Abstract
The ocular surface ectoderm (OSE) is essential for the development of the ocular surface, yet the molecular mechanisms driving its differentiation are not fully understood. In this study, we used single-cell transcriptomic analysis to explore the dynamic cellular trajectories and regulatory networks during the in vitro differentiation of embryonic stem cells (ESCs) into the OSE lineage. We identified nine distinct cell subpopulations undergoing differentiation along three main developmental branches: neural crest, neuroectodermal, and surface ectodermal lineages. Key marker gene expression, transcription factor activity, and signaling pathway insights revealed stepwise transitions from undifferentiated ESCs to fate-specified cell types, including a PAX6 + TP63 + population indicative of OSE precursors. Comparative analysis with mouse embryonic development confirmed the model's accuracy in mimicking in vivo epiblast-to-surface ectoderm dynamics. By integrating temporal dynamics of transcription factor activation and cell-cell communication, we constructed a comprehensive molecular atlas of the differentiation pathway from ESCs to distinct ectodermal lineages. This study provides new insights into the cellular heterogeneity and regulatory mechanisms of OSE development, aiding the understanding of ocular surface biology and the design of cell-based therapies for ocular surface disorders.