CRX Expression in Pluripotent Stem Cell-Derived Photoreceptors Marks a Transplantable Subpopulation of Early Cones

Abstract

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

Human iPSC differentiation to retinal organoids in response to IGF1 and BMP4 activation is line- and method-dependent

“…Induced pluripotent stem cell (IPSC)-derived retinal organoids have been shown to have a wide range of applications, including the study of human retinogenesis, 1-3 disease modeling, 4,5 drug discovery, 6,7 and cell therapy. [8][9][10] Numerous protocols have been developed for the generation of retinal organoids that follow basic developmental principles of forebrain development and eye formation. Despite the ability of these protocols to give rise to laminated retinal organoids, variability in the propensity of iPSCs to give rise to various retinal cell types is often reported.…”

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“…Previously, the research team headed by Majlinda Lako (Newcastle University, Newcastle‐upon‐Tyne, UK) generated a CRX‐green fluorescent protein reporter hESC line, with CRX a key transcription factor in retinal development with predominant expression in photoreceptor precursors . Now, the team returns with a Stem Cells article that employs this reporter line to investigate the transcriptional profile of differentiated CRX‐expressing photoreceptor precursors and the engraftment of these cells in a mouse model of retinitis pigmentosa characterized by the rapid degeneration of photoreceptors . Collin et al began by differentiating reporter hESCs into retinal organoids and then sorting/enriching CRX‐expressing photoreceptor precursors.…”

“…However, there remains the need to enhance the efficiency and scalability of retinal organoid production, to optimize differentiation protocols, to characterize the retinal cell types within the organoids, and improve the clinical applicability of the produced retinal cells. In our second Feature Article this month from Stem Cells , Collin et al report that CRX expression marks a population of homogenous photoreceptor precursors committed to an early cone phenotype that make connections with host interneurons and mature into cones when transplanted into degenerated retinae . A Related Article from Stem Cells Translational Medicine by Zhu et al supports the advancement of cell replacement therapy for preclinical studies and clinical applications by describing the generation of photoreceptors from a current Good Manufacturing Practice (cGMP) compliant hiPSC line employing a novel small‐molecule induction protocol .…”

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“…5The development of human embryonic (hESC) and induced pluripotent (hiPSC) stem cell-derived retinal organoids that exhibit the characteristics of the whole laminated neural retina6,7 has been driven by the lack of animal models that fully recapitulate the structure and function of the human retina. The continual development of retinal organoid formation protocols has allowed advancements in multiple areas, including the study of human retinogenesis, disease modeling, drug discovery, and importantly, cell therapy 8. Indeed, the availability of various sources of hESCs/hiPSCs that comply with regulatory requirements for human therapies may facilitate the derivation of cells for retinal therapies in the near future; however, the protocols involved still require additional optimization to foster the efficient and cost-effective generation of specific cell types within the retinal organoid.…”