Stem cell–replacement therapies have been proposed as a potential tool to treat sensorineural hearing loss by aiding the regeneration of spiral ganglion neurons (SGNs) in the inner ear. However, transplantation procedures have yet to be explored thoroughly to ensure proper cell differentiation and optimal transplant procedures. We hypothesized that the aggregation of human embryonic stem cell (hESC)–derived otic neuronal progenitor (ONP) cells into a multicellular form would improve their function and their survival in vivo post-transplantation. We generated hESC–derived ONP spheroids—an aggregate form conducive to differentiation, transplantation, and prolonged cell survival—to optimize conditions for their transplantation. Our findings indicate that these cell spheroids maintain the molecular and functional characteristics similar to those of ONP cells, which are upstream in the SGN lineage. Moreover, our phenotypical, electrophysiological, and mechanical data suggest an optimal spheroid transplantation point after 7 days of in vitro three-dimensional (3D) culture. We have also developed a feasible transplantation protocol for these spheroids using a micropipette aided by a digital microinjection system. In summary, the present work demonstrates that the transplantation of ONP cells in spheroid form into the inner ear through micropipette 7 days after seeding for 3D spheroid culture is an expedient and viable method for stem cell replacement therapies in the inner ear.

中文翻译：

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源自人类胚胎干细胞的三维耳神经元祖细胞球体

干细胞替代疗法已被提议作为一种潜在的工具，通过帮助内耳螺旋神经节神经元 (SGN) 的再生来治疗感觉神经性听力损失。然而，移植程序尚未彻底探索，以确保适当的细胞分化和最佳移植程序。我们假设人类胚胎干细胞 (hESC) 衍生的耳神经元祖 (ONP) 细胞聚集成多细胞形式将改善它们的功能和它们在体内的存活率移植后。我们生成了 hESC 衍生的 ONP 球体——一种有助于分化、移植和延长细胞存活的聚集形式——以优化其移植条件。我们的研究结果表明，这些细胞球体保持了与 SGN 谱系上游的 ONP 细胞相似的分子和功能特征。此外，我们的表型、电生理学和机械数据表明，体外7 天后的最佳球体移植点三维（3D）文化。我们还使用由数字显微注射系统辅助的微量移液器为这些球体开发了一种可行的移植协议。总之，目前的工作表明，在 3D 球体培养播种后 7 天，通过微量移液器将球体形式的 ONP 细胞移植到内耳是内耳干细胞替代疗法的一种方便且可行的方法。