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A three-dimensional retinal tissue (3D-retina) is a promising graft source for retinal transplantation therapy. We previously demonstrated that embryonic stem cells (ESCs) can generate 3D-retina  in vitro  using a self-organizing stem cell culture technique known as SFEBq. Here we show an optimized culture method for 3D-retina generation from feeder-free human pluripotent stem cells (hPSCs). Although feeder-free hPSC-maintenance culture was suitable for cell therapy, feeder-free hPSC-derived aggregates tended to collapse during 3D-xdifferentiation culture. We found that the initial hPSC state was a key factor and that preconditioning of the hPSC state by modulating TGF-beta and Shh signaling improved self-formation of 3D-neuroepithelium. Using the preconditioning method, several feeder-free hPSC lines robustly differentiated into 3D-retina. In addition, changing preconditioning stimuli in undifferentiated hPSCs altered the proportions of neural retina and retinal pigment epithelium, important quality factors for 3D-retina. We demonstrated that the feeder-free hiPSC-derived 3D-retina differentiated into rod and cone photoreceptors  in vitro  and  in vivo . Thus, preconditioning is a useful culture methodology for cell therapy to direct the initial hPSC state toward self-organizing 3D-neuroepithelium.

Preconditioning the Initial State of Feeder-free Human Pluripotent Stem Cells Promotes Self-formation of Three-dimensional Retinal Tissue