SummaryThe plasticity of pluripotent stem cells provides new possibilities for studying development, degeneration, and regeneration. Protocols for the differentiation of retinal organoids from embryonic stem cells have been developed, which either recapitulate complete eyecup morphogenesis or maximize photoreceptor genesis. Here, we have developed a protocol for the efficient generation of large, 3D-stratified retinal organoids that does not require evagination of optic-vesicle-like structures, which so far limited the organoid yield. Analysis of gene expression in individual organoids, cell birthdating, and interorganoid variation indicate efficient, reproducible, and temporally regulated retinogenesis. Comparative analysis of a transgenic reporter for PAX6, a master regulator of retinogenesis, shows expression in similar cell types in mouse in vivo, and in mouse and human retinal organoids. Early or late Notch signaling inhibition forces cell differentiation, generating organoids enriched with cone or rod photoreceptors, respectively, demonstrating the power of our improved organoid system for future research in stem cell biology and regenerative medicine.

The mechanisms limiting neuronal regeneration in mammals and their relationship with reactive gliosis are unknown. Müller glia (MG), common to all vertebrate retinas, readily regenerate neuron loss in some species, but normally not in mammals. However, experimental stimulation of limited mammalian retina regeneration has been reported. Here, we use a mouse retina organ culture approach to investigate the MG responses at different mouse ages. We found that MG undergo defined spatio-temporal changes upon stimulation. In EGF-stimulated juvenile postmitotic retinas, most MG upregulate cell-cycle regulators (Mcm6, Pcna, Ki67, Ccnd1) within 48 h ex vivo; some also express the neurogenic factors Ascl1, Pax6, and Vsx2; up to 60% re-enter the cell cycle, some of which delaminate to divide mostly apically; and the majority cease to proliferate after stimulation. A subpopulation of MG progeny starts to express transcription factors (Ptf1a, Nr4a2) and neuronal (Calb1, Calb2, Rbfox3), but not glial, markers, indicating neurogenesis. BrdU-tracking, genetic lineage-tracing, and transgenic-reporter experiments suggest that MG reprogram to a neurogenic stage and proliferate; and that some MG progeny differentiate into neuronal-like cells, most likely amacrines, no photoreceptors; most others remain in a de-differentiated state. The mouse MG regeneration potential becomes restricted, dependent on the age of the animal, as observed by limited activation of the cell cycle and neurogenic factors. The stage-dependent analysis of mouse MG revealed similarities and differences when compared with MG-derived regeneration in fish and chicks. Therefore, the mouse retina ex vivo approach is a potential assay for understanding and overcoming the limitations of mammalian MG-derived neuronal regeneration. Postmitotic MG in mouse retina ex vivo can be stimulated to proliferate, express neurogenic factors, and generate progeny expressing neuronal or glial markers. This potential regenerative competence becomes limited with increasing mouse age.

Comparison of photoreceptor grafts in preclinical models with incomplete or complete photoreceptor loss, showed differential transplant success with effective and impaired integration, respectively. Thus, Cpfl1/Rho-/- mice represent a potential benchmark model resembling patients with severe retinal degeneration to optimize photoreceptor replacement therapies.

Once human photoreceptors die, they do not regenerate, thus, photoreceptor transplantation has emerged as a potential treatment approach for blinding diseases. Improvements in transplant organization, donor cell maturation, and synaptic connectivity to the host will be critical in advancing this technology for use in clinical practice. Unlike the unstructured grafts of prior cell-suspension transplantations into end-stage degeneration models, we describe the extensive incorporation of induced pluripotent stem cell (iPSC) retinal organoid–derived human photoreceptors into mice with cone dysfunction. This incorporative phenotype was validated in both cone-only as well as pan-photoreceptor transplantations. Rather than forming a glial barrier, Müller cells extended throughout the graft, even forming a series of adherens junctions between mouse and human cells, reminiscent of an outer limiting membrane. Donor-host interaction appeared to promote polarization as well as the development of morphological features critical for light detection, namely the formation of inner and well-stacked outer segments oriented toward the retinal pigment epithelium. Putative synapse formation and graft function were evident at both structural and electrophysiological levels. Overall, these results show that human photoreceptors interacted readily with a partially degenerated retina. Moreover, incorporation into the host retina appeared to be beneficial to graft maturation, polarization, and function.

No abstract