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

Abstract

Proprietary differentiation media limit compositional transparency and experimental reproducibility. In this study, we developed a chemically defined limbal differentiation medium (CDLDM) with fully disclosed supplementation for generating induced pluripotent stem cell (iPSC)-derived limbal epithelial stem cell-like cells (iLESCs) and compared it with the proprietary CnT-30 medium. Both conditions generated epithelial-like cells expressing the limbal/basal epithelial markers P63, ABCG2, CK14, and CK15, accompanied by reduced expression of pluripotency-associated markers. No significant difference was detected in the proportion of P63-positive cells between CDLDM-iLESCs and CnT-30-iLESCs (P = 0.61), although RT-qPCR and transcriptomic analyses revealed protocol-associated differences in individual lineage markers and biological programs. Compared with CnT-30-iLESCs, CDLDM-iLESCs exhibited lower collagen-, extracellular matrix-remodeling-, and muscle-related transcriptional signatures. To examine stromal influences within the CDLDM system, a non-contact corneal stromal cell co-culture model was established. Stromal co-culture was associated with changes in cell morphology and transcriptional programs related to extracellular matrix organization, glycolysis, hypoxia responses, and antioxidant signaling. The nuclear fluorescence fractions of HIF-1α, NRF2, and HO-1 were increased in co-cultured cells, while Western blotting demonstrated significantly increased HO-1 protein expression but only a nonsignificant upward trend in total NRF2 expression. Finally, CDLDM-iLESCs, CDLDM-CO-iLESCs, and CnT-30-iLESCs were subjected to downstream air-liquid interface differentiation in serum-containing corneal epithelial cell differentiation medium. At later differentiation stages, all three groups showed reduced P63 and ABCG2 signals and detectable CK3 and CK12 expression, consistent with acquisition of a more differentiated corneal epithelial marker profile. Collectively, CDLDM provides a disclosed and customizable medium formulation for the iLESC differentiation phase and supports downstream investigation of stromal interactions and corneal epithelial differentiation.