<b>Generation of iPSC-Derived Limbal Epithelial Stem Cells via a Transparent Defined Protocol Optimized by Stromal Cell Co-Culture</b>

Purpose: To establish a scientifically transparent, chemically defined protocol for generating induced pluripotent stem cell (iPSC)-derived limbal epithelial stem cells (iLESCs) to address the manufacturing limitations associated with proprietary commercial media. Methods: We developed a chemically defined limbal differentiation medium (CDLDM) with fully disclosed supplementation. Differentiation efficiency and purity were compared against a proprietary standard (CnT-30). The protocol was optimized using a corneal stromal cell (CSC) co-culture system. Finally, the developmental plasticity of the generated iLESCs was validated through a subsequent prolonged terminal differentiation phase. Results: CDLDM-derived cells robustly expressed limbal markers (P63, ABCG2) and achieved differentiation efficiency equivalent to the proprietary control while yielding superior epithelial lineage purity. CSC co-culture maintained iLESC identity while upregulating hypoxia-inducible factor-1α (HIF-1α) and nuclear factor erythroid 2-related factor 2 (NRF2) pathways, promoting metabolic reprogramming, cytoskeletal spreading, and off-target lineage suppression. Upon subsequent terminal differentiation, these iLESCs successfully matured, evidenced by decreased stemness markers and the emergence of mature corneal markers (CK3 and CK12). Conclusions: This transparent platform efficiently generates high-purity iLESCs capable of terminal corneal maturation. Furthermore, it elucidates the stromal-mediated metabolic and adhesive signaling essential for maintaining stem cell quality.