Mapping oto-pharyngeal development in a human inner ear organoid model

ABSTRACT Inner ear development requires the coordination of cell types from distinct epithelial, mesenchymal and neuronal lineages. Although we have learned much from animal models, many details about human inner ear development remain elusive. We recently developed an in vitro model of human inner ear organogenesis using pluripotent stem cells in a 3D culture, fostering the growth of a sensorineural circuit, including hair cells and neurons. Despite previously characterizing some cell types, many remain undefined. This study aimed to chart the in vitro development timeline of the inner ear organoid to understand the mechanisms at play. Using single-cell RNA sequencing at ten stages during the first 36 days of differentiation, we tracked the evolution from pluripotency to various ear cell types after exposure to specific signaling modulators. Our findings showcase gene expression that influences differentiation, identifying a plethora of ectodermal and mesenchymal cell types. We also discern aspects of the organoid model consistent with in vivo development, while highlighting potential discrepancies. Our study establishes the Inner Ear Organoid Developmental Atlas (IODA), offering deeper insights into human biology and improving inner ear tissue differentiation.

Since our last publication on the inner ear organoid protocol (Koehler et al., 2017), we have made several modifications and validated the protocol with various cell lines (van der Valk et al., 2023).However, this report focuses specifically on the use of the WTC cell line.The detailed methods can be found in (Zhang et al., 2021).

Basal medium optimization:
We updated the composition of certain media elements used during differentiation.Previously, we used a Chemically Defined Medium (CDM).However, we found that batch-to-batch variability caused by pipetting errors or reagent stability made CDM unsuitable.Therefore, we substituted individually prepared CDM for commercially available Essential 6 (E6) media (Gibco, A1516401) due to its fully defined and stable components.With E6 media as a basal media, we can supplement with additional small molecule and growth factor components tailored to our specific needs.

Matrigel optimization:
We optimized our use of Matrigel (Corning Matrigel Growth Factor Reduced,354230) as a culture component.Previously, we embedded cell aggregates in droplets of 100% Matrigel on the bottom of a petri dish and then submerged the droplets in medium.However, this can be a time-consuming process and leads to significant Matrigel use.We have found that we can introduce Matrigel to cell aggregates by adding Matrigel to the media at a 1% concentration and then suspending cell aggregates in floating culture.This approach has shown robust inner ear organoid generation.

Long-term culture format:
We previously used a spinner flask for long-term culture of cell aggregates but have switched to using individual wells of a low adhesion 24-well plate (Thermo Scientific, 174930) for long-term culture.This modification allows us to follow individual aggregates over time and makes it easier to track experimental groups.We place a single aggregate in each well and then place the plate on an orbital shaker.

CHIR 99021 utilization:
We updated the timing and concentrations of our CHIR-99021 (CHIR; Stemgent, 04-0004-10) treatments in culture.On day 8, we treat cell aggregates with 3 μM of CHIR.Then, on day 10, we add CHIR again at the same concentration.On day 12, we transition to floating culture but ensure that the 3 μM concentration of CHIR is maintained as aggregates are transitioned to fresh media.On day 15, we perform a half-medium change, but also add CHIR to maintain the concentration at 3 μM.Finally, on day 18 of culture, we perform a half-medium change but do not supplement the media with CHIR.We do not use CHIR as a media component again during continued long-term culture beyond day 18.OEP cell cluster of day 0 through day 36, there appears to be a spectrum of expression: the lower area expresses PAX8, and the upper portion ISL1.This highlighted the diversity of cell types contained in the otic epithelium cell cluster and suggested a broad spectrum of otic vesicle patterning was represented.To confirm the transcriptomic analysis, we performed immunohistochemistry on day 36 samples and observed some vesicles that were ISL + but not PAX8 + (F, G) and some vesicles that were PAX8 + but not ISL1 + .
Table S1.List of the top differentially 100 genes in the comparison of CHIR-treated (UP) vs control (DOWN) in the day 13 otic population (OEPD1, OEPD2, OEP, HC)

Fig
Fig. S3.IODA Reproducibility and Pluripotency Markers.(A) UMAPs as result of mapping by Symphony of day 6 organoids from two experiments using the hiPSC line WTC-DSP and one experiment using the WA25 hESC line.The bar plot displays the relative cell type contribution per experiment.By comparing cell type contributions across multiple experimenters and cell lines, it was clear that there were differences

Fig. S4 .
Fig. S4.Cell Signaling Analyses Across Time Points and Cell Types.(A) Gene expression of targets of the TGFβ, FGF, Hedgehog, Notch, retinoic acid, and WNT signaling pathways in the whole dataset per time point.(B) Gene expression per cell type of members of the TGFβ,FGF, retinoic acid, Hedgehog, Notch, and WNT signaling pathways per cell cluster.Signaling pathway members that showed no expression were excluded.Annotations highlight a select group of expected and novel insights.RDH10 expression citation(Ono et al., 2020).

Fig. S5 .
Fig. S5.Epidermal and Amnion Markers at Days 6 and 8, NE to NCC Transition, Glial Marker Expression in NCC, and the Working Model of Lineage Segregation.(A) Density plots displaying expression of marker genes for surface ectoderm and epidermis, as well as marker genes for amnion cells in day 6 to day 8 data.(B) Shared FOXD3 expression between NE and NCC in day 3 through day 6 data.(C) Density plots displaying expression of marker genes for glial cells in the NCC cluster (day 0 to day 36).(D) Illustration demonstrating the development of surface ectoderm and neuroectoderm derivatives after supplementing day 3 organoids with FGF and inhibiting BMP.

Fig. S6 .
Fig. S6.Spatial Analysis of Placodal and Pharyngeal Marker Genes and Proteins.(A) Distinct expression of posterior placodal marker GBX2 and anterior placodal marker OTX2 in day 12 organoids by RNAScope.(B) Density plots showing expression of pharyngeal markers NKX2-5 and VGLL2 in the pPPE cluster (day 0 to day 36 data).(C) Illustration of NKX2-5 pharyngeal expression in the developing mouse embryo, based on(Zhang et al., 2014).(D)

Fig. S7 .
Fig. S7.HOX Gene Analysis.(A) HOX gene expression analysis showed that the mesenchyme (cySE, ctSE, tME, and MYO) clusters expressed low levels of HOX genes, which can help to determine the axial level of development.Additionally, the lack of HOX gene expression in the NCC was consistent with past analyses of the lack of HOX gene expression in cranial neural crest cells.(B) The HOX gene expression in the cySE and ctSE clusters started around day 6 and decreased around day 8, whereas HOX expression in tME and MYO clusters increased around day 30-36 of the culture.(C) Density plots of HOX gene expression in the day 0 through day 36 data.

Fig. S8 .
Fig. S8.Spatial Analysis of Periotic Mesenchyme.(A) Considering that POU3F4 + mesenchyme plays an important role in axon guidance during inner ear development, we stained organoid samples at day 36 for the neuronal marker, TUBB3, and POU3F4.A higher magnification image revealed TUBB3 + axons and POU3F4 + mesenchyme surrounding a developing inner ear organoid.(B) In addition to immunohistochemistry analysis, we also examined transcript expression and found OTOR and POU3F4 expression in the fME mesenchymal cluster.TUBB3 was expressed in the neuronal cell cluster.Note the POU3F4 expression in NEP and the CNS-like portion of the NEU cluster, consistent with its role in neurodevelopment.Scale bars 100 μm.

Fig. S9 .
Fig. S9.HC Maturation and Segregation of OEP in Cochlear versus Vestibular and Sensory versus Nonsensory Domains.(A) Dynamic expression of genes in hair cells that are known to have a temporal expression during hair cell development.(B) Considering the role that TBX2 plays in inner ear patterning, we analyzed our dataset for TBX2 expression and found otic epithelium expression.To confirm otic TBX2 expression, we performed in situ hybridization with the RNAScope platform on day 36 samples (C) and found TBX2 expression throughout the epithelium.(D) Cochlear versus vestibular marker gene expression in the OEP.(E) Within the

Movie 1 .
Representative DSP-GFP iPSC-Derived Aggregates Undergoing Inner Ear Organoid Induction.A 24-hour time-lapse recording of three organoids between days 11-12.Z-stacked images were taken every hour.Note the evaginating vesicle-like structures reminiscent of invaginating otic vesicles in the embryo.Movie 2. Wholemount Immunostaining of Inner Ear Organoids Derived From DSP-GFP iPSCs.A representative day 80 organoid specimen was wholemount immunostained with antibodies for MYO7A and TUBB3 using a modified SHIELD protocol.The imaged region contains otic organoids with sensory epithelia and dense innervation.

Table S2 .
List of antibodies used for immunocytochemistryClick here to download TableS1Click here to download TableS2