Fig. 2

Multizone ocular progenitor cells expanded the neuroectoderm, surface ectoderm and neural crest cell regions. A Representative phase-contrast images of CB30 hiPSC differentiation toward multizone ocular progenitor cells (mzOPCs). The images of mzOPCs at 10 (D10), 20 (D20) and 30 (D30) DIV show the eye-field primordial clusters that develop in neuroectoderm (NE), surface ectoderm (SE), retinal pigment epithelium (RPE) and neural retina (NR) (n > 8 independent experiments). Scale bars: 500 µm. B Immunofluorescence images of hiPSCs expressing the pluripotency stem cell markers NANOG, TRA-1-81, OCT4, SSEA3, SOX2, SSEA4 and TRA-1-60 (a–c). mzOPCs at 10 DIV (d–f) and at day 20 (g–i) expressed NE-specific markers PAX6, NRL, and MITF; SE-specific markers CK19 and p63; and neural crest (NC)-markers SOX9, SOX10 and p75-NGFR. At day 30 (j–l), mzOPCs consisted of differentiated ocular clusters, including NR (RAX, TUJ1, PAX6); RPE (MITF); surface epithelial cells (CK19) and neural crest cells (SOX9). Nuclei were stained in DAPI. Scale bars: 100 µm in a–c, g, h, j,k; 50 µm in e, f,l; 25 µm in d, i. C Relative gene expression detected by RT-qPCR in hiPSCs and mzOPCs at 30 DIV of eye-field transcription factors PAX6, RAX, SIX6, SE markers p63 and CK19, the RPE-specific marker MITF, and the pluripotency marker OCT4. Values are normalized to GAPDH. Data are presented as the mean ± SD (n = 3 independent experiments). Values indicated with stars are significantly different from those in hiPSCs (Student’s t-test; *p < 0.05; **p < 0.001)