AIM To investigate whether human embryonic stem cells(hESCs) could be made to attach, grow and differentiate on a human Descemet’s membrane(DM).METHODS Spontaneously differentiated hESCs were transferred onto a human corneal button with the endothelial layer removed using ocular sticks. The cells were cultured on a DM for up to 15 d. The genetically engineered hESC line expressed green fluorescent protein, which facilitated identification during the culture experiments, tissue preparation, and analysis. To detect any differentiation into human corneal endothelial-like cells, we analysed the transplanted cells by immunohistochemistry using specific antibodies.RESULTS We found transplanted cells form a single layer of cells with a hexagonal shape in the periphery of the DM. The majority of the cells were negative for octamer-binding transcription factor 4 but positive for paired box 6 protein, sodium potassium adenosine triphosphatase(NaKATPase), and Zona Occludens protein 1. In four of the 18 trials, the transplanted cells were found to express CK3, which indicates that the stem cells differentiated into corneal epithelial cells in these cases. CONCLUSION It is possible to get cells originating from hESCs to become established on a human DM, where they grow and differentiate into corneal endothelial-like cells in vitro. AIM To investigate whether human embryonic stem cells (hESCs) could be made to attach, grow and differentiate on a human Descemet’s membrane (DM). METHODS Spontaneously differentiated hESCs were transferred onto a human corneal button with the endothelial layer removed using ocular sticks. cells were cultured on a DM for up to 15 d. The genetically engineered hESC line expressed green fluorescent protein, which facilitated identification during the culture experiments, tissue preparation, and analysis. To detect any differentiation into human corneal endothelial-like cells, we analyzed the transplanted cells by immunohistochemistry using specific antibodies. RESULTS We found transplanted cells form a single layer of cells with a hexagonal shape in the periphery of the DM. The majority of the cells were negative for octamer-binding transcription factor 4 but positive for paired box 6 protein, sodium potassium adenosine triphosphatase (NaKATPase), and Zona Occludens protein 1. In four of the 18 trials, the transplanted cells were found to express CK3, which indicates that the stem cells differentiated into corneal epithelial cells in these cases. CONCLUSION It is possible to get cells originating from hESCs to become established on a human DM, where they grow and differentiate into corneal endothelial-like cells in vitro.