: To develop techniques to culture the human corneal endothelial cells and to identify the cultured human corneal endothelial cells maintain the property of stem cells by ex vivo expansion on amniotic membrane. Materials and Methods: Human donor corneas were selected for studies. To understand the possibility of human corneal endothelium contains stem cells, we set up an organ culture technique for human donor cornea. Human donor cornea was cultured in culture medium with BrdU for labeling 2 weeks then chasing for another 2 weeks. BrdU labeling retention cells will be studied under immunomicroscopy. To further study the human corneal endothelial stem cells can be preserved and cultured on amniotic membrane, human corneal endothelium was obtained from the inner part of human limbal corneal rim, when the central cornea was prepared for PkP. Human corneal endothelium was cut into a small pieces and cultured on the different kind of substrate including amniotic membrane. Series passages were performed, and markers of Na/K ATPase, P63, and ABCG2 were used for immunochemistry studies. Cultured human corneal endothelial cells finally were growth on the human corneal disc. Results: Age of human donor corneas was from 37 y/o to 70 y/o with mean 63 ± 11 y/o. The death to cultured period was from 6 days to 10 days with mean 8.4 ± 1.8 days. The cultured human corneal endothelial cells (HCEC) could be growth on the substrate with basement membrane substance. Serial passages from each HCEC were performed and results with 4.5 ± 2.3 (2 to 9 passages) had been achieved from the aged human corneas. For the study of organ culture of human donor cornea, BrdU labeling retention cells were detected only on the peripheral area of donor corneal endothelial zone, but negative over the central corneal area. To explore the corneal endothelial cells contain stem cells on the cultured system, cultured corneal endothelial cells at different culture period was studied for Na/K ATPase, ABCG2 and P63. The positive staining of ABCG2 and P63 were detected only on the earlier 2 weeks culture period but negative on the longer one month culture period. However, Na/K ATPase detected only on the longer cultured period. When HCEC was cultured on the human corneal disc, TEM demonstrated mosaic pattern of HCEC on the cornea disc Conclusion: The ex vivo expansion of human corneal endothelial cells systems have been established. The HCEC may maintain the stem cells property on AM. Also, the cultured HCEC could be transplanted on the human corneal disc. These techniques will allow us to further study the engineering of cornea.