Patent Description:
This invention was made with government support under DC015624, DC012617, and DC013294 awarded by the National Institutes of Health. The government has certain rights in the invention.

Provided herein are methods for directing differentiation of human pluripotent stem cells into inner ear sensory epithelia and sensory neurons. More particularly, provided herein are methods for obtaining three-dimensional cultures comprising human pluripotent stem cell-derived pre-otic epithelium, otic vesicles, and inner ear sensory epithelia containing hair cells and supporting cells as well as sensory neurons innervating the sensory epithelia.

Nearly half a billion people have hearing loss world-wide, yet there are no pharmacological, genetic, or cell therapies that can treat hearing loss. This method could be used to generate human inner ear stem cells, supporting cells, hair cells and neurons for cell therapies or for use in drug discovery.

Accordingly, there remains a need in the art, for efficient, reproducible, and xenogeneic material-free methods for differentiating human pluripotent stem cells into inner ear sensory tissue suitable for clinical cell therapies. <CIT> describes a method of generating inner ear cells from pluripotent stem cells in vitro characterized by three steps, first the formation of pre-placodal ectodermal cells, then a population comprising otic progenitor cells and finally a population comprising inner ear cells. Ronaghi (<NUM>) describes the generation of inner ear hair cells from human embryonic stem cells (hESCs) using a <NUM>-day sequential protocol.

Provided herein is a method as defined in claim <NUM>.

These and other features, aspects, and advantages described herein will become better understood upon consideration of the following drawings, detailed description, and appended claims.

The present invention is based at least in part on the Inventors' discovery that human pluripotent stem cell-derived precursor cells cultured under conditions that are permissive towards differentiation and remodeling form highly uniform compositions of inner ear tissue that recapitulate the complexity and organization of human inner ear sensory epithelia and include functional hair cells. The Inventors discovered that it was possible to produce complex human tissues having the uniformity necessary for large-scale, quantitative in vitro modeling and screening applications.

Accordingly, the present invention relates to compositions including three-dimensional tissue constructs and cultures and methods of using such compositions as highly uniform models of human inner ear tissue and for screening drug candidates. In particular, provided herein are methods of efficiently and reproducibly producing and expanding complex, organized human inner ear sensory tissue suitable as a source of human hair cells for transplantation, as a model for understanding sensory deficits, and as a platform for screening drug candidates. An important advantage of the methods and systems provided herein is the ability to generate complex tissue constructs comprising multiple functional cell types from a single cell source. In addition, the methods and systems provided herein faithfully recapitulate in vivo development of complex, organized inner ear structural layers. The present invention provides a scalable and robust system for generating human inner ear sensory tissue as well as an important opportunity to study such tissues in an in vitro human model. In addition, methods of the present invention are useful for identifying materials and combinatorial strategies for human tissue engineering.

In exemplary embodiments, the methods provided herein comprise differentiating human pluripotent stem cells under conditions that promote differentiation of the pluripotent stem cells into inner ear sensory tissue. Generally, cells of inner ear sensory tissue are identified by their surface phenotype, by the ability to respond to growth factors, and being able to differentiate in vivo or in vitro into particular cell lineages.

In a first aspect, a method of obtaining human inner ear sensory tissue comprises aggregating human pluripotent stem cells into spheroids and culturing the spheroids for about three to four days in the presence of in a culture medium comprising factors that promote induction of non-neural epithelium (NNE). Such a culture medium comprises or consists essentially of the following chemically defined components: bone morphogenetic protein-<NUM> (BMP4) and an inhibitor of transforming growth factor beta (TGFβ) signaling such as, for example, SB-<NUM> ("SB"), whereby at least a subset of the pluripotent stem cells are induced to differentiate to form a core of mesodermal cells within each aggregate. Preferably, aggregates comprising a core of mesodermal cells are cultured in the presence of BMP4 and an inhibitor of TGFβ signaling (e.g., SB) for about <NUM> days to about <NUM> days. SB-<NUM><NUM> is a specific inhibitor of the activin receptor-like kinase receptors ALK5, ALK4, and ALK7. Following the <NUM> to <NUM> day culture, cells of the mesodermal core migrate to the surface of the aggregates and produce a layer of non-neural epithelium within which inner ear organoids will develop. The non-neural epithelium that produces inner ear organoids, now lining the core of the aggregate, can eventually differentiate into epidermal tissue.

Referring to <FIG>, NNE cells formed according to the culture step outlined above are cultured in the presence of a combination of Fibroblast Growth Factor (FGF) (e.g., FGF-<NUM>) and an inhibitor of bone morphogenetic protein (BMP) signaling, whereby the NNE cells differentiate into a pre-otic epithelium, also known as a otic-epibranchial progenitor domain (OEPD), from which the otic placode is derived. The OEPD is thickened relative to NNE cells cultured in the presence of a TGFB-signaling inhibitor alone and expresses a combination of posterior placode markers, such as PAX8, SOX2, TFAP2, ECAD, and NCAD. Inhibitors of BMP signaling appropriate for use according to the methods provided herein include, without limitation, LDN-<NUM> and SB-<NUM>. LDN-<NUM> is a selective BMP signaling inhibitor that inhibits the transcriptional activity of the BMP type I receptors ALK2 and ALK3.

Next, aggregates comprising pre-otic epithelium (i.e., OEPD) are embedded in a semi-solid culture medium such as, for example, a semi-solid composition of extracellular matrix proteins. The embedded aggregates are then cultured in the presence of a Wnt agonist until pre-otic epithelium self-assembles into organized otic vesicles. In some cases, aggregates comprising pre-otic epithelium are cultured in the presence of a Wnt agonist for about <NUM> days to about <NUM> days (e.g., about <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> days).

Otic vesicle-laden aggregates are further cultured for at least about <NUM> days (e.g., about <NUM> days, about <NUM> days, about <NUM> days, about <NUM> days, about <NUM> days, about <NUM> days, about <NUM> days, or more), during which inner ear organoids comprising mechanosensory cells (e.g., hair cells) are obtained. Hair cells are specialized mechanosensory receptor cells of the vertebrate inner ear and lateral line organs that mediate hearing and balance.

To form aggregates, a confluent culture of pluripotent stem cells can be chemically, enzymatically or mechanically dissociated from a surface, such as Matrigel® into clumps, aggregates, or single cells. In exemplary embodiments, the dissociated cells (as clumps, aggregates, or single cells) are plated onto a surface in a protein-free basal medium such as Dulbecco's Modified Eagle's Medium (DMEM)/F12, mTeSR™ (StemCell Technologies; Vancouver, British Columbia, Canada), and TeSR™. The full constituents and methods of use of TeSR™ are described in Ludwig et al. See, e.g., <NPL>); and <NPL>). Other DMEM formulations suitable for use herein include, e.g., X-Vivo (BioWhittaker, Walkersville, MD) and StemPro® (Invitrogen; Carlsbad, CA).

In some cases, aggregates of pluripotent stem cells are cultured in the presence of a Rho kinase (ROCK) inhibitor. Kinase inhibitors, such as ROCK inhibitors, are known to protect single cells and small aggregates of cells. See, e.g., <CIT>; and <NPL>). ROCK inhibitors are shown below to significantly increase pluripotent cell survival on chemically defined surfaces. ROCK inhibitors suitable for use herein include, but are not limited to, (S)-(+)-<NUM>-methyl-<NUM>-[(<NUM>-methyl-<NUM>-isoquinolinyl)sulfonyl]homopiperazine dihydrochloride (informal name: H-<NUM>), <NUM>-(<NUM>-isoquinolinesulfonyl)piperazine hydrochloride (informal name: HA-<NUM>), <NUM>-(<NUM>-isoquinolinesulfonyl)-<NUM>-methylpiperazine (informal name: H-<NUM>), <NUM>-(<NUM>-isoquinolinesulfonyl)-<NUM>-methylpiperazine (informal name: iso H-<NUM>), N-<NUM>-(methylamino) ethyl-<NUM>-isoquinoline-sulfonamide dihydrochloride (informal name: H-<NUM>), N-(<NUM>-aminoethyl)-<NUM>-isoquinolinesulphonamide dihydrochloride (informal name: H-<NUM>), N-[<NUM>-p-bromo-cinnamylamino)ethyl]-<NUM>-isoquinolinesulfonamide dihydrochloride (informal name: H-<NUM>), N-(<NUM>-guanidinoethyl)-<NUM>-isoquinolinesulfonamide hydrochloride (informal name: HA-<NUM>), <NUM>-(<NUM>-isoquinolinesulfonyl) homopiperazine dihydrochloride (informal name: HA-<NUM>), (S)-(+)-<NUM>-Methyl-<NUM>-glycyl-<NUM>-(<NUM>-methylisoquinolinyl-<NUM>-sulfonyl)homopiperazine dihydrochloride (informal name: glycyl H-<NUM>) and (+)-(R)-trans-<NUM>-(<NUM>-aminoethyl)-N-(<NUM>-pyridyl)cyclohexanecarboxamide dihydrochloride (informal name: Y-<NUM>). The kinase inhibitor can be provided at a concentration sufficiently high that the cells survive and remain attached to the surface. An inhibitor concentration between about <NUM> to about <NUM> can be suitable. At lower concentrations, or when no ROCK inhibitor is provided, undifferentiated cells typically detach, while differentiated cells remain attached to the defined surface.

FGF-<NUM> is an agonist of FGF signaling, and FGF signaling can be antagonized using, for example, the small molecule inhibitor PD-<NUM>. BMP4 is an agonist of BMP signaling, and BMP signaling can be antagonized using, for example, the small molecule inhibitor LDN-<NUM>. TGFβ-<NUM> and Activin A are agonists of TGFβ signaling, and TGFβ signaling can be antagonized using, for example, the small molecule inhibitor SB-<NUM>. CHIR-<NUM> is an agonist of the Wnt/β-catenin signaling pathway, and Wnt/β-catenin signaling can be antagonized using, for example, the small molecule inhibitor XAV-<NUM>. Other Wnt agonists include inhibitors/antagonists of the molecule Glycogen Synthase Kinase <NUM> (GSK3).

In some cases, the semi-solid composition of extracellular matrix proteins is a commercially available product such as Geltrex® basement membrane matrix. Geltrex® basement membrane matrix is suitable for use with human pluripotent stem cell applications using StemPro® hESC SFM or Essential <NUM>™ media systems. In other cases, the semi-solid composition comprises two or more extra cellular matrix proteins such as, for example, laminin, entactin, vitronectin, fibronectin, a collagen, or combinations thereof.

Preferably, human pluripotent stem cells are cultured in a chemically-defined basal culture medium formulation comprising the defined components of culture medium "DF3S" as set forth in <NPL>). As used herein, the terms "E7 culture medium" and "E7" are used interchangeably and refer to a chemically defined culture medium comprising or consisting essentially of DF3S supplemented to further comprise insulin (<NUM>µg/mL), transferrin (<NUM> ng/mL) and human Fibroblast Growth Factor <NUM> (FGF2) (<NUM> ng/mL). As used herein, the terms "E8 culture medium" and "E8" are used interchangeably and refer to a chemically defined culture medium comprising or consisting essentially of DF3S supplemented by the addition of insulin (<NUM>µg/mL), transferrin (<NUM> ng/mL), human FGF2 (<NUM> ng/mL), and human TGFβ1 (Transforming Growth Factor Beta <NUM>) (<NUM> ng/mL).

Any appropriate method can be used to detect expression of biological markers characteristic of cell types described herein. For example, the presence or absence of one or more biological markers can be detected using, for example, RNA sequencing, immunohistochemistry, polymerase chain reaction, qRT-PCR, or other technique that detects or measures gene expression. In exemplary embodiments, a cell population obtained according to a method provided herein is evaluated for expression (or the absence thereof) of biological markers of pre-otic epithelial cells and otic placode such as Foxi1, Dlx genes, Pax8, Pax2, Sox3, Eya1, Gata3, Gbx2, and Sox9. Quantitative methods for evaluating expression of markers at the protein level in cell populations are also known in the art. For example, flow cytometry is used to determine the fraction of cells in a given cell population that express or do not express biological markers of interest. Differentiated cell identity is also associated with downregulation of pluripotency markers such as NANOG and OCT4 (relative to human ES cells or induced pluripotent stem cells).

As used herein, "pluripotent stem cells" appropriate for use according to a method of the invention are cells having the capacity to differentiate into cells of all three germ layers. Suitable pluripotent cells for use herein include human induced pluripotent stem (iPS) cells. Disclosure of human embryonic stem cells (hESCs) is for reference only. As used herein, "embryonic stem cells" or "ESCs" mean a pluripotent cell or population of pluripotent cells derived from an inner cell mass of a blastocyst. See <NPL>). These cells express Oct-<NUM>, SSEA-<NUM>, SSEA-<NUM>, TRA-<NUM>-<NUM> andTRA-<NUM>-<NUM>, and appear as compact colonies having a high nucleus to cytoplasm ratio and prominent nucleolus. ESCs are commercially available from sources such as WiCell Research Institute (Madison, Wis. As used herein, "induced pluripotent stem cells" or "iPS cells" mean a pluripotent cell or population of pluripotent cells that may vary with respect to their differentiated somatic cell of origin, that may vary with respect to a specific set of potency-determining factors and that may vary with respect to culture conditions used to isolate them, but nonetheless are substantially genetically identical to their respective differentiated somatic cell of origin and display characteristics similar to higher potency cells, such as ESCs, as described herein. See, e.g., <NPL>).

Induced pluripotent stem cells exhibit morphological properties (e.g., round shape, large nucleoli and scant cytoplasm) and growth properties (e.g., doubling time of about seventeen to eighteen hours) akin to ESCs. In addition, iPS cells express pluripotent cell-specific markers (e.g., Oct-<NUM>, SSEA-<NUM>, SSEA-<NUM>, Tra-<NUM>-<NUM> or Tra-<NUM>-<NUM>, but not SSEA-<NUM>). Induced pluripotent stem cells, however, are not immediately derived from embryos. As used herein, "not immediately derived from embryos" means that the starting cell type for producing iPS cells is a non-pluripotent cell, such as a multipotent cell or terminally differentiated cell, such as somatic cells obtained from a post-natal individual.

Human iPS cells can be used according to a method described herein to obtain primitive macrophages and microglial cells having the genetic complement of a particular human subject. For example, it may be advantageous to obtain inner ear sensory cells that exhibit one or more specific phenotypes associated with or resulting from a particular disease or disorder of the particular mammalian subject. In such cases, iPS cells are obtained by reprogramming a somatic cell of a particular human subject according to methods known in the art. See, for example, <NPL>); <NPL>); <NPL>); <NPL>). Induced pluripotent stem cell-derived inner ear sensory tissues can be used to screen drug candidates in tissue constructs that recapitulate inner ear sensory tissue in an individual having, for example, a particular disease. Subject-specific somatic cells for reprogramming into induced pluripotent stem cells can be obtained or isolated from a target tissue of interest by biopsy or other tissue sampling methods. In some cases, subject-specific cells are manipulated in vitro prior to use in a three-dimensional tissue construct of the invention. For example, subject-specific cells can be expanded, differentiated, genetically modified, contacted to polypeptides, nucleic acids, or other factors, cryo-preserved, or otherwise modified prior to introduction to a three-dimensional tissue construct.

Preferably, human pluripotent stem cells (e.g., human ESCs or iPS cells) are cultured in the absence of a feeder layer (e.g., a fibroblast layer), a conditioned medium, or a culture medium comprising poorly defined or undefined components. As used herein, the terms "chemically defined medium" and "chemically defined cultured medium" also refer to a culture medium containing formulations of fully disclosed or identifiable ingredients, the precise quantities of which are known or identifiable and can be controlled individually. As such, a culture medium is not chemically defined if (<NUM>) the chemical and structural identity of all medium ingredients is not known, (<NUM>) the medium contains unknown quantities of any ingredients, or (<NUM>) both. Standardizing culture conditions by using a chemically defined culture medium minimizes the potential for lot-to-lot or batch-to-batch variations in materials to which the cells are exposed during cell culture. Accordingly, the effects of various differentiation factors are more predictable when added to cells and tissues cultured under chemically defined conditions. As used herein, the term "serum-free" refers to cell culture materials that are free of serum obtained from animal (e.g., fetal bovine) blood. In general, culturing cells or tissues in the absence of animal-derived materials (i.e., under xenogen-free conditions) reduces or eliminates the potential for cross-species viral or prion transmission.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs.

As used herein, "a medium consisting essentially of' means a medium that contains the specified ingredients and those that do not materially affect its basic characteristics.

As used herein, "effective amount" means an amount of an agent sufficient to evoke a specified cellular effect according to the present invention.

As used herein, "about" means within <NUM>% of a stated concentration range, density, temperature, or time frame.

The invention will be more fully understood upon consideration of the following non-limiting Examples. It is specifically contemplated that the methods disclosed are suited for pluripotent stem cells generally.

The human inner ear contains approximately <NUM>,<NUM> sensory hair cells that detect sound and movement via mechanosensitive stereocilia bundles<NUM>. Genetic mutations or environmental insults, such as loud noises, can cause irreparable damage to these hair cells, leading to dizziness or hearing loss<NUM>,<NUM>. We previously demonstrated how to generate inner ear organoids from mouse pluripotent stem cells (PSCs) using timed manipulation of the FGF, TGFβ, BMP, and Wnt signaling pathways in a 3D culture system<NUM>-<NUM>. We have shown that mouse inner ear organoids contain sensory hair cells with similar structure and function to native vestibular hair cells in the mouse inner ear<NUM>. Moreover, our past findings bolstered a working model of otic induction signaling dynamics, wherein BMP signaling activation and TGFβ inhibition initially specifies non-neural ectoderm and subsequent BMP inhibition and FGF activation induces a pre-otic fate<NUM>,<NUM>. Despite several recent attempts, a developmentally faithful approach for deriving functional hair cells from human PSCs (hPSC) has yet to be described<NUM>-<NUM>. Here, to generate human inner ear tissue from hPSCs, we first established a timeline of in vitro human inner ear organogenesis (<FIG>). The inner ear arises from the ectoderm layer and, in humans, produces the first terminally differentiated hair cells by ~<NUM> days post conception (dpc)<NUM>. Beginning with pluripotent cells in the epiblast, inner ear induction is initiated ~<NUM> dpc with formation of the ectoderm epithelium. Then, the epithelium splits into the non-neural ectoderm (also known as surface ectoderm) and the neuroectoderm (<FIG>). The non-neural ectoderm ultimately produces the inner ear as well as the epidermis of the skin; thus, in our initial experiments, we sought to establish a chemically defined 3D culture system for targeted derivation of non-neural ectoderm epithelia, from which we could derive inner ear organoids (<FIG>).

We first confirmed that dissociated human embryonic stem cells (hESCs) of the reference cell line WA25 (WiCell) aggregated well in E8 Medium containing a ROCK inhibitor, Y-<NUM>, and displayed superior uniformity and cell-survival compared to cells aggregated in a chemically-defined differentiation medium (hereafter, CDM; <FIG> and Table <NUM>).

Following a <NUM>-day incubation in E8 Medium, we transferred aggregates to CDM containing a low concentration of Matrigel and FGF-<NUM> to stimulate epithelization and ectoderm differentiation on the aggregate surface. We previously showed that a combination of BMP4 and the TGFβ inhibitor SB-<NUM> (hereafter, "SB") promoted non-neural induction from mouse PSCs (mPSCs)<NUM>. We found that combining <NUM> ng/ml BMP4 and <NUM> SB (dual SBBMP4 treatment referred to as "SBB") induces not only non-neural marker genes, such as TFAP2 and DLX3, but also the extraembryonic marker CDX2 (<FIG>; <FIG>)<NUM>. In contrast, SB treatment alone led to an increase in TFAP2 and DLX3 expression with no corresponding CDX2 expression (<FIG>). Remarkably, <NUM>% of SB treated aggregates generated TFAP2+ E-cadherin (ECAD)+ epithelium with a surface ectoderm-like morphology by days <NUM>-<NUM> of differentiation-a time scale consistent with human embryogenesis (n = <NUM> aggregates, <NUM> experiments; <FIG>; <FIG>). Over a period of <NUM> days, the epithelium expanded into a cyst composed of TFAP2+ Keratin-<NUM> (KRT5)+ keratinocyte-like cells (<FIG>). From these findings, we concluded that treating WA25 cell aggregates with SB is sufficient to induce a non-neural epithelium.

Curious whether endogenous BMP activity is sufficient for non-neural specification, we performed a co-treatment with the BMP inhibitor, LDN-<NUM> (hereafter, LSB; dual LDN/SB treatment referred to as LSB). As previously shown in hESC monolayer cultures<NUM>, LSB treatment to WA25 aggregates up-regulated neuroectoderm markers, such as PAX6 and N-cadherin (NCAD), and abolished TFAP2 and ECAD expression, suggesting that endogenous BMP signals drive non-neural conversion (<FIG>; <FIG>). To further validate our approach, we treated human iPSCs (mND2-<NUM>, WiCell) with SB and found, contrary to our results with WA25 hESCs, SB-only conditions generated PAX6+ neuroectoderm and TFAP2+ ECAD- neural crest-like cells (<FIG>). We reasoned that variation in endogenous BMP levels may underlie the different outcomes and the BMP concentration may need to be fine-tuned for each cell line. Accordingly, a low concentration of BMP4 (<NUM> ng/ml) in addition to SB (SBB) could generate TFAP2+ ECAD+ non-neural epithelium from mND2-<NUM> iPSCs (<FIG>; <FIG>). With either the SB or SBB approaches, the resulting epithelia closely resembled non-epithelia generated with mPSCs<NUM>,<NUM>. In contrast to our mouse culture, non-neural conversion occurs without off-target induction of Brachyury (BRA)+ mesendoderm cells (<FIG>). The following data were generated using either the SB (WA25) or SBB (mND2-<NUM>) approaches.

Next, we attempted to convert the non-neural epithelium into otic placode epithelium prior to keratinocyte commitment. Human cranial placodes arise at approximately <NUM>-<NUM> dpc; thus, assuming hPSCs represent cells at ~<NUM> dpc, otic placodes would develop in our culture within the first <NUM>-<NUM> days of differentiation with proper signaling modulation (<FIG>). Drawing on our previous finding that FGF activation and BMP inhibition are essential for pre-placode and otic induction from mPSC cultures, we treated day <NUM> aggregates with a combination of FGF-<NUM> and LDN (hereafter, "SBFL"). With SBFL treatment, the outer-epithelium thickened relative to SB-treated samples and expressed a combination of posterior placode markers, such as PAX8, SOX2, TFAP2, ECAD, and NCAD, indicating a phenotype similar to the otic-epibranchial progenitor domain (OEPD) from which the otic placode arises (<FIG>; <FIG>). When allowed to undergo self-guided differentiation in a minimal medium, we found that SBFL aggregates generated BRN3A+ TUJ1+ sensory-like neurons between days <NUM>-<NUM> (<FIG>). Since both the epibranchial placodes and the otic vesicles produce sensory neurons, we wondered which tissue type had developed. Notably, we did not detect expression of the otic marker PAX2 nor did we observe any vesicles in SBFL-treated aggregates, which would signify otic induction (data not shown). Thus, we concluded that SBFL treatment may be sufficient to induce epibranchial neurons, yet fails to initiate otic induction.

To promote PAX2 expression and vesicle formation, we began testing various signaling modulators (<FIG>). Although none of the conditions we tested had a detectable effect on PAX2 gene expression using qPCR analysis, extensive immunostaining drew our attention to a small population of PAX2+ PAX8+ ECAD+ cells in the epithelia of aggregates of control samples on day <NUM>, reminiscent of the otic placodes in vivo (<FIG>). We suspected that extracellular matrix could provide structural support for vesicle formation; thus, we transferred day <NUM> aggregates to Matrigel droplets in a minimal media (<FIG>). In these cultures, we observed radial production of migratory cells, but no vesicle-like structures or PAX2+ cells were apparent (<FIG>). Wnt activation seems to be essential for otic, but not epibranchial development, in vivo and can enhance the production of mouse inner ear organoids in vitro<NUM>,<NUM>-<NUM>. Remarkably, in <NUM> ± <NUM>% of Matrigel®-embedded aggregates treated with a Wnt signaling agonist, CHIR99021, between days <NUM>-<NUM> (n = <NUM>, <NUM> experiments), we witnessed epithelial protrusions reminiscent of the otic pits that precede vesicle development in vivo (<FIG>). We determined that the otic pit-like structures were PAX2+ PAX8+ SOX2+ SOX10+ JAG1+, confirming otic identity (<FIG>). Interestingly, the otic pits were accompanied by migrating TFAP2+ SLUG+ SOX10+ cranial neural crest-like cells that formed a mesenchyme around the otic pits, similar to the peri-otic mesenchyme in vivo (<FIG>).

We cultured the aggregates in stationary droplets until day <NUM>, then transferred them to a <NUM>-well plate on an orbital shaker or a spinner flask for further self-organized maturation-both formats produced comparable results. At <NUM>-<NUM> days in culture, vesicles remained visible through the surface of <NUM> ± <NUM>% aggregates examined (n = <NUM>, <NUM> experiments; <FIG><NUM>). In each aggregate we immunostained, we found multiple otic vesicles surrounding a central core epithelium that expressed the basal keratinocyte markers TFAP2 and KRT5 (<FIG>). As late as day <NUM>, we observed vesicles and otic placode-like epithelia that appeared to be partially attached or incorporated into the epidermal epithelium (<FIG>). In addition, older vesicles (><NUM> days) expressed the transcription factor FBXO2, which was recently shown to be highly specific to developing inner ear epithelia in mice (<FIG>)<NUM>.

After <NUM>-<NUM> days of incubation, vesicles with complex multi-chambered morphologies were visible through the aggregate surface (<FIG>). Remarkably, we found that a subset of vesicles in both WA25 and mND2-<NUM> derived aggregates developed epithelia containing cells expressing multiple hair cell markers, including MYO7A, PCP4, ANXA4, SOX2, and CALB2 (<FIG>-Q; <FIG>). The sensory-like epithelia also contained SOX2+ SOX10+ SPARCL1+ cells, reminiscent of supporting cells in the mammalian utricle<NUM>. The luminal cells in these epithelia had elongated morphologies with F-actin-rich apical junctions characteristic of inner ear sensory epithelia (<FIG>). The cells expressing hair cell markers also had F-actin-rich and espin (ESPN)+ apical stereocilia bundles protruding into the vesicle lumen that were associated with an acetylated-alpha-Tubulin (TUBA4A)+ kinocilium (<FIG>). Together, these findings confirm that the hPSC-derived otic vesicles generate inner ear organoids with sensory epithelia containing hair cells and supporting cells.

To facilitate live-cell imaging and electrophysiological experiments, we engineered a novel hESC reporter cell line to endogenously label hair cells with enhanced green fluorescent protein (eGFP). We used the CRISPR/Cas9 system to insert a 2A-eGFP gene cassette at the stop codon of the ATOH1 gene, which is highly expressed during hair cell induction and early maturation (<FIG>)<NUM>. We verified inner ear organoid induction from two clones containing the proper bi-allelic insertion of the 2A-eGFP cassette using our established protocol (hereafter ATOH1-2A-eGFP cells). Remarkably, as early as day <NUM>, we observed eGFP+ hair cell-like cells emerging in inner ear organoids (data not shown). We noted that the individual organoids often contained multiple discrete patches with hundreds of eGFP+ cells (<FIG>). Immunostaining with hair cell markers, such as BRN3C and ESPN, confirmed the hair cell identity of eGFP+ cells (<FIG>). Between days <NUM>-<NUM>, <NUM> ± <NUM>% of aggregates contained at least one hair cell bearing organoid (n = <NUM>, <NUM> experiments). The seemingly low efficiency of hair cell induction may be due to our inability to detect organoids deep within the aggregates or it could indicate that the endogenous signals required for sensory epithelia formation vary from aggregate-to-aggregate. Notably, all WA25 and mND2-<NUM> aggregates examined between days <NUM>-<NUM> contained organoids with SOX10+ non-sensory epithelia, suggesting that organoid induction may be highly reproducible, but non-sensory inner ear epithelia are preferentially induced (n = <NUM>, <NUM> experiments; <FIG>). The 2A-eGFP+ hair cells that develop could be maintained for over <NUM> days in floating culture and retain hair bundle morphology even after dissection and sub-culturing (<FIG>).

Finally, we wondered whether the derived hair cells functioned similar to native mammalian hair cells. Using aggregates produced from ATOH1-2A-eGFP cells, we dissected and flat-mounted inner ear organoids between differentiation days <NUM>-<NUM>. To our knowledge, these constitute the first recordings of human hair cells derived from hPSCs. The cells had large outwardly-rectifying currents, but no Na+ current (seen in developing rodent hair cells, but absent in most mature hair cells) was detected in our sample (<FIG>). The K+ current amplitudes at nominal <NUM> mV were as follows: day <NUM>: <NUM>, <NUM>, <NUM> pA; day <NUM>: <NUM>, <NUM>, <NUM> pA; day <NUM>: <NUM>, <NUM>, <NUM> pA. This is comparable to the average of <NUM> pA for day <NUM> mouse organoid hair cells. Responses to step and sinusoidal current injection (<FIG>) resembled that of rodent hair cells, with an initial peak then repolarization, and larger deflections to hyperpolarizing than depolarizing current. However, resting potential of the cells was consistently slightly higher than that seen in rodents: day <NUM>: -<NUM>, -<NUM>; day <NUM>: -<NUM>, -<NUM> mV. Possibly relatedly, the prominent sub-threshold inward rectifier current thought to be carried by Kir2. <NUM> that develops early in hair cell differentiation and is present in all rodent vestibular hair cells and mouse organoid hair cells was absent or greatly reduced in human organoid cells (<FIG>)<NUM>,<NUM>. It is possible that development, expression, function, or modulation of Kir2. <NUM> is different in human tissue. Importantly, the constricted lumen morphology seen in most ><NUM>-days-old organoids and in all of the organoids used for recording, made direct access to the hair bundles for mechanotransduction analysis challenging (<FIG>). Nonetheless, our data strongly suggest that the human organoids, like mouse inner ear organoids<NUM>, contain immature vestibular hair cells.

In conclusion, we have established a robust culture system for guiding the development of human inner ear organoids in culture (<FIG>). Our findings further support our previous model of in vitro pre-otic induction and underscore the importance of Wnt signaling in otic progenitor differentiation. Interestingly, the resulting convoluted and multi-chambered morphology of human inner ear organoids bear a remarkable resemblance to the inner ear's membranous labyrinth, which consists of a series of tubes and chambers containing sensory and non-sensory epithelia. In addition, much like mouse organoids, the hPSC-derived organoids appear to form only vestibular sensory epithelia by default; thus, additional signaling manipulation will be needed to initiate cochlear organogenesis<NUM>,<NUM>. We expect that this culture system will be a powerful tool for uncovering mechanisms of human inner ear development and testing potential inner ear therapies.

hPSC culture: Human PSCs (hESCs of the reference cell line WA25, passage <NUM>-<NUM>; mND2-<NUM> iPSCs, passage <NUM>-<NUM>) were cultured in Essential <NUM> (E8) Medium or Essential <NUM> Flex Medium (E8f) (Invitrogen) supplemented with <NUM>µg/ml Normocin (Invivogen) on recombinant human Vitronectin-N (Invitrogen)-coated <NUM>-well plates according to an established protocol<NUM>,<NUM>. At <NUM>% confluency or every <NUM>-<NUM> days, the cells were passaged at a split ratio of <NUM>:<NUM>-<NUM>:<NUM> using an EDTA solution. Both cell lines were acquired from the WiCell Research Institute and arrived with a statement of verification and authenticity. Additional validation and testing information can be found on the cell line webpages, available at wicell. org/home/stemcell-lines/catalog-of-stem-cell-lines/wa25. cmsx and wicell. org/home/stem-cell-lines/catalog-of-stem-cell-lines/mirjt7i-mnd2-<NUM>. cmsx on the World Wide Web. Cell lines were determined to be mycoplasma contamination-free using the MycoAlert Mycoplasma Detection Kit (Lonza).

hPSC differentiation. To start differentiation, hPSC cells were dissociated with StemPro Accutase (Invitrogen) and distributed, <NUM>,<NUM> cells per well, onto <NUM>-well V-bottom plates in E8 medium containing <NUM> Y-<NUM> (Stemgent) and Normocin. Following a <NUM> hour incubation, the aggregates were transferred to <NUM>-well U-bottom plates in <NUM>µl of Chemically Defined Medium (CDM) containing <NUM> ng ml-<NUM> FGF-<NUM> (Peprotech), <NUM> SB-<NUM> (Stemgent), and, for some experiments, <NUM> ng ml-<NUM> BMP4 (Stemgent), and <NUM>% Growth Factor Reduced (GFR) Matrigel (Corning) to initiate non-neural induction-i.e. differentiation day <NUM>. CDM contained a <NUM>:<NUM> mixture of F-<NUM> Nutrient Mixture with GlutaMAX (Gibco) and Iscove's Modified Dulbecco's Medium with GlutaMAX (IMDM; Gibco) additionally supplemented with <NUM>% Bovine Serum Albumin (BSA), 1X Chemically Defined Lipid Concentrate (Invitrogen), <NUM>µg ml-<NUM> Insulin (Sigma), <NUM>µg ml-<NUM> Transferrin (Sigma), <NUM> Mono-Thioglycerol, and Normocin (see Table <NUM> for a detailed formulation). After <NUM> days of incubation, <NUM>µl of CDM containing a <NUM> ng ml-<NUM> FGF-<NUM> (<NUM> ng/ml final concentration) and <NUM> LDN-<NUM> (<NUM> final concentration) was added to the pre-existing <NUM>µl of media in each well. After an additional <NUM> days (<NUM> days total), <NUM>µl of CDM was added to the media. For some experiments, CDM containing a <NUM> CHIR99021 (<NUM> final concentration; Stemgent) was added to the pre-existing <NUM>µl of media in each well-we determined that this treatment is optional for inner ear organoid production, but may improve induction of otic placode-like cells. On differentiation day <NUM>, the aggregates were pooled together and washed with freshly prepared Organoid Maturation Medium (OMM) containing a <NUM>:<NUM> mixture of Advanced DMEM:F12 (Gibco) and Neurobasal Medium (Gibco) supplemented with <NUM>. 5X N2 Supplement (Gibco), <NUM>. 5X B27 without Vitamin A (Gibco), 1X GlutaMAX (Gibco), <NUM> β-Mercaptoethanol (Gibco), and Normocin (see Table <NUM> for a detailed formulation).

The formulation set forth in Table <NUM> provides for <NUM> of medium, which should be used for <<NUM> weeks. OMM is a custom-made hybrid of two media previously used to generate cerebral and gastric organoids<NUM>,<NUM>. B27 without Vitamin A was used to limit the influence of endogenously produced retinoic acid.

The aggregates were resuspended in ice cold undiluted GFR Matrigel and placed in approximately <NUM>µl droplets on the surface of a <NUM> bacterial culture plate. After at least <NUM> minutes of incubation at <NUM>, the droplets were bathed in <NUM> of OMM containing <NUM> CHIR99021. For non-droplet otic induction, the aggregates were washed and plated individually into each well of a <NUM>-well low cell adhesion plate in OMM containing <NUM> CHIR and <NUM>% GFR Matrigel. After <NUM> days of differentiation, the CHIR was removed from the medium by washing and the droplet aggregates were moved to a floating culture. Droplets were carefully dislodged using a wide-mouth 1000P tip and transferred to <NUM> of fresh OMM in a <NUM> disposable spinner flask (Corning). Spinner flasks were maintained on an in-incubator stir plate (Thermo Scientific) at <NUM> RPM for up to <NUM> days of differentiation. For some experiments, the aggregates were maintained in individual wells of <NUM>-well low-cell adhesion plates in <NUM> of OMM on an in-incubator orbital shaker (Thermo Scientific) for up to <NUM> days.

Choice of Media Components: We used two media components that may lead to variability in results due to lack of definition or poor compatibility with human cells: GFR Matrigel and BSA. GFR (Growth Factor Reduced) Matrigel contains, < <NUM> pg ml-<NUM> FGF-<NUM>, < <NUM> ng ml-<NUM> EGF, <NUM> ng ml-<NUM> IGF-<NUM>, < <NUM> pg ml-<NUM> PDGF, < <NUM> ng ml-<NUM> NGF, and <NUM> ng ml-<NUM> TGFβ. In particular, the TGFβ in GFR Matrigel may have impacted cell fate specification on day <NUM> or later because we did not include a TGFβ inhibitor in the media during that phase of culture. GFR Matrigel was chosen because it has been shown to be a reliable inducer of self-organizing epithelia from pluripotent stem cells in 3D culture<NUM>. GFR Matrigel is a more defined alternative to Matrigel, in which the concentration of growth factors, such as Egf, Igf1, Fgf2, and TGFβ, have been minimized to levels that should have a negligible effect on cell fate specification. In Other alternatives to Matrigel include, without limitation, synthetic hydrogels and recombinant protein-based matrices that support basement membrane formation and self-organization of differentiating PSCs into epithelia. For example, a purified Laminin/Entactin complex (Corning) may be a suitable, fully chemically defined alternative<NUM>. In the CDM, BSA was chosen as a cost-effective and easy to dissolve alternative to Human Serum Albumin and Polyvinyl Alcohol (PVA), respectively. PVA has been shown to be a suitable chemically defined substitute for BSA in CDM<NUM>.

Signaling molecules and recombinant proteins. The following small molecules and recombinant proteins were used: recombinant human BMP4 (<NUM>-<NUM> ng ml-<NUM>; Stemgent), human FGF-<NUM> (<NUM> ng ml-<NUM>; Peprotech), SB-<NUM> (<NUM>; Tocris Bioscience), CHIR99021 (<NUM>; Stemgent), and LDN-<NUM> (<NUM>; Stemgent).

Quantitative PCR. Analysis was performed as previously described on an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems) or a Bio-Rad CFX96 quantitative PCR machine (Bio-Rad)<NUM>. Data were normalized to L27 expression (internal control) and the fold change was calculated relative to Ct values from d0 WA25 aggregates using the ΔΔCt method. Unless stated otherwise, data represent at least <NUM> separate biological samples from separate experiments. All indicators of statistical significance refer to comparisons between a given condition and the control group. Refer to Table <NUM> for primer details.

Immunohistochemistry. Aggregates were fixed with <NUM>% paraformaldehyde for <NUM> at room temperature or at <NUM> overnight. The fixed specimens were cryoprotected with a graded treatment of <NUM>% and <NUM>% sucrose and then embedded in tissue freezing medium. Frozen tissue blocks were sectioned into <NUM> cryosections on a Leica CM-<NUM> cryostat. For immunostaining, a <NUM>% goat or horse serum in <NUM>% Triton X-<NUM>1X PBS solution was used for blocking, and a <NUM>% goat or horse serum in <NUM>% Triton X-<NUM>1X PBS solution was used for primary/secondary antibody incubations. Alexa Fluor conjugated anti-mouse, rabbit, or goat IgG (Invitrogen) were used as secondary antibodies. Prolong Gold with DAPI (Thermo Scientific) was used to mount the samples and visualize cellular nuclei. For wholemount staining, a similar staining paradigm was used; however, the Triton X-<NUM> concentration was increased to <NUM>%, and the blocking and primary/secondary incubations were done at <NUM> on a rotating shaker for <NUM> hours and <NUM> hours, respectively. Following each incubation, the samples were subjected to three <NUM>-hour washes in 1X PBS containing <NUM>% Triton X-<NUM> at <NUM> on a rotating shaker. Wholemount samples were mounted in ScaleA2 clearing solution for <NUM>-<NUM> days or ScaleSQ(<NUM>) clearing solution for <NUM>-<NUM> hours prior to imaging<NUM>. Microscopy was performed on a Lieca DMi8 inverted microscope, a Nikon TE2000 inverted microscope, or an Olympus FV1000-MPE Confocal/Multiphoton Microscope. 3D reconstruction was performed using the Imaris <NUM> software package (Bitplane) housed at the Indiana Center for Biological Microscopy. For segmentation analysis, 2A-eGFP cells were processed using the 'Spots' module in Imaris. Classification was based on estimated size, quality and signal intensity. Objects touching the border of the image were excluded. The following build parameters were used to identify 2A-eGFP+ cell bodies: estimated XY diameter = <NUM>; estimated Z diameter = <NUM>; `Quality' above <NUM>; `distance to image border XYZ' above <NUM>; 'intensity center Ch = <NUM>' above <NUM>,<NUM>. Movies were generated in Imaris from the raw image files and compiled in Adobe Premiere Pro to add titles and text. See Table <NUM> for a list of antibodies.

Electrophysiological recordings. Human organoids were shipped at day <NUM> in cold Hibernate A medium supplemented with 1X GlutaMax, 1X B27 Supplement, and Normocin. They were replaced back into OMM on day <NUM> in an incubator at <NUM>% CO<NUM> and <NUM>. On recording days, organoids were dissected out using sharp tungsten needles (Fine Science Tools) and pinned to glass coverslips. The 2A-eGFP+ signal was used to find areas with hair cells and to target hair cells for recording. Whole-cell patch clamp was performed on the semi-intact tissue with <NUM>-<NUM> MΩ glass electrodes. Data were acquired using an Axopatch 200B amplifier (Molecular Devices), filtered at <NUM>, then digitized at <NUM> through a Digidata 1322A converter. The recording pipette solution contained (in mM): <NUM> KCl, <NUM> HEPES, <NUM> EGTA, <NUM> MgCl<NUM>, <NUM><NUM>-ATP, <NUM> CaCl<NUM>, adjusted with KOH to pH <NUM>, -<NUM> mmol kg-<NUM>. The external solution contained: <NUM> NaCl, <NUM> KCl, <NUM> NaH<NUM>PO<NUM>, <NUM> HEPES, <NUM> CaCl<NUM>, <NUM> MgCl<NUM>, <NUM> Glucose, and was supplemented with vitamins and essential amino acids (Invitrogen, Carlsbad, CA), adjusted to pH <NUM> with NaOH, ~<NUM> mmol kg-<NUM>. Recordings were compensated <NUM>% and cells were held at -<NUM> mV for voltage clamp. Averages are reported ± SEM.

Generation of ATOH1-2A-eGFP reporter cell line. gRNAs (<NUM>'-TCGGATGAGGCAAGTTAGGA-<NUM>' (SEQ ID NO: <NUM>) and <NUM>'-GTCACTGTAATGGGAATGGG-<NUM>' (SEQ ID NO: <NUM>), offset = 0bp) targeting the stop codon region of ATOH1 were cloned into pSpCas9n(BB) vectors which express Cas9n under the control of CBh promoter (Addgene #<NUM>)<NUM>. To construct the donor vector, a 2A-eGFP-PGK-Puro cassette (Addgene #<NUM>)<NUM> flanked by two 1kb homology arms PCR amplified from extracted WA25 hESC genomic DNA were cloned into a pUC19 backbone. The two gRNA vectors and the donor vector, as well as a vector expressing Cas9n under the control of CMV promoter (Addgene #<NUM>)<NUM> were transfected into WA25 hESCs with 4D Nucleofector (Lonza) using the P3 Primary Cell 4D-Nucleofector X kit and Program CB-<NUM>. After nucleofection, cells were plated in growth medium containing 1X RevitaCell (Thermo Fisher) for improved cell survival rate, and <NUM> of Scr7 (Xcessbio) for higher HDR efficiency<NUM>. <NUM>µg µl-<NUM> puromycin selection was performed for <NUM> days starting from <NUM> post-nucleofection. The PGK-Puro sub-cassette flanked by two LoxP sites was removed from the genome after puromycin selection by nucleofection of a Cre recombinase expressing vector (Addgene #<NUM>). Clonal cell lines were established by low-density seeding (<NUM>-<NUM> cells cm-<NUM>) of dissociated single hESCs followed by isolation of hESC colonies after <NUM>-<NUM> days of expansion. Genotypes of the clonal cell lines were analyzed by PCR amplification followed by gel electrophoresis, and by Sanger sequencing of total PCR amplicons or individual PCR amplicons cloned into TOPO vectors. Cell lines with bi-allelic 2A-eGFP integration were used for inner ear hair cell differentiation.

Statistical analysis. All statistics were performed using GraphPad Prism <NUM> software. A Shapiro-Wilk normality test was used prior to analysis to determine that the data had a normal distribution. Statistical significance was determined using a one-way analysis of variance (ANOVA) followed by a Dunnett's post-hoc test for multiple comparisons to a control group (e.g. vehicle treated). A Brown-Forsythe test was used to determine that the variation among sample groups was similar. No statistical test was used to predetermine sample size, the investigators were not blinded to the treatment groups, and the samples were not randomized.

Representative Data and Reproducibility. Unless stated otherwise, images are representative of specimens obtained from at least <NUM> separate experiments. For IHC analysis of aggregates between days <NUM>-<NUM>, we typically sectioned <NUM>-<NUM> aggregates from each condition in each experiment. IHC analyses for later stages of the protocol were performed on at least <NUM> aggregates from each condition per experiment. The finalized culture method was successfully replicated <NUM> times by four independent investigators using the WA25 (wild-type or ATOH1-2A-eGFP) cell line. The method, with noted modifications, was replicated <NUM> times using the mND2-<NUM> iPSC line. A replication was deemed successful by confirming pit/vesicle formation during days <NUM>-<NUM> and positively identifying inner ear organoids in at least one aggregate on days <NUM>-<NUM> of differentiation. Experiments were excluded from analysis if no pits were observed during days <NUM>-<NUM>.

In day-<NUM> aggregates, we observed patches of PAX8+ PAX2+ epithelia reminiscent of otic placodes; therefore, we assayed for culture conditions promoting otic vesicle formation. Under control conditions (DMSO), we did not observe vesicle formation (data not shown). Since otic induction is dependent on Wnt signaling, we treated <NUM>-day aggregates with a potent GSK3β inhibitor and known Wnt signaling agonist27, CHIR99021 (CHIR), between days <NUM>-<NUM>. Under these conditions, PAX8+ PAX2+ SOX10+ vesicles evaginate from the outer epithelium (-<NUM>-<NUM> vesicles per aggregate; see <FIG>). Remarkably, Islet1+ (ISL1+) neuroblasts appear to delaminate from the otic vesicles (<FIG>). We also see neuroblasts in DMSO-treated aggregates and in the non-otic interior of CHIR-treated aggregates (<FIG>, arrowheads). Thus, we hypothesized that CHIR-treated aggregates yield a mixture of otic (i.e. vesicle-derived) and epibranchial (i.e. epithelium-derived) neurons (see <FIG>). In support of this hypothesis, we confirmed that the neurogenic factors Neurog1 (NGN1), a marker of otic neurons, and Neurog2 (NGN2), a marker of epibranchial neurons, are expressed in CHIR-treated aggregates (<FIG>).

After <NUM> days of differentiation, otic induced aggregates were plated on bacterial dishes in Matrigel droplets in medium containing <NUM> CHIR. On day <NUM>, the aggregates were fixed and immunostained with antibodies for markers of sensory neurons, BRN3A and βIII-Tubulin (TUJ1). Radially oriented BRN3A+ TUJ1+ neurons produced outgrowing processes with growth cones, confirming widespread sensory neurogenesis in the organoid cultures.

As further evidence of inner ear organogenesis in our culture system, the hair cells displayed CTBP2+ puncta by day <NUM>-<NUM> of culture, indicating putative ribbon synapse-like structures (<FIG>; n = <NUM> sensory epithelia). As noted previously, we observed BRN3a-positive sensory-like neurons in cell aggregates during the vesicle formation stage. Additionally, we observed TUJ1+ neuronal processes targeted to sensory epithelia with hair cells (<FIG>). Together, our findings suggest that the human inner ear organoid model may recapitulate assembly of the sensorineural circuit between hair cells and sensory neurons.

This example describes a protocol for inducing formation of non-neural ectoderm and inner ear sensory tissue from human pluripotent stem cells. As described in greater detail in the following paragraphs, pluripotent stem cells aggregates were cultured in a medium containing Matrigel, which is rich in basement membrane proteins, to induce ectoderm development and production of ectoderm epithelium on the aggregate surface. We then used a combined treatment of bone morphogenetic protein-<NUM> (BMP4) and a transforming growth factor beta (TGFβ) inhibitor such as the small molecule SB-<NUM> ("SB") to promote non-neural differentiation in the epithelium. To further initiate inner ear induction, BMP signaling was inhibited and fibroblast growth factor (FGF) signaling was activated using recombinant FGF-<NUM> approximately <NUM> hours after the initial BMP4 and SB treatment. Remarkably, the combined treatment protocol initiated self-organization of otic vesicles that later developed into inner ear organoids containing functional vestibular sensory epithelia.

ES cell culture in E8 medium on Vitronectin-coated plates (steps <NUM>-<NUM>). We maintain our hPS cells in E8 Medium under feeder-free conditions and use EDTA for passaging (see previous protocol by Beers et al. )<NUM> We prefer this method of hPS cell maintenance because, in our hands, it reduces spontaneous differentiation with limited time and effort.

Non-neural ectoderm and pre-otic induction (steps <NUM>-<NUM>). To start differentiation, hPS cells are dissociated and distributed, <NUM>,<NUM> cells per well, onto <NUM>-well V-bottom plates. For this initial aggregation step we use E8 medium containing Y-<NUM>, a potent ROCK signaling inhibitor. ROCK signaling inhibition has been shown to limit the amount of dissociation-induced apoptosis in hPS cells. Additionally, we have found that aggregation in E8 medium helps with cell survival and leads to more uniform cell aggregates, which impacts the reproducibility of the protocol. SB treatment inhibits TGFβ to induce ectoderm development. Endogenous BMP signaling generates non-neural rather than neural ectoderm. FGF-<NUM> and LDN treatment induces pre-placodal development. By day <NUM> the outer epithelium begins to express PAX8, indicating an oticepibranchial placode (OEPD)-like character. CHIR treatment on day <NUM> induces small patches of PAX2+ cells, indicating the earliest signs of otic placode development by day <NUM>.

Otic prosensory vesicle and inner ear organoid formation (steps <NUM>-<NUM>). A low concentration of extracellular matrix proteins (e.g., Matrigel™) does not seem to be supportive of otic vesicle formation. To encourage the otic placode-like patches to evaginate and pinch off from the epithelium as an otic vesicle, we embed day <NUM> aggregates in Matrigel. The Matrigel-embedded aggregates are cured onto the surface of bacterial dishes and bathed in a serum-free medium containing N2 and B27 supplement previously shown to support tissue self-organization (hereafter, Organoid Medium). This supportive environment, combined with continued exposure to CHIR, causes numerous vesicles to bud-off of the epithelium between days <NUM>-<NUM>. Otic vesicle formation was observed in ><NUM>% of the aggregates (><NUM> aggregates) across four independent experiments. Additionally, between days <NUM>-<NUM>, neuroblasts delaminate from other parts of the epithelium and differentiate into sensory neurons. It is currently unclear whether these sensory neurons are epibranchial neurons such as those of cranial nerves VII, IX, and X, or inner ear neurons such as the vestibular or spiral ganglion neurons. Formation of a mesenchyme containing chondrocyte progenitor cells was also observed. It was unclear, however, whether the chondrocyte progenitor cells arose from ectodermal epithelium or another population of mesodermal cells.

On day <NUM>, the Matrigel®-embedded aggregates were removed from the stationary culture dish and pipetted into spinner flasks containing Organoid Medium devoid of any added growth factors or small molecules. After a total of <NUM> days, <NUM>-<NUM> vesicles were observed in each aggregate using phase contrast imaging. These vesicles expressed PAX2, PAX8, SOX2, and JAG1 protein indicating an otic cell fate. The vesicles appeared to grow slowly between days <NUM>-<NUM>, and it became difficult to observe the vesicles using phase contrast imaging during this period due to the growing density of the mesenchymal cell mass in which they are embedded. By days <NUM>-<NUM>, the vesicles were generally more apparent in the aggregate interior. The vesicles typically exhibited convoluted, multi-chambered morphologies, in contrast to the simple spherical and ovoid shaped otic vesicles. By day <NUM>, vesicles have developed MYO7A+ hair cell-like cells and were identified as inner ear organoids. Between days <NUM>-<NUM>, we observed MYO7A+ hair cells with F-actin-rich and Espin+ hair bundles, indicating a definitive hair cell identity.

Other reagents used in this exemplary protocol are set forth in Table <NUM>.

Human recombinant BMP7 stock solution (<NUM> ng/µL): In the biosafety cabinet, add <NUM>µL of sterile <NUM> HCl to <NUM>µg of BMP7; vortex the solution and spin down in a tabletop centrifuge. Store BMP4 solution in <NUM>µL aliquots at -<NUM> for <NUM> months or at -<NUM> for <NUM> year.

Human recombinant FGF-<NUM> stock solution (<NUM> ng/µL): In the biosafety cabinet, add <NUM>µL of sterile PBS or <NUM> Tris (pH <NUM>) to <NUM>µg of FGF-<NUM>; vortex the solution and spin down in a tabletop centrifuge. Store FGF-<NUM> solution in <NUM>µL aliquots at -<NUM> for <NUM> months or at - <NUM> for <NUM> year.

Human transferrin stock solution (<NUM>/ml): In the biosafety cabinet, dissolve <NUM> of recombinant human transferrin in <NUM> of IMDM. To fully dissolve, vortex the tube and place it on a rotating shaker for <NUM>-<NUM> at room temperature (RT). Store the transferrin solution in <NUM>µl aliquots at -<NUM> for <NUM> months or at -<NUM> for <NUM> year.

EDTA Solution (for passaging hES cells): In the biosafety cabinet, mix <NUM>µl of <NUM> EDTA into <NUM> DPBS. Filter sterilize the solution. EDTA solution can be stored at RT for <NUM> months.

Chemically Defined Medium (CDM): To prepare <NUM> CDM, measure out <NUM> BSA in a <NUM> bottle. Dissolve the BSA in <NUM> F-<NUM> Nutrient Mixture +GlutaMAX, <NUM> IMDM +GlutaMAX, <NUM> chemically-defined lipid concentrate, <NUM>µL Insulin, <NUM>µL Transferrin, and <NUM>µL <NUM>-thioglycerol. Sterile filter using a low-protein binding filter. CDM should be used for up to <NUM> weeks and stored at <NUM>. Add Normocin to the media just before use at a dilution of <NUM>µl per <NUM> of CDM. See Table <NUM> for a quick reference recipe.

Differentiation CDM: In a <NUM> conical tube, add <NUM>µL of ice cold Geltrex to <NUM> of ice cold CDM (<NUM>% final concentration). Vortex the tube well to fully dissolve the Geltrex. Place <NUM> of this solution in a new <NUM> conical tube. Add <NUM>µL of FGF-<NUM> (<NUM> ng/mL final concentration) and <NUM>µL of SB-<NUM> (<NUM> final concentration). Vortex the tube well to mix. Differentiation CDM should be made fresh on day <NUM> of differentiation. Use the remaining <NUM> of CDM +Geltrex to wash the aggregates before plating.

Organoid Maturation Medium (OMM): To prepare <NUM> OMM, combine <NUM> Advanced DMEM/F12, <NUM> Neurobasal Medium, <NUM>µL B-<NUM> Supplement without vitamin A, <NUM>µL GlutaMAX, <NUM>µL N2-supplement, and <NUM>µL Normocin in a sterile <NUM> conical tube. OMM can be used for up to <NUM> weeks if stored at <NUM>. See Table <NUM> for a quick reference recipe.

hES cell differentiation (day -<NUM> to day <NUM>): aggregation:.

Differentiation day <NUM>: transfer to differentiation CDM:.

Differentiation day <NUM>: addition of FGF-<NUM> and LDN <NUM> (FGF/LDN):.

Differentiation day <NUM>: transition to static ECM culture:.

Differentiation day <NUM>: transition to spinner flask.

Claim 1:
A method of obtaining a three-dimensional composition comprising human inner ear sensory tissue, the method comprising the steps of:
(a) culturing human induced pluripotent stem cell aggregates in a culture medium comprising FGF-<NUM>, BMP4 and a small molecule inhibitor of Transforming Growth Factor Beta (TGFβ) signaling for four days, wherein the small molecule inhibitor of TGFβ is SB431542;
(b) further culturing the cultured aggregates of (a) in the presence of a Fibroblast Growth Factor (FGF) and a small molecule inhibitor of BMP signaling for <NUM> days, wherein the small molecule inhibitor of BMP is LDN-<NUM>;
(c) contacting the further cultured aggregates of (b) to a Wnt agonist for <NUM> days, wherein the Wnt agonist is an inhibitor of GSK3, whereby cells within the contacted aggregates differentiate into pre-otic epithelial cells;
(d) embedding the pre-otic epithelial cells in a semi-solid culture medium comprising extracellular matrix protein and a basement membrane extract;
(e) culturing the embedded pre-otic epithelial cells in the presence of a Wnt agonist for <NUM> days, wherein the Wnt agonist is an inhibitor of GSK3; and
(f) culturing the embedded, pre-otic epithelial cells of (e) for at least a further <NUM>-<NUM> days under conditions that promote self-assembly of embedded pre-otic epithelial cells into otic vesicles , whereby a three-dimensional composition comprising human inner ear sensory tissue is obtained.