Fig. 3

Characteristics of C-Kit⁺ cells obtained from hEROs. a C-Kit expression in 30-day sorted C-Kit⁺ cells at passage 0 (P0). b Comparison of cell doubling time among C-Kit⁺ cells sorted from 30-day hEROs (n = 3 independent experiments/group). c Representative putative clone formation by P3 C-Kit⁺ cells at 2 weeks shown through the limited dilution colony-forming efficiency assay. d–h Expression and quantitative analysis of RPC markers and the proliferation marker Ki67 in C-Kit⁺ cells at different passages. (n = 3 independent experiments/group). i–m Retinal cell differentiation of C-Kit⁺ cells into photoreceptors was visualized by immunostaining for recoverin and rhodopsin. Bipolar cells were identified by PKCα staining, Müller cells were identified by GS staining, and retinal ganglion cells were identified by Tuj1 staining. n, o Flow cytometry analysis of the expression of embryonic markers, including Nanog and OCT4, in P3 C-Kit⁺ cells. (n = 3 independent experiments). p, q Tumorigenesis test of P3 C-Kit⁺ cells and human embryonic stem cells (hESCs). No tumor formation was detected in the inguinal region 3 months after C-Kit⁺ cell injection into 6 SCID mice (black arrow in p). After hESCs injection, 4/6 animals displayed tumor formation (black arrow in q) (n = 6 animals/group). Data are presented as mean ± SD. Scale bars, 25 μm (a, d–g, i–m), 400 μm (c)