This study developed a chemically defined limbal differentiation medium (CDLDM) with fully disclosed composition for generating iPSC-derived limbal epithelial stem cell-like cells (iLESCs), compared it to the proprietary CnT-30 medium, and examined the effect of non-contact corneal stromal cell co-culture on iLESC transcriptional programs and downstream air-liquid interface differentiation.
No significant difference was found in P63-positive cell proportions between CDLDM-iLESCs and CnT-30-iLESCs (P = 0.61); both expressed limbal/basal markers (P63, ABCG2, CK14, CK15). Stromal co-culture increased HO-1 protein expression significantly and upregulated pathways for ECM organization, glycolysis, hypoxia, and antioxidant signaling. All three iLESC groups showed reduced P63/ABCG2 and detectable CK3/CK12 after air-liquid interface differentiation, consistent with corneal epithelial maturation.
Full paper text was inaccessible (publisher paywall), so granular quantitative data and methodological details could not be verified. The study uses cell-line-level in vitro models without in vivo functional validation of the generated iLESCs. NRF2 upregulation was nonsignificant by Western blot, limiting conclusions about this pathway.
CDLDM offers a transparent, customizable alternative to proprietary media for iLESC generation that achieves comparable P63+ yield, which may improve reproducibility across labs working on corneal stem cell therapies. Stromal co-culture should be considered when modeling the limbal niche, as it meaningfully alters iLESC transcriptional programs.
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