Recruitment of endogenous progenitors is critical during tissue repair. The inner ear utricle requires mechanosensory hair cells (HCs) to detect linear acceleration. After damage, non-mammalian utricles regenerate HCs via both proliferation and direct transdifferentiation. In adult mammals, limited transdifferentiation from unidentified progenitors occurs to regenerate extrastriolar Type II HCs. Here, we show that HC damage in neonatal mouse utricle activates the Wnt target gene Lgr5 in striolar supporting cells. Lineage tracing and time-lapse microscopy reveal that Lgr5+ cells transdifferentiate into HC-like cells in vitro. In contrast to adults, HC ablation in neonatal utricles in vivo recruits Lgr5+ cells to regenerate striolar HCs through mitotic and transdifferentiation pathways. Both Type I and II HCs are regenerated, and regenerated HCs display stereocilia and synapses. Lastly, stabilized β-catenin in Lgr5+ cells enhances mitotic activity and HC regeneration. Thus Lgr5 marks Wnt-regulated, damage-activated HC progenitors and may help uncover factors driving mammalian HC regeneration.

Sensory hair cells are mechanoreceptors required for hearing and balance functions. From embryonic development, hair cells acquire apical stereociliary bundles for mechanosensation, basolateral ion channels that shape receptor potential, and synaptic contacts for conveying information centrally. These key maturation steps are sequential and presumed coupled; however, whether hair cells emerging postnatally mature similarly is unknown. Here, we show that in vivo postnatally generated and regenerated hair cells in the utricle, a vestibular organ detecting linear acceleration, acquired some mature somatic features but hair bundles appeared nonfunctional and short. The utricle consists of two hair cell subtypes with distinct morphological, electrophysiological and synaptic features. In both the undamaged and damaged utricle, fate-mapping and electrophysiology experiments showed that Plp1 + supporting cells took on type II hair cell properties based on molecular markers, basolateral conductances and synaptic properties yet stereociliary bundles were absent, or small and nonfunctional. By contrast, Lgr5 + supporting cells regenerated hair cells with type I and II properties, representing a distinct hair cell precursor subtype. Lastly, direct physiological measurements showed that utricular function abolished by damage was partially regained during regeneration. Together, our data reveal a previously unrecognized aberrant maturation program for hair cells generated and regenerated postnatally and may have broad implications for inner ear regenerative therapies.

The results suggest that reduced visual-motor integration may contribute to poor spatial organization of written work.

To date, the development of bioreactors for the study of red blood cells (RBCs, daily transfused in the case of disease or hemorrhage) has focused on hematopoietic stem cells. Despite the fact that mature RBCs are enucleated and do not expand, they possess complex cellular and metabolic pathways, as well as post-translation modification signaling and gas-exchange regulation. In order to dynamically study the behavior of RBCs and their signaling pathways under various conditions, a small-scale perfusion bioreactor has been developed. The most advanced design developed here consists of a fluidized bed of 7.6 mL containing 3·109 cells and perfused at 8.5 μL/min. Mimicking RBC storage conditions in transfusion medicine, as a proof-of-concept, we investigated the ex vivo aging of RBCs under both aerobic and anaerobic conditions. Hence, RBCs stored in saline-adenine-glucose-mannitol (SAGM) were injected in parallel into two bioreactors and perfused with a modified SAGM solution over 14 days at room temperature under air or argon. The formation of a fluidized bed enabled easy sampling of the extracellular medium over the storage period used for the quantitation of glucose consumption and lactate production. Hemolysis and microvesiculation increased during aging and were reduced under anaerobic (argon) conditions, which is consistent with previously reported findings. Glucose and lactate levels showed expected trends, i.e., decreased and increased during the 2-week period, respectively; whereas extracellular glucose consumption was higher under aerobic conditions. Metabolomics showed depletion of glycolsis and pentose phosphate pathway metabolites, and an accumulation of purine metabolite end-products. This novel approach, which takes advantage of a fluidized bed of cells in comparison to traditional closed bags or tubes, does not require agitation and limit shear stress, and constantly segragates extracellular medium from RBCs. It thus gives access to several difficult-to-obtain on- and off-line parameters in the extracellular medium. This dynamic bioreactor system does not only allow us to probe the behavior of RBCs under different storage conditions, but it also could be a powerful tool to study physiological or pathological RBCs exposed to various conditions and stimuli.

Mammalian inner ear hair cell loss leads to permanent hearing and balance dysfunction. In contrast to the cochlea, vestibular hair cells of the murine utricle have some regenerative capacity. Whether human utricular hair cells regenerate remains unknown. Here we procured live, mature utricles from organ donors and vestibular schwannoma patients, and present a validated single-cell transcriptomic atlas at unprecedented resolution. We describe previously unknown markers of 25 sensory and non-sensory cell types, with genes of hair cell and supporting cell subtypes displaying striking divergence between mice and humans. We further uncovered transcriptomes unique to hair cell precursors, which we validated to be 14-fold more robust in vestibular schwannoma utricles, representing ongoing regeneration in humans. Lastly, trajectory analysis of the supporting cell-hair cell axis revealed 5 distinct patterns of dynamic gene expression and associated pathways, including mTOR signaling and synaptogenesis. Our dataset constitutes a foundational resource, accessible via a web-based interface, serving to advance knowledge of the normal and diseased human inner ears and tools to stimulate human inner ear regeneration.