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Motokazu Tsujikawa, Erika Kimura, Yoshinori Oie, Takeshi Soma, Susumu Hara, Ryuhei Hayashi, Shin Hatou, Satoru Yoshida, Shigeto Shimmura, Kohji Nishida; The effect of retinoic acid to induce one of the perioculer neural crest. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1391.
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© ARVO (1962-2015); The Authors (2016-present)
Corneal endothelium (CE) is developed from neural crest (NC) cells. NC emerges at the interface between neural and non-neural ectoderm and migrates extensively to form skeleton, cornea, teeth, thymus adrenal glands, and other tissue. To develop CE from human induced pluripotent stem (iPS) cells, we developed a protocol to derive NC cells from human iPS cells. However, these NC cells do not express cranial and especially periocular NC markers. In this research, we investigated the effect of retinoic acid to induce one of the perioculer NC markers, PITX2.
iPS cells (253G1) were cultured for weeks by Sphere culture without serum, and NC cells were isolated by FACS using p75NTR as a marker. These NC cells were seeded on dishes with retinoic acid in various concentrations. The cells were harvested and the expression of PITX2 was quantified by RT-PCR method. To obtain highest expression of PITX2, the concentration and the duration of RA application was modified. We also investigate NC markers, p75NTR and SOX10.
PITX2 expression level showed a peak 5 to 10 days after application of RA. (<0.01% of GAPDH expression level before application, 0.95% of that in 6 days after application, respectively, Fig. 1)Expression of NC markers, such as p75NTR and SOX10 was decreased.
RA is a key factor which determines the cell fate from NC cells to perioculer NC cells.
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