Takeshi Soma

Co-ordinated ocular development from human iPS cells and recovery of corneal function.

The eye is a complex organ with highly specialized constituent tissues derived from different primordial cell lineages. The retina, for example, develops from neuroectoderm via the optic vesicle, the corneal epithelium is descended from surface ectoderm, while the iris and collagen-rich stroma of the cornea have a neural crest origin. Recent work with pluripotent stem cells in culture has revealed a previously under-appreciated level of intrinsic cellular self-organization, with a focus on the retina and retinal cells. Moreover, we and others have demonstrated the in vitro induction of a corneal epithelial cell phenotype from pluripotent stem cells. These studies, however, have a single, tissue-specific focus and fail to reflect the complexity of whole eye development. Here we demonstrate the generation from human induced pluripotent stem cells of a self-formed ectodermal autonomous multi-zone (SEAM) of ocular cells. In some respects the concentric SEAM mimics whole-eye development because cell location within different zones is indicative of lineage, spanning the ocular surface ectoderm, lens, neuro-retina, and retinal pigment epithelium. It thus represents a promising resource for new and ongoing studies of ocular morphogenesis. The approach also has translational potential and to illustrate this we show that cells isolated from the ocular surface ectodermal zone of the SEAM can be sorted and expanded ex vivo to form a corneal epithelium that recovers function in an experimentally induced animal model of corneal blindness.

Clinical features and management of cytomegalovirus corneal endotheliitis: analysis of 106 cases from the Japan corneal endotheliitis study

Aims The purpose of this study is to elucidate the clinical manifestations and the current treatment status of cytomegalovirus (CMV) endotheliitis via a large case series obtained from a national survey conducted in Japan. Methods The Japan Corneal Endotheliitis Study Group proposed diagnostic criteria for CMV endotheliitis based on a viral examination by PCR of aqueous humour, in combination with clinical manifestations. A national survey was then retrospectively conducted among 1160 members of the Japan Cornea Society. The study reviewed the patient profiles, clinical manifestations, and treatment modalities of individuals who met the diagnostic criteria for CMV endotheliitis. Results The study included 109 eyes of 106 patients. Mean patient age was 66.9±10.9 years (85 males (80.2%), 21 females (19.8%)). Patients were commonly diagnosed with anterior uveitis and ocular hypertension prior to confirmation of CMV endotheliitis. Coin-shaped lesions were observed in 70.6%, and linear keratic precipitates in 8.3% of the patients, respectively. 95% of cases were treated with anti-CMV drugs. Conclusions CMV endotheliitis is most common in middle-aged and elderly men. CMV endotheliitis should be suspected when patients present with corneal endotheliitis involving coin-shaped lesions accompanied by anterior uveitis and ocular hypertension.

Ultrahigh-resolution imaging of human donor cornea using full-field optical coherence tomography

A feasibility study of ultrahigh-resolution full-field optical coherence tomography (FF-OCT) for a subcellular-level imaging of human donor corneas is presented. The FF-OCT system employed in this experiment is based on a white light interference microscope, where the sample is illuminated by a thermal light source and a horizontal cross-sectional (en face) image is detected using a charge coupled device (CCD) camera. A conventional four-frame phase-shift detection technique is employed to extract the interferometric image from the CCD output. A 95-nm-broadband full-field illumination yields an axial resolution of 2.0 microm, and the system covers an area of 850 microm x 850 microm with a transverse resolution of 2.4 microm using a 0.3-NA microscope objective and a CCD camera with 512 x 512 pixels. Starting a measurement from the epithelial to the endothelial side, a series of en face images was obtained. From detected en face images, the epithelial cells, Bowmans layer, stromal keratocyte, nerve fiber, Descemets membrane, and endothelial cell were clearly observed. Keratocyte cytoplasm, its nuclei, and its processes were also separately detected. Two-dimensional interconnectivity of the keratocytes is visualized, and the keratocytes existing between collagen lamellaes are separately extracted by exploiting a high axial resolution ability of FF-OCT.

Expression of membrane-associated mucins in cultivated human oral mucosal epithelial cells.

Purpose: Membrane-associated mucins present in the cells of the ocular surface epithelium (MUC1, -4, and -16) are believed to contribute to the maintenance of the health and wet surface of epithelial cells. Recent studies have reported the use of cultivated oral mucosal epithelial sheet transplantation for reconstruction of the ocular surface. We studied the expression of membrane-associated mucins in cultivated human oral mucosal epithelial sheets and compared it with that in human ocular surface epithelial cells. Methods: Specimens (3 × 3 mm) of oral mucosal tissue were harvested from healthy volunteers. The oral mucosal epithelial cells obtained from the specimens, the corneal epithelial cells, or an oral mucosal cell line (KB cells) were cultured together with mitomycin C-treated 3T3 feeder cells on temperature-responsive culture surfaces for 2 weeks to produce stratified cell sheets. Reverse transcriptase-polymerase chain reaction was used to determine the expression of membrane-associated mucins (MUC1, -3, -4, -12, -13, -15, -16, and -17) in these cells. Results: MUC1, -4, and -16 but not -3, -12, -13, -15, or -17 mRNA was detected in the oral mucosal epithelial sheet and in the corneal epithelial sheet. KB cells, although unable to produce a stratified cell sheet, showed expression of MUC1, -12, -13, and -16 mRNA. Conclusions: The membrane-associated mucins of the ocular surface, MUC1, -4, and -16, are expressed in human oral mucosal epithelial sheets and corneal epithelium. These membrane-associated mucins may therefore contribute to ocular surface reconstruction after oral mucosal epithelial sheet transplantation for patients with severe ocular surface disorders.

Effect of diquafosol ophthalmic solution on the optical quality of the eyes in patients with aqueous-deficient dry eye

To investigate the short‐ and long‐term effects of diquafosol ophthalmic solution on the optical quality of the eyes in patients with aqueous‐deficient dry eye.

Effect of non-invasive tear stability assessment on tear meniscus height.

To investigate the effect of non‐invasive tear stability assessment with forced eye opening on the lower tear meniscus.

Ocular forward light scattering and corneal backward light scattering in patients with dry eye.

PURPOSE To evaluate ocular forward light scattering and corneal backward light scattering in patients with dry eye. METHODS Thirty-five eyes in 35 patients with dry eye and 20 eyes of 20 healthy control subjects were enrolled. The 35 dry eyes were classified into two groups according to whether superficial punctate keratopathy in the central 6-mm corneal zone (cSPK) was present or not. Ocular forward light scattering was quantified with a straylight meter. Corneal backward light scattering from the anterior, middle, and posterior corneal parts was assessed with a corneal densitometry program using the Scheimpflug imaging system. RESULTS Both dry eye groups had significantly higher intraocular forward light scattering than the control group (both P<0.05). The dry eye group with cSPK had significantly higher values in anterior and total corneal backward light scattering than the other two groups. Moderate positive correlations were observed between the cSPK score and corneal backward light scattering from the anterior cornea (R=0.60, P<0.001) and corneal backward light scattering from the total cornea (R=0.54, P<0.001); however, no correlation was found between cSPK score and ocular forward light scattering (R=0.01, P=0.932). CONCLUSIONS Ocular forward light scattering and corneal backward light scattering from the anterior cornea were greater in dry eyes than in normal eyes. Increased corneal backward light scattering in dry eye at least partially results from cSPK overlying the optical zone.

Effect of instillation of eyedrops for dry eye on optical quality.

PURPOSE To investigate the effects of viscosity and suspensibility of eyedrops for dry eye by evaluating an eyedrop with one of the solutions or no solution (0.3% sodium hyaluronate ophthalmic solution, 3% diquafosol ophthalmic solution, and 2% rebamipide ophthalmic suspension) on ocular higher-order aberrations (HOAs) and forward light scatter. METHODS We evaluated ocular HOAs and forward light scatter before and 1, 5, and 10 minutes after instillation of three eyedrops for dry eye in 15 healthy subjects. Saline served as the control. The HOAs were measured for a 4-mm pupil using a wavefront sensor. The obtained aberration data were analyzed in the central 4-mm diameter for total HOAs up to the sixth-order Zernike polynomials. Forward light scatter was quantified with a straylight meter. RESULTS A significant increase was seen in the HOAs 1 minute after instillation of the three eyedrops for dry eye; the HOAs recovered to the baseline level thereafter. When 0.3% sodium hyaluronate was compared with 2% rebamipide and 3% diquafosol, the HOAs increased significantly (P < 0.01 for both comparisons) immediately after instillation. A significant increase in forward light scatter occurred 1 minute after instillation of rebamipide suspension and returned to the preinstillation level 5 minutes after instillation. No significant changes in forward light scatter occurred after instillation of 3% diquafosol or 0.3% sodium hyaluronate. CONCLUSIONS Quantitative serial measurement of HOAs and forward light scatter showed that the temporal reduction in optical quality may be attributed mainly to increased HOAs after instillation of highly viscous 0.3% sodium hyaluronate ophthalmic solution and to increased forward light scatter after instillation of 2% rebamipide ophthalmic suspension in healthy subjects.

Human adipose tissue‐derived mesenchymal stem cells as a novel feeder layer for epithelial cells

We examined a novel human feeder cell layer of mesenchymal stem cells harvested from human adipose tissues. Gene expression analyses and colony‐forming assay with human primary epithelial cells showed that the adipose tissue‐derived mesenchymal stem cells produced various factors to support epithelial stem/progenitor maintenance and cell growth. Using the mesenchymal stem cells as novel feeder layers, transplantable epithelial cell sheets could be effectively generated ex vivo on temperature‐responsive cell‐culture surfaces. Copyright

Differential expression of MUC16 in human oral mucosal epithelium and cultivated epithelial sheets

Cultivated oral mucosal epithelial sheet transplantation is a new surgical strategy to treat severe ocular surface disorders such as chemical burns, ocular cicatricial pemphigoid, and Stevens-Johnson syndrome. MUC16 is thought to be the most important membrane-associated mucin on the ocular surface because it forms a protective barrier on the epithelial cell surface. In this study, we studied MUC16 expression in mRNA and protein levels and compared the expression patterns between cultivated oral mucosal epithelial cell sheet and oral mucosal tissue. Specimens (5x5 mm) of oral mucosal tissue harvested from healthy volunteers were used. The oral mucosal epithelial cells were cultured on temperature-responsive culture dishes to generate stratified cell sheets. Cultivated oral mucosal epithelial cells formed three- to five-cell thick stratified sheets for 2 weeks. Scanning electron micrographs revealed that the apical surfaces of the oral mucosal tissue and the oral mucosal sheets were covered with dense microvilli/microplicae. Real-time PCR showed significantly more MUC16 transcripts in the cultivated oral mucosal sheets and corneal epithelial sheets than in the oral mucosal tissue (P=0.023 and 0.008, respectively, Mann-Whitney rank sum test). These findings were confirmed by immunohistochemical examination using an MUC16 antibody to the protein. MUC16 protein was localized to the apical cells of the oral mucosal sheets, but the human oral mucosal tissue did not express MUC16 protein in any cell layers. In this study, interestingly, the expression of membrane-associated mucin MUC16 differs between human oral mucosal epithelia and cultivated epithelial sheets. MUC16 expressed in the oral mucosal sheets may contribute to ocular surface reconstruction after oral mucosal sheet transplantation.