Replacement of the cochlear sensory epithelium using stem cell-derived inner ear organoids

摘要 感音神经性耳聋在退伍军人中非常普遍，但目前还没有生物治疗方法 使耳蜗和听力完全恢复。干细胞疗法恢复听力成立 很好的承诺。多能干细胞生成内耳器官的新进展及我们的研究进展 为接收外来细胞而调整耳蜗条件的开创性努力已经为未来铺平了道路。然而， 将这些方法结合起来仍然存在几个主要障碍。最重要的是，缺乏一个连贯的 确定正确的供体细胞并根据耳蜗损伤的严重程度进行调整的策略。在这 建议，我们专注于干细胞治疗严重受损的耳蜗，在极端或慢性 损伤使Corti器官减少为扁平的间质细胞上皮，到目前为止，这些细胞被证明难以 再生的遗传途径。这种方法可能是恢复完整的 长期听力损失的老年退伍军人的感觉上皮细胞。内耳有机化合物的开发方案 从多能干细胞中产生潜在的捐赠者细胞，一直到胚胎发育的轨迹。值得注意的是， 我们已经开发了从这些培养的非耳源性细胞中分离耳源性囊泡前体细胞的方法，但 囊泡成熟为感觉上皮细胞需要一种复杂的商业细胞外基质 高效的体外分化。在这个提议中，我们测试了多能耳祖细胞将是 需要替换严重受损的耳蜗中的毛细胞和支持细胞。该提案将进行测试 移植供体细胞在分化的不同阶段的影响，并检查 细胞外基质在促进供体细胞存活和成熟中的作用成功完成这些研究将 为治疗严重听力损失开辟了可行的生物治疗的新途径。

ABSTRACT Sensorineural hearing loss is highly prevalent in military Veterans, but there is currently no biological treatment enabling full restoration of the cochlea and hearing perception. Hearing restoration by stem cell therapy holds great promise. New advances in the generation of inner ear organoids from pluripotent stem cells and our pioneering efforts to condition the cochlea for receiving foreign cells have paved the way forward. However, there remain several major obstacles to combining these approaches. Foremost, there is a lack of a coherent strategy for identifying the right donor cells and tailoring these to the severity of injury in the cochlea. In this proposal, we are focused on stem cell therapy for the severely damaged cochlea, where extreme or chronic injury has reduced the organ of Corti to a flat epithelium of stromal cells that have so far proven refractory to genetic approaches to regeneration. This approach may be the only viable strategy for restoring the full sensory epithelium in older Veterans with long-term hearing loss. Protocols for developing inner ear organoids from pluripotent stem cells produce potential donor cells all along the otic developmental trajectory. Notably, we have developed methods to isolate otic vesicle progenitors from non-otic cells in these cultures, but maturation of the vesicles into sensory epithelia requires a complex, commercial extracellular matrix for efficient differentiation in vitro. In this proposal, we test the hypothesis that multipotent otic progenitors will be required to replace both hair cells and supporting cells in the severely damaged cochlea. The proposal tests the impact of implanting donor cells at various stages of differentiation and examines the role of the extracellular matrix in promoting donor cell survival and maturation. Successful completion of these studies will open new paths toward a viable biological treatment for severe hearing loss.