Seiichi Yokoo

Human corneal epithelial equivalents for ocular surface reconstruction in a complete serum-free culture system without unknown factors.

PURPOSE To establish a culture technique for human corneal epithelial equivalents that do not require fetal bovine serum (FBS), feeder cells, or bovine pituitary extracts and compare this system with conventional culture medium with FBS and mouse 3T3 fibroblasts. METHODS Human corneal limbal tissue from donor corneas was dissociated on denuded amniotic membranes and then cultured for 3 weeks in feeder-cell- and serum-free medium containing epidermal growth factor and B-27. Then, the cell sheet was evaluated by light microscopy, immunohistochemistry, and electron microscopy. The epithelial proliferative capacity was compared between serum- and feeder-cell-free medium and conventional medium. The cultured cell sheets were transplanted onto the denuded rabbit ocular surface to cover the resected area. RESULTS A stratified cell sheet expressing cytokeratin-3 and -12 was grown in serum- and feeder-cell-free medium without unknown growth factors. The epithelial proliferative capacity in feeder-cell- and serum-free medium determined by WST-1 and colony-forming efficiency was significantly higher than that in conventional medium. Scanning and transmission electron microscopy showed well-formed stratified epithelium with clear cell boundaries, microvilli, and hemidesmosomal/desmosomal junctions. The transplanted cell sheets remained transparent without epithelial defects during the follow-up period. CONCLUSIONS This method using serum- and feeder-cell-free medium not containing unknown growth factors allows the highly proliferative culture of human corneal epithelium. It avoids exposure of the corneal epithelial equivalent to FBS and animal feeder cells, thus minimizing the risk of contamination by pathogens that could transmit diseases to recipients.

Adhesion, migration, and proliferation of cultured human corneal endothelial cells by laminin-5.

PURPOSE To investigate the expression of laminin-5 (LM5) and its receptors by human corneal endothelial cells (HCECs) and whether recombinant human LM5 influences adhesion, proliferation, and migration of cultured HCECs. METHODS The expression of LM5 and its receptors was examined in human donor corneas by immunohistochemistry, reverse transcription-polymerase chain reaction, and flow cytometry. HCECs cultured under serum-free conditions were used for analysis of the biological effects of LM5. Changes in HCEC adhesion and proliferation due to LM5 were evaluated by counting the number of cells. HCEC migration was assessed by quantifying the percentage of wound closure in the wound-healing assay with an image-processing and -analysis software program. RESULTS Adult HCECs expressed the LM5 receptor α3β1 integrin, but not LM5 itself. Significantly more cells became adherent to recombinant LM5 (1.0 μg/mL)-coated dishes than to uncoated dishes in the cell adhesion assay. The proliferation of cultured HCECs was moderately promoted by LM5 (1.0 μg/mL) and soluble LM5 (20 ng/mL and 50 ng/mL) in the cell proliferation assay. A significantly higher percentage of wound closure was obtained with medium containing soluble LM5 than with control medium in the wound-healing assay. CONCLUSIONS HCECs express the LM5 receptor α3β1 integrin. Recombinant LM5 promotes adhesion, migration, and moderate proliferation of cultured HCECs. It may be a critical factor in promoting HCEC culture and may contribute to the practical use of tissue-engineered HCECs.

A mouse model of allogeneic corneal endothelial cell transplantation.

Objectives:We analyzed survival, therapeutic response, and prognostic factors in patients with HIV-related Hodgkin lymphoma (HL) treated or not with highly active antiretroviral therapy (HAART). Methods:This study included 104 patients with HL, treated (n = 83) or not (n = 21) with HAART. Outcomes and prognostic factors of complete remission (CR), overall survival (OS), and disease-free survival (DFS) were assessed by an intention-to-treat analysis of all patients who received at least 1 chemotherapy course. Results:No differences were found between groups at baseline in the specific characteristics of HIV and HL. The proportion of patients receiving appropriate-for-stage therapy for HL was similar for both groups. The CR rates in the HAART (−) and HAART (+) groups were 14 (70%) of 20 versus 71 (91%) of 78 (P = 0.023). The median OS in the HAART (−) group was 39 months (95% confidence interval [CI]: 0 to 89) and was not reached in the HAART (+) group (P = 0.0089). The median DFS in the HAART (−) group was 85 months (95% CI: 73 to 97) and was not reached in the HAART (+) group (P = 0.129). Factors independently associated with CR by logistic regression analysis were appropriate-for-stage therapy of HL, HAART, and baseline CD4 count ≥100 cells/μL. CR was the only factor independently associated with OS by Cox regression analysis. Conclusions:The achievement of CR was independently associated with appropriate-for-stage therapy for HL, with HAART, and with a baseline CD4 count ≥100 cells/μL. The only variable independently associated with OS was the achievement of CR.PURPOSE Corneal endothelial cell (CEC) transplantation should become clinically applicable in the near future. However, the immunologic changes after allo-CEC transplantation are poorly understood at present. We tried to establish a mouse model of allogeneic CEC transplantation for immunologic studies. METHODS Benzalkonium chloride was injected into the anterior chamber of the eyes of recipient BALB/c mice to create bullous keratopathy. Full-thickness corneal transplantation was performed by using 4 types of corneas: BALB/c corneas (isograft group), BALB/c corneas denuded of CEC (no endothelium group), C3H/He mouse corneas (allograft group), and corneas reconstituted by seeding immortalized C3H/He mouse CECs onto BALB/c corneas denuded of endothelium (CEC allograft group). Eyes were observed with an operating microscope for 4 weeks after transplantation and were subjected to histologic examination and fluorescein microscopy. RESULTS All corneal grafts were transparent in the isograft group (n = 12), whereas none of the grafts were clear by 4 weeks after transplantation in the no endothelium group (n = 13). Corneal grafts were transparent at 4 weeks in 75% of the CEC allograft group (n = 12). The histologic rejection rate was 0% in the CEC allograft group, which was significantly lower than in the allograft group (67%; n = 18; P < 0.01). CONCLUSIONS We established a mouse allo-CEC transplantation model by using cultured cells. This model should be useful for studying the immunologic processes after CEC transplantation.

Inhibition of Corneal Neovascularization by Blocking the Angiotensin II Type 1 Receptor

PURPOSE To determine the role of angiotensin II type 1 receptor (AT1R) signaling in corneal neovascularization. METHODS Corneal neovascularization was induced by suturing 10-0 nylon 1 mm away from limbal vessels in C57 BJ6 mice. Angiotensinogen and its receptor (AT1R) gene expression levels were evaluated by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). The expression of angiotensin II (Ang2) and AT1R was confirmed by Western blotting and immunohistochemistry. To investigate the function of Ang2 in corneal neovascularization, infiltrating macrophages in vascularized corneas and the neovascularized area were investigated after intraperitoneal injection of an AT1R antagonist (telmisartan, 10 mg/kg). Further, corneal mRNA of VEGF, MCP-1, IL-6, ICAM-1, and TNF-alpha was examined in control and telmisartan-treated mice. RESULTS Ang2 and AT1R markedly increased in the neovascularized corneas compared with normal corneas. Ang2 and AT1R were expressed in epithelium and stromal cells (vascular endothelium, infiltrating leukocytes, and keratocytes) in neovascularized cornea at protein levels and were weakly detected in normal corneal epithelium. Infiltrating macrophages were reduced in telmisartan-treated mice on day 7 after suturing. Neovascularized area in the cornea of telmisartan-treated mice was 70% smaller than that of control mice on day 7 after suturing. A PPAR-gamma antagonist partially, but significantly, reversed the suppressive effect of telmisartan on induction of corneal neovascularization. The expression of VEGF, MCP-1, IL-6, and ICAM-1 was significantly inhibited in telmisartan-treated mice. CONCLUSIONS These findings indicate that Ang2, abundantly expressed in neovascularized corneas, has a significant role in inflammation-related driven corneal neovascularization. AT1R may be a therapeutic target for the suppression of corneal neovascularization.

A Novel Isolation Technique of Progenitor Cells in Human Corneal Epithelium Using Non‐Tissue Culture Dishes

The existence of adult stem cells or progenitor cells in the human corneal epithelium (i.e., self‐renewing squamous cells) has long been suggested, but these cells have not yet been isolated. Here we describe a novel isolation technique using non‐tissue culture dishes to enrich progenitor cells, which are able to reconstitute a three‐dimensional human corneal epithelial equivalent from single cells in serum‐, feeder‐, and bovine pituitary extract‐free medium. These cells showed original tissue‐committed differentiation, a high proliferative capacity, and limited self‐renewal. Laminin‐5 was measured by mass spectrometric analysis. Pretreatment of cells with anti‐laminin‐5 antibody demonstrated that laminin‐5 was important in allowing corneal epithelial progenitor cells to adhere to non‐tissue culture dishes. Hydrophilic tubes (used for cell collection throughout this study) are essential for efficient isolation of adherent corneal epithelial progenitor cells expressing laminin‐5. These findings indicate that our new technique using non‐tissue culture dishes allows the isolation of progenitor cells from human corneal limbal epithelium and that laminin‐5 has a critical role in the adhesion of these cells.

Bilateral limbal stem cell deficiency with chromosomal translocation of 3p and 9p

We describe a case of bilateral limbal stem cell defi ciency (LSCD) with systemic abnormalities, including sensorineural deafness and pituitary dwarfi sm. The patient showed balanced translocation of 3p27 and 9p13, which to our knowledge has not been reported in the literature. We performed ocular surface reconstruction by autologous cultivated conjunctival epithelial cells (CjECs) transplantation on the amniotic membrane. To the best of our knowledge, this is also the fi rst report of cultivated CjECs transplantation for bilateral LSCD in humans.