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

[摘要] Purpose: To determine whether mouseembryonic stem cell conditioned medium (ESC-CM) increases theproliferative capacity of human corneal endothelial cells (HCECs) invitro. Methods: Primary cultures of HCECs wereestablished from explants of the endothelial cell layer, including theDescemet’s membrane. Cells were cultured in human corneal endotheliummedium (CEM) containing 25% ESC-CM for the experimental group and CEMalone for the control group. Phase-contrast microscopy andreverse-transcription polymerase chain reaction (RT–PCR) were used toidentify HCECs. The eruption time and HCEC morphology were observedunder phase-contrast microscopy. We detected the protein expression ofzona occludens protein-1 (ZO-1; a tight junction protein) and the Na+-K+-ATPasebywestern blot analysis and immunocytochemistry. The mRNA expressionof the Na+-K+-ATPase, voltage-dependentanion channel 3 (VDAC3), solute carrier family 4, sodiumbicarbonate cotransporter member 4 (SLC4A4), and chloridechannel protein 3 (CLCN3) were detected by RT–PCR. To explorethe proliferation capacity of HCECs, the colony forming efficiency(CFE) was determined by Giemsa staining and the cellular proliferationmarker of Ki-67 protein (Ki-67) positive cells were detected byimmunocytochemistry and flow cytometry. Progression of the cell cycleand apoptosis were analyzed by flow cytometry. Negative regulation ofthe cell cycle, as measured by cyclin-dependent kinase inhibitor p21(p21) levels, was detected by western blot analysis andimmunocytochemistry. Results: In primary culture, HCECs inthe 25%ESC-CM group erupted with polygonal appearance on day 2, whilethose 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 6without enlargement of cell volume, while those in the CEM group wereenlarged and lost their polygonal appearance by passage 2. HCECs inboth the 25%ESC-CM and CEM groups expressed ZO-1, Na+-K+-ATPase,VDAC3,SLC4A4, and CLCN3. The number of Ki67 positive cells, CFE, andpercentage of cells entering the S and G2 phases were higherin the 25%ESC-CM group than in the CEM group. The number of apoptoticcells and p21 protein expression both decreased in the 25%ESC-CM group.Conclusions: Use of 25%ESC-CMsignificantly increased the number of proliferating cells. Theseeffects may be achieved through inhibition of p21 expression andapoptosis. These results suggested that 25%ESC-CM may be a new tool forcultivating HCECs for transplantation.

[学科分类] 生物化学/生物物理