Lee Sehee, Kubota Marie, Park Euyhyun, Heller Stefan, Im Gi Jung, Chang Jiwon
Department of Otorhinolaryngology-Head & Neck Surgery, Korea University College of Medicine, 73 Goryeodae-Ro, Seongbuk-Gu, 02841, Seoul, Republic of Korea.
Department of Otorhinolaryngology-Head & Neck Surgery, Hallym University College of Medicine, Kangnam Sacred Heart Hospital, 1, Singil-Ro, Yeongdeunpo-Gu, Seoul, 07441, Republic of Korea.
J Assoc Res Otolaryngol. 2025 Jul 16. doi: 10.1007/s10162-025-00998-x.
Permanent hearing loss primarily results from the inability of the mammalian cochlea to replace lost inner ear hair cells. However, neonatal mice exhibit a unique capacity: isolated cochlear floor cells can efficiently proliferate in vitro and form organoids that harbor new hair cells and supporting cell populations. In this study, we isolated extracellular vesicles (EVs) from organoids and analyzed the miRNAs derived from them to identify gene regulatory elements that coordinate proliferation and regeneration.
We utilized cochlear floor cells from postnatal day two mice and optimized the culture conditions to efficiently grow organoids that exhibit progenitor properties. Next, we isolated EVs from the culture media of organoids in their proliferative state. We analyzed miRNAs contained in these EVs to identify potential regulators that drive or modulate organoid cell proliferation. The miRNA sequencing data from organoid EVs were compared with miRNAs identified in EVs obtained from the culture supernatant of P2 mouse cochlear ducts.
We identified 184 miRNAs in organoid EVs and 176 miRNAs in cochlear duct EVs. A total of 122 miRNAs differed more than twofold between these groups, with 12 miRNAs (10 upregulated and 2 downregulated in organoid EVs) exhibiting statistically significant differences. The target genes of these twelve differentially expressed miRNAs are associated with pathways related to pluripotent stem cell regulation, cell proliferation, ear development, and cell fate modulation. This indicates that the miRNAs in organoid-derived EVs may impact processes associated with cell proliferation and the generation of inner ear cell types.
Our study comprehensively inventoried miRNAs contained in EVs released by growing inner ear organoids. Our differential miRNA expression analysis provides insight into regulatory mechanisms that promote cochlear floor cell proliferation and organoid formation, which could be leveraged in miRNA-based therapeutic approaches.
永久性听力损失主要是由于哺乳动物的耳蜗无法替换丢失的内耳毛细胞。然而，新生小鼠表现出一种独特的能力：分离出的耳蜗基底细胞能够在体外高效增殖，并形成含有新的毛细胞和支持细胞群体的类器官。在本研究中，我们从类器官中分离出细胞外囊泡（EVs），并分析了从中衍生的微小RNA（miRNAs），以鉴定协调增殖和再生的基因调控元件。
我们利用出生后第二天小鼠的耳蜗基底细胞，并优化培养条件，以高效培养具有祖细胞特性的类器官。接下来，我们从处于增殖状态的类器官培养基中分离出EVs。我们分析了这些EVs中所含的miRNAs，以鉴定驱动或调节类器官细胞增殖的潜在调节因子。将类器官EVs的miRNA测序数据与从P2小鼠耳蜗管培养上清液中获得的EVs中鉴定出的miRNAs进行比较。
我们在类器官EVs中鉴定出184种miRNAs，在耳蜗管EVs中鉴定出176种miRNAs。这两组之间共有122种miRNAs的差异超过两倍，其中12种miRNAs（类器官EVs中10种上调，2种下调）表现出统计学上的显著差异。这12种差异表达的miRNAs的靶基因与多能干细胞调控、细胞增殖、耳朵发育和细胞命运调节相关的途径有关。这表明类器官衍生的EVs中的miRNAs可能会影响与细胞增殖和内耳细胞类型生成相关的过程。
我们的研究全面盘点了生长中的内耳类器官释放的EVs中所含的miRNAs。我们的差异miRNA表达分析为促进耳蜗基底细胞增殖和类器官形成的调控机制提供了见解，这可用于基于miRNA的治疗方法。
