Generation and validation of a PITX2–EGFP reporter line of human induced pluripotent stem cells enables isolation of periocular mesenchymal cells

Toru Okubo, Ryuhei Hayashi, Shun Shibata, Yuji Kudo, Yuki Ishikawa, Saki Inoue, Yuki Kobayashi, Ai Honda, Yoichi Honma, Satoshi Kawasaki, Kohji Nishida

Research output: Contribution to journalArticle

Abstract

PITX2 (Paired-like homeodomain transcription factor 2) plays important roles in asymmetric development of the internal organs and symmetric development of eye tissues. During eye development, cranial neural crest cells migrate from the neural tube and form the periocular mesenchyme (POM). POM cells differentiate into several ocular cell types, such as corneal endothelial cells, keratocytes, and some ocular mesenchymal cells. In this study, we used transcription activator–like effector nuclease technology to establish a human induced pluripotent stem cell (hiPSC) line expressing a fluorescent reporter gene from the PITX2 promoter. Using homologous recombination, we heterozygously inserted a PITX2–IRES2–EGFP sequence downstream of the stop codon in exon 8 of PITX2. Cellular pluripotency was monitored with alkaline phosphatase and immunofluorescence staining of pluripotency markers, and the hiPSC line formed normal self-formed ectodermal autonomous multizones. Using a combination of previously reported methods, we induced PITX2 in the hiPSC line and observed simultaneous EGFP and PITX2 expression, as indicated by immunoblotting and immunofluorescence staining. PITX2 mRNA levels were increased in EGFP-positive cells, which were collected by cell sorting, and marker gene expression analysis of EGFP-positive cells induced in self-formed ectodermal autonomous multizones revealed that they were genuine POM cells. Moreover, after 2 days of culture, EGFP-positive cells expressed the PITX2 protein, which co-localized with forkhead box C1 (FOXC1) protein in the nucleus. We anticipate that the PITX2–EGFP hiPSC reporter cell line established and validated here can be utilized to isolate POM cells and to analyze PITX2 expression during POM cell induction.

Original languageEnglish
Pages (from-to)3456-3465
Number of pages10
JournalJournal of Biological Chemistry
Volume295
Issue number11
DOIs
Publication statusPublished - 2020 Mar 13

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Okubo, T., Hayashi, R., Shibata, S., Kudo, Y., Ishikawa, Y., Inoue, S., Kobayashi, Y., Honda, A., Honma, Y., Kawasaki, S., & Nishida, K. (2020). Generation and validation of a PITX2–EGFP reporter line of human induced pluripotent stem cells enables isolation of periocular mesenchymal cells. Journal of Biological Chemistry, 295(11), 3456-3465. https://doi.org/10.1074/jbc.RA119.010713