Background <p>Vision loss due to retinal and corneal cell degeneration is significant clinical challenge, with current cell therapies hindered by limited donor cells. To address this, we developed a streamlined platform using human induced pluripotent stem cells (hiPSCs) that integrates 2D and 3D culture techniques to simultaneously generate retinal ganglion cells (RGC) and corneal lineages.</p> Methods <p>The non-integrated hiPSCs were used to ocular cells differentiation, four time-points cells were collected for 10×Genomics sing-cell RNA sequencing to trace RGC and corneal lineage development. The confirmed differentiated window phase to pick optic vesicles for 3D ocular organoids culture, and as well as directional induced corneal epithelium in remaining 2D dish. After FACS-based cell sorting, the enriched RGC-like and corneal cells were propagated in vitro, and these cells were transplanted into optic nerve crush (ONC) and corneal damaged mice respectively to observe the regenerative repairment capacity.</p> Results <p>Through single-cell RNA sequencing, we mapped differentiation trajectories and identified surface markers-CD184 and CD171 for RGCs, and CD104 for corneal progenitors facilitating purification. In mouse models, transplanted hiPSC-derived CD184⁺CD171⁺ RGC-like cells integrated into injured retinas, enhanced host RGC survival, and restored visual function following optic nerve injury. Concurrently, hiPSC-derived CD104⁺ corneal progenitor cells exhibited self-renewal, differentiation capabilities, and accelerated corneal repair with reduced neovascularization. Additionally, this platform enables the synchronous production of retinal and corneal organoids, which are valuable for both regenerative therapy and disease modeling.</p> Conclusions <p>Our study establishes a cost-effective surface marker-based method for deriving transplantable RGC and corneal lineage cells from hiPSCs, overcoming key obstacles in ocular regenerative medicine.</p>