Time-course single-cell RNA sequencing reveals transcriptional dynamics and heterogeneity of limbal stem cells derived from human pluripotent stem cells

Abstract

Background Human pluripotent stem cell-derived limbal stem cells (hPSC-derived LSCs) provide a promising cell source for corneal transplants and ocular surface reconstruction. Although recent efforts in the identification of LSC markers have increased our understanding of the biology of LSCs, much more remains to be characterized in the developmental origin, cell fate determination, and identity of human LSCs. The lack of knowledge hindered the establishment of efficient differentiation protocols for generating hPSC-derived LSCs and held back their clinical application. Results Here, we performed a time-course single-cell RNA-seq to investigate transcriptional heterogeneity and expression changes of LSCs derived from human embryonic stem cells (hESCs). Based on current protocol, expression heterogeneity of reported LSC markers were identified in subpopulations of differentiated cells. EMT has been shown to occur during differentiation process, which could possibly result in generation of untargeted cells. Pseudotime trajectory analysis revealed transcriptional changes and signatures of commitment of hESCs-derived LSCs and their progeny—the transit amplifying cells. Conclusion Single-cell RNA-seq revealed time-course expression changes and significant transcriptional heterogeneity during hESC-derived LSC differentiation in vitro. Our results demonstrated candidate developmental trajectory and several new candidate markers for LSCs, which could facilitate elucidating the identity and developmental origin of human LSCs in vivo.

Transcriptomic Landscape and Functional Characterization of Human Induced Pluripotent Stem Cell-Derived Limbal Epithelial Progenitor Cells

“…Several candidate LEPC markers such as TP63, KRT15, CK19, CDH3, and MSX2, were similarly expressed in hiPSC-LEPC and T-LEPC. Markers for terminally differentiated corneal epithelium such as KRT3, KRT12, and IVL showed very low expression similar to earlier reports [ 52 ]. However, bulk RNA-seq data also indicated a higher expression of KRT13 in hiPSC-LEPC compared to T-LEPC, suggesting the presence of some non-LEPCs (maybe conjunctival epithelial cells) in cultures as reported earlier [ 42 , 53 ].…”

Corneal epithelial differentiation of human pluripotent stem cells generates ABCB5+ and ∆Np63α+ cells with limbal cell characteristics and high wound healing capacity

“…The results demonstrate high reproducibility of the method in our hands. Noteworthy, Sun et al [ 46 ] utilized a differentiation protocol with the same induction molecules to investigate cellular heterogeneity during differentiation of hPSC-LSCs at days 0, 7, 14 and 21, using single cell RNA sequencing. Interestingly, they reported overall very low expressions for PAX6, p63, and CK14 and distinctive expression pattern for ABCG2, demonstrating the highest peak of ABCG2 expression at day 21.…”

Single-cell transcriptomics of the ocular anterior segment: a comprehensive review

“…At 20–21 PCW, an increase in fibroblastic corneal endothelial cells was noted, suggesting these cell types serve an integral role in corneal endothelial cell proliferation that may potentially benefit endothelial wound healing. Sun et al investigated limbal stem cell (LSC) developmental trajectories in vitro at four different time points and found that pluripotent markers POU5F1 (POU class 5 homeobox 1 ) , SOX2 (SRY-Box 2), and NANOG (Nanog homeobox) were most highly expressed at the first time point (Day 0) and steadily decreased by the second time point (Day 7) [ 45 ]. TFAP2A (Transcription factor AP-2 alpha), a surface ectodermal marker, and epithelial and limbal stem cell markers were expressed by Day 7.…”