title: Characterization of rabbit limbal epithelial side population cells using RNA sequencing and single-cell qRT-PCR.
abstract: Corneal epithelial stem cells reside in the limbus, a transitional zone between the cornea and conjunctiva, and are essential for maintaining homeostasis in the corneal epithelium. Although our previous studies demonstrated that rabbit limbal epithelial side population (SP) cells exhibit stem cell-like phenotypes with Hoechst 33342 staining, the different characteristics and/or populations of these cells remain unclear. Therefore, in this study, we determined the gene expression profiles of limbal epithelial SP cells by RNA sequencing using not only present public databases but also contigs that were created by de novo transcriptome assembly as references for mapping. Our transcriptome data indicated that limbal epithelial SP cells exhibited a stem cell-like phenotype compared with non-SP cells. Importantly, gene ontology analysis following RNA sequencing demonstrated that limbal epithelial SP cells exhibited significantly enhanced expression of mesenchymal/endothelial cell markers rather than epithelial cell markers. Furthermore, single-cell quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) demonstrated that the limbal epithelial SP population consisted of at least two immature cell populations with endothelial- or mesenchymal-like phenotypes. Therefore, our present results may propose the presence of a novel population of corneal epithelial stem cells distinct from conventional epithelial stem cells.
authors.affiliation: Integrative Bioscience and Biomedical Engineering, Graduate School of Advanced Science and Engineering, Waseda University, Tokyo, Japan; Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, Tokyo, Japan.
authors.forename: Sumako
authors.initials: S
authors.lastname: Kameishi
authors.affiliation: Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, Tokyo, Japan.
authors.forename: Terumasa
authors.initials: T
authors.lastname: Umemoto
authors.affiliation: Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, Tokyo, Japan.
authors.forename: Yu
authors.initials: Y
authors.lastname: Matsuzaki
authors.affiliation: Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, Tokyo, Japan.
authors.forename: Masako
authors.initials: M
authors.lastname: Fujita
authors.affiliation: Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, Tokyo, Japan.
authors.forename: Teruo
authors.initials: T
authors.lastname: Okano
authors.affiliation: Integrative Bioscience and Biomedical Engineering, Graduate School of Advanced Science and Engineering, Waseda University, Tokyo, Japan; Department of Biology, School of Education, Waseda University, Tokyo, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Kato
authors.affiliation: Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, Tokyo, Japan. Electronic address: yamato.masayuki@twmu.ac.jp.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Yamato
mesh_terms: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2 / Q000378:metabolism; D000818:Animals; D001562:Benzimidazoles / Q000737:chemistry; D019070:Cell Lineage; D002469:Cell Separation; D020451:Contig Mapping; D042783:Endothelial Cells / Q000166:cytology; D004847:Epithelial Cells / Q000166:cytology*; D019573:Epithelium, Corneal / Q000166:cytology*; D005434:Flow Cytometry; D059014:High-Throughput Nucleotide Sequencing; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology; D010641:Phenotype; D011817:Rabbits; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017423:Sequence Analysis, RNA; D058985:Side-Population Cells / Q000166:cytology*; D013234:Stem Cells / Q000166:cytology*
chemical_list: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D001562:Benzimidazoles; C017807:bisbenzimide ethoxide trihydrochloride
keywords: Corneal epithelial stem cells; Gene expression profile; Limbal epithelial side population; RNA sequencing; Single-cell qRT-PCR