Characterization of rabbit limbal epithelial side population cells using RNA sequencing and single-cell qRT-PCR

Sumako Kameishi, Terumasa Umemoto, Yu Matsuzaki, Masako Fujita, Teruo Okano, Takashi Kato, Masayuki Yamato

Research output: Contribution to journalArticlepeer-review

5 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)704-709
Number of pages6
JournalBiochemical and Biophysical Research Communications
Volume473
Issue number3
DOIs
Publication statusPublished - 2016 May 6

Keywords

  • Corneal epithelial stem cells
  • Gene expression profile
  • Limbal epithelial side population
  • RNA sequencing
  • Single-cell qRT-PCR

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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