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. 2022 Jan 4;132(1):e146658. doi: 10.1172/JCI146658

Figure 1. Induction and characterization of corneal endothelial precursors from hESCs.

Figure 1

(A) Schematic representation of the method used for the generation of NCCs, CEPs, and CECs. For the NCC induction, hPSCs are incubated in the neural crest differentiation medium with 4 ng/mL bFGF and 1 μM RA for 5 days. For the CEP and CEC induction, the medium is changed into corneal endothelial differentiation medium with 10 μM Y27632 and 1 μM SB431542 for a subsequent 3 days and 14 days. (B) Representative immunofluorescence staining of the neural crest markers P75 and HNK-1, and corneal endothelial markers ZO-1, ATP1A1, N-cadherin, and SLC4A11 in CEPs. Nuclei were stained with DAPI. Scale bar: 50 μm. (C) Flow cytometry analysis of ATP1A1, TRA-1-60, HNK-1, and SLC4A11 in CEPs. The experiments were repeated 3 times. (D) qPCR analysis of the gene expression during the 3 stages of hESC differentiation. n = 3, **P < 0.01, *P < 0.05 by 1-way ANOVA with Tukey’s HSD test.