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.
Highlights
PITX2 (Paired-like homeodomain transcription factor 2) plays important roles in asymmetric development of the internal organs and symmetric development of eye tissues
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 periocular mesenchyme (POM) cells
We recently reported that human induced pluripotent stem cell (hiPSC) form self-formed ectodermal autonomous multizones (SEAMs) from which ocular cells, such as corneal epithelial cells, conjunctival epithelial cells, lens cells, retinal cells, and Neural crest cells (NCCs), can be derived [22, 23]
Summary
We evaluated Pitx expression in POM cells in mouse embryos at E10.5 (Fig. S1A) and E12.5 (Fig. S1B) At both stages, cells ranging from the periocular sites to primordial cells of the cornea were positive for Pitx and Foxc, but negative for Sox, a negative marker of POM cells. Cells ranging from the periocular sites to primordial cells of the cornea were positive for Pitx and Foxc, but negative for Sox, a negative marker of POM cells This finding indicated that POM cells exist in the periocular sites in E10.5 and E12.5 mouse embryos. Fluorescence intensity was stronger when EGFP was located downstream of IRES2 than when it was downstream of the 2A peptide sequences (Fig. S2B). We used a donor vector containing EGFP, IRES2, and SV40 as the GFP variant, polycistronic sequence, and polyadenylation signal, respectively, for establishing a PITX2–GFP reporter hiPSC line
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