Ryuhei Hayashi

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.

A novel method of culturing human oral mucosal epithelial cell sheet using post-mitotic human dermal fibroblast feeder cells and modified keratinocyte culture medium for ocular surface reconstruction

Background/aims To cultivate human oral mucosal epithelial cell sheets with post-mitotic human dermal fibroblast feeder cells and modified keratinocyte culture medium for ocular surface reconstruction. Methods Human oral mucosal epithelial cells obtained from three healthy volunteers were cultured with x-ray-treated dermal fibroblasts (fibroblast group) and NIH/3T3 feeder layers (3T3 group) on temperature-responsive culture dishes. Media were supplemented using clinically approved products. Colony-forming efficiency was determined in both groups. Histological and immunohistochemical analyses were performed for cell sheets. Cell viability and purity of cell sheets were evaluated by flow cytometry. Results Colony-forming efficiency in the fibroblast group was similar to that in the 3T3 group. All cell sheets were well stratified and harvested successfully. The expression patterns of keratin 1, 3/76, 4, 10, 12, 13, 15, ZO-1 and MUC16 were equivalent in both groups. The percentage of p63-positive cells in the fibroblast group (46.1±4.2%) was significantly higher than that in the 3T3 group (30.7±7.6%) (p=0.038, t test). The cell viability and purity were similar between the two groups. Conclusion This novel culture method using dermal fibroblasts and pharmaceutical agents provides a safe cell processing system without xenogenic feeder cells for ocular surface reconstruction.

Neural crest-derived multipotent cells in the adult mouse iris stroma.

The purpose of this study was to characterize neural crest–derived cells within the adult murine iris. The iris was isolated from P0‐Cre/Floxed‐EGFP transgenic (TG) mice. The isolated iris cells formed EGFP‐positive spheres on non‐adhesive culture plates. Immunostaining showed that these EGFP‐positive spheres expressed neural crest markers including Sox10 and p75NTR, and these cells showing in vitro sphere‐forming ability were originally resided in the iris stroma (IS), in vivo. Real‐time RT‐PCR showed that the EGFP‐positive spheres expressed significantly higher levels of the neural crest markers than EGFP‐negative spheres and bone marrow–derived mesenchymal stem cells. Furthermore, the iris stromal sphere had capability to differentiate into various cell lineages including smooth muscle and cartilage. These data indicate that neural crest–derived multipotent cells can be isolated from the murine IS and expanded in sphere culture.

Induction of putative stratified epithelial progenitor cells in vitro from mouse-induced pluripotent stem cells

Induced pluripotent stem (iPS) cells generally exhibit a normal karyotype, are transcriptionally and epigenetically similar to embryonic stem (ES) cells, and maintain the potential to differentiate into derivatives of all germ layers. Recently, the use of different types of cell or tissue derived from iPS cells for transplantation has become a possibility. However, the differentiation of epithelial lineages from iPS cells has not yet been demonstrated. We attempted to establish a culture technique for the induction of epithelial progenitors from mouse iPS cells. Mouse iPS cells were cultured on dishes coated with type IV collagen in keratinocyte culture medium (KCM) supplemented with or without bone morphogenic protein-4 (BMP-4) or combined with pretreatment of retinoic acid (RA) and BMP-4 in the undifferentiated state. Markers for undifferentiated cells (Oct3/4, Nanog) and for differentiation (p63, cytokeratin14) were analyzed by immunofluorescence staining and real-time RT-PCR. Putative epithelial progenitors were successfully induced in vitro from iPS cells. These progenitors expressed p63, a transcription factor necessary for maintenance of regenerative epithelia and cytokeratin 14 constitutively present in the basal layer of stratified epithelia. Enhancement of putative epithelial progenitor commitment was observed when cultured in KCM with BMP-4 following pretreatment of RA and BMP-4. The differentiation efficiency of putative epithelial progenitors from iPS cell cultures was similar to that of ES cell cultures. This report is the first to demonstrate in vitro differentiation of iPS cells into putative epithelial progenitors. These iPS-derived putative epithelial progenitors provide a powerful tool for understanding the mechanisms of epithelial lineage differentiation.

Analysis of soluble vascular endothelial growth factor receptor-1 secreted from cultured corneal and oral mucosal epithelial cell sheets in vitro

Background: In clinical trials, eyes transplanted with cultured oral mucosal epithelial cell sheets have shown increased neovascularisation compared with eyes treated with cultured corneal epithelial cell sheets. As reported recently, soluble vascular endothelial growth factor receptor-1 (soluble VEGFr-1) is a main factor to maintain a corneal avascularity. Aim: To investigate soluble VEGFr-1 of cultured corneal epithelial cells (CCE) and cultured oral mucosal epithelial cells (COE) in vitro. Methods: Rabbit corneal and oral mucosal epithelial cells were co-cultured with mitomycin C-treated NIH/3T3 cells on culture plates. After CCE and COE were multilayered, culture medium was replaced by basal medium and incubated. Protein secretion of soluble VEGFr-1 was assessed in conditioned medium from CCE and COE by ELISA. Angiogenic potential was examined by invasion, migration assays with human umbilical vein endothelial cells (HUVECs) in addition to recombinant soluble VEGFr-1. Results: CCE secreted a significantly higher amount of soluble VEGFr-1 than did COE. Recombinant soluble VEGFr-1 significantly suppressed HUVEC migration induced by COE, without suppression in CCE. In conclusion, these findings suggest that low protein levels of soluble VEGFr-1 may lead to corneal neovascularisation after COE sheet transplantation.

Histological evaluation of mechanical epithelial separation in epithelial laser in situ keratomileusis

PURPOSE: To evaluate the effect of mechanical epithelial separation with an epikeratome on the histologic ultrastructure of epithelial flaps and stromal beds from human corneas. SETTING: Departments of Ophthalmology, Osaka University Medical School, Osaka, and Tohoku University School of Medicine, Sendai, and Institute of Advanced Biomedical Engineering and Science and Medical Research Institute, Tokyo Womens Medical University, Tokyo, Japan. METHODS: Eye‐bank eyes were deepithelialized using an Epi‐K epikeratome. Epithelial flaps and stromal beds were assessed by light and electron microscopy. Immunofluorescence staining for types IV and VII collagens, integrins α6 and β4, and laminin 5 was also performed. RESULTS: Four eyes were evaluated. On scanning electron microscopy, the cleavage planes of epithelial flaps and stromal beds were relatively smooth. On transmission electron microscopy, epithelial flaps were separated partially within the lamina fibroreticularis and partially within the lamina lucida. Immunofluorescence showed positive staining for type VII collagen and discontinuous staining for type IV collagen in stromal beds. Discontinuous linear staining for types IV and VII collagens was observed in epithelial flaps. Staining for integrins α6 and β4 was positive in some regions and discontinuous in other regions of epithelial flaps. In stromal beds, integrins α6 and β4 had a patchy expression pattern. Staining for laminin 5 was intermittently positive along the basal side of epithelial flaps and stromal beds. CONCLUSIONS: Epithelial flaps created with an epikeratome were mechanically separated partly within the lamina fibroreticularis and partly within the lamina lucida. Stromal beds had relatively smooth surfaces with no obvious trauma to Bowman layer.