Reliability of human retina organoid generation from hiPSC-derived neuroepithelial cysts.

The possible applications for human retinal organoids (HROs) derived from human induced pluripotent stem cells (hiPSC) rely on the robustness and transferability of the methodology for their generation. Standardized strategies and parameters to effectively assess, compare, and optimize organoid protocols are starting to be established, but are not yet complete. To advance this, we explored the efficiency and reliability of a differentiation method, called CYST protocol, that facilitates retina generation by forming neuroepithelial cysts from hiPSC clusters. Here, we tested seven different hiPSC lines which reproducibly generated HROs. Histological and ultrastructural analyses indicate that HRO differentiation and maturation are regulated. The different hiPSC lines appeared to be a larger source of variance than experimental rounds. Although previous reports have shown that HROs in several other protocols contain a rather low number of cones, HROs from the CYST protocol are consistently richer in cones and with a comparable ratio of cones, rods, and Müller glia. To provide further insight into HRO cell composition, we studied single cell RNA sequencing data and applied CaSTLe, a transfer learning approach. Additionally, we devised a potential strategy to systematically evaluate different organoid protocols side-by-side through parallel differentiation from the same hiPSC batches: In an explorative study, the CYST protocol was compared to a conceptually different protocol based on the formation of cell aggregates from single hiPSCs. Comparing four hiPSC lines showed that both protocols reproduced key characteristics of retinal epithelial structure and cell composition, but the CYST protocol provided a higher HRO yield. So far, our data suggest that CYST-derived HROs remained stable up to at least day 200, while single hiPSC-derived HROs showed spontaneous pathologic changes by day 200. Overall, our data provide insights into the efficiency, reproducibility, and stability of the CYST protocol for generating HROs, which will be useful for further optimizing organoid systems, as well as for basic and translational research applications.

Modeling inducible neuropathologies of the retina with differential phenotypes in organoids.

Neurodegenerative diseases remain incompletely understood and therapies are needed. Stem cell-derived organoid models facilitate fundamental and translational medicine research. However, to which extent differential neuronal and glial pathologic processes can be reproduced in current systems is still unclear. Here, we tested 16 different chemical, physical, and cell functional manipulations in mouse retina organoids to further explore this. Some of the treatments induce differential phenotypes, indicating that organoids are competent to reproduce distinct pathologic processes. Notably, mouse retina organoids even reproduce a complex pathology phenotype with combined photoreceptor neurodegeneration and glial pathologies upon combined (not single) application of HBEGF and TNF, two factors previously associated with neurodegenerative diseases. Pharmacological inhibitors for MAPK signaling completely prevent photoreceptor and glial pathologies, while inhibitors for Rho/ROCK, NFkB, and CDK4 differentially affect them. In conclusion, mouse retina organoids facilitate reproduction of distinct and complex pathologies, mechanistic access, insights for further organoid optimization, and modeling of differential phenotypes for future applications in fundamental and translational medicine research.

HBEGF-TNF induce a complex outer retinal pathology with photoreceptor cell extrusion in human organoids.

Human organoids could facilitate research of complex and currently incurable neuropathologies, such as age-related macular degeneration (AMD) which causes blindness. Here, we establish a human retinal organoid system reproducing several parameters of the human retina, including some within the macula, to model a complex combination of photoreceptor and glial pathologies. We show that combined application of TNF and HBEGF, factors associated with neuropathologies, is sufficient to induce photoreceptor degeneration, glial pathologies, dyslamination, and scar formation: These develop simultaneously and progressively as one complex phenotype. Histologic, transcriptome, live-imaging, and mechanistic studies reveal a previously unknown pathomechanism: Photoreceptor neurodegeneration via cell extrusion. This could be relevant for aging, AMD, and some inherited diseases. Pharmacological inhibitors of the mechanosensor PIEZO1, MAPK, and actomyosin each avert pathogenesis; a PIEZO1 activator induces photoreceptor extrusion. Our model offers mechanistic insights, hypotheses for neuropathologies, and it could be used to develop therapies to prevent vision loss or to regenerate the retina in patients suffering from AMD and other diseases.

Transplanted human cones incorporate into the retina and function in a murine cone degeneration model.

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.

Mouse Retinal Organoid Growth and Maintenance in Longer-Term Culture.

Using retinal organoid systems, organ-like 3D tissues, relies implicitly on their robustness. However, essential key parameters, particularly retinal growth and longer-term culture, are still insufficiently defined. Here, we hypothesize that a previously optimized protocol for high yield of evenly-sized mouse retinal organoids with low variability facilitates assessment of such parameters. We demonstrate that these organoids reliably complete retinogenesis, and can be maintained at least up to 60 days in culture. During this time, the organoids continue to mature on a molecular and (ultra)structural level: They develop photoreceptor outer segments and synapses, transiently maintain its cell composition for about 5-10 days after completing retinogenesis, and subsequently develop pathologic changes - mainly of the inner but also outer retina and reactive gliosis. To test whether this organoid system provides experimental access to the retina during and upon completion of development, we defined and stimulated organoid growth by activating sonic hedgehog signaling, which in patients and mice in vivo with a congenital defect leads to enlarged eyes. Here, a sonic hedgehog signaling activator increased retinal epithelia length in the organoid system when applied during but not after completion of development. This experimentally supports organoid maturation, stability, and experimental reproducibility in this organoid system, and provides a potential enlarged retina pathology model, as well as a protocol for producing larger organoids. Together, our study advances the understanding of retinal growth, maturation, and maintenance, and further optimizes the organoid system for future utilization.

Optimized Adeno-Associated Virus Vectors for Efficient Transduction of Human Retinal Organoids.

The most widely used vectors for gene delivery in the retina are recombinant adeno-associated virus (rAAV) vectors. They have proven to be safe and effective in retinal gene therapy studies aimed to treat inherited retinal dystrophies, although with various limitations in transduction efficiency. Novel variants with modified capsid sequences have been engineered to improve transduction and overcome limitations of naturally occurring variants. Although preclinical evaluation of rAAV vectors based on such novel capsids is mostly done in animal models, the use of human induced pluripotent stem cell (hiPSC)-derived organoids offers an accessible and abundant human testing platform for rAAV evaluation. In this study, we tested the novel capsids, AAV9.GL and AAV9.NN, for their tropism and transduction efficiency in hiPSC-derived human retinal organoids (HROs) with all major neuronal and glial cell types in a laminated structure. These variants are based on the AAV9 capsid and were engineered to display specific surface-exposed peptide sequences, previously shown to improve the retinal transduction properties in the context of AAV2. To this end, HROs were transduced with increasing concentrations of rAAV9, rAAV9.GL, or rAAV9.NN carrying a self-complementary genome with a cytomegalovirus-enhanced green fluorescent protein (eGFP) cassette and were monitored for eGFP expression. The rAAV vectors transduced HROs in a dose-dependent manner, with rAAV9.NN achieving the highest efficiency and fastest onset kinetics, leading to detectable eGFP signals in photoreceptors, some interneurons, and Müller glia already at 2 days post-transduction. The potency-enhancing effect of the NN peptide insert was replicated when using the corresponding AAV2-based version (rAAV2.NN). Taken together, we report the application of an HRO system for screening novel AAV vectors and introduce novel vector candidates with enhanced transduction efficiency for human retinal cells.

The Mouse Retinal Organoid Trisection Recipe: Efficient Generation of 3D Retinal Tissue from Mouse Embryonic Stem Cells.

The introduction of stem cell-based technologies for the derivation of three-dimensional retinal tissues, the so-called retinal organoids, offers many new possibilities for vision research: Organoids facilitate studies on retinal development and in vitro retinal disease modeling, as well as being valuable for drug testing. Further, retinal organoids also provide an unlimited cell source for cell replacement therapies. Here, we describe our protocol for efficiently differentiating large, stratified retinal organoids from mouse embryonic stem cells: unbiased manual dissection of the developing retinal organoid at an early stage into three evenly sized neuroepithelial portions (trisection step) doubles the yield of high-quality organoids. We also describe some useful applications of the protocol, e.g., generation of rod- or cone-enriched retinal organoids, AAV transfection, and cell birth dating. In addition, we provide details of how to process retinal organoids for single organoid gene expression analysis, immunohistochemistry, and electron microscopy.

Stem Cell-Derived Photoreceptor Transplants Differentially Integrate Into Mouse Models of Cone-Rod Dystrophy.

PURPOSE: Preclinical studies on photoreceptor transplantation provided evidence for restoration of visual function with pluripotent stem cells considered as a potential source for sufficient amounts of donor material. Adequate preclinical models representing retinal disease conditions of potential future patients are needed for translation research. Here we compared transplant integration in mouse models with mild (prominin1-deficient; Prom1-/-) or severe (cone photoreceptor function loss 1/rhodopsin-deficient double-mutant; Cpfl1/Rho-/-) cone-rod degeneration. METHODS: For photoreceptor transplant production, we combined the mouse embryonic stem cell retinal organoid system with rhodopsin-driven GFP cell labeling by recombinant adeno-associated virus (AAV). Organoid-derived photoreceptors were enriched by CD73-based magnetic-activated cell sorting (MACS) and transplanted subretinally into wild-type, Prom1-/- and Cpfl1/Rho-/- hosts. The survival, maturation, and synapse formation of donor cells was analyzed by immunohistochemistry. RESULTS: Retinal organoids yielded high photoreceptor numbers that were further MACS-enriched to 85% purity. Grafted photoreceptors survived in the subretinal space of all mouse models. Some cells integrated into wild-type as well as Prom1-/- mouse retinas and acquired a mature morphology, expressing rod and synaptic markers in close proximity to second-order neurons. In contrast, in the novel Cpfl1/Rho-/- model with complete photoreceptor degeneration, transplants remained confined to the subretinal space, expressed rod-specific but only reduced synaptic markers, and did not acquire mature morphology. CONCLUSIONS: 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.

Retinal Organoids from Pluripotent Stem Cells Efficiently Recapitulate Retinogenesis.

The 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.

Age-dependent Müller glia neurogenic competence in the mouse retina.

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.