Improved method for generating inner ear organoids

Abstract

The invention relates to a method of generating endolymph-producing epithelial cells and in vitro use of a hedgehog signalling inhibitor for differentiation of inner ear progenitor cells into endolymph-producing epithelial cells; in vitro endolymph-producing epithelial cells and an inner ear organoid comprising endolymph-producing epithelial cells. Also provided is in vitro use of an inner ear organoid in drug discovery; the endolymphproducing epithelial cells for use in a method of treating diseases of the inner ear; and a method of testing one of more therapeutic agents using these cells or organoids. Lastly, also provided is a kit and an organoid maturation medium. [Figure 3]

Description

Field of the Invention The invention relates to a method of generating endolymph-producing epithelial cells and in vitro use of a hedgehog signalling inhibitor for differentiation of inner ear progenitor cells into endolymph-producing epithelial cells; in vitro endolymph-producing epithelial cells and an inner ear organoid comprising endolymph-producing epithelial cells. Also provided is in vitro use of an inner ear organoid in drug discovery; the endolymph- producing epithelial cells for use in a method of treating diseases of the inner ear; and a method of testing one of more therapeutic agents using these cells or organoids. Lastly, also provided is a kit. Background The inner ear can be divided in to an organ of hearing (the cochlea) and an organ for balance (the vestibular system). lnner ear organoids derived from human pluripotent stem cells are useful in vitro tools for investigating inner ear development and understanding the onset, progression, and potential therapeutical targeting of inner ear disorders. However, existing protocols for generating inner ear organoids fail to recapitulate all of the cell types present within the inner ear. This decreases the translational capacity and utility of these models. Summary of the Invention Current methods used in the art fail to produce endolymph-generating epithelia. The present invention provides a method of generating endolymph generating epithelia present in both the vestibular system and the cochlea. The present invention provides a method of generating dark cell epithelia of the vestibular system and marginal cells of the stria vascularis of the cochlea. No current method used in the art produces these cells. In a first aspect the invention provides a method of generating endolymph-producing epithelial cells, the method comprising: a) obtaining inner ear progenitor cells; b) adding a hedgehog pathway inhibitor to cause differentiation of the one or more inner ear progenitor cells to form endolymph-producing epithelial cells. In a further aspect, the invention provides an inner ear organoid comprising endolymph- producing epithelial cells, optionally wherein the endolymph-producing epithelial cells were obtained by the method described above. In a further aspect, the invention provides in vitro use of the inner ear organoid described above in drug discovery. In a further aspect, the invention provides isolated endolymph-producing epithelial cells, optionally obtained by the method described above. In a further aspect, the invention provides the isolated endolymph-producing epithelial cells for use in a method of treating a disease of the inner ear wherein the method comprises transplanting the endolymph-epithelial cells into the subjects ear. In a further aspect, the invention provides a method of testing one or more therapeutic agents, the method comprising: a) providing endolymph-producing epithelial cells as described above or an inner ear organoid as described above; b) contacting the cells or organoid with at least one therapeutic agent; c) detecting one or more changes in the cells or organoid; d) determining the effects of the therapeutic agent based on the absence or presences of the one or more changes. In a further aspect, the invention provides a kit comprising: a) a hedgehog inhibitor; and any one or more of the following: b) i) cell culture media; and / or ii) otic progenitor cells; and / or iii) a Wnt activator; and / or iv) a hedgehog activator and a Wnt inhibitor. In a further aspect, the invention provides organoid maturation media comprising a hedgehog inhibitor. In a further aspect, the invention provides in vitro use of a hedgehog signalling inhibitor for differentiation of inner ear progenitor cells into endolymph-producing epithelial cells. Detailed description General terms Endolymph-producing epithelial cells Endolymph is the fluid found in the membranous labyrinth of the inner ear. The main component of this extracellular fluid is potassium and the endolymph plays an important role in hearing and balance. Endolymph-producing epithelial cells may be vestibular endolymph producing epithelia such as dark cell epithelium. Vestibular dark cells are localised within the utricle, ampullae, and the common crus of the semicircular canals and play an important role in maintaining the high potassium and low sodium content of the endolymph. Endolymph producing cells may also be cochlear endolymph producing epithelia such as the stria vascularis. The stria vascularis is a highly vascular tissue that lines the medial aspect of the lateral wall of the cochlea. Marginal cells form the most medial layer of the stria vascularis and face the endolymph of the cochlea. Marginal cells, together with the intermediate and basal layer of the stria vascularis, are responsible for the active transport of potassium in to the endolymph and support the high potassium gradient of the inner ear. Stem cells and progenitor cells A progenitor cell is a biological cell that can differentiate into a specific cell type. These are formed by culturing stem cells, for example pluripotent stem cells or multipotent (also referred to as tissue-resident) stem cells. The progenitor cell is an inner ear progenitor cell. These are also known in the field as otic progenitor cells. Otic progenitor cells are a specialized group of multipotent cells derived from the otic placode and / or otic vesicle, the embryonic structure that gives rise to the inner ear. These progenitor cells have the potential to differentiate into various cell types of the inner ear, including sensory cells, and endolymph-producing cells. Derivative cells from otic progenitor cells can also be utilised. Otic progenitor cells may be identified by markers such as PAX2, PAX8, SOX2, DLX5, GATA3, OC90 or OTX2. Organoid The term organoid is used to refer to self-organized three-dimensional tissue cultures. Organoids may include artificial, in vitro three-dimensional structures made to mimic or resemble the functional and / or histological structure of an organ or portion thereof, such as the inner ear. The term "organoid" includes spheroids or cell clusters formed from suspension cell cultures. Such organoids may be derived from stem cells. For example, organoids may be derived from tissue specific stem cells, embryonic stem cells or induced pluripotent stem cells. Embryonic stem cells may be human embryonic stem cells. The endolymph producing cells and organoid comprising these cells are human cells and organoids. m The culture process generally comprises three stages: - A first stage where stem cells (e.g. pluripotent stem cells) are cultured; - A second stage where small molecules and growth factors encourage the development of the stem cells into progenitor cells; and - A third stage where the organoid self-organises and matures. The hedgehog signalling pathway inhibitor is added after the second stage, to inner ear progenitor cells. Vlth regards to the first stage, as standardly carried out in the field, this stage generally comprises enzymatically disassociating the stem cells, e.g. human pluripotent stem cell colonies, to embryoid bodies before proceeding to the second stage. An example of the timeline of stages 2 and 3 are shown in Figure 2A. Vlth regards to the second stage, growth factors and other molecules are added, for example, for 15 days, up to 24 days, to the stem cells to cause differentiation into inner ear progenitor cells. Differentiation into inner ear progenitor cells can be by various methods known in the field. For example, adding any one or more of the following: 1. TGF-ß inhibitor, optionally SB-431542 2. Fibroblast Growth Factor family, optionally recombinant protein basic FGF 3. TGF-ß superfamily of growth factors, optionally recombinant protein BMP-4 4. BMP signalling inhibitor, optionally LDN-193189 5. Wnt activator, optionally the GSK3 inhibitor CHIR99021 6. Hedgehog activator, optionally purmorphamine 7. Wnt inhibitor, optionally IWP-2 For example, 1-5 may be used in an optimised protocol for producing inner ear progenitor cells ahead of adding cyclopamine to result in vestibular endolymph producing epithelial cells. For example, 1-7 may be used in an optimised protocol for producing inner ear progenitor cells ahead of adding cyclopamine to result in cochlear endolymph producing cells. The initial time period in the above media may be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 days of differentiation before the hedgehog inhibitor is added. For example, for vestibular endolymph producing cells, numbers 1-3 above may be added for an initial time period of approximately 1-8 days, for example approximately 3 days. This may be followed by the addition of 2 and 4 for a further 1-8 days, for example approximately 5 days. This may be followed by the addition of number 5 at approximately 8 days after the addition of numbers 1-3. Incubation with number 5 may be for approximately 6-13 days, for example for 10 days. For example, for cochlear endolymph producing cells, the same protocol may be followed up to number 5. Number 6 may then be added approximately 12 days after the addition of numbers 1- 3. Incubation with number 6 may be for approximately 6-15 days, for example 12 days. Number 7 may be added approximately 18 days after the addition of numbers 1-3. Number 5 may be removed before adding number 7. Incubation with number 7 may be for approximately 3-9 days, for example 6 days. A table setting out an example of molecules and times of addition is given below. This differentiation at this second stage results in the formation of the otic placode and vesicle, i.e. the formation of inner ear progenitor cells. Other methods for producing inner ear progenitor cells may also be used. Table 1. Time added after Example of molecule obtaining pluripotent stem cells Day 0, for approximately TGF-B inhibitor SB-431542 1-8 days in duration, e.g. Fibroblast Growth bFGF 3 days Factor family TGF-B superfamily of BMP-4 growth factors Around Day 3, e.g. for BMP signalling inhibitor LDN-193189 approximately 1-8 days Fibroblast Growth bFGF in duration, e.g. 5 days Factor family Around Day 8 Wnt activator CHIR99021 approximately, e.g. for approximately 2-7 days in duration, e.g. 4 days Around Day 12 Wnt activator CHIR99021 approximately, e.g. in Organoid maturation approximately 3-9 days media in duration, e.g. 6 days Around Day 12 Hedgehog activator Purmorphamine (in addition to a __- approximately 1-15 days in Organoid maturation approximately Day 18; and then in in duration, e.g. 12 days media combination with a Wnt inhibitor for cochlear endolymph from approximately Day 18 up to producing cells approximately Day 24 see row below) Around Day 18 Wnt inhibitor lWP-2 (after removal of a Wnt approximately, e.g. in Organoid maturation activator and in combination with a approximately 3-9 days media Hedgehog activator, e.g. in duration, e.g. 6 days purmorphamine as above). for cochlear endolymph producing cells Vlth the addition of number 5 (for vestibular cells): the Wnt activator, a maturation media may be used. For example, the maturation media may be added approximately 12 days after the addition of 1-3, for example 10-15 days after the addition of 1-3. Therefore, number 5 may be present withOMM for approximately days 12-18, that is for approximately 6 days. The maturation media (for example for cochlear endolymph producing epithelia) OMM may additionally comprise a Hedgehog activator and Wnt inhibitor. Therefore number 6 may be present for approximately 12-24 days, that is for approximately 12 days. Number 7 may be present for approximately days 18-24, that is for approximately 6 days. Therefore, the second stage comprises three different sub-stages at three different time periods. Sub-stage 1 uses culture media comprising numbers 1-3 above. Sub-stage 2 uses culture media comprising numbers 4-5 above. Sub-stage 3 uses culture media comprising number 5 (vestibular and cochlear) or numbers 5, 6 and 7 (cochlear), optionally in each case including a maturation media. The maturation media supplies essential salts, amino acids, enzymes, proteins, fatty acids, and other components necessary for the growth of the developing cell types within the organoid. The second sub-stage initiates differentiation to an early otic placode progenitor identity. The third sub-stage causes further differentiation in otic vesicle and other otic progenitor cells. Aside from the maturation media used in sub-stage 3, other basal culture media used in the other sub-stages may be any used in the field, for example E8 or E6 medium. Standard additives to such media include for example Matrigel and antibacterials. After the third sub-stage the cells are inner ear progenitor cells. After exposure to number 5 in OMM (vestibular); or numbers 5-6 then 6-7 inOMM (cochlear), above, for example at least 0 days, 1, days, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, and 15 days and optionally the maturation media, the hedgehog inhibitor is added to the cells. Number 6 (for the differentiation of cochlear endolymph producing cells) is removed fromOMM prior to hedgehog inhibitors being added to the cells. Hedgehog inhibitor The term hedgehog inhibitor refers to a compound of a molecule capable of inhibiting (that is preventing or downregulating) the hedgehog signalling pathway. The hedgehog signalling pathway may be the sonic hedgehog signalling pathway and / or sonic hedgehog protein. The hedgehog inhibitor may be any compound that inhibits the hedgehog signalling pathway. The hedgehog signalling pathway is shown in Figure 3. A hedgehog pathway inhibitor may target any one or more of the proteins or genes encoding these proteins. For example, the inhibitor may be an inhibitor that inhibits gene expression or targets mRNA destruction (i.e. an siRNA). Alternatively, the inhibitor may be one that decreases signalling through the Hedgehog pathway by affecting a further pathway, e.g. increasing signalling in the ERBB pathway inhibits the hedgehog pathway therefore increasing expression in this pathway provides a way of inhibiting the hedgehog pathway. Alternatively, the expressed hedgehog protein may be targeted by binding and the hedgehog protein and inhibiting its function. Any of the above mechanisms may be used together, or multiple enzymes / genes in the pathways may be targeted. For example, the hedgehog inhibitor may inhibit hedgehog signalling by binding to Smoothened (SMO) protein. The hedgehog inhibitor may inhibit hedgehog signalling by binding to Protein patched homolog 1 (PTCH1) receptor. The hedgehog inhibitor may be at least one of: cyclopamine, vismodegib, robotnikinin and / or forskolin. The hedgehog inhibitor is added to inner ear progenitor cells (otic progenitor cells). Ideally the inhibitor is added to otic progenitor cells. This may be before transcriptional cell fate decisions are made by the cells. This can be estimated by monitoring expression levels of early otic markers, such as PAX2 and SOX2, which are indicative of progenitor status, and observing the absence of markers for more differentiated cell types likeATOH1 (for hair cells) and NEUROG1 (for neural progenitors) for instance. When the method further comprises culturing stem cells, e.g. pluripotent cells, to obtain otic progenitor cells, the hedgehog inhibitor may be administered to the culture medium after an initial time period. For example, the hedgehog inhibitor may be administered between differentiation day 10 and day 40, preferably between day 15 and day 30. Sensory cells, that is hair cells, begin to emerge at the transcriptional level as early as day 18, and are detectable via immunohistochemistry by day 30. These sensory cell types may develop in parallel with endolymph-producing cells. Therefore, to divert and optimise the differentiation of otic progenitor cells towards an endolymph-producing state, treatment may be approximately around day 15 (before the anticipated transcriptional cell fate decisions) and continue until up to day 30 (for example, when commitment to a specific cell fate is more established at the protein level). For example, for development of dark cells, the hedgehog inhibitor may be added around day 15, for example day 12-21. For the induction of cochlear endolymph producing epithelia, the hedgehog inhibitor may be added around day 21, for example day 18-27. The cells may be incubated with the hedgehog inhibitor for at least 3 days. For example, the cells may be incubated with the hedgehog inhibitor for at least 3 days, 6 days, 9 days, 12 days, 15 days, 18 days, 21 days or 24 days. When the hedgehog inhibitor is cyclopamine, the concentration that is administered may be approximately 1 uM. For example, 0.1-10 uM. Diseases of the inner ear Various disorders can affect, or potentially affect, the endolymph-producing epithelia, leading to balance disorders and hearing loss. These include, but are not limited to, genetic inner ear disorders (such as Jervell and Lange-Nielsen syndrome and DFNB109), exposure to ototoxic drugs (e.g., aminoglycosides, loop diuretics), age-related degeneration, noise-induced hearing loss, autoimmunity, systemic diseases (such as type 2 diabetes or hypothyroidism), infections (e.g., CMV), and Menière's disease. Beyond serving as a disease model, endolymph-containing organoids are instrumental in identifying and evaluating preventative or therapeutic strategies. The cells or organoids described may therefore be used in drug discovery. For example, the cells or organoids may be used to test one or more therapeutic agents for efficacy in treating or preventing any of the above diseases. Determining the effects of the therapeutic agent may be based on any one or more of the following changes: - death or survival of cells of the organoid; - transcriptional changes; - epigenetic changes; - protein changes; - metabolic changes; - genomic changes; - post-translational protein changes; and / or - phenotypic changes. Medical use The endolymph-producing epithelial cells may also be used in a method of treating any disease of the inner ear listed above. The cells used are isolated cells, i.e. not part of the human body, in vitro cells. m The disclosed invention may be provided as a kit of parts for producing inner ear organoids with endolymph-producing epithelia comprising a hedgehog inhibitor. The kit may be sold with any of the further additives in Table 1 above, culture media, e.g. organoid maturation media, and / or cells, e.g. otic progenitor cells or stem cells. The hedgehog inhibitor may also be present in the organoid maturation media, optionally in a kit, the kit further comprising: cells; and / or a Wnt activator; and / or a hedgehog activator and a Wnt inhibitor. The maturation media may comprise basal media optionally Advanced DMEM / F12 and Neurobasal Medium, essential amino acids optionally GlutaMax, neuronal cell culture supplements optionally B-27 without vitamin A and / or N2 supplement, reducing agents and / or antioxidants optionally 2-mercaptoethanol, and antibiotics, optionally Normocin. For example, for producing endolymph producing epithelial cells, the kit may comprise a hedgehog inhibitor and maturation media additives: Wnt activator, e.g. a GSK3 inhibitor. For example, for producing cochlear endolymph producing cells, the kit may comprise a hedgehog inhibitor and maturation media additives: a hedgehog activator and a Wnt inhibitor. By Wnt activator is meant a molecule which increases signalling through the Wnt pathway. By hedgehog activator is meant a molecule which increases signalling through the hedgehog pathway. By Wnt inhibitor is meant a molecule which decreases signalling through the Wnt pathway. Throughout the specification, unless the context demands otherwise, the terms comprise or include, or variations such as comprises or comprising, includes or including will be understood to imply the method or kit includes a stated integer or group of integers, but not the exclusion of any other integer or group of integers. Each document, reference, patent application or patent cited in this text is expressly incorporated herein in their entirety by reference, which means it should be read and considered by the reader as part of this text. That the document, reference, patent application or patent cited in the text is not repeated in this text is merely for reasons of conciseness. Reference to cited material or information contained in the text should not be understood as a concession that the material or information was part of the common general knowledge orwas known in any country. Description of the Figures Figure 1 shows single-nucleus RNA transcriptomic analysis of the inner ear highlights the critical role of the Hedgehog signalling pathway in the development of vestibular dark cells and developing cochlear lateral roof cells, which give rise to the marginal cells of the stria vascularis. A. Single-nucleus RNA sequencing was conducted using the 10X Genomics platform on human fetal inner ear samples from fetal weeks 7 to 15. B. UMAP-based dimensional reduction of the Human Inner Ear Development RNAseq Atlas (HIEDRA) comprising 55,940 nuclei across 9 time points, revealing 42 distinct, color-coded clusters, with vestibular dark cells and lateral roof cells highlighted. C. Top 20 enriched marker genes for vestibular dark cells and cochlear lateral roof cells. D. Gene set enrichment analysis (GSEA) of the marker genes for vestibular dark cells and cochlear lateral roof cells, using the Environmental Information Processing dataset from the KEGG Pathway Database. E. Expression profiles of Hedgehog signalling pathway-related genes within vestibular dark cells and cochlear lateral roof cells, grouped as endolymph- producing epithelia. Figure 2 shows A. a schematic of the standard inner ear organoid differentiation protocol leading to human vestibular inner ear organoids on the top. The bottom shows the invention protocol for inducing dark cells in inner ear organoids. B. lmmunostaining of differentiation day 60 vesicles within the inner ear organoid with SOX2- positive sensory epithelium and DACH1- negative / OTX2-negative epithelium. C. lmmunostaining of a day 60 inner ear organoid which has been treated with cyclopamine between day 15 and day 30 in which in addition to SOX2- positive sensory epithelium, also DACH1-positive (SOX2- / OTX2-negative) dark cell epithelium is developing. D. Close-up of the area of otic dark cell epithelium (also SOX10-positive and CDH1-positive) reveals integration of MLANA-positive melanocytes as well as basal membrane disruption and ion channel expression. E. Validation of DACH1, SOX2 and OTX2 expression in the human fetal vestibular system (left) and cochlear (right). Figure 3 Overview of Hedgehog Signalling Pathway States and Inhibition Mechanisms. The figure illustrates three different states of Hedgehog signalling: inactive, active, and inhibited. Left Panel: Inactive Hedgehog Signalling - In the absence of Hedgehog ligands (e.g., SHH), the Patched (PTCH) receptor inhibits the Smoothened (SMO) protein, preventing downstream signalling. SUFU (Suppressor of Fused) retains GLI transcription factors in a repressive state (GLIR), leading to the degradation of GLI proteins and suppression of target gene expression. Middle Panel: Active Hedgehog Signalling - When Hedgehog ligands such as Sonic Hedgehog (SHH) bind to PTCH, the inhibition ofSMO is relieved, allowing SUFU to release GLI. Active GLI (GLIA) translocates to the nucleus and promotes the transcription of Hedgehog target genes. Right Panel: Hedgehog Pathway Inhibition - Examples of inhibitors that target various components of the pathway are shown. Robotnikinin inhibits SHH binding to PTCH, while cycIopamine and vismodegib inhibitSMO activity, preventing pathway activation. Forskolin inhibits GLI activation, thereby reducing target gene expression despite active upstream signalling. Examples Aspects of the present invention will now be illustrated by way of example only and with reference to the following experimentation. Example 1: Single-nucleus RNA sequencing analysis of the inner ear identifies signalling pathways crucial to the development of the inner ear Methods: Human Inner Ear Tissue Collection and Processing Human fetal inner ear tissue was collected in accordance with the Dutch legislation (Fetal Tissue Act, 2001) and theWMA Declaration of Helsinki guidelines. Ethical approval was obtained from the Medical Research Ethics Committee of Leiden University Medical Center (protocol registration number B19.070), and written informed consentwas obtained from the donor, adhering to the Guidelines on the Provision of Fetal Tissue by the Dutch Ministry of Health, Welfare, and Sport (revised 2018). Inner ear tissue was collected after elective termination of pregnancy through vacuum aspiration, as previously described (van Beelen et al., 2022). Embryonic or fetal age was determined via obstetric ultrasonography prior to termination, calculated as gestational age minus two weeks, with a standard error of two days. The tissue was preserved in RNAIater (AM7020, lnvitrogen) for RNAseq experiments. Human Inner Ear Tissue Dissociation As for the fetal inner ears, residual tissue was removed using a dissection microscope (M205C, Leica). Early fetal inner ears were processed with the otic capsule intact, while the otic capsule was manually removed for the FW9.2 fetal inner ear and later tissues. This dissection strategy resulted in isolating the membranous labyrinth of the cochlea and vestibular system, which was then processed for nuclei isolation. Dissociation into single nuclei followed a similar protocol as previously described (van der Valk et al., 2023), including a lysis buffer containing 0.005% Nonidet P40 substitute. Additionally, the suspension was filtered through a series of Flowmi cell strainers (40 um) and further purified using a 20 um Pluristrainer (43-10020-40, PluriSelect) to remove debris. RNA Sequencing Analysis We extended our previous dataset to generate a comprehensive RNAseq atlas of the human fetal inner ear covering developmental stages atweeks 7.1, 7.5, 8.4, 9.2, 10.0, 11.0, 11.5, 12.1, and 15.1. Sequencing libraries were prepared using the 10x Genomics Chromium platform, with protocols adapted for high-quality RNA extraction from preserved tissue. Sequencing was performed on a NovaSeq 6000 S4 flow cell (lllumina), and raw sequencing reads were processed using the Cell Ranger 6.0.1 pipeline (10x Genomics) for demultiplexing, alignment to the GRCh38 reference genome, and generation of gene expression matrices. Data Analysis and Integration Data analysis was carried out using the UniApp platform from Unicle, allowing for analysis and visualization of gene expression profiles across developmental time points. Standard quality control measures, such as filtering based on UMI counts, gene detection, and mitochondrial transcript content, were applied to ensure high-quality data. The datasetwas normalized and principal component analysis (PCA) and Uniform Manifold Approximation and Projection (UMAP) were used for dimensionality reduction and visualization of cell clusters. Differential expression analysis and marker gene identification were employed for cell type annotation across different developmental stages. RESULTS: The results are shown in Figure 1. The Human Inner Ear Development RNAseg Atlas (HIEDRA) Reveals Repression of Hedgehog Signalling in Developing Endolymph-Producing Epithelia We created a detailed single-nucleus RNAseq atlas of human fetal inner ear development, covering developmental stages from fetal weeks 7.1, 7.5, 8.4, 9.2, 10.0, 11.0, 11.5, 12.1, and 15.1 (Figure 1A). This analysis identified 42 distinct clusters, encompassing 55,940 nuclei across these nine time points, including key cell populations such as vestibular dark cells and cochlear lateral roof cells, which will differentiate into the marginal cells of the stria vascularis (Figure 1B). By combining these cell types into a group referred to as the endolymph-producing epithelia, we identified several genes enriched within this population (Figure 1C), including LRP2, LMX1A, and MEIS1, all ofwhich have known interactions with Hedgehog signalling. LRP2 functions as a clearance receptor for SHH, a Hedgehog pathway ligand, in the retina and ventral neural tube. LMX1A has been shown to repress SHH expression during midbrain development and plays opposing roles to Hedgehog signalling in the development of midbrain dopaminergic neurons. Additionally, MEIS1 is known as a promoter of PTCH1, a key component in the Hedgehog pathway. These gene expression patterns suggested a possible link between the Hedgehog pathway and the development of vestibular dark cells and cochlear lateral roof cells. In our gene set enrichment analysis (GSEA) using the Environmental Information Processing dataset from the KEGG Pathway Database, we found that the Hedgehog signalling pathway is enriched in the endolymph-producing epithelia (Figure 1D). Furthermore, ERRB signalling was also identified as enriched. lnterestingly, previous research has shown a link between ERRB signalling and Hedgehog pathway inhibition, where increased levels of ERRB pathway-related proteins can lead to the suppression of Hedgehog activity. It is important to note that the enrichment of a signalling pathway in a GSEA does not necessarily indicate active signalling in the cells or tissues, as GSEA reflects the coordinated expression of pathway-related genes rather than their direct functional activation. Finally, our analysis of Hedgehog pathway-associated gene expression in the human developing endolymph-producing epithelia revealed expression of SUFU and PTCH1, where PTCH1 is known to inhibit SMO, and SUFU acts as a negative regulator of the Hedgehog pathway (Figure 1E). Additionally, we observed expression of GLI2 and GL / 3, but not the transcriptional activator GLI1. GLI2 and GLI3 are primarily known as repressors of the Hedgehog pathway, although they can act as activators in certain contexts. Together, these findings suggest that while components of the Hedgehog pathway are present, the signalling is repressed in the developing endolymph-producing epithelia (dark cells and marginal cells). Example 2: Inner Ear Organoid Generation Containing Endolymph-Producing Epithelium Following the results of Example 1, we created an inner ear organoid containing endolymph- producing epithelium by inhibition of the hedgehog signalling pathway. The induction of inner ear organoids followed the protocol explained below. Human pluripotent stem cell (hPSC) colonies were enzymatically dissociated using Accutase, yielding a single-cell suspension in E8 medium (Gibco, A1517001) ormTESR Plus (Stemcell Technologies, 100-0276) supplemented with 20mM Y27632 (Reprocell, 04-0012-02) orCEPT Cocktail (Tocris, 7991) to inhibit apoptosis and supplemented with Normocin lnvivogen, ant-nr- 1). Cell concentration was determined using an automated cell counter (Countess Il Automated Cell Counter, Life Technologies), and cell viability was assessed using a 1:1 ratio of Trypan Blue (Gibco, 15250061) to cell suspension. A total of 3,500 cells when usingY or 2,500 cells when using CEPT Cocktail in 100 uL of suspension were seeded into each well of a 96-well U- bottom plate (ThermoFisher Scientific, 174925) with a super-low cell attachment surface. Centrifugation at 110><g for 6 minutes was performed to facilitate cell aggregation, followed by incubation at 37°C under5% CO2 for 48 hours to allow aggregate formation. During this incubation period, 100 uL of fresh E8 medium ormTESR Plus medium was added after 24 hours to dilute Y27632 if this was used. On differentiation day 0 (D0), cell aggregates were transferred individually to a new 96-well U- bottom plate with 100 uL of Essential 6 (E6) medium (Gibco, A1516401) supplemented with 100 ug / mL Normocin, 2% Matrigel Growth Factor Reduced (Corning, 354230), 10 uM SB431542 (Stemgent, 04-0010-05), 4 ng / mL basic FGF (PeproTech, 100-18B), and optimised concentrations of BMP-4 (PeproTech, 120-05; R&D Systems, 314-BPE). The aggregates were incubated for 3 days (until D3), after which 25 uL of E6 medium containing 1 uM LDN-193189 (Stemgent, 04-0074-02) and 250 ng / mL basic FGF was added per well, resulting in final concentrations of 200 nM LDN and 50 ng / mL basic FGF in a total volume of 125 uL per well. On D6, 75 uL of fresh E6 medium was added, increasing the total volume per well to 200 uL. On D8, 100 uL of the medium was replaced with fresh E6 medium containing 6 uM CHIR99021 (Stemgent, 04-0004-10) to induce otic placode formation, resulting in a final concentration of 3 uM CHIR per well. On D10, 100 uL of the medium was replaced with fresh E6 medium containing 3 uM CHIR99021. On D12, the cell aggregates were transferred to a 24-well plate with a super-low cell attachment surface (ThermoFisher Scientific, 174930) containing 500 uL of Organoid Maturation Medium (OMM), 3 uM CHIR99021, and 1% Matrigel. OMM consisted of a 1:1 mixture ofAdvanced DMEM / F12 (Gibco, 12634010) and Neurobasal Medium (Gibco, 21103049), supplemented with 1x GlutaMax (Gibco, 35050061), 0.5x B-27 Without Vitamin A (Gibco, 12587010), 0.5x N2 Supplement (Gibco, 17502048), 0.1 mM 2-mercaptoethanol (Gibco, 21985023), 100 ug / mL Normocin. The plates were placed on an orbital shaker at 65 rpm inside a 37°C incubator with 5% CO2 to maintain the suspension of the aggregates. A novel component of this protocol involves the incorporation of Cyclopamine (MedChem Express, HY-17024) into the culture medium, used here to facilitate vestibular dark cell formation. On D15, Cyclopamine was added at a final concentration of 1 uM through a full medium change withOMM containing 1% Matrigel and 3 uM CHIR. To maintain the effects of Cyclopamine, full medium changes with 500 uLOMM containing 1 uM Cyclopamine were performed on D18 , D21, D24, and D27. On D30, a full medium change was conducted using OMM without Cyclopamine, after which the culture was continued by medium changes three times a week medium with OMM. As the organoids grew and increased in size, the medium volume was gradually increased, starting from 500 uL on D12 to 1 mL per well by approximately D45 and reaching up to 1.5 mL per well by D60 and beyond. If required, the frequency of medium changes was adjusted to every other day. To facilitate cochlear marginal cell formation, cochlear identitywas achieved by treatment on D12 and D15 with 1 uM Purmorphamine (Reprocell, 04-0009) and 3 uM CHIR. On D18 aggregates were washed and treated with 1 uM Purmorphamine and 3 uM lWP-2 (Stemcell Technologies, 72122). At D21 cells were treated with 1 uM Cyclopamine and 3 uM lWP-2. lWP- 2 was removed at D24, and OMM containing only 1 uM Cyclopamine was added on D24, D27, D30, D33, D36, D39. On D42, a full medium change was conducted usingOMM without Cyclopamine, after which the culture was continued as previously described. Organoid Processing and Immunohistochemistry Organoids were washed twice with PBS and fixed overnight at 4°C using 4% formaldehyde in 0.1 M Na+ / K+-phosphate buffer (pH 7.4). For paraffin embedding, organoids were dehydrated through an ascending ethanol series (70%99%; 84050068.2500, Boom), cleared in xylene (534056, Honeywell), and embedded in paraffin wax (2079, Klinipath). Sections of 45 pm thickness were cut using a rotary microtome (HM355S, Thermo Scientific). The sections were deparaffinized in xylene, rehydrated through a descending ethanol series (96%50%), and rinsed several times in deionized water. Approximately every 10th section was selected for routine hematoxylin (40859001, Klinipath) and eosin (40829002, Klinipath) staining. For immunostaining, antigen retrieval was performed by heating the sections either in 10mM sodium citrate buffer (pH 6.0; S1804500G, Sigma-Aldrich) for 12 minutes at 97°C or, alternatively, in 1 mM EDTA buffer (pH 8.0) for 20 minutes at 97°C. Specifically for laminin immunostaining, digestion was performed using 20 ug / ml proteinase K in PBS for 5 minutes at room temperature. Sections were rinsed in a washing buffer containing 0.05% Tween-20 (H5152, Promega), then incubated for 30 minutes in a blocking solution of5% bovine serum albumin (BSA; A7030, Sigma-Aldrich) and 0.05% Tween-20 in PBS. This was followed by an overnight incubation at 4°C with the primary antibodies listed in the table below. The next day, sections were incubated at room temperature (RT) with Alexa Fluor-conjugated secondary antibodies, as specified in the table below for 2 hours. Nuclei were stained using 4',6-diamidino- 2-phenylindole (DAPI; 1:1,000; D1306, lnvitrogen), and sections were mounted with ProLong Gold Antifade Mountant (P36934, lnvitrogen). RGET COMPANY CATALOGUE DILUTION ---- ___ nkey anti-mouse lnvitrogen A21202 1:1000-2000 ---- nkey anti-mouse lnvitrogen A21203 1:1000-2000 ---- nkey anti-mouse lnvitrogen A10038 1:1000-2000 ---- nkey anti-rabbit Invitrogen A21206 1:1000-2000 ___- nkey anti-rabbit Invitrogen A21207 1:1000-2000 ___- nkey anti-rabbit Invitrogen A10043 1:1000-2000 ___- nkey anti-goat Invitrogen A11055 1:1000-2000 ___- nkey anti-goat Invitrogen A21084 1:1000-2000 ___- Results: The results are shown in Figure 2. We modified the standard inner ear organoid differentiation protocol to induce dark cell epithelium by adding 1 uM cyclopamine (final concentration) on days 15, 18, 21, 24, and 27 of differentiation (Figure 2A). In the standard protocol, SOX2-positive sensory epithelium develops, but no DACH1-positive, OTX2-negative dark cell epithelium forms (Figure 2B). However, following cyclopamine treatment, we observed the development of DACH1-positive, OTX2- negative dark cell epithelium alongside SOX2-positive sensory epithelium (Figure 2C). Further characterization of the dark cell epithelium in cycIopamine-treated organoids revealed SOX10- positive, CDH1-positive otic identity, along with MLANA-positive melanocytes integrating into the epithelium, consistent with what has been described during human vestibular dark cell epithelium development (van Beelen et al., 2022) (Figure 2D). Additionally, the expression of ion channels such as SLC26A4, ATP1 B2, and BSND confirms early commitment to dark cell epithelium (Figure 2D). The specificity of DACH1 as a marker for endolymph-producing epithelium has been validated in human inner ear tissue (Figure 2E).

Claims

1. Method for generating endolymph-producing epithelial cells, whereby the method includes: a. obtaining inner ear progenitor cells; b. the addition of a hedgehog trajectory inhibitor, in order to a differentiation causing one or more inner ear progenitor cells to produce endolymph to form producing epithelial cells.

2. Method according to conclusion 1, whereby the method additionally involves the cultivation of stem cells includes, in order to obtain inner ear progenitor cells.

3. Method in accordance with conclusions 1 to 2, whereby the endolymph-producing epithelial cells are vestibular endolymph-producing epithelial cells, optional dark cell epithelial cells; and / or where the endolymph-producing epithelial cells cochlear endolymph-producing epithelial cells are, optionally, marginal cells of the stria vascularis.

4. Method in accordance with one of the preceding claims, whereby the hedgehog inhibitor a hedgehog signaling inhibits by inhibiting a SMO expression or function, optional, where the SMO inhibitor is cyclopamine.

5. Method in accordance with one of the preceding claims, whereby the hedgehog inhibitor a hedgehog signaling inhibits by: a. inhibiting an SHH expression or function, optional where the SHH inhibitor is robotnikinine; and / or b. inhibiting a Gli expression or function, optionally where the Gli inhibitor forskolin is; and / or c. inhibiting a PTCH receptor expression or function.

6. Method in accordance with one of the preceding claims, whereby the method furthermore comprises: c) the generation of an inner ear organoid that the endolymph-producing includes epithelial cells.

7. Method in accordance with one of the preceding conclusions, whereby the inner ear progenitor cells be incubated with the hedgehog inhibitor for at least three days.

8. Inner ear organoid comprising endolymph-producing epithelial cells, optional in which the endolymph-producing epithelial cells were obtained by means of a method in accordance with one of claims 1 through 7.

9. In vitro use of an inner ear organoid pursuant to claim 8, at the discovery and the development of medicines.

10. Isolated endolymph-producing epithelial cells, optionally obtained using a method in accordance with one of claims 1 to 7.

11. Endolymph-producing epithelial cells according to claim 10, prior use in a method for treating a disease of the inner ear, whereby the method the includes transplanting the endolymphatic epithelial cells to the ear of a subject.

12. Method for testing one or more therapeutic agents, whereby the method includes: a. the provision of endolymph-producing epithelial cells in accordance with claim 10, or of an inner ear organoid according to claim 8; b. bringing the cells or the organelle into contact with at least a therapeutic agent; c. detecting one or more changes in the cells or in the organelle; d. determining the effects of the therapeutic agent, on the basis of the absence or presence of one or more changes.

13. Kit, comprising: a. a hedgehog inhibitor; and any or multiple of the following: b. i) cell culture medium; and / or ii) hearing progenitor cells; and / or ii) a Wnt activator; and / or iv) a hedgehog activator and a Wnt activator.

14. Organoid maturation medium, comprising a hedgehog inhibitor.

15. In vitro use of a hedgehog signaling inhibitor, for differentiating 5 inner ear progenitor cells into endolymph-producing epithelial cells.

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