three genes (dedicator of cytokinesis 9, neuronal differentiation 1 and activated leukocyte cell adhesion molecule) were found to have high interaction frequencies. The expression levels of 7 randomly selected miRNAs and the 3 intersection genes were further validated by reverse transcription-quantitative polymerase chain reaction. It was found that miR-10b, the Wnt signaling pathway and the 3 intersection genes may act together and serve a critical role in the transdifferentiation process. This study identified miRNAs and genes that were expressed in SESCs and CECs that may assist in uncovering its underlying molecular mechanism, as well as promote corneal tissue engineering using epidermal stem cells for clinical applications.