Cell surface markers of functional phenotypic corneal endothelial cells. Invest Ophthalmol Vis Sci 2014

PURPOSE. Cultured human corneal endothelial cells (HCECs) are anticipated to serve as an alternative to donor corneas for the treatment of corneal endothelial dysfunction. However, corneal endothelial cells (CECs) tend to exhibit fibroblastic transformation, thereby losing their functional phenotype when cultured. The purpose of this study was to investigate the usefulness of surface markers of CECs displaying fibroblastic phenotypes as a means of cell characterization. METHODS. The expression levels of 242 cell surface antigens were screened in cultured human and monkey CECs using flow cytometry. An expression intensity ratio of nonfibroblastic/ fibroblastic CECs > 2 and of fibroblastic/nonfibroblastic CECs > 2 were selected as indicating nonfibroblastic and fibroblastic markers, respectively. Nonfibroblastic and fibroblastic CECs were mixed, and CD73-positive and -negative cells were sorted using flow cytometry and further cultured. The functional phenotype of the sorted cells was evaluated according to morphology and the expression of function-related (Na þ /K þ -ATPase and ZO-1) and fibroblastic (type I collagen and fibronectin) markers. RESULTS. Flow cytometry analysis demonstrated that CD98, CD166, and CD340 are elevated in HCECs of nonfibroblastic phenotype, while CD9, CD49e, CD44, and CD73 are markers of fibroblastic phenotype HCECs. The CECs that sorted as CD73-negative exhibited normal hexagonal morphology and expressed functional markers, whereas CECs that sorted as CD73-positive exhibited the fibroblastic phenotype. CONCLUSIONS. These markers will be useful for quality control to characterize the phenotype of cells destined for tissue engineering-based therapy. In addition, this selection protocol will provide a novel method for purification of functional cells

Cultivation of corneal endothelial cells on a pericellular matrix prepared from human decidua-derived mesenchymal cells.

The barrier and pump functions of the corneal endothelium are essential for the maintenance of corneal transparency. Although corneal transplantation is the only current therapy for treating corneal endothelial dysfunction, the potential of tissue-engineering techniques to provide highly efficient and less invasive therapy in comparison to corneal transplantation has been highly anticipated. However, culturing human corneal endothelial cells (HCECs) is technically difficult, and there is no established culture protocol. The aim of this study was to investigate the feasibility of using a pericellular matrix prepared from human decidua-derived mesenchymal cells (PCM-DM) as an animal-free substrate for HCEC culture for future clinical applications. PCM-DM enhanced the adhesion of monkey CECs (MCECs) via integrin, promoted cell proliferation, and suppressed apoptosis. The HCECs cultured on the PCM-DM showed a hexagonal morphology and a staining profile characteristic of Na⁺/K⁺-ATPase and ZO-1 at the plasma membrane in vivo, whereas the control HCECs showed a fibroblastic phenotype. The cell density of the cultured HCECs on the PCM-DM was significantly higher than that of the control cells. These results indicate that PCM-DM provides a feasible xeno-free matrix substrate and that it offers a viable in vitro expansion protocol for HCECs while maintaining cellular functions for use as a subsequent clinical intervention for tissue-engineered based therapy of corneal endothelial dysfunction

Antiapoptotic effect of PCM-DM.

<p>(A) To evaluate the effect of PCM-DM on the apoptosis of MCECs, serum was removed from the culture medium to induce apoptosis. TUNEL staining was performed to evaluate DNA fragmentation during apoptosis after 24, 48, and 72 h. Scale bar: 50 µm. (B) The percentages of TUNEL-positive apoptotic cells were evaluated (<i>n</i> = 3). The experiment was performed in duplicate. *<i>p</i><0.01.</p

Characteristics of cultivated HCECs on PCM-DM.

<p>(A) An image of corneal endothelium of a normal human subject was obtained by in vivo contact specular microscopy. Scale bar: 100 µm. (B) The HCECs (passage 3) were passaged on the PCM-DM and on the control culture dish and cultured for 30 days to form a monolayer sheet. Representative phase contrast images are shown. Scale bar 100 µm. The experiments were performed in triplicate. (C) The HCECs were passaged on the noncoated plate (control), collagen 1, collagen 4, fibronectin, and PCM-DM and cultured for 30 days. The cell densities of the HCECs were evaluated by KSS-400EB software. The experiments were performed in triplicate. (D) ZO-1 and Na⁺/K⁺-ATPase at the plasma membrane was stained in the HCEC culture on the PCM-DM and on the control culture plate. Scale bar 50 µm.</p

Preparation of pericellular matrix prepared from human decidua-derived mesenchymal cells (PCM-DM) for culture substrate.

<p>(A) Schematic of the structure of human fetal membrane (FM). Human FM consists of three main layers: amniotic, chorionic, and decidual membranes. (B) Representative phase-contrast image of human decidua-derived mesenchymal cells (DMCs). The human decidual tissues were isolated from the FMs. The DMCs were isolated from the decidua and cultured with DMEM/F-12 (1:1)-based culture medium supplemented with 10% fetal bovine serum (FBS) (scale bar, 200 µm). (C) The procedure for preparation of the PCM-DM from the DMCs. The DMCs were plated at a density of 3.5×10⁴ cells/cm² onto the culture plate coated with 0.1% gelatin and cultured for 3 days after reaching confluence. They were then lysed with deoxycholate solution (0.5% sodium deoxycholate in 10 mM Tris-HCl, pH 8.0). PCM-DM coated dishes stored under semidry conditions at 4°C for less than 8 months were used for the experiments. (D) The DMCs were plated at a density of 3.5×10⁴ cells/cm² onto Corning^® Transwell^® inserts coated with 0.1% gelatin and cultured for 3 days after reaching confluence, and then lysed with deoxycholate solution. PCM-DM was evaluated by fibronectin and collagen 4 staining (scale bar, 50 µm).</p

Effect of PCM-DM on the promotion of cell proliferation and migration.

<p>(A+B) To test the proliferative potential, the MCECs cultured on the PCM-DM were immunostained with 5-ethynyl-2 Click-iT^® EdU Imaging Kits, and the percentages of EdU-positive cells were evaluated after 24 h of incubating with 10 µM EdU. The experiments were performed in duplicate. Scale bar: 100 µm; *<i>p</i><0.01. (C) The MCECs were seeded at a density of 5.0×10³ cells/well. The proliferation of the MCECs was evaluated by a BrdU incorporation assay after 24 h of incubating with 10 µM BrdU. The experiment was performed in duplicate. *<i>p</i><0.01. (D+E) The MCECs were cultured for 14 days after reaching confluence, and the monolayer cells were wounded by scratching. After 12 and 18 h, the wound distance was quantified by Image J software (<i>n</i> = 10). The experiment was performed in triplicated. *<i>p</i><0.01; scale bar: 200 µm.</p