Although the application of human embryonic stem cells (hESCs) in stem cell-replacement therapy remains promising, its potential is hindered by a low cell survival rate in post-transplantation within the inner ear. Here, we aim to enhance the in vitro and in vivo survival rate and neuronal differentiation of otic neuronal progenitors (ONPs) by generating an artificial stem cell niche consisting of three-dimensional (3D) hESC-derived ONP spheroids with a nanofibrillar cellulose hydrogel and a sustained-release brain-derivative neurotrophic factor delivery system. Our results demonstrated that the transplanted hESC-derived ONP spheroids survived and neuronally differentiated into otic neuronal lineages in vitro and in vivo and also extended neurites toward the bony wall of the cochlea 90 days after the transplantation without the use of immunosuppressant medication. Our data in vitro and in vivo presented here provide sufficient evidence that we have established a robust, reproducible protocol for in vivo transplantation of hESC-derived ONPs to the inner ear. Using our protocol to create an artificial stem cell niche in the inner ear, it is now possible to work on integrating transplanted hESC-derived ONPs further and also to work toward achieving functional auditory neurons generated from hESCs. Our findings suggest that the provision of an artificial stem cell niche can be a future approach to stem cell-replacement therapy for inner-ear regeneration. STATEMENT OF SIGNIFICANCE: Inner ear regeneration utilizing human embryonic stem cell-derived otic neuronal progenitors (hESC-derived ONPs) has remarkable potential for treating sensorineural hearing loss. However, the local environment of the inner ear requires a suitable stem cell niche to allow hESC-derived ONP engraftment as well as neuronal differentiation. To overcome this obstacle, we utilized three-dimensional spheroid formation (direct contact), nanofibrillar cellulose hydrogel (extracellular matrix), and a neurotrophic factor delivery system to artificially create a stem cell niche in vitro and in vivo. Our in vitro and in vivo data presented here provide sufficient evidence that we have established a robust, reproducible protocol for in vivo transplantation of hESC-derived ONPs to the inner ear.

中文翻译：

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通过应用具有持续释放神经营养因子递送系统的纳米原纤维纤维素水凝胶，在内耳中工程改造的三维干细胞生态位。

尽管人类胚胎干细胞（hESCs）在干细胞替代疗法中的应用前景广阔，但其潜力受到内耳移植后细胞存活率低的阻碍。在这里，我们的目标是通过生成包含三维（3D）hESC衍生的ONP球状体和纳米原纤维纤维素水凝胶的人工干细胞生态位，从而提高耳神经元祖细胞（ONP）的体外和体内存活率以及神经元分化。持续释放的脑源性神经营养因子递送系统。我们的结果表明，移植的hESC衍生的ONP椭球在体外和体内存活并神经元分化为耳神经元谱系，并且在移植后90天未使用免疫抑制剂的情况下，神经突向耳蜗骨壁延伸。这里提供的体外和体内数据提供了充分的证据，表明我们已经建立了可靠的，可重复的方案，用于将hESC衍生的ONP体内移植到内耳。使用我们的协议在内耳中创建人工干细胞生态位，现在可以进一步整合已移植的hESC衍生的ONP，还可以朝着从hESC产生的功能性听觉神经元的方向努力。我们的发现表明，提供人工干细胞小生境可能是未来用于内耳再生的干细胞置换疗法的方法。意义声明：利用人类胚胎干细胞源性耳神经元祖细胞（hESC衍生的ONPs）进行内耳再生在治疗感觉神经性听力损失方面具有巨大潜力。然而，内耳的局部环境需要合适的干细胞生态位以允许hESC衍生的ONP植入以及神经元分化。为了克服这一障碍，我们利用三维球体形成（直接接触），纳米原纤维纤维素水凝胶（细胞外基质）和神经营养因子递送系统在体外和体内人为地创建了干细胞生态位。