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Inner ear organoids derived from pluripotent stem cells could be a useful model system to study development, disease, and regeneration. However, there is considerable heterogeneity in the size, morphology, and efficiency of organoid production using standard protocols. Greater control of the culture microenvironment could decrease heterogeneity and increase the yield of organoids. Animal-derived otic vesicles show some autonomy during development and can differentiate into cochlear and vestibular domains in mesenchyme-free ex vivo culture. Therefore, we investigated whether stem cell-derived otic vesicles can autonomously generate inner ear organoids. Isolated, stem cell-derived vesicles grew into cyst-like organoids with high efficiency, over 90%, when embedded in droplets of the basement membrane matrix Matrigel. Though nearly all vesicles within the aggregate were competent to mature into organoids, the efficiency of organoid production depended on the stage of vesicle isolation and required supplementation with Matrigel.

Stem cell therapy holds great promise for treating neurodegenerative disease, but major barriers to effective therapeutic strategies remain. A complete understanding of the derived phenotype is required for predicting cell response once introduced into the host tissue. We sought to identify major axonal guidance cues present in neurons derived from the transient overexpression of neurogenin-1 (Neurog1) in mouse embryonic stem cells (ESCs). Neurog1 upregulated the netrin-1 axon guidance receptors DCC (deleted in colorectal cancer) and neogenin (NEO1). Quantitative polymerase chain reaction results showed a 2-fold increase in NEO1 mRNA and a 36-fold increase in DCC mRNA in Neurog1-induced compared with control ESCs. Immunohistochemistry indicated that DCC was primarily expressed on cells positive for the neuronal marker TUJ1. DCC was preferentially localized to the cell soma and growth-cones of induced neurons. In contrast, NEO1 expression showed less specificity, labeling both TUJ1-positive and TUJ1-negative cells as well as uninduced control cells. Axonal outgrowth was directed preferentially toward aggregates of HEK293 cells secreting a recombinant active fragment of netrin-1. These data indicate that DCC and NEO1 are downstream products of Neurog1 and may guide the integration of Neurog1-induced ESCs with target cells secreting netrin-1. Differential expression profiles for netrin receptors could indicate different roles for this guidance cue on neuronal and non-neuronal cells.