Deciphering the heterogeneity of differentiating hPSC-derived corneal limbal stem cells through single-cell RNA-sequencing [Bulk-seq]

A comprehensive understanding of the human pluripotent stem cell (hPSC) differentiation process stands as a prerequisite for the development hPSC-based therapeutics. In this study, single-cell RNA-sequencing (scRNA-seq) was performed to decipher the heterogeneity during differentiation of three hPSC lines towards corneal limbal stem cells (LSCs). The scRNA-seq data revealed the presence of nine clusters, among which five clusters followed the anticipated differentiation path of LSCs. The remaining four clusters were linked to previously undescribed cell states that were annotated as either mesodermal or undifferentiated subpopulations, and their prevalence was hPSC line-dependent. Distinct cluster-specific marker genes identified in this study were confirmed by immunofluorescence analysis and employed to purify hPSC-derived LSCs, which effectively minimized the variation in the line-dependent differentiation efficiency. In summary, scRNA-seq offered molecular insights into the heterogeneity of hPSC-LSC differentiation, allowing a data-driven strategy to be adopted for consistent and robust generation of LSCs, essential for future advancement toward clinical translation. iPSC1 (n=2) and hESC1 (n=2) were differentiated into induced limbal stem cells, cells were FACs selected on AREG6+, ITGA6+ and PODXL- and cultured for an additional 15 days. Cells were subjected to bulkRNA sequencing to quantify marker gene expression in the induced limbal stem cells originating from iPSCs and hESC.