Single-cell transcriptomics identifies limbal stem cell population and cell types mapping its differentiation trajectory in limbal basal epithelium of human cornea

Li, Dequan; Kim, Sangbae; Li, Jinmiao; Gao, Qianmiao; Choi, Jongsu; Bian, Fang; Hu, Jiaoyue; Zhang, Yun; Li, Jin; Lu, Rong; Li, Yumei; Pflugfelder, Stephen C.; Miao, Hongyu; Chen, Rui

doi:10.1016/j.jtos.2020.12.004

Cited by 73 publications

(72 citation statements)

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“…A similar approach was applied to the ocular anterior segment in mouse, leading to identification of a novel marker for stem and early transit amplifying cells, TXNIP [ 14 ]. While this work was under review, two studies reported the single cell transcriptomic analysis of human limbal basal epithelium [ 15 ] and murine corneal non-myelinating Schwann cells [ 16 ]. Our study complements and expands these published data by providing a comprehensive single cell atlas of the whole developing and adult human cornea and adjacent conjunctiva that defines their development, limbal progenitor cells and their interactions with immune cells.…”

Section: Introductionmentioning

confidence: 99%

A single cell atlas of human cornea that defines its development, limbal progenitor cells and their interactions with the immune cells

et al. 2021

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Purpose Single cell (sc) analyses of key embryonic, fetal and adult stages were performed to generate a comprehensive single cell atlas of all the corneal and adjacent conjunctival cell types from development to adulthood. Methods Four human adult and seventeen embryonic and fetal corneas from 10 to 21 post conception week (PCW) specimens were dissociated to single cells and subjected to scRNA- and/or ATAC-Seq using the 10x Genomics platform. These were embedded using Uniform Manifold Approximation and Projection (UMAP) and clustered using Seurat graph-based clustering. Cluster identification was performed based on marker gene expression, bioinformatic data mining and immunofluorescence (IF) analysis. RNA interference, IF, colony forming efficiency and clonal assays were performed on cultured limbal epithelial cells (LECs). Results scRNA-Seq analysis of 21,343 cells from four adult human corneas and adjacent conjunctivas revealed the presence of 21 cell clusters, representing the progenitor and differentiated cells in all layers of cornea and conjunctiva as well as immune cells, melanocytes, fibroblasts, and blood/lymphatic vessels. A small cell cluster with high expression of limbal progenitor cell (LPC) markers was identified and shown via pseudotime analysis to give rise to five other cell types representing all the subtypes of differentiated limbal and corneal epithelial cells. A novel putative LPCs surface marker, GPHA2, expressed on the surface of 0.41% ± 0.21 of the cultured LECs, was identified, based on predominant expression in the limbal crypts of adult and developing cornea and RNAi validation in cultured LECs. Combining scRNA- and ATAC-Seq analyses, we identified multiple upstream regulators for LPCs and demonstrated a close interaction between the immune cells and limbal progenitor cells. RNA-Seq analysis indicated the loss of GPHA2 expression and acquisition of proliferative limbal basal epithelial cell markers during ex vivo LEC expansion, independently of the culture method used. Extending the single cell analyses to keratoconus, we were able to reveal activation of collagenase in the corneal stroma and a reduced pool of limbal suprabasal cells as two key changes underlying the disease phenotype. Single cell RNA-Seq of 89,897 cells obtained from embryonic and fetal cornea indicated that during development, the conjunctival epithelium is the first to be specified from the ocular surface epithelium, followed by the corneal epithelium and the establishment of LPCs, which predate the formation of limbal niche by a few weeks. Conclusions Our scRNA-and ATAC-Seq data of developing and adult cornea in steady state and disease conditions provide a unique resource for defining genes/pathways that can lead to improvement in ex vivo LPCs expansion, stem cell differentiation methods and better understanding and treatment of oc...

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Section: Introductionmentioning

confidence: 99%

A single cell atlas of human cornea that defines its development, limbal progenitor cells and their interactions with the immune cells

et al. 2021

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“…It is The copyright holder for this preprint this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.07.451489 doi: bioRxiv preprint 11 corneal blindness. This research further complements scRNAseq analysis of the developing human cornea and of the human corneal limbus 43 .…”

Section: Discussionmentioning

confidence: 67%

A single cell transcriptome atlas reveals the heterogeneity of the healthy human cornea and identifies novel markers of the corneal limbus and stroma

Català

Gröen²,

et al. 2021

Preprint

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The cornea is the clear window that lets light into the eye. It is composed of five layers: epithelium, Bowman’s layer, stroma, Descemet’s membrane and endothelium. The maintenance of its structure and transparency are determined by the functions of the different cell types populating each layer. Attempts to regenerate corneal tissue and understand disease conditions requires knowledge of how cell profiles vary across this heterogeneous tissue. We performed a single cell transcriptomic profiling of 19,472 cells isolated from eight healthy donor corneas. Our analysis delineates the heterogeneity of the corneal layers by identifying cell populations and revealing cell states that contribute in preserving corneal homeostasis. We identified that the expression of CAV1, CXCL14, HOMER3 and CPVL were exclusive to the corneal epithelial limbal stem cell niche, CKS2, STMN1 and UBE2C were exclusively expressed in highly proliferative transit amplifying cells, and NNMT was exclusively expressed by stromal keratocytes. Overall, this research provides a basis to improve current primary cell expansion protocols, for future profiling of corneal disease states, to help guide pluripotent stem cells into different corneal lineages, and to understand how engineered substrates affect corneal cells to improve regenerative therapies.

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“…S1). 28 We constructed two scRNA-seq libraries of limbal cells from the two healthy young donors and generated transcriptomic profiles for 7096 and 10,491 cells with 823,020,840 and 861,610,927 total reads, respectively, using the 10x Genomics platform. Mean reads per cell were 115,983 and 82,128, and median genes per cell were 3257 and 2731, respectively (Supplementary Fig.…”

Section: Single-cell Transcriptomics Revealed a Unique Cell Type Representing Tacs In Heterogeneous Limbal Basal Epithelium Of Human Cornmentioning

confidence: 99%

“…6,9,10 To identify the progenitor feature of C8, all nine clusters of limbal epithelial cells were further analyzed; the other three clusters were determined to be conjunctival cells (C7) and melanocytes (C9 and C11). 28 Ten markers that have been reported to be related to cell proliferation 19,26,[30][31][32] were examined through heatmap visualization (Fig. 3A).…”

Section: A Unique Cell Type Was Identified As a Tac Population Entity Based On Expression Patterns Of Marker Genes Known To Be Related Tomentioning

confidence: 99%

“…3D). 28 Interestingly, TACs in C8 were found to express moderately high levels of putative stem/progenitor markers such as ABCB5, PROM1, POSTN, A2M, ITGA9, LEF1, ABCG2, and TCF4, whereas these marker genes were strongly upregulated in LSCs (C10) but dramatically downregulated in TDCs (C0), as determined by analyzing the log2 fold changes (all P < 0.05) of these markers in the three cell types from scRNA-seq data (Fig. 3E).…”

Section: A Unique Cell Type Was Identified As a Tac Population Entity Based On Expression Patterns Of Marker Genes Known To Be Related Tomentioning

confidence: 99%

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Single-Cell Transcriptomics Identifies a Unique Entity and Signature Markers of Transit-Amplifying Cells in Human Corneal Limbus

Kim

Zhang

et al. 2021

Invest. Ophthalmol. Vis. Sci.

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PURPOSE. Differentiated from adult stem cells (ASCs), transit-amplifying cells (TACs) play an important role in tissue homeostasis, development, and regeneration. This study aimed to characterize the gene expression profile of a candidate TAC population in limbal basal epithelial cells using single-cell RNA sequencing (scRNA-seq). METHODS.Single cells isolated from the basal corneal limbus were subjected to scRNA-seq using the 10x Genomics platform. Cell types were clustered by graph-based visualization methods and unbiased computational analysis. BrdU proliferation assays, immunofluorescent staining, and real-time reverse transcription quantitative polymerase chain reaction were performed using multiple culture models of primary human limbal epithelial cells to characterize the TAC pool. RESULTS.Single-cell transcriptomics of 16,360 limbal basal cells revealed 12 cell clusters. A unique cluster (3.21% of total cells) was identified as a TAC entity, based on its less differentiated progenitor status and enriched exclusive proliferation marker genes, with 98.1% cells in S and G2/M phases. The cell cycle-dependent genes were revealed to be largely enriched by the TAC population. The top genes were characterized morphologically and functionally at protein and mRNA levels. The specific expression patterns of RRM2, TK1, CENPF, NUSAP1, UBE2C, and CDC20 were well correlated in a time-and cycle-dependent manner with proliferation stages in the cell growth and regeneration models. CONCLUSIONS.For the first time, to the best of our knowledge, we have identified a unique TAC entity and uncovered a group of cell cycle-dependent genes that serve as TAC signature markers. The findings provide insight into ASCs and TACs and lay the foundation for understanding corneal homeostasis and diseases.

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Single-cell transcriptomics identifies limbal stem cell population and cell types mapping its differentiation trajectory in limbal basal epithelium of human cornea

Cited by 73 publications

References 65 publications

A single cell atlas of human cornea that defines its development, limbal progenitor cells and their interactions with the immune cells

A single cell atlas of human cornea that defines its development, limbal progenitor cells and their interactions with the immune cells

A single cell transcriptome atlas reveals the heterogeneity of the healthy human cornea and identifies novel markers of the corneal limbus and stroma

Single-Cell Transcriptomics Identifies a Unique Entity and Signature Markers of Transit-Amplifying Cells in Human Corneal Limbus

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