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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE248495 | GEO | 2024/06/26

REPOSITORIES: GEO

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Project description:Limbal stem cells (LSCs), known as corneal epithelial stem cells, are located at the basal epithelial layer of the corneal limbus and serve an important function in maintaining the homeostasis of the corneal epithelium. Several putative molecular markers of LSCs have been previously identified. However, the specificity of these markers remains largely controversial. To address this gap in the current understanding of LSCs, we performed a transcriptome profiling of heterogeneous corneal limbal basal cells using single-cell transcriptomics technology to identify LSCs and their exclusive markers. We isolated limbal basal cells from two young donors, constructed scRNA-seq libraries, generated RNA-sequencing data using the 10x Genomics platform, and then finally obtained the transcriptome of about 18,000 individual single-cells using Cell ranger (https://10xgenomics.com). Next, we performed quality control, filtering and data integration using Seurat package (https://satijalab.org/seurat), about 16,400 cells were retained for further downstream analysis such as dimensional reduction, unsupervised clustering, and Differentially expressed gene selection, trajectory analysis and visualization. We identified 11 unique clusters of cells assigned to a putative cell type based on published known biomarkers for differentiation, proliferation and putative epithelial stem cells. As a results, we found 2 Terminally Differentiated Cell (TDC), 3 Post-Mitotic Cell (PMC), 1 Transient Amplifying Cell (TAC), 2 Limbal Progenitor Cell (LPC), Putative Limbal Stem Cell (LSC) and 2 Melanocyte (MC) sub-cell type clusters. Furthermore, we confirmed the trajectory order of LSC differentiation for 9 clusters using Pseudotemporal and Functional PCA analysis. Lastly, we validated each assigned cell subtypes and determined their location in human corneal limbus tissue with 9 markers using RNAscope We were able to reveal the heterogeneity of corneal limbal basal epithelium by defining novel dynamic trajectories for cell types in a pseudotemporal manner. This approach allowed us to identify the distinct clusters of LSCs and progenitors with exclusively expressed markers, and might apply for translational research on regenerating a normal corneal epithelium and restoring vision.

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

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