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

• Published in: The Journal of biological chemistry Vol. 295; no. 11; pp. 3456 - 3465
• Main Authors: Okubo, Toru, Hayashi, Ryuhei, Shibata, Shun, Kudo, Yuji, Ishikawa, Yuki, Inoue, Saki, Kobayashi, Yuki, Honda, Ai, Honma, Yoichi, Kawasaki, Satoshi, Nishida, Kohji
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 13.03.2020; American Society for Biochemistry and Molecular Biology
• Subjects: Animals; Cell Biology; Cell Line; Cell Separation; Clone Cells; Ectoderm - cytology; Embryo, Mammalian - cytology; Eye - cytology; eye development; Fluorescence; Genes, Reporter; Green Fluorescent Proteins - metabolism; Homeobox Protein PITX2; Homeodomain Proteins - metabolism; Humans; induced pluripotent stem cell (iPS cell) (iPSC); Induced Pluripotent Stem Cells - cytology; Mesenchymal Stem Cells - metabolism; Mice, Inbred ICR; neural crest cell; paired-like homeodomain transcription factor 2 (PITX2); periocular mesenchyme (POM); Phenotype; Promoter Regions, Genetic - genetics; regenerative medicine; reporter gene; Reproducibility of Results; RNA Splicing - genetics; stem cells; transcription activator–like effector nuclease (TALEN); Transcription Factors - metabolism; stem cells; paired-like homeodomain transcription factor 2 (PITX2); induced pluripotent stem cell (iPS cell) (iPSC); periocular mesenchyme (POM); neural crest cell; regenerative medicine; eye development; reporter gene; transcription activator–like effector nuclease (TALEN)
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.RA119.010713

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.