Mapping the developmental potential of mouse inner ear organoids at single-cell resolution.

Inner ear organoids recapitulate development and are intended to generate cell types of the otic lineage for applications such as basic science research and cell replacement strategies. Here, we use single-cell sequencing to study the cellular heterogeneity of late-stage mouse inner ear organoid sensory epithelia, which we validated by comparison with datasets of the mouse cochlea and vestibular epithelia. We resolved supporting cell sub-types, cochlear-like hair cells, and vestibular type I and type II-like hair cells. While cochlear-like hair cells aligned best with an outer hair cell trajectory, vestibular-like hair cells followed developmental trajectories similar to in vivo programs branching into type II and then type I extrastriolar hair cells. These results highlight the transcriptional accuracy of the organoid developmental program but will also inform future strategies to improve synaptic connectivity and regional specification.

Matrigel is required for efficient differentiation of isolated, stem cell-derived otic vesicles into inner ear organoids.

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.

From Otic Induction to Hair Cell Production: Pax2EGFP Cell Line Illuminates Key Stages of Development in Mouse Inner Ear Organoid Model.

Producing hair cells of the inner ear is the major goal of ongoing research that combines advances in developmental and stem cell biology. The recent advent of an inner ear organoid protocol-resulting in three-dimensional stem cell-derived tissues resembling vestibular sensory epithelia-has sparked interest in applications such as regeneration, drug discovery, and disease modeling. In this study, we adapted this protocol for a novel mouse embryonic stem cell line with a fluorescent reporter for Pax2 expression. We used Pax2EGFP/+ organoid formation to model otic induction, the pivotal developmental event when preplacodal tissue adopts otic fate. We found upregulation of Pax2 and activation of ERK downstream of fibroblast growth factor signaling in organoid formation as in embryonic inner ear development. Pax2 expression was evident from the EGFP reporter beginning at the vesicle formation stage and persisting through generation of the sensory epithelium. The native ventralizing signal sonic hedgehog was largely absent from the cell aggregates as otic vesicles began to form, confirming the dorsal vestibular organoid fate. Nonetheless, cochlear- or vestibular-like neurons appeared to delaminate from the derived otic vesicles and formed synaptic contacts with hair cells in the organoids. Cell lines with transcriptional reporters such as Pax2EGFP/+ facilitate direct evaluation of morphological changes during organoid production, a major asset when establishing and validating the culture protocol.

Netrin-1-mediated axon guidance in mouse embryonic stem cells overexpressing neurogenin-1.

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.