Optic cup-like structures were differentiated from hESCs in vitro. The brightfield (A, C, E, G) and fluorescence (B, D, F, H) images of the differentiation process. (A, B) Undifferentiated hESCs were cultured on feeder. (C, D) Embryoid bodies were formed *3 to 4 days after suspension culture of hESCs aggregated. (E, F) Neural rosettes were differentiated within weeks after adherent culture of embryoid bodies. (G, H) Optic cuplike structures were self-organized within 2 weeks after suspension culture of the neural epithelium. Scale bar = 200 mm (in A-D, G, H), scale bar = 50 mm (in E, F). hESCs, human embryonic stem cells. Color images are available online.

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... enhanced green fluorescent protein-expressing human ES (H9 inGFPhES) cell line was gifted from the laboratory of SuChun Zhang (Waisman Center and Wicell Research Institute). Cells were maintained in ESC medium (see Supplementary Table S1 for media) for several passages for acclimation to culture conditions, and then were differentiated according to previously established methods (Supplementary Fig. S1). Partially differentiated colonies were manually removed before differentiation analysis. ...

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... hESCs were maintained in the undifferentiated state using mouse fibroblasts, and the morphology of stem cell clones were well defined (Fig. 1A, B). In particular, differentiated colonies were manually removed before subculture and differentiation analysis. The hESCs were identified by expression of the stem cell markers Nanog and Oct 3/4 ( Supplementary Fig. S2). Following our published efficient differentiation protocol for producing optic cup-like structures from hESCs, the ...

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... colonies were manually removed before subculture and differentiation analysis. The hESCs were identified by expression of the stem cell markers Nanog and Oct 3/4 ( Supplementary Fig. S2). Following our published efficient differentiation protocol for producing optic cup-like structures from hESCs, the embryoid bodies were formed on days 3-4 (Fig. 1C, D) after detaching the hESC clones from the feeder. On days 14-16, hESC-derived neural tube-like rosettes were observed (Fig. 1E, F). On days 28-35, the optic cup-like structures were formed spontaneously and self-organized (Fig. 1G, H), and the transparent neuroepithelia structures were clearly observed within ...

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... stem cell markers Nanog and Oct 3/4 ( Supplementary Fig. S2). Following our published efficient differentiation protocol for producing optic cup-like structures from hESCs, the embryoid bodies were formed on days 3-4 (Fig. 1C, D) after detaching the hESC clones from the feeder. On days 14-16, hESC-derived neural tube-like rosettes were observed (Fig. 1E, F). On days 28-35, the optic cup-like structures were formed spontaneously and self-organized (Fig. 1G, H), and the transparent neuroepithelia structures were clearly observed within ...

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... protocol for producing optic cup-like structures from hESCs, the embryoid bodies were formed on days 3-4 (Fig. 1C, D) after detaching the hESC clones from the feeder. On days 14-16, hESC-derived neural tube-like rosettes were observed (Fig. 1E, F). On days 28-35, the optic cup-like structures were formed spontaneously and self-organized (Fig. 1G, H), and the transparent neuroepithelia structures were clearly observed within ...

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... helix-loop-helix transcription factor that is specifically expressed by RPCs. This is required not only for cell cycle progression but also for early multiple retinal neuronal differentiation, including RGCs, photoreceptor, horizontal, and amacrine cells [25][26][27]. The vast majority of cells in optic cup-like structures were Math5 positive (Fig. 2A1), suggesting that these cells were ...

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... climbing pieces of the cells extracted from the optic cup-like structures were collected and detected by using double immunofluorescence. As expected, we found abundant expression of a number of transcription factors, including Math5 (Fig. 3A1) and Pax6 (Fig. 3B1). Previous studies have shown that these markers were specially expressed by RPCs. Meanwhile, all nuclei of the cells derived from hESCs were efficiently labeled with human nuclei antigen (hNu) (Fig. 3A2, B2). To characterize hNu, it is more convenient to identify and quantify the transplanting cells than the GFP ...

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... climbing pieces of the cells extracted from the optic cup-like structures were collected and detected by using double immunofluorescence. As expected, we found abundant expression of a number of transcription factors, including Math5 (Fig. 3A1) and Pax6 (Fig. 3B1). Previous studies have shown that these markers were specially expressed by RPCs. Meanwhile, all nuclei of the cells derived from hESCs were efficiently labeled with human nuclei antigen (hNu) (Fig. 3A2, B2). To characterize hNu, it is more convenient to identify and quantify the transplanting cells than the GFP cells, although the ...

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... were Math5+/Brn3b+. Nuclei were stained with hNu (green) (A2, B2) and DAPI (blue) (A3, B3). Merged images are shown in A4 and B4. Scale bar = 50 mm. Color images are available online. [31]. Therefore, on the fourth week after transplantation, a significant reduction in Brn3a-positive cells (red) were observed in the NMDA-treated retina (Fig. 4A1, B1) compared with the control group. This demonstrated that the experimental glaucoma models were made successfully. Meanwhile, many hNu-positive cells were also clearly observed in the host retina (Fig. 4A2, B2). These data showed that the transplanting cells migrated from the vitreous cavity to the retina and survived. Surprisingly, it ...

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... visual function of the host. To determine whether the RGCs could be generated by the transplanting RPCs derived from hESCs, the host retinas were analyzed by using triple immunofluorescence staining on the fifth week after transplantation (Fig. 5). Brn3a, a mature ganglion cell marker, was used to label the host residual RGCs in the mouse retina (Fig. 5A1, B1). As expected, the complementary distribution of hNupositive cells and Brn3a-positive cells was also observed in the GCL of mouse retinas (Fig. 5A4). Surprisingly, a small number of hNu-positive cells were colabeled with Brn3a (Fig. 6B1, C1, D1, E1), which indicated that the mature RGCs have been generated even although the expression ...

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... 5). Brn3a, a mature ganglion cell marker, was used to label the host residual RGCs in the mouse retina (Fig. 5A1, B1). As expected, the complementary distribution of hNupositive cells and Brn3a-positive cells was also observed in the GCL of mouse retinas (Fig. 5A4). Surprisingly, a small number of hNu-positive cells were colabeled with Brn3a (Fig. 6B1, C1, D1, E1), which indicated that the mature RGCs have been generated even although the expression level of Brn3a was lower than in the host conventional RGCs. The aforementioned data showed that the transplanting RPCs contained the ganglion cell progenitors, but the percentage is lower; some new RGCs may be differentiated from RPCs under ...

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... aforementioned data showed that the transplanting RPCs contained the ganglion cell progenitors, but the percentage is lower; some new RGCs may be differentiated from RPCs under the influence of the host retina microenvironment. Furthermore, the same results were further certified from the level of retinal sections ( Supplementary Fig. S5); the surviving transplanted cells (hNu + ) (red) were clearly observed in the host retina GCL (Supplementary Fig. S5A1, A2), and they also expressed Brn3a (pink) ( Supplementary Fig. S5B1, B2), which is a classic marker of mature RGCs, confirming their potential to differentiate into RGCs. Meanwhile, other surviving transplanting cells localized in the GCL were Brn3a negative (Fig. 6F1). ...

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... aforementioned data showed that the transplanting RPCs contained the ganglion cell progenitors, but the percentage is lower; some new RGCs may be differentiated from RPCs under the influence of the host retina microenvironment. Furthermore, the same results were further certified from the level of retinal sections ( Supplementary Fig. S5); the surviving transplanted cells (hNu + ) (red) were clearly observed in the host retina GCL (Supplementary Fig. S5A1, A2), and they also expressed Brn3a (pink) ( Supplementary Fig. S5B1, B2), which is a classic marker of mature RGCs, confirming their potential to differentiate into RGCs. Meanwhile, other surviving transplanting cells localized in the GCL were Brn3a negative (Fig. 6F1). ...

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... (hNu + ) (red) were clearly observed in the host retina GCL (Supplementary Fig. S5A1, A2), and they also expressed Brn3a (pink) ( Supplementary Fig. S5B1, B2), which is a classic marker of mature RGCs, confirming their potential to differentiate into RGCs. Meanwhile, other surviving transplanting cells localized in the GCL were Brn3a negative (Fig. 6F1). These cells may be amacrine cells or another type of ganglion cell. Either way, the complementary distribution of these cells with remaining host RGCs might be helpful for the retina. On the whole, the overall efficiency integration was poor (*5%) on the fifth week after transplantation. Excitingly, there was no teratoma appeared in ...