청각 오가노이드 연구 어디까지 진행되었나?
How Far Have We Come in Auditory Organoid Research?
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Trans Abstract
The development of auditory organoids represents a groundbreaking advancement in auditory research and regenerative medicine. These three-dimensional tissue structures, derived from pluripotent stem cells, replicate the architecture and function of the human inner ear, including the key sensory and neural cell types such as hair cells, supporting cells, and neurons. Auditory organoids enable researchers to study inner ear development, model genetic hearing disorders, and evaluate the effects of ototoxic drugs. Recent breakthroughs include the successful differentiation and functional validation of hair cell-like structures responsive to sound signals, as well as the integration of LGR5+ hair cell progenitor cells to enhance regenerative capacity. Despite significant progress, there remain many challenges of achieving full functional maturation, improving reproducibility, and scaling production for clinical applications. This emerging technology holds immense potential for understanding the inner ear biology, advancing personalized medicine, and developing novel therapies for hearing loss and balance disorders.
Introduction
The human ear plays a vital role in perceiving sound and maintaining balance. However, the ear’s intricate structure and limited regenerative capacity make the treatment of hearing loss and related conditions a significant challenge. To address these issues, auditory organoids, an innovative area in biology and medical research, have gained considerable attention. Auditory organoids are artificially created three-dimensional (3D) tissue structures that mimic the human inner ear. They are used to study the development and function of auditory cells or to explore new treatment approaches. While remarkable progress has been made in this field, there are still hurdles to overcome for commercialization.
Organoids are artificially cultured miniature organs derived from stem cells. They exhibit structural and functional characteristics similar to actual organs and are used for studying human physiological processes, drug testing, and disease modeling. Auditory organoids, specifically, aim to replicate the sensory and neural cells of the human inner ear, including partial functions of the cochlea and vestibular system.
This technology involves differentiating induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs) into 3D structures using specific growth factors. The resulting organoids include hair cells, supporting cells, and neurons (Fig. 1). Among these, hair cells, which convert sound vibrations into electrical signals, are a key focus, as their successful generation within organoids represents a major research milestone.
Representative image of auditory organoid. Auditory organoids differentiated from stem cells (nuclei in blue, hair cells in green, and supporting cells in red).
Current Research and Key Achievements
Auditory organoid research has advanced rapidly in recent years, with leading contributions from institutions in the United States, Japan, and Europe. Hair cells play a crucial role in converting sound vibrations into neural signals but cannot naturally regenerate once damaged [1]. In 2023, researchers at Harvard Medical School successfully cultured hair cell-like structures from human iPSCs within auditory organoids and demonstrated their response to stimulus. This finding holds significant potential for developing treatments for hearing loss [2-7]. Auditory organoids have emerged as critical platforms for drug development. Traditional animal models often fail to replicate the complexity of the human inner ear. By utilizing organoids, researchers can more accurately evaluate drug efficacy and toxicity using human cells. For instance, the effects of certain antibiotics and chemotherapy drugs on hair cells have been analyzed, improving the safety of these treatments Auditory organoids also enable the modeling of genetic disorders that lead to hearing loss. By manipulating specific genes, researchers can replicate genetic hearing disorders and explore gene therapy options. For example, researchers at Indiana University successfully created auditory organoids from iPSCs carrying mutations in the TMPRSS3 gene, a known cause of genetic hearing loss, and identified the mechanisms underlying the condition [8].
Discovery and Role of LGR5+ Hair Cell Progenitor Cells
LGR5+ hair cell progenitor cells, known for their regenerative potential in the inner ear, are garnering significant interest in auditory cell research. LGR5 is a marker expressed in inner ear progenitor cells with the potential to differentiate into hair cells. While mature hair cells cannot naturally regenerate, LGR5+ cells can differentiate into hair cells under specific conditions. LGR5+ cells serve as a starting point in developing auditory organoids. Researchers have cultivated these cells to generate hair cells and supporting cells within organoids. For example, activating the Wnt signaling pathway promotes the proliferation of LGR5+ cells, which can be used to model inner ear damage or test therapeutic approaches [6,9]. LGR5+ cells offer several advantages, including high regenerative capacity and the ability to model damaged inner ears more precisely (Fig. 2). In contrast, mature hair cells have limited regeneration potential and are less suitable for studying early developmental processes in the inner ear.
Hair cells differentiated in LGR5+progenitor cell. Hair cells and stereocilia strucrues in LGR5+ auditory organoid (nuclei in blue, hair cells [POU4f3] in green, and stereocilia [Espin] in red).
Key Factors in the Differentiation of Inner Ear Organoids
The development of inner ear organoids represents a remarkable advancement in regenerative medicine and auditory research. These 3D structures mimic the architecture and function of the inner ear, enabling researchers to study development, disease, and potential therapeutic applications. A key factor in the differentiation of inner ear organoids is the optimization of signaling pathways that guide the formation of specialized inner ear structures, including hair cells, supporting cells, and sensory neurons.
Role of signaling pathways
The differentiation of inner ear organoids heavily depends on precise manipulation of key signaling pathways, including Wnt, Notch, fibroblast growth factor (FGF), and bone morphogenetic protein (BMP). Each pathway plays a critical role in patterning and cell fate determination:
• Wnt signaling promotes otic placode induction and hair cell differentiation [10].
• FGF signaling is crucial for otic vesicle specification and sensory cell lineage commitment [11].
• Notch signaling maintains a balance between hair cell and supporting cell populations [12].
• BMP signaling helps establish dorsoventral polarity and regionalization within the organoid [13].
Key signaling pathways and the agents commonly used to modulate them during inner ear organoid development
The differentiation of inner ear organoids from pluripotent stem cells (PSCs) or iPSCs relies on the precise orchestration of multiple signaling pathways. These pathways mimic embryonic developmental processes to guide cells toward forming specialized inner ear structures such as hair cells, supporting cells, and neurons. Below, we review the major signaling pathways integral to the differentiation of inner ear organoids.
Wnt signaling pathway [7,14,15]
Wnt signaling is pivotal in the early stages of inner ear development, particularly in otic placode induction and cell fate specification.
• Role in differentiation: activation of the Wnt pathway, often through small molecules like CHIR99021, promotes the formation of the otic placode by enhancing ectodermal specification.
• Spatiotemporal control: precise temporal activation is critical, as prolonged or excessive Wnt signaling can lead to aberrant cell fates and non-otic structures.
• Downstream effects: Wnt signaling upregulates transcription factors such as PAX2, SOX2, and EYA1, which are essential for otic lineage commitment.
FGF signaling [16,17]
FGF signaling plays a crucial role in the specification of the otic placode and subsequent otic vesicle formation.
• FGF isoforms in use: FGF2, FGF3, and FGF10 are commonly used in differentiation protocols to induce otic-specific structures.
• Role in inner ear organoids: FGF signaling facilitates the development of sensory progenitors within the otic vesicle, leading to hair cell and supporting cell differentiation.
• Synergy with Wnt pathway: FGF and Wnt signaling work synergistically during the early stages of otic differentiation to enhance the efficiency and fidelity of inner ear organoid development.
Notch signaling pathway [18-20]
The Notch pathway governs cell fate decisions and maintains the balance between sensory hair cells and supporting cells.
• Lateral inhibition: through lateral inhibition, Notch signaling ensures that only a subset of progenitors differentiate into hair cells, while the rest become supporting cells.
• Fine-tuning differentiation: modulation of Notch signaling, using inhibitors like N-[N-(3,5-difluorophenacetyl)-lalanyl]-s-phenylglycinet-butylester (DAPT), can enhance hair cell production in inner ear organoids.
• Context-dependent effects: overactivation can inhibit hair cell formation, underscoring the importance of controlled inhibition during differentiation protocols.
BMP signaling [3,14,21]
BMP signaling is critical for dorsoventral patterning and regional specification in inner ear organoids.
• Dual role: BMP signaling promotes otic placode formation at early stages but must be fine-tuned later to prevent non-sensory cell fates.
• Interaction with FGF: BMP antagonists like noggin are often used alongside FGF to create a permissive environment for sensory cell differentiation.
Retinoic acid signaling [11]
Retinoic acid (RA) is essential for the anteroposterior patterning of the otic vesicle and differentiation of sensory and neural components.
• Role in hair cell differentiation: RA signaling enhances the formation of mechanosensory hair cells within organoids.
• Stage-specific application: RA is typically introduced during later stages of differentiation to drive maturation of sensory structures.
Matrix composition and 3D environment
The extracellular matrix (ECM) used for culturing organoids provides mechanical support and biochemical cues. Hydrogels such as Matrigel, which mimic the native ECM, play a pivotal role in shaping organoid morphology and fostering cell differentiation [22]. The 3D environment also facilitates cell-cell and cell-matrix interactions, critical for recapitulating the complex architecture of the inner ear.
Stepwise differentiation protocols
Successful inner ear organoid differentiation relies on stepwise protocols that sequentially guide PSCs or iPSCs through stages of embryonic development. Initial steps typically involve [3,14]:
• Induction of ectodermal progenitors.
• Specification into otic placode-like structures.
• Differentiation into otic vesicles and sensory cell types.
Optimal timeline for pathway modulators based on prior publications
The timing and dosage of pathway modulators are critical, as they must mimic the spatiotemporal cues of embryonic development to yield fully functional inner ear structures.
Day 0-3: pluripotency and early induction
Key focus: establish ectodermal lineage.
Factors:
• Wnt activation (e.g., CHIR99021): promotes ectodermal specification and otic placode induction [14].
• FGF (e.g., FGF3, FGF10): induces otic lineage by synergizing with Wnt signaling [14].
Day 4-7: otic placode formation
Key focus: transition from ectoderm to otic progenitors.
Factors:
• Wnt activation (continued): drives otic placode differentiation and upregulates transcription factors (e.g., PAX2, SOX2) [14].
• FGF signaling: reinforces otic fate and enhances sensory progenitor development.
- Low BMP activity: prevents non-otic ectodermal fates.
- BMP inhibitors (e.g., Noggin, LDN-193189): used in combination.
Day 8-12: otic vesicle development
Key focus: regional specification of otic vesicle into sensory and non-sensory regions.
Factors:
• Notch activation (e.g., Jagged1/delta-like ligands): promotes supporting cell fates while suppressing excessive hair cell differentiation [18-20].
• BMP activation (e.g., BMP4): supports patterning and cochlear structure formation [3,14,21].
Day 13-18: sensory cell specification
Key focus: formation of hair cells and supporting cells.
Factors:
• RA: drives sensory hair cell differentiation and neural specification.
• Notch inhibition (e.g., DAPT): enhances hair cell formation by inhibiting lateral inhibition [18-20].
Day 19-30: maturation and neural integration
Key focus: maturation of hair cells, neural connections, and organoid complexity.
Factors:
• Hedgehog activation (e.g., SAG): supports cochlear morphogenesis and non-sensory structure formation.
• Transforming growth factor-β modulation (e.g., SB-431542): assists in ECM remodeling and advanced structural maturation [23].
Emerging Technologies and Tools
The integration of bioengineering and advanced imaging techniques has further enhanced the differentiation of inner ear organoids. Single-cell RNA sequencing allows detailed characterization of cell populations within the organoids, enabling researchers to fine-tune protocols for higher fidelity and efficiency [2,4]. Photobiomodulation is a technique to improved differentiation and proliferation of neural stem cells [24-26]. This non-invasive technique has been proved to increase the differentiation of inner ear organoid [27,28]. Further analysis to validate this approach is necessary.
Challenges and Obstacles
Despite the promising advancements, several technical and ethical challenges remain in auditory organoid research. Current auditory organoids can replicate only partial functions of the inner ear. The actual inner ear comprises diverse structures, including the cochlea, vestibular organs, and auditory nerve, which interact intricately. Recreating these interactions in vitro is a significant challenge. Additionally, the maturity of organoids often falls short compared to real organs, with differentiation into specific inner or outer hair cells remaining a key hurdle. Developing technologies to ensure the stable, large-scale production of auditory organoids is essential. While small-scale production in laboratories suffices for research purposes, clinical and commercial applications require mass production and standardized quality control. For instance, auditory organoids derived from iPSCs have shown limited differentiation efficiency into hair cells. Although organoids based on LGR5+ progenitor cells exhibit higher differentiation rates, current methods rely on harvesting these cells from animal models, necessitating the development of cell lines for scalable use. Research involving stem cells continues to raise ethical concerns. While iPSC technology offers an alternative to ESCs, ethical issues surrounding the procurement and use of cellular resources still require careful consideration.
Future Prospects
Yet, neural functionality of these auditory organoids, especially integrative sensory – neuron function which can be demonstrated by synaptic connections and neural firings in electrophysiological assays is not established. Several studies showed good patch clamp outcomes which were solitary sensory cell responses. Nevertheless, to ensure the appropriate functionality, it is essential to prove the sensory – neural connections in various auditory organoids. In recent study, coculture outcomes which showed good synaptic connections have demonstrated more refiled differentiation of detailed hair cell morphology and function [29]. In addition, several applications of biomaterials have been shown to advance the neural integration [30] which could increase the functionality of auditory organoid.
Auditory organoids hold immense potential as innovative tools for studying and treating hearing disorders. The following areas show great promise. Creating patient-specific auditory organoids using their own cells could lead to more effective treatments. For example, patient-derived iPSCs can be used to model genetic hearing disorders and design personalized drug or gene-editing therapies. A long-term goal is the transplantation of lab-grown auditory organoids into patients to restore lost hearing function. Research is ongoing to address challenges related to immune rejection and ensuring stable organoid function in vivo. Organoid-based drug testing offers cost-effective and time-saving advantages, providing precise data for developing new treatments for hearing loss. Although auditory organoid technology is still in its infancy, its potential to transform biology and medicine is vast. With continued advancements, this technology could bring new hope to millions of people suffering from hearing loss.
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Acknowledgments
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