Critical roles of FGF, RA, and WNT signalling in the development of the human otic placode and subsequent lineages in a dish

Tsubasa Saeki; Sho Yoshimatsu; Mitsuru Ishikawa; Chung-Chau Hon; Ikuko Koya; Shinsuke Shibata; Makoto Hosoya; Chika Saegusa; Kaoru Ogawa; Jay W Shin; Masato Fujioka; Hideyuki Okano

doi:10.1016/j.reth.2022.04.008

Critical roles of FGF, RA, and WNT signalling in the development of the human otic placode and subsequent lineages in a dish

Regen Ther. 2022 May 16:20:165-186. doi: 10.1016/j.reth.2022.04.008. eCollection 2022 Jun.

Authors

Tsubasa Saeki¹, Sho Yoshimatsu¹, Mitsuru Ishikawa¹, Chung-Chau Hon^{2

3}, Ikuko Koya³, Shinsuke Shibata^{1

4

5}, Makoto Hosoya⁶, Chika Saegusa^{6

7}, Kaoru Ogawa⁶, Jay W Shin^{2

3}, Masato Fujioka^{6

7}, Hideyuki Okano¹

Affiliations

¹ Department of Physiology, Keio University School of Medicine, 35 Shinanomachi Shinjuku-ku, Tokyo 160-8582, Japan.
² RIKEN Center for Life Science Technologies, Division of Genomic Technologies, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.
³ RIKEN Center for Integrative Medical Sciences, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.
⁴ Electron Microscope Laboratory, Keio University School of Medicine, 35 Shinanomachi Shinjuku-ku, Tokyo 160-8582, Japan.
⁵ Division of Microscopic Anatomy, Graduate School of Medical and Dental Sciences, Niigata University, 1-7587 Asahimachi- Dori, Chuo-ku, Niigata City, Niigata 951-8510, Japan.
⁶ Department of Otorhinolaryngology, Head and Neck Surgery, Keio University School of Medicine, 35 Shinanomachi Shinjuku-ku, Tokyo 160-8582, Japan.
⁷ Department of Molecular Genetics, Kitasato University School of Medicine, 1-15-1, Kitasato, Minami-ku, Sagamihara, Kanagawa, 252-0374, Japan.

Abstract

Introduction: Efficient induction of the otic placode, the developmental origin of the inner ear from human pluripotent stem cells (hPSCs), provides a robust platform for otic development and sensorineural hearing loss modelling. Nevertheless, there remains a limited capacity of otic lineage specification from hPSCs by stepwise differentiation methods, since the critical factors for successful otic cell differentiation have not been thoroughly investigated. In this study, we developed a novel differentiation system involving the use of a three-dimensional (3D) floating culture with signalling factors for generating otic cell lineages via stepwise differentiation of hPSCs.

Methods: We differentiated hPSCs into preplacodal cells under a two-dimensional (2D) monolayer culture. Then, we transferred the induced preplacodal cells into a 3D floating culture under the control of the fibroblast growth factor (FGF), bone morphogenetic protein (BMP), retinoic acid (RA) and WNT signalling pathways. We evaluated the characteristics of the induced cells using immunocytochemistry, quantitative PCR (qPCR), population averaging, and single-cell RNA-seq (RNA-seq) analysis. We further investigated the methods for differentiating otic progenitors towards hair cells by overexpression of defined transcription factors.

Results: We demonstrated that hPSC-derived preplacodal cells acquired the potential to differentiate into posterior placodal cells in 3D floating culture with FGF2 and RA. Subsequent activation of WNT signalling induced otic placodal cell formation. By single-cell RNA-seq (scRNA-seq) analysis, we identified multiple clusters of otic placode- and otocyst marker-positive cells in the induced spheres. Moreover, the induced otic cells showed the potential to generate hair cell-like cells by overexpression of the transcription factors ATOH1, POU4F3 and GFI1.

Conclusions: We demonstrated the critical role of FGF2, RA and WNT signalling in a 3D environment for the in vitro differentiation of otic lineage cells from hPSCs. The induced otic cells had the capacity to differentiate into inner ear hair cells with stereociliary bundles and tip link-like structures. The protocol will be useful for in vitro disease modelling of sensorineural hearing loss and human inner ear development and thus contribute to drug screening and stem cell-based regenerative medicine.

Keywords: 2D, two-dimensional; 3D, three-dimensional; BMP, bone morphogenetic protein; CHIR, CHIR-99021; EGF, epidermal growth factor; FGF, fibroblast growth factor; Floating culture; Hair cell; Human pluripotent stem cell; KSR, KnockOut serum replacement; LDN, LDN-193189; OCT, optimal cutting temperature; Otic placode; PBS, phosphate-buffered saline; RA, retinoic acid; SB, SB-431542; SEM, scanning electron microscope; SF, serum free; Single cell RNA-seq; TGFβ, transforming growth factor-β; hESCs, human embryonic stem cells; hPSCs, human pluripotent stem cells; qPCR, quantitative PCR; scRNA-seq, single-cell RNA-sequencing.