Journal Club: Profiling of microRNAs inside extracellular vesicles isolated from mouse inner ear organoids and comparative mouse cochlea.

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Today's journal article

Lee S, Kubota M, Park E, Heller S, Im GJ, Chang J. Analysis of miRNAs from Inner Ear Organoid-Derived Extracellular Vesicles. 

Why I picked this article

Sensorineural hearing loss caused by damage to the cochlea and/or auditory nerve is irreversible. This is because the mature mammalian cochlea cannot readily replace lost or damaged cells in the cochlea. However, the young immature cochlea used to have some capacity for regeneration; during the development of the cochlea, cells in the greater epithelial ridge (GER) contain some cells that can become new auditory hair cells. Taking advantage of this property, researchers can make an organoid, or a ball-like cluster of cells, from GER cells. GER-cell organoids can later generate hair cells and supporting cells in vitro. This research investigates GER-cell organoids, focusing on the extracellular vesicles produced by these organoids. 

Small extracellular vesicles (EVs) are small vesicles secreted by various cells for the purpose of cell-to-cell communication. EVs often carry small molecules such as microRNAs, short non-coding RNAs; those molecules can influence cells to promote cell proliferation or differentiation. This research investigates what miRNAs are packaged into EVs by proliferating inner ear organoids, and how they differ from EVs shed by native neonatal cochlea. If GER-cell organoids in a proliferative state release EVs with a characteristic miRNA cargo, that cargo could be both a readout of the regenerative state and a handle to modulate it.

Some of the research findings

GER organoid model: 
  • Source tissue: postnatal day 2 mice of ICR strain. 
  • Cochleae were extracted, and the organ of Corti was dissociated to obtain cells from GER. 
  • Dissociated cells were seeded at 1.0, 2.5, 5.0 and 7.5 x 10,000 cells/ml concentration in 12-well suspension culture dishes.
  • Culture conditions optimised to form organoids with progenitor properties (DMEM/F-12, N-2 supplement, B-27 supplement and ampicillin. 
  • Proliferations were stimulated with 20 ng/mL EGF, 10 ng/mL FGF2, 50 ng/mL IGF1, 3 μM CHIR99021, 500 μM valproic acid, 100 μg/mL 2-phospho-Lascorbic acid and 2 μM TGF-β RI kinase inhibitor.
  • EVs were isolated using Exo2DTM-EV isolation kit, EXOSOMEplus.
  • The positive presence of exosome markers, CD9 and CD63, were used to confirm vesicles as EV.  
GER organoid culture optimisation:
  • 2.5 × 10,000 cells/mL concentration was most efficient at producing organoids, producing ~60 organoids per 10,000 cells. 
  • Of solid and hollow organoids, solid organoids are more effective at producing hair cell-like cells; 6 x more organoids were generated as solid organoids by day 7. 
  • Cells in day 7 culture showed markers of proliferation such as Ki-67 and EdU. 
Part of Fig 2B. Mouse GER organoid, green = proliferation marker, red = ear cell marker, blue = cell nuclei. Lee et al. (2025)

EV collection and profiling: 
  • EVs were collected from culture supernatants of organoids in a proliferative state. 
    • The average EV size = 184.5 ± 29.5 nm.
    • The average EV concentration = 7.3 × 1000,000,000 particles/mL
    • Small-RNA sequencing identified 184 miRNAs in organoid-EVs.
  • EVs from culture supernatants of P2 mouse cochlear ducts.
    • The average EV size = 276.1 ± 16.8 nm.
    • The average EV concentration = 11.7 x 1000,000,000 particles/mL
    • 176 miRNAs in cochlear-duct EVs.
  • EVs from the organoids vs P2 cochlea
    • 122 miRNAs differed by more than twofold between the groups.
    • 12 reached statistical significance.
      • 10 up in organoid-EVs: mmu-let-7 g-5p,  mmu-let-7e-5p, mmu-miR-25-3p, mmu-let-7a-5p, mmu-miR-151-3p, mmu-let-7f-5p, mmu-miR-21a-5p, mmu-miR-200b-5p, mmu-miR-29a-3p, mmu-miR-421-3p
      • 2 down in organoid-EVs: mmu-mir-706 and mmu-mir-1895
  • Pathway enrichment of predicted targets pointed to pluripotent stem-cell regulation, cell proliferation, ear development, and cell-fate modulation—consistent with EVs reflecting the organoids’ growth state.

Haruna's takeaway

Organoids are like little artificial, self-growing 3D cultures with huge potential. Research work about 3D organoids gets me very excited. This research provides some simple but valuable comparisons between the intact cochlea and proliferating organoids. It appears that each of the identified microRNAs could have multiple target genes to regulate, so figuring out how to use these EVs and microRNAs packaged in EVs may be complicated. I would love to try organoid culture experiment one day... 

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This is Haruna's 96/100 of the 100-day challenge to post a science blog article every day! I love inner ear biology & cochlear physiology.