AND SENSORY NEURONS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 62/244,568, filed October 21, 2015, which is incorporated herein by reference as if set forth in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR

DEVELOPMENT

[0002] 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.

FIELD OF THE INVENTION

[0003] 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.

BACKGROUND OF THE INVENTION

[0004] 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.

[0005] 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. SUMMARY OF THE INVENTION

[0006] In a first aspect, provided herein is a method of obtaining human pre-otic epithelial cells. As described herein, the method comprises (a) culturing human pluripotent stem cell aggregates in a culture medium comprising a Bone Morphogenetic Protein (BMP) and an inhibitor of Transforming Growth Factor Beta (TGFP) signaling for about eight to about 10 days; (b) further culturing the cultured aggregates of (a) in the presence of a Fibroblast Growth Factor (FGF) and an inhibitor of BMP signaling for about 4 days; and (b) contacting the further cultured aggregates of (b) to a Wnt agonist for about 4 days; whereby cells within the contacted aggregates differentiate into pre-otic epithelial cells. The FGF can be FGF-2. The BMP can be BMP2, BMP4, or BMP7. The inhibitor of BMP signaling can be LDN-193189. The inhibitor of TGFpi -mediated signaling can be selected from the group consisting of SB431542 and A-83-01. The Wnt agonist can be an inhibitor of GSK3. The inhibitor of GSK3 is selected from the group consisting of CHIR99021, lithium chloride (LiCl), and 6-bromoindirubin-3'-oxime (BIO).

[0007] In another aspect, provided herein is a method of obtaining a three-dimensional composition comprising human inner ear sensory tissue, the method comprising the steps of (a) embedding human pre-otic epithelial cells obtained according to the method of claim 1 in a semisolid culture medium comprising extracellular matrix protein; and (b) culturing the embedded pre-otic epithelial cells in the presence of a Wnt agonist for about 40 to about 60 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. The Wnt agonist can be an inhibitor of GSK3, where the inhibitor of GSK3 is selected from the group consisting of CHIR99021, lithium chloride (LiCl), and 6-bromoindirubin-3'-oxime (BIO). The extracellular matrix can be a basement membrane extract (BME). The three-dimensional composition can comprise one or more mechanosensory cells. The three-dimensional

composition can comprise one or more sensory neuron cells. The three-dimensional composition cam comprise one or more sensory neuron cells that form synaptic connections with

mechanosensory cells.

[0008] These and other features, aspects, and advantages described herein will become better understood upon consideration of the following drawings, detailed description, and appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIGS. 1A-1N demonstrate an exemplary protocol for step-wise induction of otic placode-like epithelia. a, Overview of mammalian ectoderm development in the otic placode cranial region, b, Timeline for key events of human otic induction. Day 0 on the timeline indicates the approximate stage of development represented by hPSC: -12 dpc. c, Differentiation strategy for non-neural ectoderm (NNE), otic-epibranchial progenitor domain (OEPD), and otic placode induction. Potentially optional or cell line-dependent treatments are denoted in parentheses, d, qPCR analysis on day 2 of differentiation of WA25 cell aggregates treated with DMSO (Control), 10 μΜ SB, or 10 μΜ SB + 10 ng/ml BMP4, denoted as SBB. Gene expression was normalized to undifferentiated hESCs; n = 3 biological samples, 2 technical repeats;

*P<0.05, ** <0.01, *** <0.001; error bars = max/min. e, f, Representative TFAP2, ECAD, and PAX6 expression in WA25 aggregate treated with 10 μΜ SB or with 200 nM LDN + 10 μΜ SB for 6 days, g, TFAP2, ECAD, and PAX6 expression in mND2-0 iPSCs treated with 10 μΜ SB + 2.5 ng/ml BMP4 (SBB) on day 6. h, i, Representative image of a SB-treated WA25 aggregate on day 8: live (h) and immunostained with PAX8 and TFAP2 antibodies (i). When comparing morphology in panels (h) and (i) note that the outer-epithelium crumples into the aggregate core during the cryosectioning process, j, k, Representative image of a SB-treated WA25 aggregate on day 8 after treatment with 50 ng/ml FGF-2 and 200 nM LDN (SBFL) on day 4: live (j) and immunostained with PAX8 and TFAP2 antibodies (k). 1-n, WA25 SBFL- treated aggregates on day 12. The outer-epithelium contains PAX8 ⁺ ECAD ⁺ cells (1) and occasional patches of PAX8 ⁺ PAX2 ⁺ otic placode-like cells (m, n). The specimens shown were treated with 25 μΐ of additional CDM on day 8. Scale bars, 100 μιη (e-m), 50 μιη (n).

[00010] FIGS. 2A-2H demonstrate undifferentiated WA25 hESCs, cell aggregation, and initial non-neural ectoderm differentiation analysis, a, b, WA25 cells maintained on Vitronectin- N-coated plates in E8 medium express markers of primed pluripotent stem cells, c, Overview of differentiation strategy and experimental conditions, d, e, Aggregation of single-celled hESCs in E8 + 20 μΜ Y-27632 produced less cellular debris than aggregation in CDM + 20 μΜ Y-27632. f, Relative to undifferentiated cells, pluripotency markers were significantly down-regulated by day 2 in all conditions except vehicle control, g, The non-neural markers TFAP2 and DLX3 were upregulated in SB and SBB conditions, h, The mesendoderm markers BRA and EOMES were upregulated by BMP4 (B) treatment. Gene expression was normalized to dO aggregates. For statistical tests treatement values were compared to control (CTRL) values; n = 3 biological samples, 2 technical replicates; *P<0.05, ** <0.01, ***P<0.001, ns = not significant; error bars are max/min. Scale bars, 100 μπι (a, d, e), 25 μπι (b).

[00011] FIGS. 3A-3G demonstrate non-neural induction using WA25 hESCs. a, b, Overview of differentiation strategy, c-e, Aggregate diameter (c) and circularity (d) over time in culture. The circularity of the aggregates decreases overtime as the outer epithelium crumples (e). f, g, Representative day 4 aggregate showing a nearly complete lack of OCT4-expressing cells, SOX2/ECAD-expressing cells in the core, and TFAP2/ECAD-expressing cells in the outer-core and epithelium. Error bars are max/min. Scale bars, 100 μπι.

[00012] FIGS. 4A-4R demonstrate that Wnt signaling activation initiates self-organization and maturation of inner ear organoids containing vestibular-like hair cells, a, Inner ear organoid induction strategy. Day 12 aggregates were embedded in Matrigel droplets to support vesicle formation, b-d, In CHIR-treated samples, but not DMSO (Control) samples, otic pit-like structures evaginate from the outer-epithelium (d). e-i, Between days 14-35, pits and vesicles expressed otic specific markers, such as SOX10, SOX2, JAG1, PAX8, PAX2, and FBX02. The epithelium from which vesicles arise begins to express the epidermal keratinocyte marker KRT5 by day 35 (h). j, By day 40-60, the aggregates contain multiple organoids and, typically, a single epidermal unit visible under DIC imaging. Inner ear organoids are distinguishable by a defined epithelium with -25-40 μπι apparent thickness and a lumen (j inset), k, Inner ear organoids are typically oriented around the epidermal unit and contain sensory epithelia with ANXA4 ⁺ PCP4 ⁺ hair cells. The luminal surface of organoids is actin-rich, as denoted by phalloidin staining (k"). l-o, Hair cells are MY07A ⁺ SOX2 ⁺ , and supporting cells are SOX2 ⁺ . F-actin-rich hair bundles protrude from the hair cells into the lumen (n, o; asterisks denote hair bundle location in m). p, q, m D2-0 iPSC-derived sensory epithelia have a similar morphology to WA25 hESC-derived sensory epithelia and contain PCP4 ⁺ ANXA4 ⁺ hair cells. SOX10 is expressed throughout the supporting and non-sensory epithelial cell populations, but not in hair cells (p). Supporting cells express the utricle supporting cell marker SPARCLl (q). r, Hair cells in organoids have ESPN ⁺ hair bundles with a single acetylated-tubulin (TUBA4A) ⁺ kinocilium. Scale bars, 200 μπι (j), 100 μπι (b, c, e), 50 μπι (g, h, k), 25 μπι (d, f, i, 1, m, p), 10 μπι (n, q), 5 μπι (r), 2.5 μπι (o).

[00013] FIGS. 5A-5K demonstrate that hESC-derived hair cells have similar

electrophysiological properties to those of native hair cells, a, ATOHl-2A-eGFP CRISPR design. The two guide RNAs (blue, with PAM sequence in red) direct Cas9n to make two nicks (red triangles) near the stop codon (underlined with pink background) of ATOH1. The resulting DNA double strand break is repaired by the donor vector, which has a 2A-eGFP-PGK-Puro cassette and lkb left and right homology arms (LHA and RHA). The LoxP-flanked PGK-Puro sub-cassette is subsequently removed by Cre recombinase. In ATOH1 expressing hair cells, eGFP is transcribed along with ATOH1. b-d, Representative live cell images of 2A-eGFP ⁺ hair cells in 50- and 100-day-old inner ear organoids. In panel (b), cartilage nodule (cn) and asterisks denote separate 2A-eGFP ⁺ hair cell patches. The asterisk in panel (c) denotes the approximate location of the hair cells in panel (d). e, Expression of BRN3C in 140-day-old eGFP ⁺ hair cells, f, Expression of ESPN in the hair bundles of 100-day-old eGFP ⁺ hair cells, g, Human organoid hair cells displayed prominent outward currents (d64). h, The cell responded to current injection with rectifying voltage deflections with features similar to those seen in mouse vestibular type II hair cells, i, Human organoid hair cells were able to follow sinusoidal stimuli with an initial peak on the rising phase (excerpt of frequency sweep), j, Although voltage-gated currents were slightly smaller at this point in culture, the gross voltage dependence closely resembled that seen in mouse hair cells (non-leaky recordings pooled for days 64-67, comparison from P4 mouse utricle), k, However, the inward rectifier current, seen below -80 mV in mouse utricular and organoid cells, was not prominent in the human organoid cells. Scale bars, 200 μπι (b), 100 μπι (c), 25 μιη (e), 5 μιη (d, f).

[00014] FIGS. 6A-6E demonstrate that SB-treated aggregates generate keratinocytes. a, Overview of non-neural and keratinocyte induction process, b-d, In 3D culture, non-neural ectoderm induction is accompanied by characteristic morphological changes. By days 6-8, the epithelium separates from the core, forming a translucent sphere (c). The epithelium remains connected to the core via spoke-like structures (a, c). After 20 days, spokes from the core are absent, and the epithelial sphere is typically filled with cellular debris (d). e, The day 20 epithelium contains TFAP2+ KRT5+ cells, indicative of epidermal keratinocytes. Scale bars, 250 μπι (b-d), 100 μπι (e), 5 μπι (e').

[00015] FIGS. 7A-7D demonstrate that non-neural ectoderm induction in SB-treated WA25 aggregates is due to endogenous BMP signaling, a, Overview of experiment to test whether endogenous BMP signaling influence non-neural induction, b-d, LSB treatment leads to NCAD expression throughout the aggregates. Note that a subpopulation of NCAD+ PAX6+ cells (see FIG. IE) do appear in the core of SB treated aggregates. Scale bars, 50 μιη.

[00016] FIGS. 8A-8G demonstrate non-neural ectoderm induction with mND2-0 iPSCs. a, b, WA25 cells maintained on Vitronectin-N-coated plates in E8 medium express markers of primed pluripotent stem cells, c, Overview of differentiation strategy and experimental conditions. Other BMP concentrations were tested in a preliminary experiment (1.25, 2.5, 5, 10, 20, 40 ng/ml), and 2.5 ng/ml was selected as the minimum concentration that produced the morphological changes (i.e., translucent sphere) seen in SB-treated WA25 cells (see FIG. 6C). d, Representative images of SB- or SBB-treated aggregates between days 0-6. e, f, SB-treated aggregates generate PAX6+ NCAD+ epithelia, TFAP2+ migratory cells, and few ECAD+ cells by day 6, suggesting a heterogeneous mix of neuroectoderm and neural crest cells, g, Endogenous BMP signaling is insufficient for non-neural conversion in mND2-0 iPSCs; thus, additional BMP4 is necessary. Scale bars, 100 μπι (d), 25 μπι (a, b, e, f).

[00017] FIGS. 9A-9C demonstrate that non-neural ectoderm induction occurs without off- target induction of mesendodermal cells. Representative Brachyury (BRA)

immunohistochemistry in day 4 aggregates treated with 10 ng/ml BMP4 (a), 10 μΜ SB (b), and 10 μΜ SB + 2.5 ng/ml BMP4 (c) on day 0. Scale bars, 50 μιη.

[00018] FIGS. 10A-10D demonstrate induction of OEPD-like epithelium in WA25 hESC and mND2-0 iPSC aggregates by FGF-2 and LDN ("FL") treatment, a, b, SOX2, TFAP2, and ECAD are expressed throughout SB-treated WA25 aggregates on day 8 following FL treatment on day 4. PAX8 expression is restricted to the outer-epithelium. A unique characteristic of the epibranchial and otic placodes is the co-expression of ECAD and NCAD. Here, NCAD expression was observed throughout the aggregate, except for the interior-most core, c, d, iPSCs treated with SBB never express PAX8. FL treatment on day 4 induces a thicker outer-epithelium morphology and expression of PAX8. ECAD and TFAP2 are expressed throughout the

SBB+FL(d4)-treated iPSC aggregates. Scale bars, 100 μιη.

[00019] FIGS. 11 A-l IF demonstrate that OEPD induced aggregates spontaneously generate sensory-like neurons in a minimal medium floating culture, a, Overview of the experiment. SBFL-treated aggregates were transferred to OMM on day 8 of differentiation, b-d, On day 20, the aggregates are composed of patches of BRN3A+ TUJ1+ HUC+ neurons surrounding a ECAD+ epithelium. Neuronal patches were typically associated with PAX8+ epithelium. When aggregates were plated in Matrigel™ droplets they produced neurite outgrowths (e). f, BRN3 A+ neurons emerging from a PAX8+ ECAD+ epithelium is consistent with epibranchial placode neurogenesis; however, these data do not directly establish the PAX8+ ECAD+ epithelium as the origin of the sensory neurons. Scale bars, 100 um (b, c, d), 50 μιη (e).

[00020] FIGS. 12A-12C demonstrate WNT and FGF signaling modulation and PAX8/PAX2 expression during days 8-10 of differentiation, a, These qPCR data are representative of one exploratory experiment focused on identifying signaling modulators that could increase PAX2 expression following OEPD induction, b, c, FGF inhibition using, PD- 173074 likely inhibits PAX2 expression, as would be expected based on developmental studies (Groves et al.,

Development 139, 245-257 (2012)). By contrast, the WNT inhibitor, XAV939, and WNT agonist, CHIR99021, only had a modest positive or negative impact on PAX2 expression compared to a DMSO controls. Based on these results and extensive immunostaining for PAX2 expression, we reasoned that the OEPD epithelium may require more time before it will be responsive to otic inductive cues. Thus, we changed strategies by lengthening the initial culture phase to 12 days (with addition of fresh media on day 8) and testing the effect of transitioning the aggregates to Matrigel™ droplets.

[00021] FIGS. 13A-13G demonstrate that otic vesicles evaginate radially around a core epithelium of epidermal keratinocytes. a-c, Serial sections through a day 35 aggregate showing the internal organization of epidermal and otic vesicle epithelia. KRT5 expression is restricted to the epidermis, whereas EC AD is expressed in both epidermis and otic vesicle epithelial cells. Arrowheads label SOX10+ otic vesicles in (a). Note the CNC-like SOX10+ TFAP2- and

SOX10+ TFAP2+ cells in the mesenchymal layer of the aggregate. The pair of vesicles highlighted in panel (c) are labeled, at lower magnification and different orientation, in panel (b). The vesicles seen in (c) were shown to co-express the otic vesicle markers SOX10, PAX2, FBX02, JAG1, and EC AD. d-g, A day 35 aggregate that was wholemount immunostained for EC AD to reveal the epidermal core and surrounding otic vesicles. Scale bars, 250 μπι (d, f), 100 μιη (a, b), 25 μιη (c, g).

[00022] FIGS. 14A-14E demonstrate that inner ear organoids generate vestibular-like sensory epithelia. a, Day 48 aggregate with three visible inner ear organoids (arrowheads). Note that this specimen was derived from a separate experiment than the specimen seen in FIG. 2J. b, Organoid hair cells express the type II vestibular and inner cochlear hair cell marker CALB2. c, Cross-section through a sensory epithelium showing expression of SPARCL1 throughout the supporting cells. SOX2 is expressing in both supporting cells and PCP4+ hair cells, d-f, SOX10 is expressed throughout the supporting and non-sensory epithelial cells. F-actin-rich

circumferential belts were observed in both sensory and nonsensory epithelial. Scale bars, 100 μπι (a, d, e), 25 μιη (b, c), 10 μιη (f).

[00023] FIG. 15 presents a comparison of human inner ear induction studies. 2D, two dimensional. 3D, three dimensional. OEPs: otic epithelial progenitors. Note: In Chen et al. (2012) protocol, the efficiency of OEPs generation is dependent on the cell line, plating density and the degree of cell separation. In Ronaghi et al. (2014) protocol, the 1.79% hair cell-like cell generation efficiency was calculated from cells with mid-level Atohl-nGFP expression (19.8% ^χ 9.03%) = 1.79%)), which is believed to be "an indicator of a potential hair cell phenotype". If also considering cells with high Atohl-nGFP expression and cells negative for Atohl-nGFP expression, the hair cell-like cell generation efficiency is 5.89% (77.1% x 5.24% + 19.8% x 9.03% + 3.1% 2.08% = 5.89%).

[00024] FIGS. 16A-16C demonstrate otic neurogenesis, (a-b) SOX2+ otic vesicles and pits are typically associated with ISL1+ TUJ1+ neuroblasts (day 14). The epithelia of otic vesicles appear to be highly neurogenic at this stage, with weak TUJl staining throughout, (c) NGNl and NGN2 are expressed at day 14 in CHIR-treated samples; normalized to undifferentiated PSCs (n = 3). Scale, 100 (a), 20 (b) μιη.

[00025] FIGS. 17A-17C demonstrate sensory neuron outgrowth and maturation. HTR-treated organoids plated on Matrigel produce TUJ1+ BRN3 A+ neurons with a bipolar morphologies. Growth cone (inset in b). Scale, 500 (a), 50 (b), 10 (c).

[00026] FIGS. 18A-18C demonstrate that inner ear organoid-derived hair cells have ribbon synapse-like structures and are innervated by sensory neurons. A-B, CTBP2-positive puncta in WA25 hESC-derived PCP4-posivie hair cells on day 60 of differentiation. C, Representative image of neurons innervating an inner ear organoid sensory epithelium. Scale bars, 100 μπι (C), 25 μιη (A, B).

[00027] FIG. 19 depicts the ATOHl-2A-eGFP-PGK-Puro donor plasmid sequence (SEQ ID NO:21).

[00028] FIG. 20 depicts the genomic sequence of the homozygous/bi-allelic ATOH1-2A- eGFP cell line at the ATOH1 locus (SEQ ID NO:22). DETAILED DESCRIPTION

[00029] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference in their entirety as if each individual publication, patent, and patent application was specifically and individually indicated to be incorporated by reference.

[00030] 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.

[00031] 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.

[00032] Methods

[00033] 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.

[00034] 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-4 (BMP4) and an inhibitor of transforming growth factor beta (TGFP) signaling such as, for example, SB-431542 ("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 TGFP signaling (e.g., SB) for about 8 days to about 10 days. SB-431542 is a specific inhibitor of the activin receptor-like kinase receptors ALK5, ALK4, and ALK7. Following the 8 to 10 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.

[00035] Referring to FIG. 1, 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-2) 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-193189 and SB-431542. LDN-193189 is a selective BMP signaling inhibitor that inhibits the transcriptional activity of the BMP type I receptors ALK2 and ALK3.

[00036] 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 10 days to about 14 days (e.g., about 10, 11, 12, 13, or 14 days).

[00037] Otic vesicle-laden aggregates are further cultured for at least about 40 days (e.g., about 40 days, about 45 days, about 50 days, about 60 days, about 65 days, about 70 days, about 75 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.

[00038] 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., Ludwig T, et al., "Feeder-independent culture of human embryonic stem cells," Nat. Methods 3 :637-646 (2006); and Ludwig T, et al., "Derivation of human embryonic stem cells in defined conditions," Nat. Biotechnol. 24: 185-187 (2006), each of which is incorporated herein by reference as if set forth in its entirety. Other DMEM formulations suitable for use herein include, e.g., X-Vivo (BioWhittaker, Walkersville, MD) and StemPro ^® (Invitrogen; Carlsbad, CA).

[00039] 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., US Patent Application Publication No.

2008/0171385, incorporated herein by reference as if set forth in its entirety; and Watanabe K, et al, "A ROCK inhibitor permits survival of dissociated human embryonic stem cells," Nat.

Biotechnol. 25:681-686 (2007). 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)-(+)-2-methyl-l-[(4-methyl-5- isoquinolinyl)sulfonyl]homopiperazine dihydrochloride (informal name: H-1152), l-(5- isoquinolinesulfonyl)piperazine hydrochloride (informal name: HA-100), l-(5- isoquinolinesulfonyl)-2-methylpiperazine (informal name: H-7), l-(5-isoquinolinesulfonyl)-3- methylpiperazine (informal name: iso H-7), N-2-(methylamino) ethyl-5-isoquinoline- sulfonamide dihydrochloride (informal name: H-8), N-(2-aminoethyl)-5- isoquinolinesulphonamide dihydrochloride (informal name: H-9), N-[2-p-bromo- cinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (informal name: H-89), N-(2- guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride (informal name: HA-1004), l-(5- isoquinolinesulfonyl) homopiperazine dihydrochloride (informal name: HA- 1077), (S)-(+)-2- Methyl-4-glycyl-l-(4-methylisoquinolinyl-5-sulfonyl)homopipe razine dihydrochloride (informal name: glycyl H-l 152) and (+)-(R)-trans-4-(l-aminoethyl)-N-(4- pyridyl)cyclohexanecarboxamide dihydrochloride (informal name: Y-27632). 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 3 μΜ to about 10 μΜ 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.

[00040] FGF-2 is an agonist of FGF signaling, and FGF signaling can be antagonized using, for example, the small molecule inhibitor PD-173074. BMP4 is an agonist of BMP signaling, and BMP signaling can be antagonized using, for example, the small molecule inhibitor LDN- 193189. TGFP-l and Activin A are agonists of TGFP signaling, and TGFP signaling can be antagonized using, for example, the small molecule inhibitor SB-431542. CHIR-99021 is an agonist of the Wnt/p-catenin signaling pathway, and Wnt/p-catenin signaling can be antagonized using, for example, the small molecule inhibitor XAV-939. Other Wnt agonists include inhibitors/antagonists of the molecule Glycogen Synthase Kinase 3 (GSK3).

[00041] 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 8™ 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.

[00042] 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 Chen et al., Nature Methods 8:424-429 (2011), which is incorporated by reference herein as if set forth in its entirety. 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 (20 μg/mL), transferrin (10.67 ng/mL) and human Fibroblast Growth Factor 2 (FGF2) (100 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 (20 μg/mL), transferrin (10.67 ng/mL), human FGF2 (100 ng/mL), and human TGFpi (Transforming Growth Factor Beta 1) (1.75 ng/mL).

[00043] 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 Foxil, Dlx genes, Pax8, Pax2, Sox3, Eyal, 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).

[00044] 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 embryonic stem cells (hESCs) and human induced pluripotent stem (iPS) cells. 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 Thomson et al, Science 282: 1145-1147 (1998). These cells express Oct-4, SSEA-3, SSEA- 4, TRA-1-60 andTRA-1-81, 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., Yu et al., Science 318: 1917-1920 (2007).

[00045] 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-4, SSEA-3, SSEA-4, Tra-1-60 or Tra-1-81, but not SSEA-1). 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.

[00046] 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, Yu et al, Science 324(5928):797-801 (2009); Chen et al, Nat. Methods 8(5):424-9 (2011); Ebert et al, Nature 457(7227):277-80 (2009); Howden et al, Proc. Natl. Acad. Sci. U. S. A. 108(16):6537-42 (2011). 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.

[00047] 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 (1) the chemical and structural identity of all medium ingredients is not known, (2) the medium contains unknown quantities of any ingredients, or (3) 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.

[00048] 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. Although any methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein.

[00049] 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.

[00050] As used herein, "effective amount" means an amount of an agent sufficient to evoke a specified cellular effect according to the present invention.

[00051] As used herein, "about" means within 5% of a stated concentration range, density, temperature, or time frame.

[00052] 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. All papers and patents disclosed herein are hereby incorporated by reference as if set forth in their entirety. EXAMPLES

[00053] Example 1 - Generation of inner ear organoids with functional hair cells from human pluripotent stem cells

[00054] The human inner ear contains approximately 20,000 sensory hair cells that detect sound and movement via mechanosensitive stereocilia bundles ¹ . Genetic mutations or environmental insults, such as loud noises, can cause irreparable damage to these hair cells, leading to dizziness or hearing loss ^{2 3} . We previously demonstrated how to generate inner ear organoids from mouse pluripotent stem cells (PSCs) using timed manipulation of the FGF, TGF , BMP, and Wnt signaling pathways in a 3D culture system ^4"6 . 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 ⁷ . Moreover, our past findings bolstered a working model of otic induction signaling dynamics, wherein BMP signaling activation and TGF inhibition initially specifies non-neural ectoderm and subsequent BMP inhibition and FGF activation induces a pre-otic fate ^{8 9} . Despite several recent attempts, a developmental^ faithful approach for deriving functional hair cells from human PSCs (hPSC) has yet to be described ^10"15 . Here, to generate human inner ear tissue from hPSCs, we first established a timeline of in vitro human inner ear organogenesis (FIGS. 1A, IB). The inner ear arises from the ectoderm layer and, in humans, produces the first terminally differentiated hair cells by -52 days post conception (dpc) ¹⁶ . Beginning with pluripotent cells in the epiblast, inner ear induction is initiated -12 dpc with formation of the ectoderm epithelium. Then, the epithelium splits into the non-neural ectoderm (also known as surface ectoderm) and the neuroectoderm (FIGS. 1A, IB). 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 (FIGS. 1A-1C).

[00055] We first confirmed that dissociated human embryonic stem cells (hESCs; WA25 cell line, WiCell) aggregated well in E8 Medium containing a ROCK inhibitor, Y-27632, and displayed superior uniformity and cell-survival compared to cells aggregated in a chemically- defined differentiation medium (hereafter, CDM; FIGS. 2A-2H and Table 1). Table 1. Chemically-Defined Differentiation Medium (CDM)

[00056] Following a 2-day incubation in E8 Medium, we transferred aggregates to CDM containing a low concentration of Matrigel and FGF-2 to stimulate epithelization and ectoderm differentiation on the aggregate surface. We previously showed that a combination of BMP4 and the TGFP inhibitor SB-431542 (hereafter, "SB") promoted non-neural induction from mouse PSCs (mPSCs) ⁶ . We found that combining 10 ng/ml BMP4 and 10 μΜ SB (dual SB/BMP4 treatment referred to as "SBB") induces not only non-neural marker genes, such as TFAP2 and DLX3, but also the extraembryonic marker CDX2 (FIG. ID; FIGS. 3A-3G) ¹⁷ . In contrast, SB treatment alone led to an increase in TFAP2 and DLX3 expression with no corresponding CDX2 expression (FIG. ID). Remarkably, 100% of SB treated aggregates generated TFAP2 ⁺ E- cadherin (ECAD) ⁺ epithelium with a surface ectoderm-like morphology by days 4-6 of differentiation— a time scale consistent with human embryogenesis (n = 15 aggregates, 3 experiments; FIGS. IB-IE; FIGS. 3A-3G). Over a period of 20 days, the epithelium expanded into a cyst composed of TFAP2 ⁺ Keratin-5 (KRT5) ⁺ keratinocyte-like cells (FIGS. 6A-6E). From these findings, we concluded that treating WA25 cell aggregates with SB is sufficient to induce a non-neural epithelium.

[00057] Curious whether endogenous BMP activity is sufficient for non-neural specification, we performed a co-treatment with the BMP inhibitor, LDN-193189 (hereafter, LSB; dual LDN/SB treatment referred to as LSB). As previously shown in hESC monolayer cultures ¹⁸ , 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. IF; FIGS. 7A-7D). To further validate our approach, we treated human iPSCs (mND2-0, 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 (FIGS. 8A-8G). 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 (2.5 ng/ml) in addition to SB (SBB) could generate TFAP2 ⁺ ECAD ⁺ non-neural epithelium from m D2-0 iPSCs (FIG. 1G; FIGS. 8A-8G). With either the SB or SBB approaches, the resulting epithelia closely resembled non- epithelia generated with mPSCs ^5,6 . In contrast to our mouse culture, non-neural conversion occurs without off-target induction of Brachyury (BRA) ⁺ mesendoderm cells (FIGS. 9A-9C). The following data were generated using either the SB (WA25) or SBB (m D2-0) approaches.

[00058] Next, we attempted to convert the non-neural epithelium into otic placode epithelium prior to keratinocyte commitment. Human cranial placodes arise at approximately 18-24 dpc; thus, assuming hPSCs represent cells at -12 dpc, otic placodes would develop in our culture within the first 6-12 days of differentiation with proper signaling modulation (FIG. IB).

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 4 aggregates with a combination of FGF-2 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 (FIGS. 1H-1K; FIGS. 10A-10D). When allowed to undergo self-guided differentiation in a minimal medium, we found that SBFL aggregates generated BRN3A ⁺ TUJ1 ⁺ sensory-like neurons between days 10-30 (FIGS. 11A-11F). 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.

[00059] To promote PAX2 expression and vesicle formation, we began testing various signaling modulators (FIGS. 12A-12C). 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 12, reminiscent of the otic placodes in vivo (FIGS. 1L-1N). We suspected that extracellular matrix could provide structural support for vesicle formation; thus, we transferred day 12 aggregates to Matrigel droplets in a minimal media (FIG. 4A). In these cultures, we observed radial production of migratory cells, but no vesicle-like structures or PAX2 ⁺ cells were apparent (FIG. 2B). 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 ⁴ ' ^19'21 . Remarkably, in 90.9 + 5.2% of Matrigel®-embedded aggregates treated with a Wnt signaling agonist, CHIR99021, between days 12-16 (n = 84, 7 experiments), we witnessed epithelial protrusions reminiscent of the otic pits that precede vesicle development in vivo (FIG. 4C). We determined that the otic pit-like structures were PAX2 ⁺ PAX8 ⁺ SOX2 ⁺ SOX10 ⁺ JAG1 ⁺ , confirming otic identity (FIGS. 4D-4H). 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 (FIGS. 4C-4F).

[00060] We cultured the aggregates in stationary droplets until day 18, then transferred them to a 24-well plate on an orbital shaker or a spinner flask for further self-organized maturation— both formats produced comparable results. At 20-30 days in culture, vesicles remained visible through the surface of 71.7 + 23.3% aggregates examined (n = 37, 3 experiments; FIGS. 13A- 13G). In each aggregate we immunostained, we found multiple otic vesicles surrounding a central core epithelium that expressed the basal keratinocyte markers TFAP2 and KRT5 (FIGS. 4G-4I). As late as day 35, we observed vesicles and otic placode-like epithelia that appeared to be partially attached or incorporated into the epidermal epithelium (FIG. 4H). In addition, older vesicles (>30 days) expressed the transcription factor FBX02, which was recently shown to be highly specific to developing inner ear epithelia in mice (FIG. 4I) ²² .

[00061] After 40-60 days of incubation, vesicles with complex multi-chambered

morphologies were visible through the aggregate surface (FIG. 43). Remarkably, we found that a subset of vesicles in both WA25 and m D2-0 derived aggregates developed epithelia containing cells expressing multiple hair cell markers, including MY07A, PCP4, ANXA4, SOX2, and CALB2 (FIGS. 4K-Q; FIGS. 14A-14E). The sensory-like epithelia also contained SOX2 ⁺ SOX10 ⁺ SPARCL1 ⁺ cells, reminiscent of supporting cells in the mammalian utricle ²³ . The luminal cells in these epithelia had elongated morphologies with F-actin-rich apical junctions characteristic of inner ear sensory epithelia (FIGS. 4L-40). 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 (FIGS. 4M-4P, 4R). Together, these findings confirm that the hPSC-derived otic vesicles generate inner ear organoids with sensory epithelia containing hair cells and supporting cells.

[00062] 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. 5A) ⁷ . 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 ATOHl-2A-eGFP cells). Remarkably, as early as day 39, 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 (FIGS. 5B-5D).

Immunostaining with hair cell markers, such as BRN3C and ESPN, confirmed the hair cell identity of eGFP ⁺ cells (FIGS. 5E, 5F). Between days 60-100, 17.4 + 4.0% of aggregates contained at least one hair cell bearing organoid (n = 146, 6 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 mND2-0 aggregates examined between days 60-100 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 = 17, 4 experiments; FIG. 14E). The 2A-eGFP ⁺ hair cells that develop could be maintained for over 150 days in floating culture and retain hair bundle morphology even after dissection and sub-culturing (FIGS. 5B-F).

[00063] Finally, we wondered whether the derived hair cells functioned similar to native mammalian hair cells. Using aggregates produced from ATOHl-2A-eGFP cells, we dissected and flat-mounted inner ear organoids between differentiation days 63-67. 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 (FIGS. 5G, 5J). The K ⁺ current amplitudes at nominal 100 mV were as follows: day 63 : 399, 747, 340 pA; day 64: 4695, 2538, 2609 pA; day 67: 5198, 6528, 6127 pA. This is comparable to the average of 6099 pA for day 22 mouse organoid hair cells. Responses to step and sinusoidal current injection (FIGS. 5H, 51) 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 64: -43, -45; day 67: -48, -49 mV. Possibly relatedly, the prominent sub-threshold inward rectifier current thought to be carried by Kir2.1 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. 5K) ²⁴ ' ²⁵ . It is possible that development, expression, function, or modulation of Kir2.1 is different in human tissue. Importantly, the constricted lumen morphology seen in most >60- days-old organoids and in all of the organoids used for recording, made direct access to the hair bundles for mechanotransduction analysis challenging (FIGS. 5B-5E). Nonetheless, our data strongly suggest that the human organoids, like mouse inner ear organoids ²⁵ , contain immature vestibular hair cells.

[00064] In conclusion, we have established a robust culture system for guiding the development of human inner ear organoids in culture (FIG. 15). 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 ^{6 25} . 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.

[00065] Methods and Materials

[00066] hPSC culture: Human PSCs (WA25 hESCs, passage 22-50; mND2-0 iPSCs, passage 28-46) were cultured in Essential 8 (E8) Medium or Essential 8 Flex Medium (E8f) (Invitrogen) supplemented with 100 μg/ml Normocin (Invivogen) on recombinant human Vitronectin-N (Invitrogen)-coated 6-well plates according to an established protocol ' . At 80% confluency or every 4-5 days, the cells were passaged at a split ratio of 1 : 10-1 :20 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/stem-cell-lines/catalog-of-stem- cell-lines/wa25.cmsx and wicell.org/home/stem-cell-lines/catalog-of-stem-cell-lines/m irjt7i- mnd2-0.cmsx on the World Wide Web. Cell lines were determined to be mycoplasma contamination-free using the MycoAlert Mycoplasma Detection Kit (Lonza).

[00067] hPSC differentiation. To start differentiation, hPSC cells were dissociated with StemPro Accutase (Invitrogen) and distributed, 5,000 cells per well, onto 96-well V-bottom plates in E8 medium containing 20 μΜ Y-27632 (Stemgent) and Normocin. Following a 48 hour incubation, the aggregates were transferred to 96-well U-bottom plates in 100 μΐ of Chemically Defined Medium (CDM) containing 4 ng ml ^-1 FGF-2 (Peprotech), 10 μΜ SB-431542

(Stemgent), and, for some experiments, 2.5 ng ml ^"1 BMP4 (Stemgent), and 2% Growth Factor Reduced (GFR) Matrigel (Corning) to initiate non-neural induction— i.e. differentiation day 0. CDM contained a 50:50 mixture of F-12 Nutrient Mixture with GlutaMAX (Gibco) and Iscove's Modified Dulbecco's Medium with GlutaMAX (IMDM; Gibco) additionally supplemented with 0.5% Bovine Serum Albumin (BSA), IX Chemically Defined Lipid Concentrate (Invitrogen), 7 μg ml ^"1 Insulin (Sigma), 15 μg ml ^"1 Transferrin (Sigma), 450 μΜ Mono-Thioglycerol, and Normocin (see Table 1 for a detailed formulation). After 4 days of incubation, 25 μΐ of CDM containing a 250 ng ml ^"1 FGF-2 (50 ng/ml final concentration) and 1 μΜ LDN-193189 (200 nM final concentration) was added to the pre-existing 100 μΐ of media in each well. After an additional 4 days (8 days total), 25 μΐ of CDM was added to the media. For some experiments, CDM containing a 18 μΜ CHIR99021 (3 μΜ final concentration; Stemgent) was added to the pre-existing 125 μΐ 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 12, the aggregates were pooled together and washed with freshly prepared Organoid Maturation Medium (OMM) containing a 50:50 mixture of Advanced DMEM:F12 (Gibco) and Neurobasal Medium (Gibco) supplemented with 0.5X N2 Supplement (Gibco), 0.5X B27 without Vitamin A (Gibco), IX GlutaMAX (Gibco), 0.1 mM β-Mercaptoethanol (Gibco), and Normocin (see Table 3 for a detailed formulation). Table 3. Organoid Maturation Medium (OMM)

[00068] The formulation set forth in Table 3 provides for 50 mL of medium, which should be used for <2 weeks. OMM is a custom-made hybrid of two media previously used to generate cerebral and gastric organoids ⁶ ' ⁷ . B27 without Vitamin A was used to limit the influence of endogenously produced retinoic acid.

[00069] The aggregates were resuspended in ice cold undiluted GFR Matrigel and placed in approximately 25 μΐ droplets on the surface of a 100 mm bacterial culture plate. After at least 30 minutes of incubation at 37°C, the droplets were bathed in 10 ml of OMM containing 3 μΜ CHIR99021. For non-droplet otic induction, the aggregates were washed and plated individually into each well of a 24-well low cell adhesion plate in OMM containing 3 μΜ CHIR and 1% GFR Matrigel. After 18 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 75 ml of fresh OMM in a 125 ml disposable spinner flask (Corning). Spinner flasks were maintained on an in-incubator stir plate (Thermo Scientific) at 65 RPM for up to 180 days of differentiation. For some experiments, the aggregates were maintained in individual wells of 24-well low-cell adhesion plates in 1 ml of OMM on an in-incubator orbital shaker (Thermo Scientific) for up to 140 days.

[00070] 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, < 0.1 pg ml ^-1 FGF-2, < 0.5 ng ml ^"1 EGF, 5 ng ml ^"1 IGF-1, < 5 pg ml ^"1 PDGF, < 0.2 ng ml ^_1 NGF, and 1.7 ng ml ^"1 TGF . In particular, the TGF in GFR Matrigel may have impacted cell fate specification on day 12 or later because we did not include a TGFP 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 ²⁶ . GFR Matrigel is a more defined alternative to Matrigel, in which the concentration of growth factors, such as Egf, Igfl, Fgf2, and TGF , 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 ²⁷ . 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 ²⁸ .

[00071] Signaling molecules and recombinant proteins. The following small molecules and recombinant proteins were used: recombinant human BMP4 (2.5-10 ng ml ^"1 ; Stemgent), human FGF-2 (25 ng ml ^"1 ; Peprotech), SB-431542 (10 μΜ; Tocris Bioscience), CHIR99021 (3 μΜ; Stemgent), and LDN-193189 (200 nM; Stemgent).

[00072] 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) ⁶ . Data were normalized to L27 expression (internal control) and the fold change was calculated relative to Ct values from dO WA25 aggregates using the AACt method. Unless stated otherwise, data represent at least 3 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 4 for primer details.

Table 4. qPCR Primers

Reverse TTCTTGGGCTTCCCATTCAC (SEQ ID NO:4)

TFAP2 Non-neural Ectoderm

Forward TTTCAGCCATGGACCGTCA (SEQ ID NO:5)

Reverse GGGAGATTGACCTACAGTGC (SEQ ID NO:6)

CDX2 Extraembryonic

Forward GGGCTCTCTGAGAGGCAGGT (SEQ ID NO:7)

Reverse CCTTTGCTCTGCGGTTCTG (SEQ ID NO: 8)

BRA Mesendoderm

Forward TGCTTCCCTGAGACCCAGTT (SEQ ID NO: 9)

Reverse GATCACTTCTTTCCTTTGCATCAAG (SEQ ID

NO: 10)

OCT4 Pluripotency

Forward AGTGAGAGGCAACCTGGAGA (SEQ ID NO: 11)

Reverse ACACTCGGACCACATCCTTC (SEQ ID NO: 12)

NANOG Pluripotency

Forward CATGAGTGTGGATCCAGCTTG (SEQ ID NO: 13)

Reverse CCTGAATAAGCAGATCCATGG (SEQ ID NO: 14)

EOMES Mesendoderm

Forward CAACATAAACGGACTCAATCCCA (SEQ ID NO: 15)

Reverse ACCACCTCTACGAACACATTGT (SEQ ID NO: 16)

[00073] Immunohistochemistry. Aggregates were fixed with 4% paraformaldehyde for 20 min at room temperature or at 4°C overnight. The fixed specimens were cryoprotected with a graded treatment of 15% and 30% sucrose and then embedded in tissue freezing medium. Frozen tissue blocks were sectioned into 12 μπι cryosections on a Leica CM-1860 cryostat. For immunostaining, a 10% goat or horse serum in 0.1% Triton X-100 IX PBS solution was used for blocking, and a 3% goat or horse serum in 0.1% Triton X-100 IX 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-100 concentration was increased to 0.5%, and the blocking and primary/secondary incubations were done at 37°C on a rotating shaker for 24 hours and 48 hours, respectively. Following each incubation, the samples were subjected to three 1-hour washes in IX PBS containing 0.5% Triton X-100 at 37°C on a rotating shaker. Wholemount samples were mounted in ScaleA2 clearing solution for 1-2 days or ScaleSQ(5) clearing solution for 1-2 hours prior to imaging ²⁹ . 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 8 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 = 3.50 μπι; estimated Z diameter = 7.00 μπι; 'Quality' above 20.0;

'distance to image border XYZ' above 0.001 μπι; 'intensity center Ch = above 1,500. Movies were generated in Imaris from the raw image files and compiled in Adobe Premiere Pro to add titles and text. See Table 5 for a list of antibodies.

Table 5. Antibodies

PAX6 Mouse DSHB PAX6 1 :5

ANXA4 Mouse R&D Systems AF4146 1 :50

PCP4 Rabbit Sana Cruz SC74816 1 :50 espin (ESPN) Rabbit Gift from James Bartles Not applicable 1 :50

[00074] Electrophysiological recordings. Human organoids were shipped at day 62 in cold Hibernate A medium supplemented with IX GlutaMax, IX B27 Supplement, and Normocin. They were replaced back into OMM on day 63 in an incubator at 5% CO2 and 37°C. 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 4-5 ΜΩ glass electrodes. Data were acquired using an Axopatch 200B amplifier (Molecular Devices), filtered at 5000 Hz, then digitized at 20 kHz through a Digidata 1322A converter. The recording pipette solution contained (in mM): 135 KC1, 5 HEPES, 5 EGTA, 2.5 MgCk, 2.5 K2- ATP, 0.1 CaCh, adjusted with KOH to pH 7.4, -285 mmol kg ^"1 . The external solution contained: 137 NaCl, 5.8 KC1, 0.7 NaH ₂ P0 ₄ , 10 HEPES, 1.3 CaCl ₂ , 0.9 MgCl ₂ , 5.6 Glucose, and was supplemented with vitamins and essential amino acids (Invitrogen, Carlsbad, CA), adjusted to pH 7.4 with NaOH, -310 mmol kg ^"1 . Recordings were compensated 40% and cells were held at -66 mV for voltage clamp. Averages are reported ± SEM.

[00075] Generation of ATOHl-2A-eGFP reporter cell line. gRNAs (5 ' - TCGGATGAGGC AAGTT AGGA-3 ' (SEQ ID NO: 17) and 5'-

GTC ACTGT AATGGGAATGGG-3 ' (SEQ ID NO: 18), offset = Obp) targeting the stop codon region of ATOH1 were cloned into pSpCas9n(BB) vectors which express Cas9n under the control of CBh promoter (Addgene #48873) ³⁰ . To construct the donor vector, a 2A-eGFP-PGK- Puro cassette (Addgene #31938) ¹⁷ flanked by two lkb 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 #41816) ¹⁸ were transfected into WA25 hESCs with 4D Nucleofector (Lonza) using the P3 Primary Cell 4D-Nucleofector X kit and Program CB-150. After nucleofection, cells were plated in growth medium containing IX RevitaCell (Thermo Fisher) for improved cell survival rate, and Ι μΜ of Scr7 (Xcessbio) for higher HDR efficiency ³¹ . 0.5 μg μΐ ^"1 puromycin selection was performed for 10 days starting from 48h 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

#13775). Clonal cell lines were established by low-density seeding (1 - 3 cells cm ^"2 ) of dissociated single hESCs followed by isolation of hESC colonies after 5-7 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.

[00076] Statistical analysis. All statistics were performed using GraphPad Prism 7 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.

[00077] Representative Data and Reproducibility. Unless stated otherwise, images are representative of specimens obtained from at least 3 separate experiments. For IHC analysis of aggregates between days 0-12, we typically sectioned 3-6 aggregates from each condition in each experiment. IHC analyses for later stages of the protocol were performed on at least 2 aggregates from each condition per experiment. The finalized culture method was successfully replicated 15 times by four independent investigators using the WA25 (wild-type or ATOHl-2A-eGFP) cell line. The method, with noted modifications, was replicated 3 times using the mND2-0 iPSC line. A replication was deemed successful by confirming pit/vesicle formation during days 12-18 and positively identifying inner ear organoids in at least one aggregate on days 50-100 of

differentiation. Experiments were excluded from analysis if no pits were observed during days 12-18.

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Identification and characterization of mouse otic sensory lineage genes. Frontiers in cellular neuroscience 9, 79 (2015).

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[00079] Example 2 - Modeling Otic Neurogenesis

[00080] In day-12 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 12-day aggregates with a potent GSK3P inhibitor and known Wnt signaling agonist27, CHIR99021 (CHIR), between days 12-18. Under these conditions, PAX8+ PAX2+ SOX10+ vesicles evaginate from the outer epithelium (-10-30 vesicles per aggregate; see FIGS. 4A-4R). Remarkably, Isletl+ (ISL1+) neuroblasts appear to delaminate from the otic vesicles (FIGS. 16A-16B). We also see neuroblasts in DMSO-treated aggregates and in the non-otic interior of CHIR-treated aggregates (FIG. 16A, 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. 4A). In support of this hypothesis, we confirmed that the neurogenic factors Neurogl (NGN1), a marker of otic neurons, and Neurog2 (NGN2), a marker of epibranchial neurons, are expressed in CHIR-treated aggregates (FIG. 16C)

[00081] After 12 days of differentiation, otic induced aggregates were plated on bacterial dishes in Matrigel droplets in medium containing 3 μΜ CHIR. See FIGS. 17A-17C. On day 22, the aggregates were fixed and immunostained with antibodies for markers of sensory neurons, BRN3A and BIII-Tubulin (TUJl). Radially oriented BRN3A+ TUJ1+ neurons produced outgrowing processes with growth cones, confirming widespread sensory neurogenesis in the organoid cultures.

[00082] As further evidence of inner ear organogenesis in our culture system, the hair cells displayed CTBP2 ⁺ puncta by day 60-70 of culture, indicating putative ribbon synapse-like structures (FIGS. 18A-18B; n = l 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. 18C). Together, our findings suggest that the human inner ear organoid model may recapitulate assembly of the sensorineural circuit between hair cells and sensory neurons.

[00083] Example 3 - Exemplary Protocol for In Vitro Production of Inner Ear Organoids

[00084] 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-4 (BMP4) and a transforming growth factor beta (TGFP) inhibitor such as the small molecule SB-431542 ("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-2 approximately 24 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.

[00085] Methods [00086] ES cell culture in E8 medium on Vitronectin-coated plates (steps 1-7). We maintain our hPS cells in E8 Medium under feeder-free conditions and use EDTA for passaging (see previous protocol by Beers et al.)l We prefer this method of hPS cell maintenance because, in our hands, it reduces spontaneous differentiation with limited time and effort.

[00087] Non-neural ectoderm and pre-otic induction (steps 8-27). To start differentiation, hPS cells are dissociated and distributed, 5,000 cells per well, onto 96-well V-bottom plates. For this initial aggregation step we use E8 medium containing Y-27632, 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 TGFB to induce ectoderm development. Endogenous BMP signaling generates non-neural rather than neural ectoderm. FGF-2 and LDN treatment induces pre-placodal development. By day 8 the outer epithelium begins to express PAX8, indicating an oticepibranchial placode (OEPD)-like character. CHIR treatment on day 8 induces small patches of PAX2+ cells, indicating the earliest signs of otic placode development by day 12.

[00088] Otic prosensory vesicle and inner ear organoid formation (steps 28-43). 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 12 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 12-18. Otic vesicle formation was observed in >95% of the aggregates (>100 aggregates) across four independent experiments. Additionally, between days 12-18, 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. [00089] On day 18, 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 22 days, 10-30 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 22- 35, 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 35-40, 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 45, vesicles have developed MY07A ⁺ hair celllike cells and were identified as inner ear organoids. Between days 60-80, we observed MY07A ⁺ hair cells with F-actin-rich and Espin ⁺ hair bundles, indicating a definitive hair cell identity.

[00090] Materials and Reagents

[00091] hPSC culture: Human PSCs (WA25 hESCs, passage 22-50; m D2-0 iPSCs, passage 28-46) were cultured in Essential 8 (E8) Medium or Essential 8 Flex Medium (E8f) (Invitrogen) supplemented with 100 μg/ml Normocin (Invivogen) on recombinant human Vitronectin-N (Invitrogen)-coated 6-well plates according to an established protocol ^{12 13} . At 80% confluency or every 4-5 days, the cells were passaged at a split ratio of 1 : 10-1 :20 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/stem-cell-lines/catalog-of-stem- cell-lines/wa25.cmsx and wicell.org/home/stem-cell-lines/catalog-of-stem-cell-lines/m irjt7i- mnd2-0.cmsx on the World Wide Web. Cell lines were determined to be mycoplasma contamination-free using the MycoAlert Mycoplasma Detection Kit (Lonza).

[00092] Other reagents used in this exemplary protocol are set forth in Table 6.

Table 6. REAGENTS

Ham's F-12 Nutrient Mixture (Gibco, cat. no. 31765-035)

Iscove's Modified Dulbecco's Medium (IMDM) (Gibco, cat. no. 31980-030)

Advanced DMEM/F12 (Gibco, cat. no. 12491-015)

2-Mercaptoethanol (Gibco, cat. no. 21985-023)

Normocin (Invivogen, cat. no. ant-nr-1) N2 Supplement (Gibco, cat. no. 17502-048)

GlutaMax (Gibco, cat. no. 35050-079)

Geltrex (Invitrogen, cat. no. A1413302)

Vitronectin (Invitrogen, cat. no. A 14700)

Chemically-defined lipid concentrate (Invitrogen, cat. no. 11905-031)

Albumin from bovine serum (BSA; Sigma, cat. no. A 1470)

Insulin solution (Sigma, cat. no. 19278)

Human Transferrin (Sigma, cat. no. T8158)

1-thioglycerol (Sigma, cat. no. M1753)

Human BMP-7 (Peprotech, cat. no. 120-03B)

Human FGF-3 (Biolegend, cat. no. 558002)

FGF-2 (Peprotech, cat. no. 100-18B)

SB-431542 in solution (Stemgent, cat. no. 04-0010-05)

LDN-193189 in solution (Stemgent, cat. no. 04-0074-02), a light sensitive solution. Store in the dark at -20°C per the manufacture's recommendation.

Neurobasal Medium (Gibco, cat. no. 21103-049)

B27 Supplement minus Vitamin A (Gibco, cat. no. 12587-010)

CHIR99021 in solution (Stemgent, cat. no. 04-0004-02)

Y-27632 in solution (Stemgent, cat. no. 04-0012-02)

TrypLE Express (Gibco, cat. no. 12605010)

UltraPure 0.5 M EDTA, pH 8.0 (Invitrogen, cat. no. 15575-020)

Dulbecco's Phosphate Buffered Saline (DPBS; Gibco, cat. no. 12605010)

Dimethyl Sulfoxide (DMSO; Sigma, cat. no. D8418)

Paraformaldehyde (PFA, Electron Microscopy Sciences, cat. no. 15710)

Normal goat serum (Vector Laboratories, cat. no. S-1000)

Triton X-100 (Sigma, cat. no. T8787)

Antibodies required for desired characterization

[00093] Culture Medium and Stock Solutions

[00094] Human recombinant BMP7 stock solution (100 ng^L): In the biosafety cabinet, add 100 [iL of sterile 4 mM HCl to 10 μg of BMP7; vortex the solution and spin down in a tabletop centrifuge. Store BMP4 solution in 5 μL aliquots at -20°C for 6 months or at -80°C for 1 year. [00095] Human recombinant FGF-2 stock solution (200 ng/[iL): In the biosafety cabinet, add 250 [iL of sterile PBS or 5 mM Tris (pH 7.6) to 50 μg of FGF-2; vortex the solution and spin down in a tabletop centrifuge. Store FGF-2 solution in 6 μΐ _^ aliquots at -20°C for 6 months or at - 80°C for 1 year.

[00096] Human transferrin stock solution (20 mg/ml): In the biosafety cabinet, dissolve 100 mg of recombinant human transferrin in 5 ml of IMDM. To fully dissolve, vortex the tube and place it on a rotating shaker for 5-10 min at room temperature (RT). Store the transferrin solution in 150 μΐ aliquots at -20°C for 6 months or at -80°C for 1 year.

[00097] EDTA Solution (for passaging hES cells): In the biosafety cabinet, mix 50 μΐ of 0.5 M EDTA into 50 ml DPBS. Filter sterilize the solution. EDTA solution can be stored at RT for 6 months.

[00098] Chemically Defined Medium (CDM): To prepare 200 mL CDM, measure out 1 g BSA in a 250 mL bottle. Dissolve the BSA in 100 mL F-12 Nutrient Mixture +GlutaMAX, 100 mL IMDM +GlutaMAX, 2 mL chemically-defined lipid concentrate, 140 μΕ Insulin, 150 μΕ Transferrin, and 8 μΕ 1-thioglycerol. Sterile filter using a low-protein binding filter. CDM should be used for up to 2 weeks and stored at 4°C. Add Normocin to the media just before use at a dilution of 2 μΐ per 1 mL of CDM. See Table 1 for a quick reference recipe.

[00099] Differentiation CDM: In a 50 mL conical tube, add 450 μΕ of ice cold Geltrex to 34.5 mL of ice cold CDM (1.25% final concentration). Vortex the tube well to fully dissolve the Geltrex. Place 30 mL of this solution in a new 50 mL conical tube. Add 0.6 μΐ _^ of FGF-2 (4 ng/mL final concentration) and 30 μΐ _^ of SB-431542 (10 μΜ final concentration). Vortex the tube well to mix. Differentiation CDM should be made fresh on day 0 of differentiation. Use the remaining 5 mL of CDM +Geltrex to wash the aggregates before plating.

[000100] Organoid Maturation Medium (OMM): To prepare 50 mL OMM, combine 24.5 mL Advanced DMEM/F12, 24.5 mL Neurobasal Medium, 500 μΕ B-27 Supplement without vitamin A, 500 GlutaMAX, 250 μΕ N2-supplement, and 100 μΕ Normocin in a sterile 50 mL conical tube. OMM can be used for up to 2 weeks if stored at 4°C. See Table 3 for a quick reference recipe.

[000101] Procedures

[000102] hES cell maintenance and passaging: 1. In the biosafety cabinet, dilute 60 [iL of Vitronectin solution in 6 mL of DPBS. Add 1 mL of diluted vitronectin to each well of a 6-well culture plate. Incubate at RT for at least 1 hr and in the mean time proceed to step 2.

2. Equilibrate at least 20 mL of E8 Medium containing Normocin to RT.

3. To begin dissociation of the 70-80% confluent hES cells, aspirate the spent E8 Medium from one well and wash twice with DPBS.

4. Add 1 mL of EDTA solution and incubate in a 37°C incubator for 4-8 min. Under a microscope, confirm that holes have formed in the center of the colonies.

5. During the EDTA incubation, remove the vitronectin solution from the 6-well plate and quickly add 1.5 mL of E8 Medium to each well before the surface dries.

6. Collect the dissociated cells into 6 mL of E8 Medium. CRITICAL You may need to vigorously triturate the medium to remove the cell clusters from the plate. Do not over pipet the cells, as this will cause apoptosis. Care should be taken to not introduce air bubbles into the medium.

7. Plate the cells on the prepared 6-well plate (from step 5). Select plating density based on when you next wish to split the cells. We typically add 500 mL (passage in 2-3 days) or 250 [iL (passage in 4-5 days) of cell suspension to each well. Incubate cells at 37°C in 5.0% CO2 until ready for passage or use in an experiment.

[000103] hES cell differentiation (day -2 to day 0): aggregation:

8. In the biosafety cabinet, prepare conical tubes containing 22 mL and 10 mL of E8 Medium with Normocin. Add 44 μΕ of Y-27632 to the 22 mL tube (20 μΜ final concentration; hereafter, E8-Y20) and 10 μΕ of Y-27632 to the 10 mL tube (10 μΜ final concentration;

hereafter, E8-Y10).

9. When cells are 70-80% confluent, aspirate the E8 Medium from one well and wash three times with DPBS at RT.

10. Add 350 μΕ of TrypLE and incubate at 37°C for 2-4 min. Following incubation, shake the plate horizontally to break up the cells. Under a microscope, confirm that the cells have rounded and are detached from the surface of the plate.

11. Collect the dissociated cells into 5 mL of E8-Y10 Medium and transfer them into a 15 mL conical tube. 12. Break the cell clumps into single-cells by pipetting with a P1000 tip. Pellet the cells, by centrifugation at 200 g for 5 minutes.

13. Completely remove the supernatant and resuspend the cell pellet in 1 mL of E8-Y10 Medium.

14. Forcefully pipet 1 mL of E8-Y10 Medium through a cell-strainer-top test tube to prime the strainer. Pipet the 1 mL of hES cell suspension drop-wise onto the cell strainer. Next, pipet 1 mL of E8-Y10 Medium drop-wise onto the cell strainer. There should be 3 mL in the test tube. This step is important for ensuring that the cells are completely dissociated into single cells.

15. Mix the cell suspension by pipetting with a PI 000 tip and determine the concentration of cells using a hemocytometer or automated cell counter.

16. Dilute the appropriate volume of cell suspension in the 22 mL of E8-Y20 Medium to acquire a final concentration of 50,000 cells per mL (i.e., l . lxlO ⁶ total cells). For example, if the cell suspension contains lxlO ⁶ cells per mL, dilute 1.1 mL of cell suspension (l . lxlO ⁶ divided by lxlO ⁶ ) in 20.9 mL of E8-Y20 Medium. Invert several times to mix.

17. Pour the cell suspension into a reservoir and aliquot 100 μΐ of cell suspension into each well of two 96-well V-bottom plates using a multichannel pipette.

18. Spin down the plates at 120 g for 5 min at RT.

19. Place the plates in a 37°C incubator with 5.0% C02 for 48 hrs. Add 50 μΐ of fresh E8 Medium (not containing Y-27632) to each well after 24 hrs.

[000104] Differentiation day 0: transfer to differentiation CDM:

20. Prepare 30 ml of Differentiation CDM containing Geltrex, FGF-2, and SB-431542. Allow the media to equilibrate to RT.

21. With the multichannel pipette set to 125 μΐ (i.e. 25 μΐ less than the volume of each well), carefully the aggregates from each well of the 96-well plate from step 18. Deposit the aggregates in a bacterial dish and then transfer them to a 2-ml tube.

22. Wash the aggregates at least 3 times with CDM to completely remove traces of E8 Medium.

23. Resuspend the aggregates in differentiation CDM and transfer them to a new bacterial dish.

24. Using a P200 pipette, individually transfer each aggregate to each well of two 96-well U-bottom plates in 100 μΐ of media. It may be difficult to see the aggregates at this stage. We typically place the bacterial dish on a non-reflective white surface (e.g., a Kimwipe) to enhance the contrast of the aggregates.

25. Return plates to the incubator for 4 days. Observe the morphology of the aggregates daily.

[000105] Differentiation day 4: addition of FGF-2 and LDN-193189 (FGF/LDN) :

26. In a 15-ml conical tube, add FGF-2 and LDN-193189 to the CDM at a 5X

concentration. For example, we add 6.25 μΐ of 200 ng/μΐ FGF-2 and 0.5 iL of 10 mM LDN- 193189 to 5 ml of CDM.

27. Add 25 μΐ of CDM containing FGF/LDN to each well of the plate from step 24. Each well now contains 125 μΐ of medium with a final concentration of 50 ng/ml FGF-2 and 200 nM LDN-193189. Incubate cells for a further 4 days.

28. In a 15-ml conical tube, add CHIR99021 to the CDM at a 6X concentration. For example, we add 9 μΐ of 10 mM CHIR99021 to 5 ml of CDM.

29. Add 25 μΐ of CDM containing CHIR to each well of the plate from step 26. Each well now contains 125 μΐ of medium with a final concentration of 3 μΜ CHIR. Incubate cells for a further 4 days.

[000106] Differentiation day 12: transition to static ECM culture:

30. About 2 hrs prior to performing the following steps, remove a 1-ml aliquot of Geltrex from the freezer and allow it to thaw completely on ice or in the refrigerator.

31. Prepare 50 ml of organoid medium in a 50-ml conical tube.

32. Using a cut PI 000 pipette tip, transfer the aggregates from each well of the plate in step 28 into a 2-ml tube.

33. Allow the aggregates to settle at the bottom of the tube and then carefully aspirate the media.

34. Wash the aggregates with 1-2 ml of RT organoid medium at least 3 times. Remove as much of the medium as possible following the final wash.

35. Resuspend the aggregates in ice-cold Geltrex. Set the pipette to 20 μΐ and individually transfer the aggregates to a 100 mm bacterial dish. We typically add 20-30 droplets to each plate. Gentle shaking the plate in different directions helps to flatten and adhere the droplets to the surface of the plate. Geltrex will begin to polymerize after 10-15 minutes at RT. It is recommended to keep a small container of ice in biosafety cabinet where the aggregate/Geltrex mixture can be placed between pipetting steps.

36. Incubate the plates without medium for 30 minutes at 37°C.

37. Add 10-12 ml of organoid medium containing CHIR. Add the orgnaoid medium slowly as to not detach the droplets from the plate. It is fine if some droplets may detach.

38. Incubate cells for a further 6 days. Perform a half media change after 3 days.

[000107] Differentiation day 18: transition to spinner flask:

39. Using a wide-mouth P1000 tip, scrape-off and pipette-up each aggregate from the surface of the bacterial dish. Transfer the aggregates to a 2-ml tube.

40. Wash the aggregates 3 times with organoid medium.

41. Pour 50-75 ml of organoid medium into a 125-ml spinner flask.

42. Add the aggregates to the spinner flask. Confirm that the side ports are opened by a ¼ turn and place the spinner flask in the incubator on the stirrer plate. Set the stirrer controller at 60-65 rpm.

43. Incubate cells for a further 22-42 days. Completely change the medium every week. For longer experiments, aggregates can be maintained in the spinner flask indefinitely. To monitor development, we periodically transfer the aggregates to a bacterial dish using wide- mouth PI 000 and image using an inverted microscope.

[000108] Timing:

Steps 1-7, Maintenance and passaging hES cells: 30 min

Steps 8-19, hES cell differentiation: Aggregation: 40 min

Steps 20-25, Differentiation day 0: Transfer to Differentiation CDM: 1 hr

Steps 26-27, Differentiation day 4: Addition of FGF-2 and LDN-193189: 20 min

Steps 28-29, Differentiation day 8: Addition of CHIR99021 : 20 min

Steps 30-38, Differentiation day 12: Transition to static ECM culture: 45 min

Steps 39-43, Differentiation day 18: Transition to bioreactor: 30 min

Steps 1-43, Total time to generate inner ear organoids: 45-60 days to >1 yr