Defining developmental trajectories of prosensory cells in human inner ear organoids at single-cell resolution

Abstract

The inner ear sensory epithelia contain mechanosensitive hair cells and supporting cells. Both cell types arise from SOX2-expressing prosensory cells, but the mechanisms underlying the diversification of these cell lineages remain unclear. To determine the transcriptional trajectory of prosensory cells, we established a SOX2-2A-ntdTomato human embryonic stem cell line using CRISPR/Cas9, and performed single-cell RNA-sequencing analyses with SOX2-positive cells isolated from inner ear organoids at various time points between differentiation days 20 and 60. Our pseudotime analysis suggests that vestibular type II hair cells arise primarily from supporting cells, rather than bi-fated prosensory cells in organoids. Moreover, ion channel- and ion-transporter-related gene sets were enriched in supporting cells versus prosensory cells, whereas Wnt signaling-related gene sets were enriched in hair cells versus supporting cells. These findings provide valuable insights into how prosensory cells give rise to hair cells and supporting cells during human inner ear development, and may provide a clue to promote hair cell regeneration from resident supporting cells in individuals with hearing loss or balance disorders.

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

“…Our initial analysis and previous reports 18 , 81 , 82 , 83 indicate that the majority of in vitro generated HCLCs differentiate into vestibular phenotypes by default. To elucidate the phenotypic variation of the vestibular-like HCLCs, we calculated enrichment scores for perinatal extrastriolar type II, extrastriolar type I, and striolar type I HCs based on previously published data 7 using Single-Cell Signature Explorer 59 ( Figures 6 A–6C).…”

“… 84 More recently, single-cell transcriptomic evaluations of organoids from human pluripotent stem cells support the general notion that organoid HCLCs default to a vestibular-like fate. 82 , 83 Even so, at least one report has shown evidence of both vestibular and cochlear HC fates with only slight modifications to the original Koehler protocol 85 and modulations of HH and WNT signaling were deemed sufficient to tip the balance toward cochlear cell fates. 24 Using scRNA-seq, we profiled a total of 3,029 HCLCs and in an unbiased clustering approach we determined that a significant number of HCLCs acquired a transcriptional signature reminiscent of auditory HCs.…”

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

“…Our initial analysis and previous reports 18, 73–75 indicate that the majority of in vitro generated HCLCs differentiate into vestibular phenotypes by default. To elucidate the phenotypic variation of the vestibular like HCLCs, we calculated enrichment scores for perinatal extrastriolar Type II, extrastriolar Type I, and striolar Type I HCs based on previously published data 7 using Single-Cell Signature Explorer 58 (Figures 6A-C).…”

“…76 More recently, single-cell transcriptomic evaluations of organoids from human pluripotent stem cells support the general notion that organoid HCLCs default to a vestibular like fate. 74, 75 Even so, at least one report has shown evidence of both vestibular and cochlear HC fates with only slight modifications to the original Koehler protocol 77 and modulations of HH and WNT signaling were deemed sufficient to tip the balance towards cochlear cell fates. 24 Using scRNA-seq, we profiled a total of 3,029 HCLCs and in an unbiased clustering approach we determined that a significant number of HCLCs acquired a transcriptional signature reminiscent of auditory HCs.…”

Kremen1 regulates the regenerative capacity of support cells and mechanosensory hair cells in the zebrafish lateral line

“…Recent studies using mammalian inner ear cell cultures suggest manipulating the Notch and Wnt pathways are critical to regrowing damaged hair cells. 46 , 47 Interestingly, while there are conflicting results for upregulation and downregulation of Notch signaling as a mechanism to promote regeneration, upregulation of Wnt signaling is consistently necessary. 46 , 47 Krm1 is expressed in the support cells of the mammalian cochlea, making it a potential target for therapeutic intervention: inhibition of Krm1 function might be required for support cells to re-enter a more progenitor-like stage to then differentiate as mechanosensory hair cells to restore hearing.…”