将直接重编程的人皮肤成纤维细胞来源诱导神经干细胞移植到耳蜗鼓阶可增加听觉神经病动物模型的神经结构和中枢听觉连接

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中文摘要

背景： 人工耳蜗（CIs）为重度至极重度听力损失提供有效的康复手段，但其效果受到听觉神经元继发性退变的限制。直接转分化能够使体细胞谱系转换为神经干细胞而无需经过多能性阶段，与诱导多能干细胞相比具有降低致瘤风险、缩短重编程时间以及产生可扩增的自体患者特异性细胞等优势。Oct4是建立多能性的关键因子，但并非直接重编程所必需。加入Oct4可提高重编程效率并诱导更具“可塑性”的细胞状态，这可能有助于移植入靶器官后改善整合与功能适应。
方法： 由人皮肤成纤维细胞（HDFs）生成诱导神经干细胞（iNSCs）。我们通过转导外源性Oct4蛋白联合Sox2、Klf4和c-Myc生成iNSCs。对这些iNSCs的特性进行了表征，并在听觉神经病动物模型移植后评估其治疗潜力。进行了组织学和功能评估。
结果： 免疫染色和流式细胞术结果显示，人iNSCs表达Nestin、Sox2和Pax6。在神经干细胞允许性培养条件下，SKM的组成性表达和短暂Oct4启动可从HDFs诱导出iNSCs。分离的iNSCs在形态特征和表达标志物方面与对照人神经干细胞相似。移植的人iNSCs改善了耳蜗细胞完整性，特别是通过增强包括神经元和神经纤维在内的神经结构。尽管外周突触连接未恢复，但朝向中枢听觉通路的突触标志物显著增加。eABR反应显示，iNSC移植动物的反应在统计学上更高。
结论： 直接转分化提供了一种高效策略，可生成能够保护和修复听觉神经结构的自体iNSCs。通过改善神经健康，该方法有潜力提高CI性能、扩大长期听力损失患者的适应证，并为功能性听力恢复建立新的再生途径。

英文摘要

BACKGROUND: Cochlear implants (CIs) provide effective rehabilitation for severe-to-profound hearing loss, yet their success is limited by secondary degeneration of auditory neurons. Direct transdifferentiation, which enables lineage conversion of somatic cells into neural stem cells without a pluripotent stage, offers advantages over induced pluripotent stem cells by reducing tumorigenic risk, shortening reprogramming time, and producing expandable, patient-specific cells for autologous therapy. Oct4 is a critical factor for establishing pluripotency, but it is not required for direct reprogramming. Inclusion of Oct4 can enhance reprogramming efficiency and induce a more 'plastic' cellular state, which may facilitate improved integration and functional adaptation following transplantation into the target organ.
METHODS: Induced neural stem cells (iNSCs) were generated from human dermal fibroblasts. We generated iNSCs by transducing the exogenous Oct4 protein in combination with Sox2, Klf4, and c-Myc. The properties of these iNSCs were characterized, and their therapeutic potential was evaluated following transplantation in an animal model of auditory neuropathy. Histologic and functional assessment were performed.
RESULTS: Immunostaining and flow cytometry results showed that human iNSCs express Nestin, Sox2, and Pax6. Constitutive expression of SKM and transient Oct4 priming induced iNSCs from HDFs under NSC-permissive culture conditions. Isolated iNSCs had similar morphological features and expression markers as control human NSCs. Transplanted human iNSCs improved cochlear cellular integrity, particularly by enhancing neural structures including neurons and nerve fibers. Although peripheral synaptic connections were not restored, synaptic marker toward the central auditory pathway was significantly increased. eABR responses showed statistically higher response in iNSC transplanted animals.
CONCLUSION: Direct transdifferentiation provides an efficient strategy to generate autologous iNSCs capable of preserving and repairing auditory neural structures. By improving neural health, this approach has the potential to enhance CI performance, expand candidacy to patients with long-term hearing loss, and establish a novel regenerative avenue for functional hearing restoration.