Cornea Single-Cell Transcriptomics Identifies a Unique Entity and Signature Markers of Transit-Amplifying Cells in Human Corneal Limbus Jin-Miao Li,1,2 Sangbae Kim,3 Yun Zhang,1 Fang Bian,1 Jiaoyue Hu,1 Rong Lu,1,2 Stephen C. Pflugfelder,1 Rui Chen,3 and De-Quan Li1 1Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, Texas, United States 2State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China 3Human Genome Sequencing Center, Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, United States Correspondence: De-Quan Li, PURPOSE. Differentiated from adult stem cells (ASCs), transit-amplifying cells (TACs) play Ocular Surface Center, Cullen Eye an important role in tissue homeostasis, development, and regeneration. This study aimed Institute, Department of to characterize the gene expression profile of a candidate TAC population in limbal basal Ophthalmology, Baylor College of epithelial cells using single-cell RNA sequencing (scRNA-seq). Medicine, 6565 Fannin Street, NC-505c, Houston, TX 77030, USA; METHODS. Single cells isolated from the basal corneal limbus were subjected to scRNA-seq [email protected]. using the 10x Genomics platform. Cell types were clustered by graph-based visualization Received: February 15, 2021 methods and unbiased computational analysis. BrdU proliferation assays, immunofluo- Accepted: June 22, 2021 rescent staining, and real-time reverse transcription quantitative polymerase chain reac- Published: July 23, 2021 tion were performed using multiple culture models of primary human limbal epithelial cells to characterize the TAC pool. Citation: Li J-M, Kim S, Zhang Y, et al. Single-cell transcriptomics RESULTS. Single-cell transcriptomics of 16,360 limbal basal cells revealed 12 cell clusters. identifies a unique entity and A unique cluster (3.21% of total cells) was identified as a TAC entity, based on its less signature markers of differentiated progenitor status and enriched exclusive proliferation marker genes, with transit-amplifying cells in human 98.1% cells in S and G2/M phases. The cell cycle-dependent genes were revealed to be corneal limbus. Invest Ophthalmol largely enriched by the TAC population. The top genes were characterized morpholog- Vis Sci. 2021;62(9):36. https://doi.org/10.1167/iovs.62.9.36 ically and functionally at protein and mRNA levels. The specific expression patterns of RRM2, TK1, CENPF, NUSAP1, UBE2C,andCDC20 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 aunique TAC entity and uncovered a group of cell cycle-dependent genes that serve as TAC signa- ture markers. The findings provide insight into ASCs and TACs and lay the foundation for understanding corneal homeostasis and diseases. Keywords: cell cycle-dependent genes, cornea, epithelium, limbal stem cells, single-cell transcriptomics, transit-amplifying cells dult stem cells (ASCs), also known as somatic stem cells the transit stage, or TACs, are capable of rapidly producing A or tissue-specific stem cells, are present in a variety many differentiated cells, not only during development but of human organs and tissues.1 ASCs are small populations also during regeneration. Thus, TACs have been identified as of quiescent, slow-cycling, undifferentiated cells with self- a subpopulation intermediate between ASCs and differenti- renew ability and high proliferative potentiality.2–4 ated cells.6,9,10 ASCs may generate differentiated progeny through asym- This stem cell/TAC model of tissue homeostasis is well metric or symmetric divisions.5–8 Asymmetric cell division recognized in multiple organisms and different organs and gives rise to one stem cell and one daughter cell committed tissues, including the skin and hair follicles, hematopoi- to progenitor cells or transit-amplifying cells (TACs), which etic system, intestine, nervous system, and corneal epithe- finally differentiate into functionally mature cells, regenerat- lium.6,10 The physiological and pathological roles of TACs ing all of the cell types of the tissue where they are located. in health and disease have been increasingly investigated Symmetric division yields two identical SCs or two daugh- and are broadly recognized in the research areas of tissue ter cells committed to differentiation. The essential feature homeostasis, wound healing, and regeneration, as well as in of this “transit” cell population is their capacity to generate malignant diseases such as cancer,9,11–13 although the field many maturing cells from very few cells. The cells entering of regenerative biology has tended to be stem cell-centric. Copyright 2021 The Authors iovs.arvojournals.org | ISSN: 1552-5783 1 This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. Downloaded from iovs.arvojournals.org on 09/26/2021 Single-Cell Transcriptomics Identifies TAC Entity IOVS | July 2021 | Vol. 62 | No. 9 | Article 36 | 2 The importance of TACs and their diverse functions goes performed following the manufacturer’s protocols (10x beyond merely producing tissues. Defects in regeneration Genomics, Pleasanton, CA, USA). Single-cell suspensions at or injury repair are also likely to be caused by problems in 1000 cells/μL in PBS were loaded onto a 10x Genomics TACs, not just in stem cells. The impact of TACs on stem cells Chromium Controller to generate a single-cell Gel Bead- and their niche may represent a pathogenesis and poten- in-Emulsion (GEM). The scRNA-seq library was prepared tial therapeutic targets for halting or reversing degenerative with Chromium Single Cell 3 Reagent Kit, version 2 diseases. Understanding TAC biology is of important signifi- (10x Genomics). The product was amplified by PCR and cance not only for elucidating the fundamental principles of sequenced on Illumina NovaSeq 6000 (Illumina, San Diego, tissue development and regeneration but also for advancing CA, USA). our current treatment of regenerative and neoplastic disease. It is worth mentioning, however, that some researchers believe that TACs do not contribute to epidermal homeosta- Bioinformatic Analysis of scRNA-seq Data sis and tissue formation. The existence of a TAC cell type Cell Ranger 2.1 software (10x Genomics) with default has been challenged by studies based on a model of mouse settings was used for alignment, barcode assignment, and 14 tail epidermis, raising the question of whether TACs are an unique molecular identifier (UMI) counting of the raw entity in their own right or represent a function. This argu- sequencing data with genome reference hg19 (Supplemen- ment has not been solved because most previous research tary Table S1). on TAC characterization and identification has been done at After generating the UMI count profile, we applied Seurat tissue and bulk cell population levels. 3.0 for quality control and downstream analysis.22 A global- The cutting-edge, high-throughput, single-cell RNA- scaling normalization method, LogNormalize, was employed sequencing (scRNA-seq) technology holds the promise to to normalize the gene expression measurements for each cell revolutionize our understanding of diseases and associated by the total expression, then the result was multiplied by a biological processes due to its unprecedented resolution. scale factor (10,000 by default) and log-transformed in each It has the power to reveal new cell types, identify unique dataset. For data alignment, we selected 1000 highly variable cell states, and dissect underlying heterogeneity in a high- genes in each data matrix and performed the FindIntegratio- 15,16 throughput and unbiased manner. Single-cell transcrip- nAnchors and IntegrateData functions. Next, we performed tomics opens a new door to uncovering and identifying TAC clustering using FindClusters to identify sub-cell-type clus- populations in adult tissues. ters. The Uniform Manifold Approximation and Projection The paradigm of limbal stem cells (LSCs), TACs, and (UMAP) results for each dataset were visualized if a cluster differentiated cells in the corneal epithelium has been widely was derived from both donors. accepted for more than three decades, and many features To identify differentially expressed genes (DEGs) in each 17–20 of TACs have been characterized. However, this distinct cluster, we used the FindAllMarkers function based on the epithelial cell population in the basal cornea limbus has Wilcoxon rank-sum test in Seurat. Cell-cycle scoring was not been well defined at the molecular level. In the present performed with cell-cycle phase marker genes using the Cell- study, we performed scRNA-seq on basal limbal epithelial CycleScoring function in Seurat.23,24 cells and identified a cell cluster with increased expression of proliferation-associated genes that may represent a TAC population. Primary Human Limbal Epithelial Cultures and In VitroCellGrowthandRegenerationModels MATERIALS AND METHODS Primary human limbal epithelial cells (HLECs) were cultured using explants from donor corneal limbal rims for multi- Donor Corneal Tissues and Single-Cell Isolation ple in vitro models by our previous methods.21,25 In brief, From the Corneal Limbal Epithelial Basal Layer for growth stages, cultures were collected at about 70% or Fresh human corneoscleral tissues were obtained from the 100% confluence for