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• W3005226072 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. 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. Neural crest cells (NCCs) 2The abbreviations used are: NCCneural crest cellPOMperiocular mesenchymehiPSChuman induced pluripotent stem cellSEAMself-formed ectodermal autonomous multizoneTALENtranscription activator–like effector nucleaseEnembryonic day nMEFmouse embryonic fibroblastALPalkaline phosphataseqRT-PCRquantitative RT-PCRDMdifferentiation mediumEGFepidermal growth factorbFGFbasic fibroblast growth factorPEphycoerythrin. are multipotent stem cells generated at the border between the neural tube and surface ectoderm during early embryonic development in vertebrates (1Mayor R. Theveneau E. The neural crest.Development. 2013; 140 (23674598): 2247-225110.1242/dev.091751Crossref PubMed Scopus (187) Google Scholar). In eye development, cranial NCCs migrate to form the periocular mesenchyme (POM). POM cells in turn differentiate into a wide variety of cells, such as corneal endothelial cells (2Beebe D.C. Coats J.M. The lens organizes the anterior segment: specification of neural crest cell differentiation in the avian eye.Dev. Biol. 2000; 220 (10753528): 424-43110.1006/dbio.2000.9638Crossref PubMed Scopus (163) Google Scholar), keratocytes, iris stromal cells, ciliary muscle cells, trabecular meshwork cells, and scleral cells (3Williams A.L. Bohnsack B.L. Neural crest derivatives in ocular development: discerning the eye of the storm.Birth Defects Res. C Embryo Today. 2015; 105 (26043871): 87-9510.1002/bdrc.21095Crossref PubMed Scopus (72) Google Scholar, 4Cvekl A. Tamm E.R. Anterior eye development and ocular mesenchyme: new insights from mouse models and human diseases.Bioessays. 2004; 26 (15057935): 374-38610.1002/bies.20009Crossref PubMed Scopus (216) Google Scholar). In addition, peripheral tissues of the eyes, such as cartilage, bone, dermis, and fat, which are connected to the extraocular muscles, also originate from POM cells (5Langenberg T. Kahana A. Wszalek J.A. Halloran M.C. The eye organizes neural crest cell migration.Dev. Dyn. 2008; 237 (18498099): 1645-165210.1002/dvdy.21577Crossref PubMed Scopus (62) Google Scholar). neural crest cell periocular mesenchyme human induced pluripotent stem cell self-formed ectodermal autonomous multizone transcription activator–like effector nuclease embryonic day n mouse embryonic fibroblast alkaline phosphatase quantitative RT-PCR differentiation medium epidermal growth factor basic fibroblast growth factor phycoerythrin. PITX2 (paired-like homeodomain transcription factor 2) is one of the homeobox transcription factors that play key roles during embryogenesis. PITX2 is crucial in left–right asymmetry in visceral organs (6Shiratori H. Yashiro K. Shen M.M. Hamada H. Conserved regulation and role of Pitx2 in situs-specific morphogenesis of visceral organs.Development. 2006; 133 (16835440): 3015-302510.1242/dev.02470Crossref PubMed Scopus (81) Google Scholar), such as the heart (7Lin C.R. Kioussi C. O'Connell S. Briata P. Szeto D. Liu F. Izpisúa-Belmonte J.C. Rosenfeld M.G. Pitx2 regulates lung asymmetry, cardiac positioning and pituitary and tooth morphogenesis.Nature. 1999; 401 (10499586): 279-28210.1038/45803Crossref PubMed Scopus (474) Google Scholar, 8Campione M. Steinbeisser H. Schweickert A. Deissler K. van Bebber F. Lowe L.A. Nowotschin S. Viebahn C. Haffter P. Kuehn M.R. Blum M. The homeobox gene Pitx2: mediator of asymmetric left–right signaling in vertebrate heart and gut looping.Development. 1999; 126 (10021341): 1225-1234Crossref PubMed Google Scholar, 9Franco D. Sedmera D. Lozano-Velasco E. Multiple roles of Pitx2 in cardiac development and disease.J. Cardiovasc. Dev. Dis. 2017; 4 (29367545): E16Crossref PubMed Scopus (19) Google Scholar), lungs (7Lin C.R. Kioussi C. O'Connell S. Briata P. Szeto D. Liu F. Izpisúa-Belmonte J.C. Rosenfeld M.G. Pitx2 regulates lung asymmetry, cardiac positioning and pituitary and tooth morphogenesis.Nature. 1999; 401 (10499586): 279-28210.1038/45803Crossref PubMed Scopus (474) Google Scholar), gut (8Campione M. Steinbeisser H. Schweickert A. Deissler K. van Bebber F. Lowe L.A. Nowotschin S. Viebahn C. Haffter P. Kuehn M.R. Blum M. The homeobox gene Pitx2: mediator of asymmetric left–right signaling in vertebrate heart and gut looping.Development. 1999; 126 (10021341): 1225-1234Crossref PubMed Google Scholar, 10Welsh I.C. Thomsen M. Gludish D.W. Alfonso-Parra C. Bai Y. Martin J.F. Kurpios N.A. Integration of left–right Pitx2 transcription and Wnt signaling drives asymmetric gut morphogenesis via Daam2.Dev. Cell. 2013; 26 (24091014): 629-64410.1016/j.devcel.2013.07.019Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), and stomach, as well as in eye development (11Suh H. Gage P.J. Drouin J. Camper S.A. Pitx2 is required at multiple stages of pituitary organogenesis: pituitary primordium formation and cell specification.Development. 2002; 129 (11807026): 329-337Crossref PubMed Google Scholar). In the eyes, PITX2 is expressed in the cranial NC-derived POM cells, together with other transcription factors, such as FOXC1, FOXC2 (12Seo S. Chen L. Liu W. Zhao D. Schultz K.M. Sasman A. Liu T. Zhang H.F. Gage P.J. Kume T. Foxc1 and foxc2 in the neural crest are required for ocular anterior segment development.Invest. Ophthalmol. Vis. Sci. 2017; 58 (28253399): 1368-137710.1167/iovs.16-21217Crossref PubMed Scopus (22) Google Scholar), and LMX1B (13McMahon C. Gestri G. Wilson S.W. Link B.A. Lmx1b is essential for survival of periocular mesenchymal cells and influences Fgf-mediated retinal patterning in zebrafish.Dev. Biol. 2009; 332 (19500562): 287-29810.1016/j.ydbio.2009.05.577Crossref PubMed Scopus (44) Google Scholar), and plays important roles in ocular anterior segment development (14Evans A.L. Gage P.J. Expression of the homeobox gene Pitx2 in neural crest is required for optic stalk and ocular anterior segment development.Hum. Mol. Genet. 2005; 14 (16203745): 3347-335910.1093/hmg/ddi365Crossref PubMed Scopus (134) Google Scholar). Specifically, PITX2 is involved in the development of corneal endothelial cells (15Chen L. Martino V. Dombkowski A. Williams T. West-Mays J. Gage P.J. AP-2β is a downstream effector of PITX2 required to specify endothelium and establish angiogenic privilege during corneal development.Invest. Ophthalmol. Vis. Sci. 2016; 57 (26968737): 1072-108110.1167/iovs.15-18103Crossref PubMed Scopus (22) Google Scholar), keratocytes (16Gage P.J. Kuang C. Zacharias A.L. The homeodomain transcription factor PITX2 is required for specifying correct cell fates and establishing angiogenic privilege in the developing cornea.Dev. Dyn. 2014; 243 (25044936): 1391-140010.1002/dvdy.24165Crossref PubMed Scopus (21) Google Scholar), iris stromal cells (17Kimura M. Tokita Y. Machida J. Shibata A. Tatematsu T. Tsurusaki Y. Miyake N. Saitsu H. Miyachi H. Shimozato K. Matsumoto N. Nakashima M. A novel PITX2 mutation causing iris hypoplasia.Hum. Genome Var. 2014; 1 (27081499): 1400510.1038/hgv.2014.5Crossref PubMed Scopus (6) Google Scholar), ciliary muscles, trabecular meshwork cells, scleral cells, mesenchymal cells of the ocular glands, and peripheral connective tissues connected to the extraocular muscles (18Gage P.J. Rhoades W. Prucka S.K. Hjalt T. Fate maps of neural crest and mesoderm in the mammalian eye.Invest. Ophthalmol. Vis. Sci. 2005; 46 (16249499): 4200-420810.1167/iovs.05-0691Crossref PubMed Scopus (251) Google Scholar). Mutation of PITX2 or FOXC1 causes Axenfeld–Rieger syndrome, which manifests with dysgenesis of the anterior segment of the eyes as well as mild tooth malformation and craniofacial dysmorphism (19Seifi M. Footz T. Taylor S.A. Elhady G.M. Abdalla E.M. Walter M.A. Novel PITX2 gene mutations in patients with Axenfeld–Rieger syndrome.Acta Ophthalmol. 2016; 94 (27009473): e571-e57910.1111/aos.13030Crossref PubMed Scopus (13) Google Scholar, 20Tümer Z. Bach-Holm D. Axenfeld–Rieger syndrome and spectrum of PITX2 and FOXC1 mutations.Eur. J. Hum. Genet. 2009; 17 (19513095): 1527-153910.1038/ejhg.2009.93Crossref PubMed Scopus (163) Google Scholar). It has been suggested that POM cells can be induced from human induced pluripotent stem cells (hiPSCs) (21Lovatt M. Yam G.H. Peh G.S. Colman A. Dunn N.R. Mehta J.S. Directed differentiation of periocular mesenchyme from human embryonic stem cells.Differentiation. 2018; 99 (29239730): 62-6910.1016/j.diff.2017.11.003Crossref PubMed Scopus (10) Google Scholar). We recently reported that hiPSCs form self-formed ectodermal autonomous multizones (SEAMs) from which ocular cells, such as corneal epithelial cells, conjunctival epithelial cells, lens cells, retinal cells, and NCCs, can be derived (22Hayashi R. Ishikawa Y. Sasamoto Y. Katori R. Nomura N. Ichikawa T. Araki S. Soma T. Kawasaki S. Sekiguchi K. Quantock A.J. Tsujikawa M. Nishida K. Co-ordinated ocular development from human iPS cells and recovery of corneal function.Nature. 2016; 531 (26958835): 376-38010.1038/nature17000Crossref PubMed Scopus (117) Google Scholar, 23Hayashi R. Ishikawa Y. Katori R. Sasamoto Y. Taniwaki Y. Takayanagi H. Tsujikawa M. Sekiguchi K. Quantock A.J. Nishida K. Coordinated generation of multiple ocular-like cell lineages and fabrication of functional corneal epithelial cell sheets from human iPS cells.Nat. Protoc. 2017; 12 (28253236): 683-69610.1038/nprot.2017.007Crossref PubMed Scopus (46) Google Scholar). In SEAMs, various types of cells mimic their differentiation process to form a whole eye structure in vitro. We expect that SEAMs contain PITX2-expressing POM cells differentiated from NCCs. However, it is nearly impossible to isolate PITX2-expressing POM cells from the various cell types in culture systems without a reporter line, because POM cell-specific cell-surface markers have not been reported to date. Transcription activator–like effector nucleases (TALENs) are restriction enzymes that generate site-specific double-strand breaks in DNA, with lower nonspecific cleavage activity (24Park C.-Y. Kim J. Kweon J. Son J.S. Lee J.S. Yoo J.-E. Cho S.-R. Kim J.-H. Kim J.-S. Kim D.-W. Targeted inversion and reversion of the blood coagulation factor 8 gene in human iPS cells using TALENs.Proc. Natl. Acad. Sci. U.S.A. 2014; 111 (24927536): 9253-925810.1073/pnas.1323941111Crossref PubMed Scopus (99) Google Scholar) than CRISPR-Cas (25Fu Y. Foden J.A. Khayter C. Maeder M.L. Reyon D. Joung J.K. Sander J.D. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells.Nat. Biotechnol. 2013; 31 (23792628): 822-82610.1038/nbt.2623Crossref PubMed Scopus (1957) Google Scholar), through binding of a TAL effector to specific DNA regions (26Joung J.K. Sander J.D. TALENs: a widely applicable technology for targeted genome editing.Nat. Rev. Mol. Cell Biol. 2013; 14 (23169466): 49-5510.1038/nrm3486Crossref PubMed Scopus (930) Google Scholar). The double-strand breaks are repaired through nonhomologous end joining or homologous recombination. TALENs can be used for specific gene knockout or knockin. We previously demonstrated that a p63 knockin reporter line generated with TALEN technology could be used for detailed analysis and isolation of p63-positive cells in SEAMs (27Kobayashi Y. Hayashi R. Quantock A.J. Nishida K. Generation of a TALEN-mediated, p63 knockin in human induced pluripotent stem cells.Stem Cell Res. 2017; 25 (29179035): 256-26510.1016/j.scr.2017.10.015Crossref PubMed Scopus (3) Google Scholar). Here, we report the generation of a PITX2 reporter line of hiPSCs harboring an IRES2-EGFP sequence, using TALEN technology. We validated the reporter line in a system in which PITX2 expression is induced in pluripotent stem cells. In addition, we were able to isolate and analyze POM cells. The PITX2 reporter hiPSC line generated in this study allows robust induction and isolation of POM-derived cells and insights into the detailed mechanisms of induction of POM cells and POM cell-derived cells. We evaluated Pitx2 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 Pitx2 and Foxc1, but negative for Sox10, 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. To achieve strong GFP fluorescence upon forced expression in 293T cells, various GFP variants, polycistronic sequences, and polyadenylation (poly(A)) signals were evaluated. First, three GFPs—EGFP, EmGFP, and TurboGFP—were inserted downstream of the PITX2 and 2A peptide sequences in pEF5/FRT/V5-DEST. There were no obvious differences in intensity between these three GFPs (Fig. S2A). As polycistronic sequences, 2A peptides and IRES2 were evaluated. Fluorescence intensity was stronger when EGFP was located downstream of IRES2 than when it was downstream of the 2A peptide sequences (Fig. S2B). As for polyadenylation, poly(A) signals of bovine growth hormone, herpes simplex virus–thymidine kinase, SV40, PITX2 3′-UTR, and β-actin 3′-UTR were evaluated. SV40 and PITX2 3′-UTR yielded slightly stronger EGFP intensity than the other poly(A) signals (Fig. S2C). Based on our findings, 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. There are six splice variants of PITX2 and three isoforms (Fig. 1A). Exon 8 is common to all PITX2 variants. Thus, we added the artificial sequences downstream of PITX2 exon 8. As shown in Fig. 1B, a TAL effector recognition site in the left arm was located immediately upstream of a stop codon of PITX2, and a right-arm recognition site was designed after the PITX2 stop codon. The donor vector was designed so that IRES2–EGFP–SV40 poly(A) followed the left arm of PITX2 with a silent mutation to avoid the generation of double-strand breaks after successful site-specific double-strand break generation by the TALEN. After electroporation of the TALEN vector and donor vector into 201B7 hiPSCs, the cells were seeded on DR4 mouse embryonic fibroblasts (MEFs) for drug selection. Knockin cells were screened on G418 sulfate as outlined in Fig. 2A. Based on PCR results, colony 8 (IRES2) was selected for further recloning analysis (Fig. 2B). Colonies 5 and 7 (2A) seemed to be successfully transfected; however, they were not further analyzed because the 2A peptides yielded lower EGFP intensity as shown in Fig. S2B. Colony 8 was recloned, and six colonies were analyzed by PCR, which revealed that the construct was heterozygously introduced in all six colonies. Colony 8-2 produced a slightly stronger band intensity than the other colonies (Fig. 2C) and was therefore chosen as the best candidate PITX2–EGFP hiPSC reporter line for further analysis. The genome sequence of this line was confirmed using Sanger sequencing (Fig. S3). After passaging the PITX2 knockin hiPSC line for feeder-free culture, the cells formed round, normal colonies on iMatrix-511, as shown in Fig. 3A. In an alkaline phosphatase (ALP)–staining assay, PITX2 knockin hiPSC clone 8-2 showed a staining intensity and color similar to those of 201B7 WT hiPSCs (Fig. 3B). Expression of the pluripotent markers NANOG, OCT3/4, TRA-1–60, and SSEA-4 was evaluated by immunofluorescence analysis using specific antibodies. All these markers showed strong expression from the center to the borders of the colonies (Fig. 3C). We successfully induced SEAM structures consisting of four zones using the PITX2 knockin hiPSC line as reported previously (22Hayashi R. Ishikawa Y. Sasamoto Y. Katori R. Nomura N. Ichikawa T. Araki S. Soma T. Kawasaki S. Sekiguchi K. Quantock A.J. Tsujikawa M. Nishida K. Co-ordinated ocular development from human iPS cells and recovery of corneal function.Nature. 2016; 531 (26958835): 376-38010.1038/nature17000Crossref PubMed Scopus (117) Google Scholar, 23Hayashi R. Ishikawa Y. Katori R. Sasamoto Y. Taniwaki Y. Takayanagi H. Tsujikawa M. Sekiguchi K. Quantock A.J. Nishida K. Coordinated generation of multiple ocular-like cell lineages and fabrication of functional corneal epithelial cell sheets from human iPS cells.Nat. Protoc. 2017; 12 (28253236): 683-69610.1038/nprot.2017.007Crossref PubMed Scopus (46) Google Scholar). After SEAM induction, markers of corneal epithelial cells (p63, PAX6) lens cells (p63, α-crystallin), neuroretina (CHX10), and retinal pigment epithelial cells (MITF) were stained (Fig. 3D). The cells in zone 3 were p63- and PAX6-positive and showed cobblestone morphology, which indicated that they were corneal epithelial cells. The aggregated cells at the end of zone 2 were p63- and α-crystallin–positive lens cells. The cells in the inner area of zone 2 were CHX10-positive neuroretinal cells. The cells aggregated in the outer area of zone 2 were MITF-positive retinal pigment epithelial cells. All these structures were similar to those induced in 201B7 hiPSCs. We also evaluated gene expression patterns in SEAMs (Fig. 3E). Expression of TUBB3, a neuron marker, was substantially higher in zone 1. Expression of the neural crest cell marker SOX10 and the neural retina marker RAX was higher in zone 2. PAX6 was expressed in all zones. Epithelial markers DN-p63, CDH1, and KRT18 were highly expressed in zones 3 and 4. The lens cell marker CRYAA was the most strongly expressed in zones 3 and 4. This expression pattern was similar to that of 201B7 hiPSCs (22Hayashi R. Ishikawa Y. Sasamoto Y. Katori R. Nomura N. Ichikawa T. Araki S. Soma T. Kawasaki S. Sekiguchi K. Quantock A.J. Tsujikawa M. Nishida K. Co-ordinated ocular development from human iPS cells and recovery of corneal function.Nature. 2016; 531 (26958835): 376-38010.1038/nature17000Crossref PubMed Scopus (117) Google Scholar). To confirm that EGFP is expressed in the PITX2 knockin hiPSC line, POM cells were induced by a combination of reported induction methods (28Milet C. Monsoro-Burq A.H. Neural crest induction at the neural plate border in vertebrates.Dev. Biol. 2012; 366 (22305800): 22-3310.1016/j.ydbio.2012.01.013Crossref PubMed Scopus (113) Google Scholar, 29Roy O. Leclerc V.B. Bourget J.-M. Thériault M. Proulx S. Understanding the process of corneal endothelial morphological change in vitro.Invest. Ophthalmol. Vis. Sci. 2015; 56 (25698769): 1228-123710.1167/iovs.14-16166Crossref PubMed Scopus (56) Google Scholar, 30Mimura S. Suga M. Okada K. Kinehara M. Nikawa H. Furue M.K. Bone morphogenetic protein 4 promotes craniofacial neural crest induction from human pluripotent stem cells.Int. J. Dev. Biol. 2016; 60 (26934293): 21-2810.1387/ijdb.160040mkCrossref PubMed Scopus (16) Google Scholar, 31Fukuta M. Nakai Y. Kirino K. Nakagawa M. Sekiguchi K. Nagata S. Matsumoto Y. Yamamoto T. Umeda K. Heike T. Okumura N. Koizumi N. Sato T. Nakahata T. Saito M. et al.Derivation of mesenchymal stromal cells from pluripotent stem cells through a neural crest lineage using small molecule compounds with defined media.PLoS One. 2014; 9 (25464501): e11229110.1371/journal.pone.0112291Crossref PubMed Scopus (95) Google Scholar) (Fig. 4A). Aggregated cells were detected ∼20 days after the start of induction (Fig. 4B). The aggregated cells showed EGFP signals at day 20 (Fig. 4C). We determined EGFP and PITX2 protein expression levels using whole cell lysates by Western blotting analyses. PITX2 expression in knockin iPSCs was nearly the same as that in clone 8-2 PITX2 knockin iPSCs (Fig. 4D), and we observed robust protein expression of EGFP in PITX2 knockin iPSC clone 8-2. Next, we conducted immunofluorescence staining using anti-PITX2 antibody to evaluate whether EGFP and PITX2 are expressed simultaneously. As shown in Fig. 4E, EGFP fluorescence and PITX2 fluorescent staining were detected simultaneously. Next, we sorted and collected EGFP-positive and -negative cells by FACS (Fig. 4F). We analyzed PITX2 expression levels by quantitative RT-PCR (qRT-PCR). The population of EGFP-positive cells exhibited high PITX2 expression, whereas EGFP-negative cells hardly expressed PITX2. Moreover, FOXC1 and TFAP2B, which are markers of POM cells, were significantly more strongly expressed in EGFP-positive cells when compared with EGFP-negative cells. Conversely, SOX10, which is a negative marker of POM cells, was more strongly expressed in EGFP-negative than in EGFP-positive cells. On the other hand, the POM markers FOXC2, LMX1B, NGFR, LMX1B, and COL8A2 were not highly expressed in EGFP-positive cells (Fig. 4G). To acquire more genuine POM cells, we tried a SEAM induction method (Fig. 5A). Typical, aggregated cells emerged in SEAM zone 2 after 14 days of induction (Fig. 5B). EGFP signals were confirmed at the location of aggregated cells in SEAM zone 2 (Fig. 5C). After sorting EGFP-positive cells (Fig. 5D), they were analyzed for marker expression by qRT-PCR. All positive POM cell markers were significantly increased in EGFP-positive compared with EGFP-negative cells, and there was no difference of SOX10 expression level between them (Fig. 5E), which revealed that the EGFP-positive cells were POM cells. They were cultivated for 2 days in a culture plate using differentiation medium (DM) with Y-27632, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and retinoic acid (Fig. 5F). Cells expressed PITX2 protein, which co-localized with FOXC1 protein in the nucleus (Fig. 5G). Various GFP variants, polycistronic sequences, and poly(A) signals were evaluated in a stepwise manner to establish a PITX2–EGFP knockin hiPSC reporter line with optimum GFP expression. The GFP variants EGFP, EmGFP, and TurboGFP did not show a difference in fluorescence intensity. Unexpectedly, the IRES2 sequence yielded stronger EGFP fluorescence than the 2A peptides. The 2A peptides were added at the C terminus of PITX2, and the proline added at the N terminus of GFP might have affected GFP expression or fluorescence. Alternatively, unknown mechanisms might determine the compatibility between target gene and following polycistronic sequences. The various polyadenylation signals tested yielded slightly different EGFP expression. Interestingly, among them, poly(A) of SV40 and PITX2 3′-UTR had the strongest ability to stabilize PITX2 mRNA. The efficiency of PITX2–IRES2–EGFP knockin was 1 of 12. This was more or less as expected, but there is room for improving the knockin efficiency, for example by increasing the vector concentrations and optimizing the electroporation program. The reporter line established in this study showed normal pluripotency based on ALP staining and immunofluorescence staining of the markers analyzed in this study. We confirmed that a PITX2 knockin hiPSC line formed typical SEAMs and showed a robust ability to induce lens cells, neuroretinal cells, and retinal pigment cells, although they induced less corneal epithelial cells than 201B7 hiPSCs did (Fig. 3D). Such a difference in differentiation tendency often occurs among pluripotent stem cell lines (32Nishizawa M. Chonabayashi K. Nomura M. Tanaka A. Nakamura M. Inagaki A. Nishikawa M. Takei I. Oishi A. Tanabe K. Ohnuki M. Yokota H. Koyanagi-Aoi M. Okita K. Watanabe A. et al.Epigenetic variation between human induced pluripotent stem cell lines is an indicator of differentiation capacity.Cell Stem Cell. 2016; 19 (27476965): 341-35410.1016/j.stem.2016.06.019Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). We were able to establish PITX2-expressing cells by combining published approaches: NC induction by WNT and low BMP signaling (28Milet C. Monsoro-Burq A.H. Neural crest induction at the neural plate border in vertebrates.Dev. Biol. 2012; 366 (22305800): 22-3310.1016/j.ydbio.2012.01.013Crossref PubMed Scopus (113) Google Scholar), corneal endothelial cell induction by EGF treatment, and FGF signaling for POM cell-derived tissue induction (29Roy O. Leclerc V.B. Bourget J.-M. Thériault M. Proulx S. Understanding the process of corneal endothelial morphological change in vitro.Invest. Ophthalmol. Vis. Sci. 2015; 56 (25698769): 1228-123710.1167/iovs.14-16166Crossref PubMed Scopus (56) Google Scholar). Unfortunately, the cells did not show perfect gene expression patterns of POM cells, although they showed high FOXC1 mRNA expression and low SOX10 mRNA expression, which are characteristic for POM cells (12Seo S. Chen L. Liu W. Zhao D. Schultz K.M. Sasman A. Liu T. Zhang H.F. Gage P.J. Kume T. Foxc1 and foxc2 in the neural crest are required for ocular anterior segment development.Invest. Ophthalmol. Vis. Sci. 2017; 58 (28253399): 1368-137710.1167/iovs.16-21217Crossref PubMed Scopus (22) Google Scholar, 33Hall B.K. Pigment cells (chromatophores).The Neural Crest and Neural Crest Cells in Vertebrate Development and Evolution. Springer, Boston, MA2009: 159-177Crossref Google Scholar). However, PITX2-expressing cells induced by the SEAM method did show perfect gene expression patterns of POM cells based on qRT-PCR results (Fig. 5E). Moreover, COL8A2 and COL8A1, which are markers of POM and corneal endothelial cell, were significantly increased in EGFP-positive cells. This might indicate that they are POM cells and have a potential to differentiate into POM-derived cells. In the near future, we would like to utilize this cell line to further analyze POM cells in SEAM-inducing and other culture conditions. In conclusion, we successfully generated and validated a PITX2–IRES2–EGFP knockin hiPSC line, and we were able to isolate PITX2-expressing POM cells. These cells showed higher POM marker expression than EGFP-negative cells. The POM cells sorted in this study are a reliable tool for detailed analysis of POM-derived ocular cells. Our reporter line provides a technical platform for testing induction methods and for detailed analysis of PITX2-expressing cells and their derivatives. All animal experimental protocols in this study were in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Visual Research, with prior approval from the Animal Ethics Committee of Osaka University. E10.5 and E12.5 ICR mice were purchased from Japan SLC (Sizuoka, Japan). After the mice were euthanized with pentobarbital sodium, they were perfused with PBS. Mouse embryos were collected and were also euthanized. They were embedded in Tissue-Tek O.C.T. compound (Sakura Finetek Japan, Tokyo, Japan), frozen on dry ice, and stored at −80 °C. The m" @default.
• W3005226072 created "2020-02-14" @default.
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• W3005226072 date "2020-03-01" @default.
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• W3005226072 title "Generation and validation of a PITX2–EGFP reporter line of human induced pluripotent stem cells enables isolation of periocular mesenchymal cells" @default.
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