Enhanced survival in vitro of human corneal endothelial cells using mouse embryonic stem cell conditioned medium

PURPOSE: To determine whether mouse embryonic stem cell conditioned medium (ESC-CM) increases the proliferative capacity of human corneal endothelial cells (HCECs) in vitro. METHODS: Primary cultures of HCECs were established from explants of the endothelial cell layer, including the Descemet’s membrane. Cells were cultured in human corneal endothelium medium (CEM) containing 25% ESC-CM for the experimental group and CEM alone for the control group. Phase-contrast microscopy and reverse-transcription polymerase chain reaction (RT–PCR) were used to identify HCECs. The eruption time and HCEC morphology were observed under phase-contrast microscopy. We detected the protein expression of zona occludens protein-1 (ZO-1; a tight junction protein) and the Na(+)-K(+)-ATPase by western blot analysis and immunocytochemistry. The mRNA expression of the Na(+)-K(+)-ATPase, voltage-dependent anion channel 3 (VDAC3), solute carrier family 4, sodium bicarbonate cotransporter member 4 (SLC4A4), and chloride channel protein 3 (CLCN3) were detected by RT–PCR. To explore the proliferation capacity of HCECs, the colony forming efficiency (CFE) was determined by Giemsa staining and the cellular proliferation marker of Ki-67 protein (Ki-67) positive cells were detected by immunocytochemistry and flow cytometry. Progression of the cell cycle and apoptosis were analyzed by flow cytometry. Negative regulation of the cell cycle, as measured by cyclin-dependent kinase inhibitor p21 (p21) levels, was detected by western blot analysis and immunocytochemistry. RESULTS: In primary culture, HCECs in the 25%ESC-CM group erupted with polygonal appearance on day 2, while those in the CEM group erupted with slightly larger cells on day 3–4. HCECs in the 25%ESC-CM group could be subcultured until passage 6 without enlargement of cell volume, while those in the CEM group were enlarged and lost their polygonal appearance by passage 2. HCECs in both the 25%ESC-CM and CEM groups expressed ZO-1, Na(+)-K(+)-ATPase, VDAC3, SLC4A4, and CLCN3. The number of Ki67 positive cells, CFE, and percentage of cells entering the S and G(2) phases were higher in the 25%ESC-CM group than in the CEM group. The number of apoptotic cells and p21 protein expression both decreased in the 25%ESC-CM group. CONCLUSIONS: Use of 25%ESC-CM significantly increased the number of proliferating cells. These effects may be achieved through inhibition of p21 expression and apoptosis. These results suggested that 25%ESC-CM may be a new tool for cultivating HCECs for transplantation.