Dysfunction of Human corneal endothelial cells (HCECs) is a crucial cause of corneal blindness, and the current treatment includes partial or complete transplantation of the cornea. However, transplantation is limited by low availability of donor corneas. Therefore, bio-engineered sustainable corneal endothelial graft will be of great clinical interest. In the present study, we developed a novel serial culture media approach for the proliferation of HCECs in five conditions: (A) native HCECs, (B) culture cells in initiation medium, (C) culture cells in proliferation medium, (D) seeding of expanded cells on scaffold in proliferating medium, and (E) culture of cell/scaffold in condition D in a stabilizing medium. Each condition was performed in triplicates, except E with only one replicate. The differences in global gene expression patterns throughout the serial cultures and the experimental conditions were analyzed by transcriptome profiling through RNA sequencing. Engaged genes were mostly related to entire cell-cycle stages, including the regulators of cell cycle and cell cycle-related signaling pathways, suggesting their association with cell proliferation.