Majlinda Lako, MD

Konstantina M. Stankovic, MD

Miodrag Stojkovic, MD

To read the articles in this collection, go to HearingLoss.StemCells.com

Traditionally, STEM CELLS hosts exciting series of articles on important issues related to stem cell biology, early development, differentiation, and regenerative medicine. All parts build the complex mosaic, which should help fight debilitating human diseases, including hearing loss (HL). According to the World Health Organization, HL is a major global health problem of pandemic proportions. Four hundred sixty-six million people worldwide have disabling HL, and this number is expected to increase to 900 million people by 2050 (https://www.who.int/news-room/fact-sheets/detail/deafness-and-hearing-loss). Unfortunately, current treatments for HL are primarily limited to different devices that amplify sounds or directly electrically stimulate the auditory nerve. However, these approaches fail to correct the underlying cause and have substantial performance restrictions. To capture the most recent research, we proudly introduce this new series, which summarizes the stem cell field's progress focusing on HL.^1-5

In the past 10 years, stem cell technologies have enabled differentiation of human sensory cell types in vitro, providing novel tools to study inner ear development, model disease, and validate various therapeutic approaches.⁵ Ethically regulated access to human embryonic and fetal material and human induced pluripotent stem cells (hiPSCs) has allowed the scientific and clinical community to better understand the mechanisms orchestrating normal and abnormal early inner ear development and create conditions for targeted differentiation and genetic editing/manipulation. Animal models and personalized hiPSC lines enable studies of the molecular pathogenesis of inherited HL and HL of unknown etiology. Kempfle et al¹ used a transgenic mouse model to transiently overexpress Lin28, a neural stem cell regulator that promotes in vitro proliferation and conversion of auditory glial cells into neurons.^{6, 7} To study the effects of Lin28 on endogenous glial cells after the loss of auditory neurons in vivo, the authors produced an auditory neuropathy model. After selective damage of auditory neurons, the upregulation of the Lin28 in inner ear glial cells induced expression of neural stem cell/precursor cell markers (Hmga2, NeuroD, NeuroG, and AScl1) and subsequent conversion into neurons. However, transient postnatal upregulation of Lin28 after neural damage induced proneural gene expression and reprogramming into cells that expressed neuron-specific marker class III ß tubulin. This work elegantly demonstrates the potential of direct reprogramming of inner ear cells and their conversion into desirable sensory neurons as a regeneration therapy for neural replacement in auditory neuropathy. Marta Roccio² summarizes the generation of sensory cells through the directed differentiation of pluripotent stem cells, as well as the latest “cell conversion approaches.”² Undoubtedly, the improvement of both strategies necessitates the improvement of suboptimal in vitro growth conditions. For instance, to trigger successful differentiation and ectoderm induction toward inner ear cells, the mesendoderm fate needs to be blocked with inhibitors of TGFβ and Wnt signaling.^{8, 9} Applying directed differentiation protocols in 3D culture, or organoids, has been recently successfully translated to human pluripotent stem cells.^{10, 11} These protocols work by generating 3D aggregates of pluripotent stem cells, promoting the differentiation by providing exogenous BMP ligands.

In contrast, the otic fate is promoted using bFGF first, subsequently a Wnt signaling agonist, and the niche-specific extracellular matrix (ECM). The pivotal role of ECM in the fate determination of inner ear progenitor cells was highlighted by Xia et al.⁴ The authors concluded that the biological behavior of inner ear progenitor cells in an encapsulated culture system is critically dependent on the mechanical cues from the ECM.⁴ The authors nicely show that ECM promoted the survival and expansion of progenitor cells inducing the accumulation of Ras homolog family member A (RhoA), an essential factor in the maintenance of stem cells and their differentiation,^{12, 13} which causes the polymerization of actin cytoskeletons. These changes, in turn, resulted in increased Yes associated protein (YAP) nuclear localization and enhanced expansion of inner ear progenitor cells, partially through upregulating the canonical Wnt signaling pathway. Herewith, the authors provided the first characterization of the action of YAP as mediators of mechanotransduction signaling to promote the proliferation of inner ear progenitor cells.

Considering age-related, acoustic, ototoxic, and genetic insults, which are the most frequent causes of irreversible damage of inner ear hair cells and spiral ganglion neurons,^{14, 15} new methods of genome editing could bring additional opportunities to understand the pathogenesis of HL and identify novel therapies.³ In this review, Stojkovic et al³ summarized the CRISPR/Cas9 platform and its potential to accelerate basic and translational research in HL. One of the work's conclusions is that stem cells’ usage for therapeutic purposes still faces several significant obstacles, particularly concerning efficiency, efficacy, and safety.³ Finally, for therapeutic purposes, cultured stem cells must be precisely integrated within their microanatomical niche after being introduced into the inner ear.⁵ Whether this will be done via intraperilymphatic or intraendolymphatic injection approach, technical challenges remain,^16-20 concerning the following: (a) how to reach the ultimate destination, the Rosenthal canal, (b) how to cross the tightly sealed cochlear scala media or break the tight junction complex between hair cells and supporting cells, (c) how to surpass the endolymph, which contains a high level of potassium, and (d) how to define the balance between the intrinsic factors (stage of differentiation of grafted cells) with the extrinsic factors (host background and means of delivery). These questions are justifiably raised by Zine et al⁵ and request intensive research and technical improvements to tackle HL successfully.

Nevertheless, we are confident that the progress achieved in the past decade will unceasingly continue to flourish. Meanwhile, we are witnessing the multistep strategies, including large-scale postmortem studies with a comprehensive meta-analysis of genome, epigenome, and transcriptome of patients with specific types of HL. Banks of undifferentiated and differentiated patient-specific hiPSCs, enhanced organoid models by additional bodily systems under dynamic microfluid chips, and many other emerging approaches will provide powerful tools for accelerating drug and/or cell therapies. As the STEM CELLS journal remains curious and determined to push these research frontiers, we will be thrilled to recurrently share and celebrate with you the ground-breaking work in the field of HL. We hope that you enjoy reading this exciting series and continue to contribute to the joint mission of improving life quality for millions of people with HL worldwide.

Majlinda Lako，医学博士

Konstantina M. Stankovic，医学博士

米奥德拉格·斯托伊科维奇，医学博士

要阅读此合集中的文章，请访问 HearingLoss.StemCells.com

传统上，干细胞就与干细胞生物学、早期发育、分化和再生医学相关的重要问题发表激动人心的系列文章。所有部分都构成了复杂的马赛克，这将有助于对抗使人衰弱的人类疾病，包括听力损失 (HL)。根据世界卫生组织的说法，HL 是一个严重的全球性大流行性健康问题。全世界有 4.66 亿人患有致残性 HL，预计到 2050 年这一数字将增加到 9 亿人（https://www.who.int/news-room/fact-sheets/detail/deafness-and-听力损失）。不幸的是，目前对 HL 的治疗主要限于放大声音或直接电刺激听觉神经的不同设备。但是，这些方法无法纠正根本原因并且具有很大的性能限制。^1-5

在过去的 10 年中，干细胞技术已经实现了体外人类感觉细胞类型的分化，为研究内耳发育、模拟疾病和验证各种治疗方法提供了新的工具。⁵对人类胚胎和胎儿材料以及人类诱导多能干细胞 (hiPSC) 的道德规范访问使科学和临床界能够更好地了解协调正常和异常早期内耳发育的机制，并为靶向分化和基因编辑/操作创造条件. 动物模型和个性化的 hiPSC 系能够研究遗传性 HL 和病因不明的 HL 的分子发病机制。Kempfle 等人¹使用转基因小鼠模型瞬时过表达Lin28是一种神经干细胞调节剂，可促进听觉神经胶质细胞体外增殖和转化为神经元。^{6, 7}为了研究Lin28在体内听觉神经元丧失后对内源性神经胶质细胞的影响，作者制作了一个听觉神经病变模型。在听觉神经元选择性损伤后，内耳神经胶质细胞中Lin28的上调诱导神经干细胞/前体细胞标志物（Hmga2、NeuroD、NeuroG和AScl1）的表达并随后转化为神经元。然而，Lin28的短暂产后上调在神经损伤诱导原神经基因表达并重新编程为表达神经元特异性标志物 III 类 ß 微管蛋白的细胞后。这项工作优雅地展示了内耳细胞直接重编程及其转化为所需感觉神经元的潜力，作为听觉神经病中神经替代的再生疗法。Marta Roccio ²总结了通过多能干细胞的定向分化产生的感觉细胞，以及最新的“细胞转化方法”。²毫无疑问，这两种策略的改进都需要改进次优的体外生长条件。例如，为了触发内耳细胞的成功分化和外胚层诱导，需要用 TGFβ 和 Wnt 信号传导抑制剂阻断中内胚层的命运。^{8, 9}在 3D 培养或类器官中应用定向分化方案，最近已成功转化为人类多能干细胞。^{10, 11}这些协议通过生成多能干细胞的 3D 聚集体发挥作用，通过提供外源性 BMP 配体促进分化。

相比之下，首先使用 bFGF，然后使用 Wnt 信号激动剂和特定于生态位的细胞外基质 (ECM) 来促进耳命运。Xia 等人强调了 ECM 在内耳祖细胞命运决定中的关键作用。⁴作者得出结论，内耳祖细胞在封装培养系统中的生物学行为严重依赖于 ECM 的机械信号。⁴作者很好地表明，ECM 促进了祖细胞的存活和扩增，诱导了 Ras 同源家族成员 A (RhoA) 的积累，这是维持干细胞及其分化的重要因素，^{12, 13}这导致肌动蛋白细胞骨架的聚合。反过来，这些变化导致 Yes 相关蛋白 (YAP) 核定位增加和内耳祖细胞扩增增强，部分是通过上调经典 Wnt 信号通路。因此，作者首次描述了 YAP 作为机械转导信号介质促进内耳祖细胞增殖的作用。

考虑到与年龄相关的、听觉的、耳毒性的和遗传损伤，这些是内耳毛细胞和螺旋神经节神经元不可逆损伤的最常见原因，^14、15种新的基因组编辑方法可以为了解 HL 的发病机制带来更多机会并确定新疗法。³在这篇综述中，Stojkovic 等人³总结了 CRISPR/Cas9 平台及其加速 HL 基础和转化研究的潜力。这项工作的一个结论是，干细胞用于治疗目的仍然面临着几个重大障碍，特别是在效率、功效和安全性方面。³最后，为了治疗目的，培养的干细胞必须在被引入内耳后精确地整合到它们的微解剖生态位中。⁵无论是通过外淋巴内注射还是内淋巴内注射方法，技术挑战仍然存在，^16-20关于以下内容：（a）如何到达最终目的地，罗森塔尔运河，（b）如何穿过紧密密封的耳蜗中层或打破毛细胞和支持细胞之间的紧密连接复合体，（c）如何超越内淋巴，其中含有高水平的钾，以及（d）如何定义内在因素（移植细胞的分化阶段）与外在因素（宿主背景和递送方式）之间的平衡。^{Zine 等人5}提出了这些问题是有道理的，并要求进行深入的研究和技术改进以成功解决 HL。

尽管如此，我们有信心，过去十年取得的进展将继续蓬勃发展。与此同时，我们正在见证多步骤策略，包括大规模尸检研究，对特定类型 HL 患者的基因组、表观基因组和转录组进行全面的荟萃分析。未分化和分化的患者特异性 hiPSC 库、通过动态微流体芯片下的附加身体系统增强的类器官模型以及许多其他新兴方法将为加速药物和/或细胞疗法提供强大的工具。作为干细胞杂志保持好奇并决心推动这些研究前沿，我们将很高兴与您经常分享和庆祝 HL 领域的开创性工作。我们希望您喜欢阅读这个激动人心的系列文章，并继续为改善全球数百万 HL 患者的生活质量这一共同使命做出贡献。