US20190093072A1 - Methods of generating human inner ear sensory epithelia and sensory neurons - Google Patents

Methods of generating human inner ear sensory epithelia and sensory neurons Download PDF

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US20190093072A1
US20190093072A1 US15/769,254 US201615769254A US2019093072A1 US 20190093072 A1 US20190093072 A1 US 20190093072A1 US 201615769254 A US201615769254 A US 201615769254A US 2019093072 A1 US2019093072 A1 US 2019093072A1
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Karl R. Koehler
Eri Hashino
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Indiana University Research and Technology Corp
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Definitions

  • kits 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.
  • a method of obtaining human pre-otic epithelial cells 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 (TGF ⁇ ) 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.
  • BMP Bone Morphogenetic Protein
  • TGF ⁇ Transforming Growth Factor Beta
  • 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 TGF ⁇ 1-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).
  • a method of obtaining a three-dimensional composition comprising human inner ear sensory tissue comprising the steps of (a) embedding human pre-otic epithelial cells obtained according to the method of claim 1 in a semi-solid 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.
  • 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 ⁇ M SB, or 10 ⁇ M SB+10 ng/ml BMP4, denoted as SBB.
  • e, f Representative TFAP2, ECAD, and PAX6 expression in WA25 aggregate treated with 10 ⁇ M SB or with 200 nM LDN+10 ⁇ M SB for 6 days.
  • h, i Representative image of a SB-treated WA25 aggregate on day 8: live (h) and immunostained with PAX8 and TFAP2 antibodies (i).
  • 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). l-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 ⁇ l of additional CDM on day 8. Scale bars, 100 ⁇ m (e-m), 50 ⁇ m (n).
  • 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 ⁇ M Y-27632 produced less cellular debris than aggregation in CDM+20 ⁇ M 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.
  • 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 ⁇ m.
  • 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 FBXO2.
  • the epithelium from which vesicles arise begins to express the epidermal keratinocyte marker KRTS 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 ⁇ m 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′′).
  • Hair cells are MYO7A + 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, mND2-0 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 SPARCL1 (q).
  • Hair cells in organoids have ESPN + hair bundles with a single acetylated-tubulin (TUBA4A) + kinocilium.
  • Scale bars 200 ⁇ m (j), 100 ⁇ m (b, c, e), 50 ⁇ m (g, h, k), 25 ⁇ m (d, f, l, m, p), 10 ⁇ m (n, q), 5 ⁇ m (r), 2.5 ⁇ m (o).
  • FIGS. 5A-5K demonstrate that hESC-derived hair cells have similar electrophysiological properties to those of native hair cells.
  • a ATOH1-2A-eGFP CRISPR design.
  • the two guide RNAs blue, with PAM sequence in red
  • 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 1kb left and right homology arms (LHA and RHA).
  • LHA and RHA left and right homology arms
  • the LoxP-flanked PGK-Puro sub-cassette is subsequently removed by Cre recombinase.
  • 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.
  • 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.
  • Human organoid hair cells displayed prominent outward currents (d64).
  • 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.
  • 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+ KRTS+ cells, indicative of epidermal keratinocytes. Scale bars, 250 ⁇ m (b-d), 100 ⁇ m (e), 5 ⁇ m (e′).
  • 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. 1E ) do appear in the core of SB treated aggregates. Scale bars, 50 ⁇ m.
  • 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.
  • FIGS. 9A-9C demonstrate that non-neural ectoderm induction occurs without off-target induction of mesendodermal cells.
  • 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.
  • 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 ⁇ m.
  • FIGS. 11A-11F demonstrate that OEPD induced aggregates spontaneously generate sensory-like neurons in a minimal medium floating culture.
  • SBFL-treated aggregates were transferred to OMM on day 8 of differentiation.
  • the aggregates are composed of patches of BRN3A+ TUJ1+ HUC+ neurons surrounding a ECAD+ epithelium. Neuronal patches were typically associated with PAX8+ epithelium.
  • aggregates were plated in MatrigelTM droplets they produced neurite outgrowths (e).
  • BRN3A+ 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 ⁇ m (e).
  • 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)).
  • the WNT inhibitor, XAV939, and WNT agonist, CHIR99021 only had a modest positive or negative impact on PAX2 expression compared to a DMSO controls.
  • 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.
  • KRTS expression is restricted to the epidermis, whereas ECAD 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, FBXO2, JAG1, and ECAD.
  • d-g A day 35 aggregate that was wholemount immunostained for ECAD to reveal the epidermal core and surrounding otic vesicles. Scale bars, 250 ⁇ m (d, f), 100 ⁇ m (a, b), 25 ⁇ m (c, g).
  • 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 .
  • 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 ⁇ m (a, d, e), 25 ⁇ m (b, c), 10 ⁇ tm (f).
  • 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 TUJ1 staining throughout.
  • FIGS. 17A-17C demonstrate sensory neuron outgrowth and maturation.
  • HIR-treated organoids plated on Matrigel produce TUJ1+ BRN3A+ neurons with a bipolar morphologies. Growth cone (inset in b). Scale, 500 (a), 50 (b), 10 (c).
  • 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-positive hair cells on day 60 of differentiation.
  • C Representative image of neurons innervating an inner ear organoid sensory epithelium. Scale bars, 100 ⁇ m (C), 25 ⁇ m (A, B).
  • FIG. 19 depicts the ATOH1-2A-eGFP-PGK-Puro donor plasmid sequence (SEQ ID NO:21).
  • FIG. 20 depicts the genomic sequence of the homozygous/bi-allelic ATOH1-2A-eGFP cell line at the ATOH1 locus (SEQ ID NO:22).
  • 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.
  • 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.
  • 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.
  • 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.
  • methods of the present invention are useful for identifying materials and combinatorial strategies for human tissue engineering.
  • 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.
  • 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.
  • 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).
  • 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 (TGF ⁇ ) 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.
  • aggregates comprising a core of mesodermal cells are cultured in the presence of BMP4 and an inhibitor of TGF ⁇ 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 ALKS, ALK4, and ALK7.
  • 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.
  • FGF Fibroblast Growth Factor
  • BMP bone morphogenetic protein
  • OEPD otic-epibranchial progenitor domain
  • 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.
  • aggregates comprising pre-otic epithelium are embedded in a semi-solid culture medium such as, for example, a semi-solid composition of extracellular matrix proteins.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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, mTeSRTM (StemCell Technologies; Vancouver, British Columbia, Canada), and TeSRTM.
  • DMEM Dulbecco's Modified Eagle's Medium
  • mTeSRTM StemTeSRTM
  • TeSRTM TeSRTM
  • DMEM formulations suitable for use herein include, e.g., X-Vivo (BioWhittaker, Walkersville, Md.) and StemPro® (Invitrogen; Carlsbad, Calif.).
  • ROCK Rho kinase
  • ROCK inhibitors such as ROCK inhibitors
  • 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-1-[(4-methyl-5-isoquinolinyl)sulfonyl]homopiperazine dihydrochloride (informal name: H-1152), 1-(5-isoquinolinesulfonyl)piperazine hydrochloride (informal name: HA-100), 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (informal name: H-7), 1-(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-cinnamylamin
  • 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 ⁇ M to about 10 ⁇ M can be suitable.
  • undifferentiated cells typically detach, while differentiated cells remain attached to the defined surface.
  • 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.
  • TGF ⁇ -1 and Activin A are agonists of TGF ⁇ signaling, and TGF ⁇ signaling can be antagonized using, for example, the small molecule inhibitor SB-431542.
  • CHIR-99021 is an agonist of the Wnt/ ⁇ -catenin signaling pathway, and Wnt/ ⁇ -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).
  • 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.
  • 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.
  • human pluripotent stem cells are cultured in a chemically-defined basal culture medium formulation comprising the defined components of culture medium “DF3 S” 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.
  • 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 ⁇ g/mL), transferrin (10.67 ng/mL) and human Fibroblast Growth Factor 2 (FGF2) (100 ng/mL).
  • 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 ⁇ g/mL), transferrin (10.67 ng/mL), human FGF2 (100 ng/mL), and human TGF ⁇ 1 (Transforming Growth Factor Beta 1) (1.75 ng/mL).
  • any appropriate method can be used to detect expression of biological markers characteristic of cell types described herein.
  • 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.
  • 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.
  • 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).
  • pluripotency markers such as NANOG and OCT4 (relative to human ES cells or induced pluripotent stem cells).
  • 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).
  • ESCs are commercially available from sources such as WiCell Research Institute (Madison, Wis.).
  • 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).
  • 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.
  • 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).
  • pluripotent stem cells are not immediately derived from embryos.
  • 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, Yu et al., Science 324(5928):797-801 (2009); Chen et al., Nat.
  • 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.
  • subject-specific cells are manipulated in vitro prior to use in a three-dimensional tissue construct of the invention.
  • 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.
  • human pluripotent stem cells e.g., human ESCs or iPS cells
  • a feeder layer e.g., a fibroblast layer
  • a conditioned medium e.g., a conditioned medium
  • a culture medium comprising poorly defined or undefined components.
  • 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.
  • 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.
  • 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.
  • a medium consisting essentially of means a medium that contains the specified ingredients and those that do not materially affect its basic characteristics.
  • an effective amount means an amount of an agent sufficient to evoke a specified cellular effect according to the present invention.
  • 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 .
  • PSCs mouse pluripotent stem cells
  • 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, 1B ).
  • 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 ).
  • CDM Chemically-Defined Differentiation Medium
  • LSB dual LDN/SB treatment referred to as LSB.
  • 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. 1F ; FIGS. 7A-7D ).
  • SBFL treatment may be sufficient to induce epibranchial neurons, yet fails to initiate otic induction.
  • FIGS. 12A-12C 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 ).
  • FIGS. 4D-4H 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 ).
  • vesicles with complex multi-chambered morphologies were visible through the aggregate surface ( FIG. 4J ).
  • a subset of vesicles in both WA25 and mND2-0 derived aggregates developed epithelia containing cells expressing multiple hair cell markers, including MYO7A, 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-4O ).
  • 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 ).
  • FIGS. 5B-5D Immunostaining with hair cell markers, such as BRN3C and ESPN, confirmed the hair cell identity of eGFP + cells ( FIGS. 5E, 5F ).
  • hair cell markers such as BRN3C and ESPN
  • 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.
  • 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, 5I ) 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.
  • 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 .
  • 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 (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 ⁇ g/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.
  • 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 ⁇ M Y-27632 (Stemgent) and Normocin.
  • CDM Chemically Defined Medium
  • Peprotech 4 ng m1 ⁇ 1 FGF-2
  • 10 ⁇ M SB-431542 10 ⁇ M SB-431542
  • 2.5 ng ml ⁇ 1 BMP4 2 ng ml ⁇ 1 BMP4
  • GFR Growth Factor Reduced
  • 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), 1 ⁇ Chemically Defined Lipid Concentrate (Invitrogen), 7 ⁇ g ml ⁇ 1 Insulin (Sigma), 15 ⁇ g ml ⁇ 1 Transferrin (Sigma), 450 ⁇ M Mono-Thioglycerol, and Normocin (see Table 1 for a detailed formulation).
  • GlutaMAX GlutaMAX
  • IMDM Iscove's Modified Dulbecco's Medium with GlutaMAX
  • BSA Bovine Serum Albumin
  • Lipid Concentrate Invitrogen
  • 7 ⁇ g ml ⁇ 1 Insulin Sigma
  • 15 ⁇ g ml ⁇ 1 Transferrin Sigma
  • CDM containing a 250 ng ml ⁇ 1 FGF-2 (50 ng/ml final concentration) and 1 ⁇ M LDN-193189 (200 nM final concentration) was added to the pre-existing 100 ⁇ l of media in each well. After an additional 4 days (8 days total), 25 ⁇ l of CDM was added to the media.
  • CDM containing a 18 ⁇ M CHIR99021 (3 ⁇ M final concentration; Stemgent) was added to the pre-existing 125 ⁇ l 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.
  • Organoid Maturation Medium containing a 50:50 mixture of Advanced DMEM:F12 (Gibco) and Neurobasal Medium (Gibco) supplemented with 0.5 ⁇ N2 Supplement (Gibco), 0.5 ⁇ B27 without Vitamin A (Gibco), 1 ⁇ GlutaMAX (Gibco), 0.1 mM B-Mercaptoethanol (Gibco), and Normocin (see Table 3 for a detailed formulation).
  • OMNI 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.
  • the aggregates were resuspended in ice cold undiluted GFR Matrigel and placed in approximately 25 ⁇ l 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 OMNI containing 3 ⁇ M 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 OMNI containing 3 ⁇ M 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.
  • GFR 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 ⁇ .
  • the TGF ⁇ in GFR Matrigel may have impacted cell fate specification on day 12 or later because we did not include a TGF ⁇ 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, Igf1, Fgf2, and TGF ⁇ , have been minimized to levels that should have a negligible effect on cell fate specification.
  • 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.
  • a purified Laminin/Entactin complex may be a suitable, fully chemically defined alternative 27 .
  • 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 .
  • 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 ⁇ M; Tocris Bioscience), CHIR99021 (3 ⁇ M; Stemgent), and LDN-193189 (200 nM; 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) 6 . Data were normalized to L27 expression (internal control) and the fold change was calculated relative to Ct values from d0 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.
  • Alexa Fluor conjugated anti-mouse, rabbit, or goat IgG were used as secondary antibodies.
  • Prolong Gold with DAPI was used to mount the samples and visualize cellular nuclei.
  • DAPI Thermo Scientific
  • 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 1 ⁇ PBS containing 0.5% Triton X-100 at 37° C. on a rotating shaker.
  • Electrophysiological recordings Human organoids were shipped at day 62 in cold Hibernate A medium supplemented with 1 ⁇ GlutaMax, 1 ⁇ B27 Supplement, and Normocin. They were replaced back into OMM on day 63 in an incubator at 5% CO 2 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 M ⁇ 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 MgCl 2 , 2.5 K 2 -ATP, 0.1 CaCl 2 , adjusted with KOH to pH 7.4, ⁇ 285 mmol kg ⁇ 1 .
  • the external solution contained: 137 NaCl, 5.8 KCl, 0.7 NaH 2 PO 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, Calif.), 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.
  • a 2A-eGFP-PGK-Puro cassette (Addgene #31938) 17 flanked by two 1 kb 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.
  • 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.
  • images are representative of specimens obtained from at least 3 separate experiments.
  • 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 ATOH1-2A-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.
  • Islet1+ (ISL1+) neuroblasts appear to delaminate from the otic vesicles ( FIGS. 16A-16B ).
  • FIGS. 16A-16B We also see neuroblasts in DMSO-treated aggregates and in the non-otic interior of CHIR-treated aggregates ( FIG. 16A , arrowheads).
  • CHIR-treated aggregates yield a mixture of otic (i.e. vesicle-derived) and epibranchial (i.e. epithelium-derived) neurons (see FIG. 4A ).
  • NNN1 neurogenic factors Neurogl
  • NNN2 Neurog2
  • otic induced aggregates were plated on bacterial dishes in Matrigel droplets in medium containing 3 ⁇ M CHIR. See FIGS. 17A-17C .
  • the aggregates were fixed and immunostained with antibodies for markers of sensory neurons, BRN3A and ⁇ III-Tubulin (TUJ1).
  • BRN3A+ TUJ1+ neurons produced outgrowing processes with growth cones, confirming widespread sensory neurogenesis in the organoid cultures.
  • This example describes a protocol for inducing formation of non-neural ectoderm and inner ear sensory tissue from human pluripotent stem cells.
  • 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.
  • BMP4 bone morphogenetic protein-4
  • TGF ⁇ transforming growth factor beta
  • FGF fibroblast growth factor
  • ES cell culture in E8 medium on Vitronectin-coated plates (steps 1-7).
  • 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 8-27.
  • hPS cells are dissociated and distributed, 5,000 cells per well, onto 96-well V-bottom plates.
  • 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.
  • 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.
  • the outer epithelium begins to express PAX8, indicating an oticepibranchial placode (OEPD)-like character.
  • OEPD oticepibranchial placode
  • CHIR treatment on day 8 induces small patches of PAX2+ cells, indicating the earliest signs of otic placode development by day 12.
  • Otic prosensory vesicle and inner ear organoid formation steps 28-43.
  • a low concentration of extracellular matrix proteins e.g., MatrigelTM
  • MatrigelTM extracellular matrix proteins
  • 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 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.
  • 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.
  • 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.
  • 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.
  • day 45 vesicles have developed MYO7A + hair cell-like cells and were identified as inner ear organoids.
  • days 60-80 we observed MYO7A + hair cells with F-actin-rich and Espin + hair bundles, indicating a definitive hair cell identity.
  • 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 ⁇ g/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.
  • Vitronectin Invitrogen, cat. no. A14700
  • Chemically-defined lipid concentrate Invitrogen, cat. no. 11905-031
  • Albumin from bovine serum BSA; Sigma, cat. no. A1470
  • Insulin solution Sigma, cat. no. I9278
  • Human Transferrin Sigma, cat. no. T8158
  • 1-thioglycerol Sigma, cat. no. M1753
  • Human BMP-7 Human FGF-3 (Biolegend, cat. no. 558002)
  • FGF-2 Peprotech, cat. no. 100-18B
  • SB-431542 in solution (Stemgent, cat. no.
  • Human recombinant BMP7 stock solution (100 ng/ ⁇ L): In the biosafety cabinet, add 100 ⁇ L of sterile 4 mM HCl to 10 ⁇ g of BMP7; vortex the solution and spin down in a tabletop centrifuge. Store BMP4 solution in 5 ⁇ L aliquots at ⁇ 20° C. for 6 months or at ⁇ 80° C. for 1 year.
  • Human recombinant FGF-2 stock solution 200 ng/ ⁇ L: In the biosafety cabinet, add 250 ⁇ L of sterile PBS or 5 mM Tris (pH 7.6) to 50 ⁇ g of FGF-2; vortex the solution and spin down in a tabletop centrifuge. Store FGF-2 solution in 6 ⁇ L aliquots at ⁇ 20° C. for 6 months or at ⁇ 80° C. for 1 year.
  • 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 ⁇ l aliquots at ⁇ 20° C. for 6 months or at ⁇ 80° C. for 1 year.
  • EDTA Solution for passaging hES cells: In the biosafety cabinet, mix 50 ⁇ l of 0.5 M EDTA into 50 ml DPBS. Filter sterilize the solution. EDTA solution can be stored at RT for 6 months.
  • CDM Chemically Defined Medium
  • Differentiation CDM In a 50 mL conical tube, add 450 ⁇ L 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 ⁇ L of FGF-2 (4 ng/mL final concentration) and 30 ⁇ L of SB-431542 (10 ⁇ M 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.
  • Organoid Maturation Medium To prepare 50 mL OMM, combine 24.5 mL Advanced DMEM/F12, 24.5 mL Neurobasal Medium, 500 ⁇ L B-27 Supplement without vitamin A, 500 ⁇ L GlutaMAX, 250 ⁇ L N2-supplement, and 100 ⁇ L 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.
  • step 1 dilute 60 ⁇ L 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.
  • step 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 ⁇ L (passage in 4-5 days) of cell suspension to each well. Incubate cells at 37° C. in 5.0% CO 2 until ready for passage or use in an experiment.
  • hES cell differentiation (day -2 to day 0): aggregation:
  • Steps 1-43 Total time to generate inner ear organoids: 45-60 days to >1 yr

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