Generation and validation of a PITX2–EGFP reporter line of human induced pluripotent stem cells enables isolation of periocular mesenchymal cells
| Autor: | Yuki Ishikawa, Yuki Kobayashi, Shibata Shun, Ai Honda, Yuji Kudo, Kohji Nishida, Yoichi Honma, Toru Okubo, Saki Inoue, Satoshi Kawasaki, Ryuhei Hayashi |
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| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
Cell type RNA Splicing Green Fluorescent Proteins Induced Pluripotent Stem Cells Cell Separation Eye Biochemistry Fluorescence Cell Line 03 medical and health sciences stomatognathic system Genes Reporter Ectoderm Animals Humans Promoter Regions Genetic Induced pluripotent stem cell Molecular Biology Homeodomain Proteins Mice Inbred ICR Reporter gene 030102 biochemistry & molecular biology Chemistry Mesenchymal stem cell Reproducibility of Results Neural crest Mesenchymal Stem Cells Cell Biology Cell sorting Embryo Mammalian Clone Cells Cell biology stomatognathic diseases Phenotype 030104 developmental biology Cell culture sense organs Stem cell Transcription Factors |
| Zdroj: | J Biol Chem |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.ra119.010713 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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