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Summary Phagocytosis is a key function in various cells throughout the body. A deficiency in photoreceptor outer segment (POS) phagocytosis by the retinal pigment epithelium (RPE) causes vision loss in inherited retinal diseases and possibly age-related macular degeneration. To date, there are no effective therapies available aiming at recovering the lost phagocytosis function. Here, we developed a high-throughput screening assay based on RPE derived from human embryonic stem cells (hRPE) to reveal enhancers of POS phagocytosis. One of the hits, ramoplanin (RM), reproducibly enhanced POS phagocytosis and ensheathment in hRPE, and enhanced the expression of proteins known to regulate membrane dynamics and ensheathment in other cell systems. Additionally, RM rescued POS internalization defect in Mer receptor tyrosine kinase (MERTK) mutant hRPE, derived from retinitis pigmentosa patient induced pluripotent stem cells. Our platform, including a primary phenotypic screening phagocytosis assay together with orthogonal assays, establishes a basis for RPE-based therapy discovery aiming at a broad patient spectrum., Graphical Abstract, Highlights • A phenotypic screening assay based on human RPE and POS was established • Screen identifies ramoplanin as an inducer of POS phagocytosis in hESC-RPE • Ramoplanin upregulates expression of genes involved in POS ensheathment • Ramoplanin rescues POS phagocytosis defect in retinitis pigmentosa RPE, In this work, Schreiter et al. describe the establishment of a screening platform consisting of primary phenotypic assay and secondary assays based on hPSC-derived RPE. Using this platform ramoplanin was identified as a hit that upregulates POS phagocytosis in wild-type RPE and rescues phagocytosis defect in disease RPE.

Summary Tight regulation of the balance between self-renewal and differentiation of neural stem cells is crucial to assure proper neural development. In this context, Notch signaling is a well-known promoter of stemness. In contrast, the bifunctional brain-enriched microRNA miR-9/9∗ has been implicated in promoting neuronal differentiation. Therefore, we set out to explore the role of both regulators in human neural stem cells. We found that miR-9/9∗ decreases Notch activity by targeting NOTCH2 and HES1, resulting in an enhanced differentiation. Vice versa, expression levels of miR-9/9∗ depend on the activation status of Notch signaling. While Notch inhibits differentiation of neural stem cells, it also induces miR-9/9∗ via recruitment of the Notch intracellular domain (NICD)/RBPj transcriptional complex to the miR-9/9∗_2 genomic locus. Thus, our data reveal a mutual interaction between bifunctional miR-9/9∗ and the Notch signaling cascade, calibrating the delicate balance between self-renewal and differentiation of human neural stem cells., Graphical Abstract, Highlights • MiR-9/9∗ regulate Notch signaling by targeting NOTCH2 and HES1 • Notch directly regulates transcription of the miR-9_2 genomic locus • Notch-miR-9 reciprocal regulation calibrates NSC self-renewal and differentiation, In this article, Brüstle and colleagues show that a mutual interaction between microRNA-9 and Notch calibrates the delicate balance between self-renewal and differentiation of human neural stem cells (hNSC). While Notch maintains stemness and proliferation of hNSC, it also directly induces expression of miR-9, which in turn promotes hNSC differentiation by targeting NOTCH2 and HES1.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder. We screened 751 familial ALS patient whole-exome sequences and identified six mutations including p.D40G in the ANXA11 gene in 13 individuals. The p.D40G mutation was absent from 70,000 control whole-exome sequences. This mutation segregated with disease in two kindreds and was present in another two unrelated cases (P = 0.0102), and all mutation carriers shared a common founder haplotype. Annexin A11-positive protein aggregates were abundant in spinal cord motor neurons and hippocampal neuronal axons in an ALS patient carrying the p.D40G mutation. Transfected human embryonic kidney cells expressing ANXA11 with the p.D40G mutation and other N-terminal mutations showed altered binding to calcyclin, and the p.R235Q mutant protein formed insoluble aggregates. We conclude that mutations in ANXA11 are associated with ALS and implicate defective intracellular protein trafficking in disease pathogenesis.

Summary 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., Graphical Abstract, Highlights • Efficient protocol for pluripotent stem cell-derived retinal organoidogenesis • Temporal gene-expression profile analysis of individual organoids • Comparative hPAX6GFP expression in murine retinal organoids and mouse retina • Timed Notch inhibition results in cone- or rod-photoreceptor-enriched organoids, Karl and colleagues established an efficient protocol for mouse embryonic stem cell (ESC)-derived generation of large 3D retinal organoids independent of optic-vesicle evagination or eyecup formation. Retinogenesis was characterized by temporal gene-expression analysis of individual organoids, cell birthdating experiments, and comparison of hPAX6GFP reporter expression in organoids and mice in vivo. Timed Notch inhibition enabled generation of cone- or rod-photoreceptor-enriched organoids.