Death of photoreceptors is a common cause of age-related and inherited retinal dystrophies, and thus their replenishment from renewable stem cell sources is a highly desirable therapeutic goal. Human pluripotent stem cells provide a useful cell source in view of their limitless self-renewal capacity and potential to not only differentiate into cells of the retina but also self-organize into tissue with structure akin to the human retina as part of three-dimensional retinal organoids. Photoreceptor precursors have been isolated from differentiating human pluripotent stem cells through application of cell surface markers or fluorescent reporter approaches and shown to have a similar transcriptome to fetal photoreceptors. In this study, we investigated the transcriptional profile of CRX-expressing photoreceptor precursors derived from human pluripotent stem cells and their engraftment capacity in an animal model of retinitis pigmentosa (Pde6brd1), which is characterized by rapid photoreceptor degeneration. Single cell RNA-Seq analysis revealed the presence of a dominant cell cluster comprising 72% of the cells, which displayed the hallmarks of early cone photoreceptor expression. When transplanted subretinally into the Pde6brd1 mice, the CRX⁺ cells settled next to the inner nuclear layer and made connections with the inner neurons of the host retina, and approximately one-third of them expressed the pan cone marker, Arrestin 3, indicating further maturation upon integration into the host retina. Together, our data provide valuable molecular insights into the transcriptional profile of human pluripotent stem cells-derived CRX⁺ photoreceptor precursors and indicate their usefulness as a source of transplantable cone photoreceptors. Stem Cells  2019;37:609–622

Diseases affecting the retina, the light-sensitive extension of the central nervous system, account for approximately 26% of global blindness. Human pluripotent stem cells have been shown to differentiate into various retinal cell types, including photoreceptors, which can be enriched by cell surface or fluorescent molecule tagging approaches. Molecular heterogeneity of photoreceptor precursors derived from human pluripotent stem cells and their capacity to engraft into a fast degenerative model of retinitis pigmentosa have been investigated. Data show that photoreceptor precursors characterized by CRX expression are largely homogenous and committed to an early cone phenotype. Upon transplantation into degenerated retinae, these precursors settle into the appropriate layer, make connections with the host interneurons, and mature into cones. Future work is needed to assess at the functional level whether the transplanted cells are able to restore vision in degenerative models of retinal disease.