N-Cadherin is expressed by putative stem/progenitor cells and melanocytes in the human limbal epithelial stem cell niche.

Corneal epithelial stem cells are known to be localized to the basal layer of the limbal epithelium, providing a model system for epithelial stem cell biology; however, the mechanisms regarding the maintenance of these stem cells in their specialized niche remain poorly understood. N-cadherin is a member of the classic cadherin family and has previously been demonstrated to be expressed by hematopoietic stem cells. In the present study, we demonstrate that N-cadherin is expressed by putative stem/progenitor cells, as well as melanocytes, in the human limbal epithelial stem cell niche. In addition, we demonstrate that upon in vitro culture using 3T3 feeder layers, loss of N-cadherin expression occurs with cell proliferation. These results indicate that N-cadherin may be a critical cell-to-cell adhesion molecule between corneal epithelial stem/progenitor cells and their corresponding niche cells in the limbal epithelium.

Functional human corneal endothelial cell sheets harvested from temperature-responsive culture surfaces.

This study reports a new method for fabricating bioengineered human corneal endothelial cell sheets suitable for ocular surgery and repair. We have initially cultured human corneal endothelial cells on type IV collagen-coated dishes and, after several passages, expanded cells were then seeded onto novel temperature-responsive culture dishes. Four weeks after reaching confluence, these cultured endothelial cells were harvested as intact monolayer cell sheets by simple temperature reduction without enzymatic treatment. Scanning electron microscopy indicated that these cells were primarily hexagonal with numerous microvilli and cilia, similar to the native corneal endothelium. The Na+, K+-ATPase pump sites were located at the cell borders as in vivo. Moreover, cell densities and numbers of pump sites were identical to those of in vivo human corneal endothelium under optimized conditions. A 3H-ouabain binding analysis demonstrated a linear proportionality for cell pump density between confluent cell densities of 575 cells/mm2 and 3070 cells/mm2. We also confirmed Na+, K+-ATPase activity in the sheets in vitro. Xenograft transplantation results showed that the fabricated sheets retain their function of maintaining proper stromal hydration in vivo. We have established a regimen to culture and proliferate human corneal endothelial cells and fabricate endothelial sheets ex vivo morphologically and functionally similar to the native corneal endothelium. Our results support the value of harvested cell sheets for clinical applications in ocular reconstructive surgery in patients with ocular endothelial decompensation.

Structural characterization of bioengineered human corneal endothelial cell sheets fabricated on temperature-responsive culture dishes.

For the purpose of corneal regenerative medicine, we fabricated human corneal endothelial cell sheets on temperature-responsive dishes, which could be non-invasively harvested as intact, transplantable sheets by simply reducing the culture temperature. Cells demonstrated hexagonal cell shape with numerous microvilli and cilia, and also exhibited abundant cytoplasmic organelles similar to these cells in vivo. Immunofluorescence for type IV collagen and fibronectin revealed that abundant extracellular matrix (ECM) was deposited on the basal surface throughout culture, and the deposited ECM was harvested along with the cell sheets by reducing culture temperature to 20 degrees C. Faint ECM remnants were observed on the dish surfaces after cell sheet detachment. Immunofluorescence for ZO-1 showed that tight junctions were established between cells, and immunoblotting indicated that intact ZO-1 was maintained during cell sheet harvest, while conventional proteolytic cell harvest methods resulted in the degradation of ZO-1. These results suggest that these transplantable corneal endothelial cell sheets can be applied to treat patients with damaged corneas.

Human limbal epithelium contains side population cells expressing the ATP-binding cassette transporter ABCG2.

Many types of organ-specific stem cells have been recently shown to exhibit a side population (SP) phenotype based on their ability to efflux Hoechst 33342 dye. Because stem cells from corneal epithelium reside in the basal layer of the limbal epithelium, the purpose of this study was to examine whether the limbal epithelium contains SP cells. The ATP-binding cassette transporter Bcrp1/ABCG2 is reported to contribute to the SP phenotype in cells from several diverse sources. Here we show data from fluorescence-activated cell sorting and real-time quantitative RT-PCR analysis showing that harvested limbal epithelial cells contain SP cells expressing ABCG2. Immunofluorescence revealed that a portion of limbal epithelial basal cells expressed ABCG2. Data indicate that ABCG2 positive limbal epithelial cells are putative corneal epithelial stem cells.

Effects of multipurpose solutions (MPS) for hydrogel contact lenses on gap-junctional intercellular communication (GJIC) in rabbit corneal keratocytes.

To ensure the effects of multipurpose solutions (MPS) for hydrogel contact lenses on the cornea, the inhibitory activity of three types of MPS on corneal cells has been evaluated with the use of scrape loading and dye transfer assay (SLDT assay) and Western blotting on rabbit corneal keratocytes (RC4). In SLDT assay, MPS-A and poloxamine showed dose-dependent inhibitory activity, suggesting the inhibitory action of MPS-A and poloxamine to gap junctional intercellular communication (GJIC) in the tested cells. Moreover, after treatment with MPS-A, the GJIC was initially inhibited within 4 h, and thereafter gradually turned to an approximately 60% level of the initial value. When MPS-A was removed from the incubation media after exposure of the cells, the recovery of GJIC was time dependent and returned to approximately initial levels at 8 h. Complete recovery was established after approximately 24 h. These findings suggested that the inhibitory action of MPS-A on corneal keratocytes was reversible. This inhibition was accompanied by a decrease in the quantity of connexin-43, which is a major protein constituting the gap junctional channel of these cells, and its change in the phosphorylation state. Taken together, it was suggested that MPS-A interacts with connexin-43, inducing an inhibitory action on GJIC.