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Development of the Otic (Inner Ear) Organoid with Exploring Differentiation-Promoting Factors

  • 김한태 (Hantai Kim, Ajou University Graduate School of Medicine)

초록/요약

Organoids, also known as miniaturized organs, are developed from pluripotent stem cells (PSCs) using 3-dimensional culture techniques. These structures mimic the microstructure and biological function of the target organ. They are being generated for almost every organ, including the brain, retina, thyroid, lungs, stomach, liver, intestines, kidneys, and more. Organoids serve as a valuable tool for studying developmental processes of the organ and have promising applications in regenerative medicine and drug development. The first otic organoid (also called inner ear organoid) was developed from mouse embryonic stem cells (mESCs) in 2013, followed by successful differentiation from human induced pluripotent stem cells (hiPSCs) in 2017. These organoids involve hair cells (HCs) crucial for inner ear function, with stereocilia that have a morphology consistent with their function as ion channels. However, limitations still exist. The differentiated HCs in otic organoids exhibit characteristics of vestibular HCs rather than cochlear (auditory) HCs, which limits their use in studying hearing loss. Additionally, the yields of production are not yet stable. As result, the otic organoid generation technique developed so far is still not mature. Given that current protocols for generating otic organoids only lead to otocyst (also known as otic vesicle) formation, it is probable that additional signaling factors are required for more complex differentiation. Consequently, this study aims not only to successfully differentiate otic organoids using existing protocols but also to investigate methods for enhancing their generation efficiency. The development of otic organoids is manipulated by bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), and Wnt agonists; they are typically used up to an in vitro time of 8-10 days (D8-10). In this study, we aim to investigate whether treatment with additional materials at later stages (post D8-10) can further expedite the differentiation of otic organoids or enhance their efficiency. In previous studies, it has been demonstrated that combining DAPT, the Notch pathway inhibitor, with CHIR99021, the Wnt agonist, can facilitate the differentiation of supporting cells (SCs) and HCs. The objective of this study was to examine the effectiveness of DAPT and CHIR99021 on the differentiation of otic organoids. Initially, we treated the aggregates with 10 μM DAPT and 5 μM CHIR99021 on D12-18. Immunohistochemical staining revealed that some otic organoids exhibited a thicker, more abundant formation of MyoVIIa, a marker of HCs, in the inner portion of the organoid. However, qRT-PCR results did not demonstrate a significant difference in total MyoVIIa expression among all otic organoids produced. Alternatively, treatment with 5 μM DAPT and 2.5 μM CHIR99021 on D28-34 significantly increased the expression of MyoVIIa and Math1, markers of HCs, in qRT-PCR. Nevertheless, while MyoVIIa was visibly expressed in a structured form sole in the otocyst area at the earlier D12-18 treatment, at D28-34 treatment, MyoVIIa expression was dispersed throughout the aggregate. The current method only utilizes a small portion of the cell aggregate to form organoid portion. However, this study shows that the remaining portion of the aggregate also has the potential to differentiate into HCs or SCs. Treatment with DAPT and CHIR99021 at D28-34 seems to induce the conversion of the remainder of the aggregate to HCs, which explains the scattered expression of MyoVIIa observed in immunohistochemical staining. This finding may offer a promising strategy for generating larger quantities of HCs for potential use in cell therapy. Further research is necessary to identify this possibility.

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목차

I. INTRODUCTION 1
II. STUDY 1: Otic Organoid Generation 19
1. Summary of the previous protocol for the generation 19
2. Materials and methods 20
2-1. Maintenance of mESCs 20
2-2. Development of otic organoids 20
2-3. Immunohistochemistry 24
3. Results 24
3-1. Cell aggregates 24
3-2. Hair cell-like cells 27
3-3. Stereocilia and other microstructures 27
III. STUDY 2: Exploration of the Factors 30
1. Finding the promoting factors 30
2. Materials and methods 30
2-1. Maintenance of mESCs and the development 30
2-2. Treatment of DAPT and CHIR-99021 30
2-3. Immunohistochemistry 31
2-4. qRT-PCR 31
3. Results 33
3-1. Treatment between D8 and D12 33
3-2. Treatment between D12 and D18 35
3-3. Treatment between D28 and D34 35
IV. DISCUSSION 38
1. Development of mature otic organoids 38
2. Application of DAPT and CHIIR-99021 to otic organoids 41
3. Application and future direction of otic organoids 44
3-1. Otic organoids as cell therapy 44
3-2. Inner ear organoid-on-a-chip 44
3-3. Otic organoid vesus cochlear implantation 45
V. REFERENCES 48

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