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Galectin and Myc enable cochlear progenitor expansion in vitro and in vivo

Marie KubotaiD, Julia M. AbitboliD, Paul K Lee, Maggie S. MaterniD, Sonia Bustos Barocio, Taha Adnan JaniD, Alan Gi-Lun ChengiD, Stefan HelleriD

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Abstract

The neonatal cochlear epithelium harbors regenerative capacity, attributable to the transient greater epithelial ridge (GER). After injury, GER cells re-enter the cell cycle, migrate into the damaged organ of Corti, and differentiate into sensory or supporting cells in vivo; in culture, they proliferate to form inner ear organoids. The mechanisms underlying this competence remain unclear. Here, we generated organoids from mouse GER cells and performed single-cell transcriptomics at organoid initiation. Analysis revealed extracellular matrix reorganization with prominent involvement of galectins. Pharmacological inhibition reduced organoid growth, whereas overexpression of galectin-1 or Myc enhanced growth, identifying both factors as regulators of cochlear progenitor expansion. Moreover, Myc overexpression conferred organoid-forming capacity in post-neonatal cochlear epithelial cells after hearing onset, thereby extending the time window for regenerative competence. In vivo, organ of Corti ablation induced galectin-1 upregulation and GER proliferation, suppressed by galectin-1 blockade, establishing a model to rekindle proliferative potential in mature cochlear cells.

In-depth summary

Claude Sonnet 4.6 · xhigh effort · 7 Nov 2026 · from the full text of version 2 (24 Sept 2026) · CC BY 4.0

Single-cell profiling of mouse greater epithelial ridge (GER) organoids points to galectin-1 and Myc as drivers of cochlear progenitor expansion. Myc overexpression also yields organoids from post-hearing-onset cochlear epithelium.

Neonatal GER cells can re-enter the cell cycle after injury and form organoids, but the mechanisms behind this competence remain unclear. The paper uses an organoid single-cell atlas to nominate galectin-1 and Myc, then tests them in organoids and in a mouse cell-ablation model.

Key results

  1. ShownSmart-seq2 profiling of GER-derived organoids on days 1, 3 and 7 gave seven Seurat clusters. Proliferation (Mki67) appeared by day three, and cells at the onset of proliferation (clusters 4-6) showed extracellular matrix (ECM) and cell-junction signatures, including Lgals1 and Lgals3.

    966 cell libraries, 768 sequenced, 658 passed QC. A second clustering (CellTrails, 13 clusters) overlapped: 35 of 39 upregulated genes were shared. FACS Gate 1 enriched GER cells at 93% purity. · Single cell collection from purified GER…; Identification of candidate genes that mediate…; Figure legends

  2. ShownSmall-molecule inhibitors of galectin-1 (OTX008), galectin-3 (GB1107) and Myc (10058-F4) reduced organoid formation and growth. Integrin inhibitors and enkephalin or naltrexone treatment had no significant effect.

    In cochlear duct cultures the viable cell mass assay was significant at 10 µM for OTX008 and GB1107 and at 30 µM for 10058-F4. In FACS-enriched GER cells it was significant at 5 µM for OTX008 and GB1107 and at 40 µM for 10058-F4. · Pharmacological inhibition of galectins and Myc…

  3. ShownAAV-mediated overexpression of Lgals1 or Myc, but not Lgals3, increased organoid growth from P2 cochlear duct cells. Myc gave an early Mki67 rise but lower cell viability at 9 days post-infection.

    Lgals1 mRNA was about 8-fold higher at 9 dpi; Myc mRNA was 15-fold higher at 3 dpi, then about 10-fold. AAV-Myc cultures were about 77% viable vs >90% for control and AAV-Lgals1. AAV-Myc raised organoid number but not viable cell mass. · Overexpression of galectin-1 and Myc increases…

  4. ShownMyc overexpression conferred organoid-forming capacity on EpCAM-enriched cochlear epithelial cells from P14-P17 mice, after hearing onset. Galectin-1 overexpression did not.

    AAV-myc significantly increased organoid number, size and live-cell mass vs AAV-EGFP; AAV-lgals1 did not differ from control. Viable cell mass was based on three experiments. · Myc, but not galectin-1, promotes organoid…; Figure legends

  5. SuggestedAfter Lgr5-DTR ablation in neonatal mice, Lgals1 (and Myc in medial GER) rose in the GER. Intraperitoneal OTX008 reduced Ki67-positive proliferating GER cells, including with a single P2 dose.

    scRNA-seq of 3,773 cells from P4 damage and control cochleae; galectin-1 protein domain expanded medially to laterally. OTX008 at 10 mg/kg significantly reduced Ki67-positive cells in the GER vs the damage control. · Galectin-1 is upregulated in the GER after…; In vivo inhibition of galectin-1 suppresses GER…

Methods, data and reproducibility

Smart-seq2 single-cell RNA-seq of FACS-purified GER-derived organoids at three early time points nominated candidate genes. These were tested by pharmacological inhibition and AAV overexpression in organoids, then checked in vivo with 10x scRNA-seq, immunostaining and OTX008 in an Lgr5-DTR damage model.

  • Smart-seq2 scRNA-seq of GER-derived organoids at culture days 1, 3 and 7 (FACS-purified P2 GER cells) · mouse · n = 966 libraries; 768 sequenced; 658 cells passed QC · GSE326100 and GSE291228 are deposited for the study; mapping to datasets not stated
  • 10x Genomics scRNA-seq of P4 cochlear cells from Lgr5-DTR damage model and control mice · mouse · n = 3,773 cells · GSE291228
  • Native P2 cochlear floor scRNA-seq used for comparison with organoids · mouse
  • Organoid assays (inhibitors, AAV overexpression) from P2 cochlear duct cells and P14-P17 EpCAM-enriched cells · mouse · n = N = 3-4 independent experiments for most viable cell mass assays
Methods & tools
Seurat 5.0.1, CellTrails 1.16.0, STAR, RSEM, Ingenuity Pathway Analysis, Harmony, DoubletFinder, Cell Ranger v6.0.0, AAV serotype DJ, OTX008, GB1107, 10058-F4, CellTiter 96 viable cell mass assay, AnimalTFDB 4.0
Data & accessions
https://umgear.org/
Reagents & environment
Seurat 5.0.1 (organoids) and 5.1.0 (in vivo); CellTrails 1.16.0; Cell Ranger v6.0.0; STAR and RSEM against mm10 GENCODE vM17. R is given as 4.2.0 for statistics and v4.5.0 for code availability.
Assay conditions & compute
Alignment ran on the Stanford Sherlock cluster; no time or memory figures stated.
Reproducibility gaps
No custom code was generated; analyses followed existing package vignettes. GEO accessions are given but not matched to individual datasets.
Limitations (5)
  • Endpoint inhibitor assays cannot separate reduced proliferation from impaired cell survival, so they are interpreted as contributions to organoid formation and growth. · stated by the authors
  • The OTX008 effect in vivo may reflect direct galectin-1 inhibition in the cochlea or an indirect systemic effect. It also partly mitigated Fabp7-positive cell loss. · stated by the authors
  • AAV experiments used dissociated cochlear duct cells rather than purified GER cells, so non-GER contributions to organoids cannot be fully excluded. Prolonged Myc overexpression reduced viability. · stated by the authors
  • The results do not establish galectins or Myc as therapeutic targets, since both are linked to oncogenesis and would need controlled, transient activation. · stated by the authors
  • Loss-of-function evidence rests on small-molecule inhibitors only, with no genetic knockdown or knockout. The organoid atlas is a manually picked Smart-seq2 dataset of 658 cells. · noted in this summary

Read this if you study inner ear regeneration or organoid models of progenitor proliferation and want candidate regulators, such as galectin-1 and Myc, with in vitro and in vivo tests.

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