Methods of characterizing cell populations for auditory conditions

Abstract

Provided herein methods for characterizing populations of auditory cells obtained by inducing differentiation of pluripotent cells to assume auditory fates, as well as methods of making and using populations of auditory cells.

Description

Attorney Docket No. LCTI-005 / 001WG 43694-03305METHODS OF CHARACTERIZING CELL POPULATIONS FOR AUDITORY CONDITIONSRELATED APPLICATIONS

[0001] This application claims priority to. and benefit of, U.S. Provisional Application No.63 / 748,602, filed on January 23, 2025, the contents of which are incorporated by reference in their entirety herein.BACKGROUND

[0002] More than 5% of the population in industrialized nations have significant auditory or hearing loss conditions that range in severity from modest difficulty with speech comprehension to profound deafness. Hearing loss is age-related, as about 4% of people under 45 years old and about 34% of those over 65 years old have debilitating hearing loss. In most cases, the cause is related to degeneration and death of hair cells and their associated spiral ganglion neurons that reside within the inner ear cochlea. No treatment options exist that overcome auditory' neuron loss.

[0003] There thus exists a need in the art for additional methods to address hearing loss. The disclosure provides compositions comprising populations of auditory cells suitable for administering to a subject as a cell therapy to address hearing loss, and methods of making and characterizing same.SUMMARY

[0004] The disclosure provides a method of characterizing a population of auditory cells derived by in vitro differentiation of pluripotent cells, the method comprising: (a) performing single cell RNA sequencing on the population of auditory' cells, thereby determining the expression of a plurality of genes by individual cells in the population, (b) generating a two-dimensional representation of the similarity and / or difference of gene expression of individual cells of the population based on expression of at least a subset of genes in the plurality of genes, (c) partitioning the two-dimensional representation into a plurality' of clusters, wherein the plurality of clusters comprises 1-3 major clusters and one or more minor clusters, (d) identifying a set of genes that are upregulated in at least 50% of cells of the 1-3 major clusters, and (e) identifying a percentage of cells in the population that express the set of genes, thereby characterizing the population of auditory cells.Attorney Docket No. LCTI-005 / 001WO 43694-03305

[0005] In some embodiments, step (d) comprises identifying a set of genes that are upregulated in at least 60% of cells of the 1-3 major clusters.

[0006] In some embodiments, the method comprises (f) selecting a population of auditory cells wherein at least 50% of the cells in the population express the set of genes identified at (d). In some embodiments, at least 60% of the cells in the population express the set of genes identified at (d).

[0007] In some embodiments, step (b) comprises (i) providing single cell RNA sequencing data for individual cells in the population of auditory cells, wherein each cell is associated wi th expression values for the plurality of genes based on read counts from the single cell RNA sequencing data, (ii) performing principal component analysis, thereby producing a first reduced-dimensionality representation of the single cell RNA sequencing data for individual cells, (iii) determining, for each cell, one or more neighboring cells based on a plurality of principal component scores for each cell, (iv) generating a weighted graph, wherein vertices correspond to individual cells in the population of cells and wherein edge weights correspond to a degree of similarity between the individual cells, and (v) embedding the weighted graph in two-dimensional space, whereby preservation of topological relationships from the weighted graph is maximized, thereby generating a second reduced-dimensionality representation of the single cell RNA sequencing data for individual cells.

[0008] In some embodiments, step (b) comprises uniform manifold approximation and projection (UMAP).

[0009] In some embodiments, step (c) comprises a community detection algorithm.

[0010] In some embodiments, expression of the set of genes identified at (d) is upregulated in cells of the 1-3 major clusters compared to cells of the one more minor clusters.

[0011] In some embodiments, the 1-3 major clusters consists of two clusters.

[0012] In some embodiments, the one or more minor clusters comprise 1-4 clusters. In some embodiments, less than 10% of cells in the population are in any individual minor cluster. In some embodiments, 20% or less of cells in the population are in the one or more minor clusters.

[0013] In some embodiments, at least 80% of cells in the population are in the 1-3 major clusters.

[0014] In some embodiments, the method comprises repeating steps (a)-(d) at least once using a second population of auditory cells, and wherein the set of genes identified at step (d) for the population of auditory cells and the second population of auditory cells are the same.Attorney Docket No. LCTI-005 / 001WO 43694-03305 In some embodiments, the population of auditory cells and the second population of auditory cells are independently derived by in vitro differentiation of pluripotent cells.

[0015] In some embodiments, the set of genes comprises between 30 and 70 genes. In some embodiments, the set of genes comprises about 50 genes. In some embodiments, the set of genes comprises 53 genes.

[0016] In some embodiments, the set of genes comprise genes selected from the group of genes disclosed in Tables 5-6.

[0017] In some embodiments, the set of genes comprises one or more of catenin alpha 2 (CTNNA2), cadherin 4 (CDH4), AC 109466.1, carbohydrate sulfotransferase 11 (CHST11), TOX high mobility group box family member 3 (TOX3), ephrin A5 (EFNA5), NALCN channel auxiliary factor 1 (FAM155A), phospholipase C eta 1 (PLCHI), transmembrane 131 like (TMEM131L), AAAATPase AFG2A (SPATA5), NCK associated protein 5 (NCKAP5), contactin associated protein 2 (CNTNAP2), retinoic acid receptor beta (RARB), calcium / calmodulin dependent protein kinase ID (CAMKID), aspartate beta-hydroxylase (ASPH), teneurin transmembrane protein 2 (TENM2), dihydropyrimidinase like 3 (DPYSL3), neural EGFL like 2 (NELL2), phytanoyl-CoA 2-hydroxylase interacting protein like (PHYHIPL), microtubule affinity regulating kinase 1 (MARK1), GLI2 GLI family zinc finger 2 (GLI2), roundabout guidance receptor 2 (ROBO2), collagen ty pe IV alpha 2 chain (COL4A2), zinc finger and BTB domain containing 16 (ZBTB16), ring finger protein 24 (RNF24), thymocyte selection associated high mobility group box (TOX), laminin subunit alpha 1 (LAMA1), cysteine and tyrosine rich 1 (CYYR1), microtubule crosslinking factor 2 (SOGA), FYVE, RhoGEF and PH domain containing 4a (FYVE), RhoGEF and PH domain containing 4 (FGD4), neuroligin 1 (NLGN1), neuron navigator 1 (NAVI), matrix metallopeptidase 16 (MMP16), arkadia (RNF111) C-terminal like ring finger ubiquitin ligase 2C (RNF165), sodium channel and clathrin linker 1 (SCLT1), fibroblast growth factor 13 (FGF13), SOX2 overlapping transcript (SOX2-OT), leucine rich repeats and immunoglobulin like domains 1 (LRIG1), ELAV like RNA binding protein 3 (ELAVL3), fidgetin, microtubule severing factor (FIGN), erb-b2 receptor tyrosine kinase 4 (ERBB4), podoplanin (PDPN), adenosine deaminase RNA specific B2 (ADARB2), MMS22 like, DNA repair protein (MMS22L), CUGBP Elav-like family member 2 (CELF2), neuromedin U (NMU), tetraspanin 18 (TSPAN18), chromosome 1 open reading frame 21 (Clorf21), WD repeat and HMG-box DNA binding protein 1 (WDHD1), neuronal cell adhesion molecule (NRCAM), splA / ry anodine receptor domain and SOCS box containing 4 (SPSB4), formin homology 2 domain containing 3 (FHOD3) or polypeptide N-acetylgalactosaminyltransferase 13Attorney Docket No. LCTI-005 / 001WD 43694-03305 (GALNT13). In some embodiments, the set of genes comprises at least 10 genes, at least 20 genes, at least 30 genes, at least 40 genes, at least 50 genes or all genes selected from the group consisting of CTNNA2, CDH4, AC109466.1, CHST11, TOX3, EFNA5, FAM155A, PLCHI, TMEM131L, SPATA5, NCKAP5, CNTNAP2, RARB, CAMKID, ASPH, TENM2, DPYSL3, NELL2, PHYHIPL, MARK1, GLI2, ROBO2, COL4A2, ZBTB16, RNF24, TOX, LAMA1, CYYR1, SOGA. FGD4, NLGN1, NAVI, MMP16, RNF165, SCLT1. FGF13, SOX2-OT. LRIG1, ELAVL3, FIGN, ERBB4. PDPN, ADARB2, MMS22L, CELF2, NMU, TSPAN18, Clorf21, WDHD1, NRCAM, SPSB4, FHOD3 and GALNT13

[0018] In some embodiments, the cells in in the 1-3 major clusters comprise mid otic neuronal progenitor (ONP) cells, late ONP cells, auditory neuron progenitors, mature auditory neurons, spiral ganglion neurons or a combination thereof.

[0019] In some embodiments, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% or no cells in the population express RNA of one or more pluripotent stem cell markers. In some embodiments, the one or more pluripotent stem cell markers are selected from the group consisting of POU class 5 homeobox 1 (POU5F1), Nanog, lin-28 homolog A (LIN28A), DNA methyltransferase 3 beta (DMNT3B), cripto, EGF-CFC family member (TDGF1) and LINE1 type transposase domain containing 1 (L1TD1).

[0020] In some embodiments, the population of auditory cells selected at step (f) is suitable for administration to a subject with an auditory condition.

[0021] The disclosure provides a method of characterizing a population of auditory cells comprising: (a) performing single-cell RNA sequencing on a plurality of cells in the population of auditory cells; and (b) determining a percentage of cells in the population that express one or more genes set forth in Table 5 or 6.

[0022] In some embodiments of the method, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% of the cells in the population express one or more genes set forth in Table 5 or 6. In some embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% of the cells in the population express at least 15, at least 20, at least 25, at least 50, at least 100, at least 200, at least 300. at least 400, at least 500, at least 600 or all of the genes set forth in Table 5. In some embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% of the cells in the population express at least 15, at least 20, at least 25, at least 30, at least 40, at least 50 or all of the genes selected from the group consisting of CTNNA2. CDH4. AC 109466.1, CHST11, TOX3, EFNA5, FAM155A, PLCHI, TMEM131L, SPATA5, NCKAP5,Attorney Docket No. LCTI-005 / 001WD 43694-03305 CNTNAP2, RARB, CAMKID, ASPH, TENM2, DPYSL3, NELL2, PHYHIPL, MARK1, GLI2, R0B02, COL4A2, ZBTB16, RNF24, TOX, LAMA1, CYYR1, SOGA, FGD4, NLGN1, NAVI, MMP16, RNF165, SCLT1, FGF13, SOX2-OT, LRIG1, ELAVL3, FIGN, ERBB4, PDPN, ADARB2, MMS22L, CELF2, NMU, TSPAN18, Clorf21, WDHD1, NRCAM, SPSB4, FHOD3 and GALNT13.

[0023] In some embodiments of the method, the expression of the one or more genes is determined by measuring RNA or protein encoded by the one or more genes. In some embodiments, the RNA is measured by single-cell RNA sequencing or RT-qPCR. In some embodiments, the protein is measured by flow cytometry or immunocytochemistry.

[0024] In some embodiments of the method, the population of cells comprises mid otic neuronal progenitor (ONP) cells, late ONP cells, auditory neuron progenitors, mature auditory neurons, spiral ganglion neurons or any combination thereof.

[0025] In some embodiments of the method, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% or no cells in the population express one or more pluripotent stem cell markers. In some embodiments, the one or more pluripotent stem cell markers are selected from the group consisting of POU class 5 homeobox 1 (POU5F1), Nanog, lin-28 homolog A (LIN28A), DNA methyltransferase 3 beta (DMNT3B), cripto, EGF-CFC family member (TDGF1) and LINE1 type transposase domain containing 1 (L1TD1). In some embodiments, expression of the one or more pluripotent stem cells markers is determined by RNA expression. In some embodiments, determining RNA expression comprises single-cell RNA sequencing or RT-qPCR.

[0026] In some embodiments of the method, (a) greater than or equal to 70% of the cells in the population express both Nestin and SOX2; (b) greater than or equal to 80% of the cells in the population express (3 tubulin III; (c) greater than or equal to 5% of the cells in the population express TrkB; and (d) less than or equal to 1% of the cells in the population express TRA-1-60 and / or SSEA5. In some embodiments, (a) greater than or equal to 70% of the cells in the population express SOX2 and Nestin, (b) less than 5% of the cells in the population express Myo7A, and (c) greater than 80% of the cells in the population express [3 tubulin III. In some embodiments, (d) a fold change in GLUA2 mRNA expression compared to a undifferentiated population of pluripotent cells is between 1000 and 6000, and (e) a fold change in TRKB mRNA expression compared to a undifferentiated population of pluripotent cells is between 1000 and 8000. In some embodiments, greater than or equal to 15% of the cells in the population express TrkB, and / or (ii) a fold change in TRKB mRNA expression compared to a undifferentiated population of pluripotent cells is between 1000 and 8000. InAttorney Docket No. LCTI-005 / 001WG 43694-03305 some embodiments, less than or equal to 0.1% of the cells in the population express TRA-1-60 and / or SSEA5. In some embodiments, less than or equal to 20% of the cells in the population express PAX8 and / or PAX2, or the population of cells do not significantly express PAX2 and / or PAX8. In some embodiments, greater than or equal to 10% of the cells in the population express GluA4. In some embodiments, less than or equal to 5% of the cells in the population express Myo7A. In some embodiments, greater than or equal to 50% of the cells in the population express CD 133.

[0027] In some embodiments of the method, (a) greater than or equal to 85% of the cells in the population express both Nestin and SOX2; (b) greater than or equal to 90% of the cells in the population express tubulin III; (c) greater than or equal to 15% of the cells in the population express TrkB; (d) less than or equal to 10% of the cells in the population express Myo7A; and (e) less than or equal to 0.1% of the cells in the population express TRA-1-60 and / or SSEA5.

[0028] In some embodiments of the method, (a) between about 70% to 100% of the cells in the population express both Nestin and SOX2; (b) between about 80% to 100% of the cells in the population express 0 tubulin III; (c) between about 5% to 80% of the cells in the population express TrkB; and (d) between 0 to about 0.1% of the cells in the population express TRA-1-60 and / or SSEA5.

[0029] In some embodiments of the method, between about 10% to 95% of the cells in the population express GluA4.

[0030] In some embodiments, between 0% to about 30% of cells in the population express Myo7A. In some embodiments, less than 5% of cells in the population express Myo7A.

[0031] In some embodiments of the method, the population of auditory cells is produced by a method comprising: (a) obtaining a culture of undifferentiated pluripotent cells; and (b) culturing the undifferentiated pluripotent cells under culture conditions sufficient to induce differentiation of the pluripotent cells into the population of auditory cells.

[0032] The disclosure provides a population of auditory cells characterized by the methods of the disclosure.

[0033] The disclosure provides a population of auditory cells characterized by the methods of the disclosure, wherein at least 50% of the cells in the population express one or more genes set forth in Table 5 or 6. In some embodiments, at least 50% of the cells are mid ONP cells, late ONP cells, auditory neuron progenitors, mature auditory neurons, spiral ganglion neurons any combination thereof.Attorney Docket No. LCTI-005 / 001WO 43694-03305

[0034] The disclosure provides a pharmaceutical composition, comprising the population of auditory cells of the disclosure, and a pharmaceutically acceptable carrier, diluent or excipient.

[0035] The disclosure provides a method of treating an auditory condition in a subject, comprising administering the pharmaceutical composition of the disclosure.

[0036] The disclosure provides use of the pharmaceutical composition of the disclosure for the treatment of an auditory condition in a subject.

[0037] The disclosure provides use of the pharmaceutical composition of the disclosure for use in the manufacture of a medicament for the treatment of an auditory condition in a subject.

[0038] The disclosure provides a method of isolating or enriching a population of target auditory cells comprising: (a) providing a population of cells comprising target auditory cells and at least one additional cell type; (b) contacting the cells with a detectable label that binds to a cell surface marker selected from the group consisting of CD24, TMEFF1, JAM2, PR0M1, LRP4, NLGN1, GPM6A, PDPN, GABRB3. CDH20, BOC. CDH4. PCDH11X, DPP10, GRIA4, DCC, LRIG1, NRCAM, NEGRI, ADAM22, ADGRL2. PTPRN2, NRXN1, CELSR2, NTRK2 and NTRK3; and (c) sorting the cells using the detectable label.

[0039] In some embodiments of the methods of isolating or enriching a population of target auditory cells, the sorting comprises flow cytometry. In some embodiments, the at least one additional cell type comprises pluripotent cells.BRIEF DESCRIPTION OF THE DRAWINGS

[0040] FIG. 1A and FIG. IB demonstrate the differentiation process of ANP1 cells from Human Embryonic Stem Cells (hESCs) to late otic neural progenitors (ONP)Zmature and / or early auditory neurons (FIG. 1A) and markers of hESCs, neural progenitors, ectoderm and late otic neuronal progenitors (ONP), and AN late ONP as a function of differentiation time (FIG. IB). As time progresses, the elimination of hESC markers and the expression of otic neuronal progenitor markers can be seen.

[0041] FIG. 1C is a schematic and a graph showing monitoring of ANP1 differentiation using in-process controls starting from a single pluripotent stem cell line.

[0042] FIGS. 2A-2B are schematic depictions of various assessments for ANP1 cell therapy product quality control, including bio-analytical and functional assessments, which can be used to characterize the clinical applicability of the product. FIG. 2 shows a snapshot of inAttorney Docket No. LCTI-005 / 001WO 43694-03305 vitro analytical method development designed to monitor the differentiation process and the final cell therapy product.

[0043] FIG. 3 is a flow cytometry plot showing expression of SOX2 and Nestin in ANP1 cells produced by the methods of Example 1.

[0044] FIG. 4 is a flow cytometry plot showing expression ofTra-1-60 and SSEA-5 in ANP1 cells produced by the methods of Example 1.

[0045] FIG. 5A is a diagram that shows the timeline of directed differentiation of ANP1 cells during the manufacturing process.

[0046] FIG. 5B is a pair of images and a diagram that show the directed differentiation of ANP 1 cells during the manufacturing process, which mimics natural otic development.

[0047] FIG. 6 shows bioinformatic analysis of ANP1 cells using Uniform Manifold Approximation and Projection (UMAP) graphs based on Principal Component Analysis (PCA) including auditory neuron related gene sets from publicly available data sets. Shading represents cells expressing key auditory neuron related gene sets. Bioinformatics reveals expression of key auditory neuron related genes distributed among ANP1 final product.

[0048] FIG. 7 is a pair of plots showing ANP1 cell clustering using UMAP for an initial trial batch and the first production batch of ANP1 cells.

[0049] FIGS. 8A-8B is a trio of plots (FIG. 8A) and a table (FIG. 8B) showing ANP1 cell clustering using UMAP. FIG. 8A shows plots showing ANP1 cell clustering using UMAP for three production batches of ANP1 cells. FIG. 8B shows the percentage of the total cells in the main clusters (marked C#1 and C#0) that hold the plurality of the cells, and percentage of cells in the secondary clusters (C#2-C#4).

[0050] FIG. 9 shows the process for identifying the genes that can be used to verify ANP1 cell population composition based on single cell RNA-seq and UMAP clustering analysis.

[0051] FIG. 10 is a Venn diagram showing the number of genes shared between production batches (runs #1-3) as described in Example 5.

[0052] FIG. 11 is a plot showing the average percentage of cells that are positive for 53 common genes in the main clusters (see C#0 and C#1 in FIGS. 7 and 8A), versus the secondary clusters (see C#3 or C#3 and C#4 in FIGS. 7 and 8A).DETAILED DESCRIPTIONIntroduction

[0053] The disclosure is based, at least on part, on the finding that single-cell RNA sequencing can be used to characterize populations of auditory cells, and to identify markersAttorney Docket No. LCTI-005 / 001WO 43694-03305 that can be used to characterize populations of auditory cells, which are suitable for administration to subjects as cell therapies to treat auditory conditions. The use of single-cell RNA sequencing and the markers identified herein can be used to comprehensively and rapidly characterize populations of auditory cells produced by in vitro differentiation of pluripotent cells. This ensures consistency during manufacturing and between production runs, and quality of cell therapy products.

[0054] The ear is composed of four main sections: the external ear, middle ear, inner ear, and the transmission pathway to the hearing center in the brain. The inner ear is a capsule of very dense bone containing a fluid that communicates with the middle ear. Small bones within the middle ear (the malleus, incus, and stapes) transmit sound energy from the tympanic membrane to the oval window at the entrance to the cochlea of the inner ear. The action of the stapes at the oval window exerts pressure on the fluid within the cochlea. The pressure is transmitted through the cochlea, ultimately causing a second window, the round window to oscillate. A basilar membrane that defines the fluid-filled chambers of the cochlea then transmits the oscillations to the organ of Corti. Hair cells are located in the epithelial lining of the inner ear (z.e., in the cochlear organ of Corti), as well as in the vestibular sensory epithelia of the saccular macula, the utricular macula, and the cristae of the three semicircular canals of the labyrinth. The cochlear hair cells send signals to the cochlear spiral ganglion, and the clustered neuronal cell bodies convey those signals to the cochlear nucleus of the brain stem.

[0055] Mechanosensitive sensory hair cells are the basis of our senses of hearing and balance. Our inner ear harbors about 13,000 - 15,000 cochlear and about the same number of vestibular sensory hair cells, which are the mechanoreceptors of our senses of hearing and balance. Because of their paucity, molecular studies on hair cells have been limited, and consequently, the molecular basis of their function is unknown. Aside from being scarce, hair cells are also sensitive to mechanical and chemical insults. Death or damage to the sensory cells makes up 90% of all hearing loss in humans. Acoustical overstimulation, chemotherapy, aminoglycoside drug side effects, the effects of aging, and increasingly noisy environments contribute to the deterioration of hearing over time. As a result, hundreds of millions of patients worldwide are permanently debilitated by hearing loss and balance problems. The main reason for the permanence of these chronic disorders is the fact that mammalian cochlear hair cells do not spontaneously regenerate and that the limited regeneration observed in the vestibular system is inadequate to restore function.

[0056] Auditory neuropathy is a hearing disorder in which the inner ear successfully detects sound but has a problem with sending signals from the ear to the brain. Current state of theAttorney Docket No. LCTI-005 / 001WG 43694-03305 art medical knowledge suggests that auditory neuropathies play a substantial role in hearing impairments and deafness. Damage of auditory neurons is now being reported for a variety of auditory neuropathy spectrum disorders, including those associated with aging. Hearing depends on a series of complex steps that change sound waves in the air into electrical signals. The auditory nerve then carries these signals to the brain. Outer hair cells help amplify sound vibrations entering the inner ear from the middle ear. When hearing is working normally, the inner hair cells convert these vibrations into electrical signals that travel as nerve impulses to the brain, where the brain interprets the impulses as sound. Auditory neuropathy can be caused by a number of factors including: (i) damage to the auditory neurons that transmit sound information from the inner hair cells - specialized sensory cells in the inner ear - to the brain; (ii) damage to the inner hair cells themselves; (iii) inherited genes with mutations or suffering damage to the auditory system, either of which may result in faulty connections between the inner hair cells and the auditory nerve, which leads from the inner ear to the brain; or (iv) damage to the auditory nerve itself. Researchers are still seeking effective treatments for those affected with auditory’ neuropathy and other auditory- disorders.

[0057] Several protocols have been developed for differentiation of human pluripotent stem cells, such as human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) into sensory cells of the ear that can be used in cellular therapy for treating hearing loss. While these methods have been successful in generating sensory cells of the ear. challenges remain with respect to qualify, seal abilify, and cost of goods associated with translating the existing protocols to a clinical commercial-scale production process of such sensory cells.

[0058] Hidden hearing loss (HHL) refers to a condition ty pically arising from damage to the inner ear (that is, the cochlea), generally caused by exposure to loud noise, aging, or other factors. HHL can go undetected by conventional audiograms, as the changes leading to the condition arise from aberrant signaling between the cochlear ner e connecting the brain and ear. and not within the sensory hair cells that convert sound waves into electrochemical signals that such tests are designed to assess. An example treatment for HHL is a hearing aid, such as a cochlear implant. Additional treatment options are needed.

[0059] Cochlear implants are small electronic devices that can be used to provide a sense of sound to subjects with particular kinds of auditory conditions. The implant consists of an internal portion, and an internal portion. The external portion sits behind the ear, and the internal portion is surgically implanted. Cochlear implants typically have the followingAttorney Docket No. LCTI-005 / 001WO 43694-03305 components: (a) a microphone, configured to pick up sound from the environment; (b) a processor, which selectively processes sounds picked up by the environment, such as speech sounds; (c) a transmitter and receiver or stimulator, which are configured to receive signals from the processor and convert them to electrical impulses which are then transmitted to the electrode array; and (d) an electrode array, which is a group of electrodes that receives electrical impulses from the transmitter and / or stimulator, and sends them to different regions of the auditory nerve. The auditors' nerve sends these signals to the brain, which recognizes the signals as sound. Cochlear implants can bypass damaged portions of the ear, and directly stimulate the auditors' nerve. However, where hair cells and / or neurons in the cochlea are damaged, the efficacy of the cochlear implant may be reduced or inhibited. There thus exists a need for methods that can enhance the efficacy of cochlear implants. Accordingly, the disclosure provides compositions comprising auditory’ cells, which, when administered to a subject, can enhance the efficacy of cochlear implants. It is thought that by administering such compositions to the inner ear, the cells of the composition can repopulate damaged or missing cells in the inner ear and thereby enhance activity of the cochlear implant.Advantages of the instant cell therapy include, but are not limited to, an established delivery route to the inner ear, scalable production (z.e., modalities, substrates, and harvesting) to provide a reduced cost of goods, and established preclinical models for testing.Definitions

[0060] This description is not intended to be a detailed catalog of all the different ways in which the disclosure may be implemented, or all the features that may be added to the instant disclosure. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. Thus, the disclosure contemplates that in some embodiments of the disclosure, any feature or combination of features set forth herein can be excluded or omitted. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure, which do not depart from the instant disclosure. In other instances, well-known structures, interfaces, and processes have not been shown in detail in order not to unnecessarily obscure the invention. It is intended that no part of this specification be construed to affect a disavowal of any part of the full scope of the invention. Hence, the following descriptions are intended to illustrate some particular aspects of the disclosure, and not to exhaustively specify all permutations, combinations and variations thereof.Attorney Docket No. LCTI-005 / 001WO 43694-03305

[0061] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in the description of the disclosure herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure.

[0062] All publications, patent applications, patents and other references cited herein are incorporated by reference in their entireties.

[0063] Unless the context indicates otherwise, it is specifically intended that the various features of the disclosure described herein can be used in any combination. Moreover, the present disclosure also contemplates that in some embodiments of the disclosure, any feature or combination of features set forth herein can be excluded or omitted.

[0064] Methods disclosed herein can comprise one or more steps or actions for achieving the described method. The method steps and / or actions may be interchanged with one another without departing from the scope of the present invention. In other words, unless a specific order of steps or actions is required for proper operation of the aspect, the order and / or use of specific steps and / or actions may be modified wi thout departing from the scope of the present invention.Table 1. Abbreviations""Attorney Docket No. LCTI-005 / 001WO 43694-03305

[0065] As used in the description of the disclosure and the appended claims, the singular forms "a," "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0066] As used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

[0067] The terms "about" and "approximately" as used herein when referring to a measurable value such as a percentages, density, volume and the like, is meant to encompass variations of ± 10%, ± 5%, ± 1%, ± 0.5%. or even ± 0.1% of the specified amount.

[0068] As used herein, phrases such as "between X and Y" and "between about X and Y" should be interpreted to include X and Y. As used herein, phrases such as "between about X and Y" mean "between about X and about Y" and phrases such as "from about X to Y" mean "from about X to about Y."

[0069] A “ceir as used herein, refers to a cell carrying out metabolic or other function sufficient to preserve or replicate its genomic DNA. A cell can be identified by well-known methods in the art including, for example, presence of an intact membrane, staining by a particular dye, abi 1 i ty to produce progeny or, in the case of a gamete, ability to combine with a second gamete to produce a viable offspring. Cells may include prokaryotic and eukaryotic cells. Prokaryotic cells include but are not limited to bacteria. Eukaryotic cells include but areAttorney Docket No. LCTI-005 / 00fWO 43694-03305 not limited to yeast cells and cells derived from plants and animals, for example mammalian, insect (e.g., spodoptera) and human cells. Cells may be useful when they are naturally nonadherent or have been treated not to adhere to surfaces, for example by trypsinization.

[0070] “Comprising” or “comprises” is intended to mean that the compositions and methods include the recited elements, but not excluding others. “Consisting essentially of’ when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination for the stated purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude other materials or steps that do not materially affect the basic and novel characteristic(s) of the claimed invention.“Consisting of’ shall mean excluding more than trace elements of other ingredients and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0071] An “effective amount” is an amount sufficient for a composition to accomplish a stated purpose relative to the absence of the composition (e.g. achieve the effect for which it is administered, treat a disease, reduce enzyme activity, increase enzyme activity, reduce a signaling pathway, or reduce one or more signs or symptoms of a disease or condition). An example of an “effective amount” is an amount sufficient to contribute to the treatment, prevention, or reduction of a symptom or symptoms of a disease, which could also be referred to as a “therapeutically effective amount.” A “reduction” of a symptom or symptoms (and grammatical equivalents of this phrase) means decreasing the severity or frequency of the symptom(s), or elimination of the symptom(s). For example, a therapeutically effective amount can be an amount sufficient to enhance the function of a cochlear implant. The skilled artisan will appreciate that function can be enhanced by about 5%, 10%, 15%, 20%, 25%, 30%, 35%. 40%. 45%. 50%. 55%. 60%. 65%. 70% or more by any appropriate measure, and be considered a therapeutically effective amount.

[0072] A “prophylactically effective amount” of a composition (e.g., the composition comprising cells described herein) is an amount of the composition that, when administered to a subject, will have the intended prophylactic effect, e.g.. preventing or delaying the onset (or reoccurrence) of an injury, disease, pathology or condition, or reducing the likelihood of the onset (or reoccurrence) of an injury, disease, pathology, or condition, or their symptoms. The full prophylactic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a prophylactically effective amount may be administered in one or more administrations.Attorney Docket No. LCTI-005 / 001WO 43694-03305

[0073] An “activity decreasing amount,” as used herein, refers to an amount of antagonist required to decrease the activity of an enzyme relative to the absence of the antagonist. A “function disrupting amount,” as used herein, refers to the amount of antagonist required to disrupt the function of an enzy me or protein relative to the absence of the antagonist.

[0074] The exact amounts will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art. Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy , 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins). For any composition described herein, the therapeutically effective amount can be initially determined from cell culture assays. Target concentrations will be those concentrations of active composition(s) (e.g, cell concentration or number) that are capable of achieving the methods described herein, as measured using the methods described herein or known in the art.

[0075] “Control” or “control experiment” is used in accordance with its plain ordinary meaning and refers to an experiment in which the subjects or reagents of the experiment are treated as in a parallel experiment except for omission of a procedure, reagent, or variable of the experiment. In some instances, the control is used as a standard of comparison in evaluating experimental effects. In some embodiments, a control is the measurement of the activity of a protein in the absence of a composition as described herein (including embodiments and examples).

[0076] As used herein, "implantation" or "transplantation" refers to the administration of a cell population or device into a target tissue using a suitable delivery technique, (e.g., administering a population of cells using an injection device, or a cochlear implant using an appropriate surgical technique).

[0077] “Pharmaceutically acceptable excipient” and “pharmaceutically acceptable carrier” refer to a substance that aids the administration of an active agent to and absorption by a subject and can be included in the compositions of the present disclosure without causing a significant adverse toxicological effect on the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solutions, lactated Ringer’s, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer's solution), alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethy cellulose, polyvinyl pyrrolidine, and colors, and the like. Such preparations can be sterilized and, ifAttorney Docket No. LCTI-005 / 001WO 43694-03305 desired, mixed with auxiliary' agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and / or aromatic substances and the like that do not deleteriously react with the compositions of the disclosure. One of skill in the art will recognize that other pharmaceutical excipients are useful in the present disclosure.

[0078] As used herein, a “‘patient” or "subject" refers to a living organism suffering from or prone to a disease or condition that can be treated by administration of a pharmaceutical composition, or an implantable biodegradable scaffold as provided herein. Non-limiting examples include humans, other mammals, bovines, rats, mice, dogs, monkeys, goat, sheep, cows, deer, and other non-mammalian animals. In some embodiments, a patient is human.

[0079] As used herein, a "subject in need thereof refers to an animal or a human having damaged tissue in the central nervous system. In an embodiment, an animal or a human is experiencing a loss of auditory' function.

[0080] As used herein, "treatment" or "treating," with respect to a condition or a disease, is an approach for obtaining beneficial or desired results including preferably clinical results after a condition or a disease manifests in a subject. Beneficial or desired results with respect to a disease include, but are not limited to, one or more of the following: improving a condition associated with a disease, curing a disease, lessening severity' of a disease, delaying progression of a disease, alleviating one or more symptoms associated with a disease, increasing the quality’ of life of one suffering from a disease, prolonging survival, and any combination thereof. Likewise, for purposes of this disclosure, beneficial or desired results with respect to a condition include, but are not limited to, one or more of the following: improving a condition, curing a condition, lessening severity of a condition, delaying progression of a condition, alleviating one or more symptoms associated with a condition, increasing the quality of life of one suffering from a condition, prolonging survival, and any combination thereof.

[0081] As used herein, "pluripotent stem cell" or "pluripotent cell" refers to a cell that has the ability to differentiate into all types of cells in an organism. Pluripotent cells are capable of forming teratomas and of contributing to ectoderm, mesoderm, or endoderm tissues in a living organism. Examples of pluripotent stem cells are embryonic stem (ES) cells, embryonic germ stem (EG) cells, and induced pluripotent stem (iPS) cells.

[0082] As used herein, "embry onic stem cell" or "ES cell" refers to a cell that a) can selfrenew, b) can differentiate to produce all types of cells in an organism, and c) is derived from the inner cell mass of the blastula of a developing organism. ES cells can be cultured over aAttorney Docket No. LCTI-005 / 001WO 43694-03305 long period of time while maintaining the abi 1 i ty to differentiate into all types of cells in an organism. In culture. ES cells typically grow as flat colonies with large nucleo-cytoplasmic ratios, defined borders and prominent nuclei. In addition, ES cells express stage-specific embryonic antigen (SSEA) 5 (SSEA-5), POU class 5 homeobox 1 (Oct-4), Nanog homeobox (Nanog), SSEA-3, SSEA-4, Tumor rejection antigen 1-60 (TRA-1-60), TRA-1-81 antigen (TRA-1-81), and Alkaline Phosphatase, but not SSEA-1. Examples of methods of generating and characterizing ES cells may be found in, for example, U.S. Pat. No. 7,029,913. U.S. Pat. No. 5,843,780, and U.S. Pat. No. 6,200,806, the disclosures of which are incorporated herein by reference.

[0083] As used herein, "embryonic germ stem cell", “embry onic germ cell" or "EG cell refers to a cell that a) can self-renew. b) can differentiate to produce all types of cells in an organism, and c) is derived from germ cells and germ cell progenitors, e.g. primordial germ cells, i.e. those that would become sperm and eggs. Embryonic germ cells (EG cells) are thought to have properties similar to embry onic stem cells as described above. Examples of methods of generating and characterizing EG cells may be found in, for example, U.S. Pat. No. 7,153,684; Matsui, Y„ et al., (1992) Cell 70:841; Shamblott, M„ et al. (2001) Proc. Natl. Acad. Sci. USA 98: 113; Shamblott, M., et al. (1998) Proc. Natl. Acad. Sci. USA, 95:13726; and Koshimizu, U., et al. (1996) Development, 122:1235, the disclosures of which are incorporated herein by reference.

[0084] As used herein, "induced pluripotent stem cell" or "iPS cell" refers to a cell that a) can self-renew, b) can differentiate to produce all types of cells in an organism, and c) is derived from a somatic cell. iPS cells have an ES cell-like morphology, growing as flat colonies with large nucleo-cytoplasmic ratios, defined borders and prominent nuclei. In addition, iPS cells express one or more key pluripotency markers known by one of ordinary skill in the art, including but not limited to Alkaline Phosphatase, SSEA3, SSEA4, SRY (sex determining region Y)-box transcription factor 2 (Sox2), Oct-4, Nanog, TRA-1-60, TRA-1-81, teratocarcinoma-derived growth factor 1 (TDGF1), DNA methyltransferase 3 beta (Dnmt3b), forkhead box D3 (FoxD3). growth differentiation factor 3 (GDF3), cytochrome P450 family 26 subfamily A member 1 (Cyp26al), telomerase reverse transcriptase (TERT), and ZFP42 zinc finger protein (zfp42). iPS cells may be generated by providing the cell with "reprogramming factors", i.e. one or more, i.e. a cocktail, of biologically active factors that act on a cell to alter transcription, thereby reprogramming a cell to pluripotency. These reprogramming factors may be provided to the cells individually or as a single composition, that is, as a premixed composition, of reprogramming factors. The factors may be provided atAttorney Docket No. LCTI-005 / 001WO 43694-03305 the same molar ratio or at different molar ratios. The factors may be provided once or multiple times in the course of culturing the cells of the subject invention. Examples of methods of generating and characterizing iPS cells may be found in, for example. Application Nos. US20090047263, US20090068742, US20090191159, US20090227032, US20090246875, and US20090304646, the disclosures of which are incorporated herein by reference.

[0085] It is appreciated that commercially available stem cells can also be used in aspects and embodiments of the present disclosure. Human ES cells may be purchased from the NIH human embryonic stem cells registry, www.grants.nih.govstem_cells / or from other hESC registries. Non-limiting examples of commercially available embryonic stem cell lines include Hl, HAD-C 102, BGO 1, BG02, BG03, BG04. CY12, CY30, CY92, CY1O, TE03. TE32, CHB-4, CHB-5, CHB-6, CHB-8, CHB-9, CHB-10, CHB-11, CHB-12, ESI (including ESI-013, ESI-014, ESI-017, ESI-027, ESI-035, ESI-049, ESI-051 and ESI-053), HUES 1, HUES 2, HUES 3, HUES 4, HUES 5, HUES 6, HUES 7, HUES 8, HUES 9, HUES 10, HUES 11, HUES 12, HUES 13, HUES 14, HUES 15. HUES 16, HUES 17, HUES 18, HUES 19. HUES 20, HUES 21. HUES 22. HUES 23, HUES 24. HUES 25, HUES 26, HUES 27. HUES 28, CyT49, RUES3, WAO 1, UCSF4, NYUES 1, NYUES2, NYUES3, NYUES4, NYUESS, NYUES6, NYUES7, UCLA 1, UCLA 2, UCLA 3, WA077 (H7), WA09 (H9), WA 13 (H13), WA14 (H14), HUES 62, HUES 63, HUES 64, CT I, CT2, CT3, CT4, MA135, Eneavour-2, WIBR 1, WIBR2, WIBR3, WIBR4, WIBRS, WIBR6, HUES 45, Shef 3, Shef 6, BINheml9, BJNhem20, SAGO 1, and SAOO1.

[0086] As used herein, the term “microcarrier” or “MC” refers to a suspendible support matrix that allows adherent cells to grow in dynamic or static cell culture, and can stay in suspension with gentle mixing. Microcarriers can be composed of including, but not limited to, polystyrene, surface-modified polystyrene, chemically modified polystyrene, cross-linked dextran, cellulose, acrylamide, collagen, alginate, gelatin, glass, DEAE-dextran, or a combination thereof. Microcarriers can be coated with a biological support matrix, including, but not limited to, laminin. Matrigel®, collagen, poly -lysine, poly-L-lysine, poly-D-lysine. vitronectin, fibronectin, tenascin, dextran, a peptide, or a combination thereof. Many different types of microcarriers are commercially available, including, but not limited to, HyQSphere (HyClone™), Hillex (SoloHill Engineering), and Low Concentration Synthemax® II (Coming) brands. Microcarriers can be made from cross-linked dextran such as the Cytodex® brand (GE Healthcare). Microcarriers can be spherical and smooth, can have microporous surfaces, such as CYTOPORE™ brand (GE Healthcare), and / or can be rod-Attorney Docket No. LCTI-005 / 001WO 43694-03305 shaped carriers such as DE-53 (Whatman™). Microcarriers can be impregnated with magnetic particles that may help in cell separation from beads (e.g., GEM particles from Global Cell Solutions). Chip-based microcarriers such as the pHex product (Nunc) provide a flat surface for cell growth while maintaining the high surface to volume ratio of traditional microcarriers. The properties of microcarriers may significantly affect expansion rates and cell multi- or pluripotency.

[0087] As used herein, “dynamic culture” refers to cell cultivation that, unlike cell cultivation performed in static conditions (e.g., petri dishes), is conducted with intentional active motion to enhance mass transfer and mechanotransductive effects (e.g., bioreactors) which often results in higher numbers of functional cells. For example, in dynamic differentiation processes, bioreactors directly apply mechanical forces to generate physiologic conditions and enhance differentiation towards a specific cell lineage. In dynamic culture, cells may also have a more homogenous environment, that diffusion alone cannot provide in static culture. For example, cells that are grown in the vessel periphery vs. vessel inner areas. Further, static culture may generate various biologically separate niches, as it sustains microenvironments with various cell densities, that are not sustainable in Dynamic culture.

[0088] As used herein, “dynamic two-dimensional” or “dynamic 2D” refers to a cell that are grown and form a monolayer on microcarriers. For example, cells cultured in dynamic culture (e.g. bioreactor) with suspended adhesive agents (e.g., microcarriers) that allow attachment of the cells to form dynamic 2D culture.

[0089] As used herein, “dynamic three-dimensional” or “dynamic 3D” refers to cells that are grown as aggregates in suspension, such as a cell culture in an artificially created environment in which biological cells are permitted to grow or interact with their surroundings in all three dimensions. Unlike 2D environments, a 3D cell culture allows cells in vitro to grow in all directions, similar to how they would in vivo. These three-dimensional cultures can be, for example, grown in bioreactors, small capsules in which the cells can grow into spheroids, or 3D cell aggregates.

[0090] As used herein, "somatic cell" refers to any cell in an organism that, in the absence of experimental manipulation, does not ordinarily give rise to all types of cells in an organism. In other words, somatic cells are cells that have differentiated sufficiently that they will not naturally generate cells of all three germ layers of the body, i.e. ectoderm, mesoderm and endoderm. For example, somatic cells would include both neurons and neural progenitors, the latter of which may be able to self-renew and naturally give rise to all or some cell types ofAttorney Docket No. LCTI-005 / 001WD 43694-03305 the central nervous system but cannot give rise to cells of the mesoderm or endoderm lineages.

[0091] As used herein, "endoderm" refers to the germ layer formed during animal embryogenesis that gives rise to the gastrointestinal tract, respiratory tract, endocrine glands and organs, certain structures of the auditory system, and certain structures of the urinary system.

[0092] As used herein, "mesoderm" refers to the germ layer formed during animal embryogenesis that gives rise to muscles, cartilage, bones, dermis, the reproductive system, adipose tissue, connective tissues of the gut, peritoneum, certain structures of the urinary' system, mesothelium, notochord, and spleen.

[0093] As used herein, "ectoderm" refers to the germ layer formed during animal embryogenesis that gives rise to the nervous system, tooth enamel, epidermis, hair, nails, and linings of mucosal tissues. During embryogenesis, the embryonic ectoderm is patterned into lineage progenitors for neural plate, neural crest, placodes and epidermis. “Non-neuronal ectoderm” or “non-neural ectoderm” refers to ectodermal cells that will form non-neuronal structures, such as epidermis.

[0094] As used herein, "anterior ectoderm" refers to the region of the ectodermal germ layer at the anterior, or "rostral", end of the embry o, i.e. towards the head region. Anterior ectoderm comprises pre-placodal ectoderm and adjacent tissues such as presumptive early ectoderm, presumptive neural crest, and neural tissue. Ectoderm may be induced to become anterior ectoderm by contact with rostralizing factors such as IGF1 or insulin.

[0095] As used herein, "pre-placodal ectoderm" refers to the narrow band of cells in the anterior ectoderm that surrounds the anterior neural plate at the end of gastrulation and that gives rise to cranial placodes, which in turn give rise to the paired sensory structures of the head. Pre-placodal ectoderm cells may express detectable levels of one or more of markers including but not limited to Neurotrophin receptor (CD271 / NGFR / p75NTR), fibroblast growth factor receptor 1 (FGFR1), fibroblast grow th factor receptor 2 (FGFR2), fibroblast growth factor receptor 3 (FGFR3). SIX homeobox 1 (SIX1), SIX homeobox 4 (SIX4), eyes absent homolog I (EYA1), and eyes absent homolog 2 (EYA2). Pre-placodal ectodermal cells are competent to respond to otic induction, that is, the induction of otic progenitor cells by culturing in the presence of FGFs, resulting in the upregulation of p75, Pax8, Pax2, GATA3 and SoxlO expression. Cells expressing pre-placodal ectodermal markers, and which have pre-placodal ectodermal characteristics, can be induced from undifferentiated pluriplotent stem cells using the methods described herein.Attorney Docket No. LCTI-005 / 001WO 43694-03305

[0096] As used herein, "otic progenitor cells" or "otic neural progenitor cells” refers to a somatic cell that a) can self-renew, and b) can differentiate to give rise to inner ear sensory hair cells, auditory neurons, and supporting cells. Otic progenitor cells grow as spheres of cells when cultured in non-adherent conditions, or as clusters of cells when cultured in adherent conditions. Furthermore, otic progenitor cells may express detectable levels of one or more of the following markers: paired box 2 (PAX2), paired box 8 (PAX8), distal -less homeobox 5 (DLX5), orthodenticle homeobox 2 (OTX2), eyes absent homolog 1 (EYA1), SlX homeobox 1 (SIX1), jagged 1 (JAG1), fibroblast growth factor receptor 1 (FGFR1). Other markers include forkhead box 13 (FOXI3), SRY-box 2 (SOX2), NOTCH1, delta-like 1 (DELTA1), bone morphogenetic protein 7 (BMP7), T-box 1 (TBX1), GATA binding protein 3 (GATA3), forkhead box D3 (FOXD3), hairy / enhancer-of-split related with YRPW motif 1 (HEY1), hairy / enhancer-of-split related with YRPW motif 2 (HEY2), hairy and enhancer of split 1 (HES1), haiiy7and enhancer of split 6 (HES6), Activin receptor (ACTIVIN-R), H6 family homeobox 3 (NKX5.1), Claudin 8 (CLDN8), Claudin 14 (CLDN14). Otic neural progenitor cells can be divided into early, mid, and late otic progenitor cells based on marker expression, as described herein.

[0097] As used herein, "stromal cells" refers to connective tissue cells of any organ, e.g. fibroblasts, pericytes, endothelial cells, etc.

[0098] As used herein, "sensory neuronal progenitor cells" refers to self-renewing, multipotent cells that first generate the radial glial progenitor cells that generate the neurons and glia of the nervous system of all animals during embryonic development. While sensory neuronal progenitor cells can be naturally occurring, their cellular composition differs from the cells induced from undifferentiated pluripotent stem cells using the methods disclosed herein.

[0099] '‘Cochlear nerve” or ‘'auditory nerve” refers to a nerve that relays auditory sensory information from the cochlea of the inner ear to the brain. In humans, there are on average about 30,000 nerve fibers within the cochlear nen e. The cell bodies of the neurons of the cochlear nen e lie within the cochlea, and collectively form the spiral ganglion.

[0100] As used herein, "inner ear sensory hair cells" or simply "hair cells" refers to the mechanosensory hair cells of the cochlea (the auditory system) and of the saccule, utricle, crista ampularis, and semicircular canals (the vestibular system), which contribute to detecting and amplifying sound and to maintaining balance, respectively. Hair cells resemble columnar cells, each with a hair bundle of stereocilia at the apical surface. The deflection of the stereocilia opens mechanically gated ion channels that allow small, positively chargedAttorney Docket No. LCTI-005 / 001WO 43694-03305 ions (primarily potassium and calcium) to enter the hair cell. Unlike many other electrically active cells, the hair cell itself does not fire an action potential. Rather, the influx of positive ions depolarizes the cell, resulting in a receptor potential. As such, hair cells typically show a graded electrical response rather than action potential spikes typical of other neurons. Hair cells may express detectable levels of one or more of the following markers: atonal homolog 1 (Atohl / MATHl / HATHl), myosin VI (MY06), myosin VIIA (MY07A), Espin (ESPN), myosin heavy chain 3 (MYH2), cadherin23 (CDH23), protocadherinl5 (PCDH15), otoferlin (OTOF), and prestin (SLC26A5).

[0101] As used herein, "inner ear supporting cells", or simply "supporting cells" refers to the cells that contribute to the complex structural and functional properties of the cochlea, e.g.. Deiters' (phalangeal) cells. Hensen's cells. Claudius cells. Boettcher cells, pillar cells, marginal cells, and the like, and of the saccule, utricle, crista ampularis, and semicircular canals. Supporting cells are identifiable by short microvilli at their apical cell surface. In addition, they are found in close proximity to hair cells, i. e. they are found directly adjacent to hair cells, as clusters with hair cells. Supporting cells may express detectable levels of one or more of the following markers: cyclin-dependent kinase inhibitor IB (CDKN1B, p27 (KIP1)), prospero homeobox 1 (PROXI), otoancorin (OTO A), musashi homolog 1 (MSI1), SRY-box 2 (SOX2), gap junction protein beta 2, 26 kDa (Connexin 26), gap junction protein beta 6, kDa (Connexin30), gap junction protein alpha 1, 43 kDa (Connexin43), hairy / enhancer-of-split related with YRPW motif 2 (HEY2).

[0102] As used herein, “auditory neuron,” “auditory neurons” (abbreviated AN), “auditory' cell” or “auditory cells” refers, or refer, to sensory cell populations of the ear including, but not intended to be limited to, one or more of hair cells, supporting cells, otic progenitor cells, auditory’ neuron progenitors, sensory neuronal progenitor cells, and the like. The term “auditory7cells” may, in some cases, refer to a mixed population of cells encompassing any combination of the cell types described above, in any ratio.

[0103] As used herein, “ANP,” “ANP1” and similar terms refer to a population of auditory cells derived by in vitro differentiation of pluripotent cells, such as induced pluripotent stem cells or embryonic stem cells, using the methods described herein. The ANP1 cells can include mid otic neuronal progenitor (ONP) cells, late ONP cells, auditory neuron progenitors (ANP), mature auditory7neurons (AN), spiral ganglion neurons or any combination thereof. The ANP1 cells can be characterized using the methods as described herein.Attorney Docket No. LCTI-005 / 001WO 43694-03305

[0104] As used herein, “auditory disorder"’ or “auditory condition” or “hearing disorder” or “hearing condition” refers to conditions or disorders including but not intended to be limited to conductive hearing loss, sensorineural hearing loss, central hearing loss, mixed hearing loss, auditory neuropathy spectrum disorder, central auditory processing disorder and tinnitus.

[0105] As used herein, “conductive hearing loss” refers to the impaired transmission of sound waves through the external ear canal to the bones of the middle ear.

[0106] As used herein, “sensorineural hearing loss” refers to a pathologic change in structures with the inner ear or in the acoustic nerve.

[0107] As used herein, “central hearing loss” refers to a pathologic condition above the junction of the acoustic nerve and the brainstem.

[0108] As used herein, “mixed hearing loss” refers to a subject having both conductive hearing loss and sensorineural hearing loss.

[0109] As used herein, “hidden hearing loss” (or HHL) refers to a subject having hearing loss resulting from damage to the auditor}’ nerve / auditory neurons.

[0110] As used herein, “auditory neuropathy spectrum disorder” refers to a type of sensorineural hearing loss where the auditory nen e fails to send consistent messages to the auditory’ centers of the brain. Auditory’ neuropathy is a challenging hearing disorder in which the inner ear successfully detects sound but has a problem with sending signals from the ear to the brain, currently accounting for approximately 10% of cases of sensorineural hearing loss (SNHL) in children. Current state of the art medical knowledge suggests that auditory neuropathies play a substantial role in hearing impairments and deafness. Hearing depends on a series of complex steps that change sound waves in the air into electrical signals. The auditory nerve then carries these signals to the brain. Auditory neuropathy can be caused by a number of factors including damage to the auditory neurons or loss of these neurons, a variety^ of genetic mutations, and viral infections. Researchers are still seeking effective treatments for those affected with auditory neuropathy.

[0111] As used herein “central auditory processing disorder” refers to deficits in the neural processing of auditory information in the central auditory nervous system.Methods of Characterizing Populations of Auditory Cells

[0112] The disclosure provides methods of characterizing the populations of auditory' cells produced by the methods of the disclosure, i.e. differentiated from pluripotent cells using the methods described herein. The methods described herein can determine if theAttorney Docket No. LCTI-005 / 001WG 43694-03305 population of auditory cells is a substantially pure mixture of mid ONP cells, late ONP cells, auditory neuron progenitors, mature auditory neurons and / or spiral ganglion neurons, and is substantially or entirely free of pluripotent cells or one or more intermediate cell types produced during the differentiation process.

[0113] In addition, the methods provide quantitative bioinformatic tools to verify reproducibility of the cell composition for each batches. The methods provide a gene set for auditory cells. The protein products of these genes can have extracellular epitopes (surface markers) that can be used for developing cell or protein based analytic methods, as well as selection or isolation of specific auditory subpopulations by cell separation methods such as flow cytometry (FCM).

[0114] In some embodiments, the methods comprise performing single-cell RNA sequencing on a plurality of cells in the population of auditory cells. In some embodiments, the plurality of cells comprises at least 1,000, at least 2,000, at least 3,000, at least 5,000, at least 10,000, at least 15,000, at least 20,000, at least 20,000 cells, at least 25,000 cells, at least 30,000. at least 40.000 cells, at least 50,000 cells or at least 100,000 cells. In some embodiments, the plurality of cells comprises at least 5,000 cells. In some embodiments, the plurality of cells comprises at least 10,000 cells. In some embodiments, the plurality of cells comprises at least 20,000 cells. In some embodiments, the RNA sequencing generates at least 10,000 reads per cell, at least 20,000 reads per cell, at least 30,000 reads per cell, at least 40.000 reads per cell, at least 50,000 reads per cell, at least 100,000 reads per reads per cell, at least 150,000 reads per cell or at least 200,000 reads per cell. In some embodiments, the RNA sequencing generates at least 50,000 reads per cell. In some embodiments, the RNA sequencing generates at least 100,000 reads per cell. In some embodiments, the RNA sequencing generates at least 150,000 reads per cell. The reads per cell can be mapped to transcripts, and used to measure gene expression. In some embodiments, the expression of at least 500, at least 1,000, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least 3,500, at least 4,000, at least 5,000 or at least 6,000 genes, or any range therebetween, is measured.

[0115] In some embodiments, the methods comprise determining the percentage of cells in the population that express one or more genes, set forth in Table 5 or 6. The genes in Tables 5 and 6 are upregulated in mid ONP cells, late ONP cells, auditory neuron progenitors, mature auditor)' neurons and / or spiral ganglion neurons. Thus, the percentage of cells that express combinations of these genes determines the percentage of cells in the population ofAttorney Docket No. LCTI-005 / 001WG 43694-03305 auditory cells that have differentiated into mid ONP cells, late ONP cells, auditory neuron progenitors, mature auditory neurons and / or spiral ganglion neurons.

[0116] In some embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% of the cells in the population express or more genes set forth in Table 5 or 6. The percentage of cells that express one or more genes set forth in Table 5 or 6 can be determined, inter alia, by singlecell RNA sequencing, in which individual reads can be mapped to the RNAs encoded by the genes in Table 5 or 6, and to individual cells, for example using cell-specific barcodes added during the sequencing I i brary preparation process which are sequenced along with portions of the encoded RNAs. Alternatively, the percentage of cells that express the one or more genes, set forth in Table 5 or Table 6, can be determined by methods such as flow cytometry (FCM) to assess percentage of cells positive to the protein product of these genes, which is within the skill of the person of ordinary skill in the art.

[0117] In some embodiments, a plurality' of cells, e.g. at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or any range therebetween) of cells in the population express one or more genes set forth in Table 5 or 6. In some embodiments, at least 40% of the cells in the population express or more genes set forth in Table 5 or 6. In some embodiments, at least 50% of the cells in the population express one or more genes set forth in Table 5 or 6. In some embodiments, at least 60% of the cells in the population express one or more genes set forth in Table 5 or 6. In some embodiments, at least 70% of the cells in the population express one or more genes set forth in Table 5 or 6. In some embodiments, at least 85% of the cells in the population express one or more genes set forth in Table 5 or 6. In some embodiments, at least 90% of the cells in the population express one or more genes set forth in Table 5 or 6. In some embodiments, at least 95% of the cells in the population express one or more genes set forth in Table 5 or 6. In some embodiments, at least 97% of the cells in the population express one or more genes set forth in Table 5 or 6. In some embodiments, at least 99% of the cells in the population express one or more genes set forth in Table 5 or 6.

[0118] In some embodiments, a plurality of cells, e.g. at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or any range therebetween of cells in the population express at least 20, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600 or all of the genes set forth in Table 5. In some embodiments, a plurality of cells (e.g. at least 60%) in the population express at least 20 of the genes set forth in Table 5. In some embodiments, aAttorney Docket No. LCTI-005 / 001WO 43694-03305 plurality of cells (e.g. at least 40%) in the population express at least 50 of the genes set forth in Table 5. In some embodiments, a plurality of cells (e.g. at least 50%) in the population express at least 50 of the genes set forth in Table 5. In some embodiments, a plurality of cells (e.g. at least 60%) in the population express at least 50 of the genes set forth in Table 5. In some embodiments, a plurality of cells (e.g. at least 60%) in the population express at least 100 of the genes set forth in Table 5. In some embodiments, a plurality of cells (e.g. at least 60%) in the population express at least 200 of the genes set forth in Table 5. In some embodiments, a plurality of cells (e.g. at least 60%) in the population express at least 300 of the genes set forth in Table 5. In some embodiments, a plurality of cells (e.g. at least 60%) in the population express at least 400 of the genes set forth in Table 5. In some embodiments, a plurality of cells (e.g. at least 60%) in the population express at least 500 of the genes set forth in Table 5. In some embodiments, a plurality of cells (e.g. at least 60%) in the population express at least 600 of the genes set forth in Table 5. In some embodiments, a plurality' of cells (e.g. at least 60%) in the population express all of the genes set forth in Table 5.

[0119] In some embodiments, a plurality of cells, e.g. at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or any range therebetween of cells in the population express at least 10, at least 15, at least 20, at least 25 or all of the genes selected from the group consisting of one or more of catenin alpha 2 (CTNNA2), cadherin 4 (CDH4), AC 109466.1, carbohydrate sulfotransferase 11 (CHST11), TOX high mobility group box family member 3 (TOX3), ephrin A5 (EFNA5), NALCN channel auxiliary factor 1 (FAM155A), phospholipase C eta 1 (PLCHI), transmembrane 131 like (TMEM131L), AAAATPase AFG2A (SPATA5), NCK associated protein 5 (NCKAP5), contactin associated protein 2 (CNTNAP2), retinoic acid receptor beta (RARB), calcium / calmodulin dependent protein kinase ID (CAMKID), aspartate betahydroxylase (ASPH), teneurin transmembrane protein 2 (TENM2), dihydropyrimidinase like 3 (DPYSL3), neural EGFL like 2 (NELL2), phytanoyl-CoA 2-hydroxylase interacting protein like (PHYHIPL). microtubule affinity regulating kinase 1 (MARK1), GLI2 GLI family zinc finger 2 (GLI2), roundabout guidance receptor 2 (ROBO2), collagen type IV alpha 2 chain (COL4A2), zinc finger and BTB domain containing 16 (ZBTB16), ring finger protein 24 (RNF24), thymocyte selection associated high mobility group box (TOX), laminin subunit alpha 1 (LAMA1), cysteine and tyrosine rich 1 (CYYR1), microtubule crosslinking factor 2 (SOGA). FYVE. RhoGEF and PH domain containing 4a (FYVE), RhoGEF and PH domain containing 4 (FGD4), neuroligin 1 (NLGN1), neuron navigator 1 (NAVI), matrixAttorney Docket No. LCTI-005 / 001WG 43694-03305 metallopeptidase 16 (MMP16), arkadia (RNF111) C-terminal like ring finger ubiquitin ligase 2C (RNF165), sodium channel and clathrin linker 1 (SCLT1). fibroblast growth factor 13 (FGF13), SOX2 overlapping transcript (SOX2-OT), leucine rich repeats and immunoglobulin like domains 1 (LRIG1), ELAV like RNA binding protein 3 (ELAVL3), fidgetin, microtubule severing factor (FIGN), erb-b2 receptor tyrosine kinase 4 (ERBB4), podoplanin (PDPN), adenosine deaminase RNA specific B2 (ADARB2), MMS22 like, DNA repair protein (MMS22L), CUGBP Elav-like family member 2 (CELF2), neuromedin U (NMU), tetraspanin 18 (TSPAN18), chromosome 1 open reading frame 21 (Clorf21), WD repeat and HMG-box DNA binding protein 1 (WDHD1), neuronal cell adhesion molecule (NRCAM), splA / ry anodine receptor domain and SOCS box containing 4 (SPSB4), formin homology 2 domain containing 3 (FHOD3) and polypeptide N-acetylgalactosaminyltransferase 13 (GALNT13). In some embodiments, a plurality of cells (e.g. at least 40% or at least 50%) in the population express at least 10 of the genes selected from the group consisting of CTNNA2, CDH4, AC109466.1, CHST11, TOX3, EFNA5, FAM155A, PLCHI, TMEM131L, SPATA5, NCKAP5, CNTNAP2. RARB, CAMKID, ASPH, TENM2. DPYSL3, NELL2. PHYHIPL, MARK1, GLI2, ROBO2, COL4A2. ZBTB16, RNF24, TOX. LAMAL CYYRL SOGA, FGD4, NLGN1, NAVI, MMP16, RNF165, SCLT1, FGF13, SOX2-OT, LRIG1, ELAVL3, FIGN, ERBB4, PDPN, ADARB2, MMS22L, CELF2, NMU, TSPAN18, Clorf21, WDHD1, NRCAM, SPSB4, FHOD3 and GALNT13. In some embodiments, a plurality of cells (e.g. at least 60%) in the population express at least 15 of the genes selected from the group consisting of CTNNA2, CDH4, AC 109466.1, CHST11, TOX3, EFNA5, FAM 155 A, PLCHI, TMEM131L, SPATA5, NCKAP5, CNTNAP2, RARB, CAMKID, ASPH, TENM2, DPYSL3, NELL2, PHYHIPL, MARK1, GLI2, ROBO2, COL4A2, ZBTB16, RNF24, TOX, LAMAL CYYR1, SOGA. FGD4, NLGN1, NAVI, MMP16, RNF165, SCLTL FGF13, SOX2-OT, LRIG1, ELAVL3, FIGN, ERBB4, PDPN, ADARB2, MMS22L, CELF2, NMU, TSPAN18, Clorf21, WDHD1, NRCAM, SPSB4, FHOD3 and GALNT13.In some embodiments, a plurality of cells (e.g. at least 60%) in the population express at least 10 of the genes selected from the group consisting of CTNNA2, CDH4, AC109466.1, CHST11, TOX3. EFNA5, FAM155A, PLCH1, TMEM131L, SPATA5, NCKAP5, CNTNAP2, RARB. CAMKID, ASPH, TENM2, DPYSL3, NELL2, PHYHIPL, MARK1, GLI2, ROBO2, COL4A2, ZBTB16, RNF24, TOX, LAMA1, CYYR1, SOGA, FGD4, NLGN1, NAVI, MMP16, RNF165, SCLT1, FGF13, SOX2-OT, LRIG1, ELAVL3, FIGN, ERBB4, PDPN, ADARB2, MMS22L, CELF2. NMU, TSPAN18, Clorf21, WDHD1, NRCAM. SPSB4, FHOD3 and GALNT13. In some embodiments, a plurality of cells (e.g. at least 60%) in theAttorney Docket No. LCTI-005 / 001WO 43694-03305 population express at least 15 of the genes selected from the group consisting of CTNNA2, CDH4, AC109466.1, CHST11, T0X3, EFNA5, FAM155A. PLCHI, TMEM131L, SPATA5, NCKAP5, CNTNAP2, RARB, CAMKID, ASPH, TENM2, DPYSL3, NELL2, PHYHIPL, MARK1, GLI2, R0B02, COL4A2, ZBTB16, RNF24, TOX, LAMA1, CYYR1, SOGA, FGD4, NLGN1, NAVI, MMP16, RNF165, SCLT1, FGF13, SOX2-OT, LRIG1, ELAVL3, FIGN, ERBB4, PDPN, ADARB2, MMS22L, CELF2. NMU, TSPAN18, Clorf21, WDHD1, NRCAM, SPSB4, FHOD3 and GALNT13. In some embodiments, a plurality of cells (e.g. at least 60%) in the population express at least 20 of the genes selected from the group consisting of CTNNA2, CDH4, AC109466.1, CHST11, TOX3, EFNA5, FAM155A, PLCHI, TMEM131L, SPATA5, NCKAP5, CNTNAP2, RARB, CAMKID, ASPH, TENM2, DPYSL3, NELL2, PHYHIPL, MARK1, GLI2, ROBO2. COL4A2, ZBTB16, RNF24, TOX, LAMA1, CYYR1, SOGA, FGD4, NLGN1, NAVI, MMP16, RNF165, SCLT1, FGF13, SOX2-OT, LRIG1, ELAVL3, FIGN, ERBB4, PDPN, ADARB2, MMS22L, CELF2, NMU, TSPAN18, Clorf21, WDHD1, NRCAM, SPSB4, FHOD3 and GALNT13. In some embodiments, a plurality of cells (e.g. at least 60%) in the population express at least 25 of the genes selected from the group consisting of CTNNA2, CDH4, AC109466.1. CHST1L TOX3. EFNA5, FAM155A, PLCHI, TMEM131L, SPATA5, NCKAP5, CNTNAP2, RARB, CAMKID, ASPH, TENM2, DPYSL3, NELL2, PHYHIPL, MARK1, GLI2, ROBO2, COL4A2, ZBTB16, RNF24, TOX, LAMA1, CYYR1, SOGA, FGD4, NLGN1, NAVI, MMP16, RNF165. SCLT1, FGF13, SOX2-OT, LRIGL ELAVL3, FIGN. ERBB4, PDPN, ADARB2, MMS22L, CELF2, NMU, TSPAN18, Clorf21, WDHD1, NRCAM, SPSB4, FHOD3 and GALNT13. In some embodiments, a plurality of cells (e.g. at least 40%, at least 50%, or at least 60%) in the population express all of CTNNA2, CDH4, AC109466.1, CHSTH. TOX3, EFNA5, FAM155A, PLCHI, TMEM131L, SPATA5, NCKAP5, CNTNAP2, RARB, CAMKID, ASPH, TENM2, DPYSL3, NELL2, PHYHIPL, MARK!, GLI2, ROBO2, COL4A2, ZBTB16, RNF24, TOX, LAMA1, CYYR1, SOGA, FGD4, NLGN1, NAVI, MMP16, RNF165, SCLT1, FGF13, SOX2-OT, LRIG1, ELAVL3, FIGN, ERBB4, PDPN, ADARB2, MMS22L, CELF2, NMU, TSPAN18, ClorfZl, WDHD1, NRCAM. SPSB4, FHOD3 and GALNTI3.

[0120] In some embodiments, the cells in the population that express the one or more genes set forth in Table 5 or 6 are mid otic neuronal progenitor (ONP) cells, late ONP cells, auditory neuron progenitors, mature auditory neurons, spiral ganglion neurons or any combination thereof.Attorney Docket No. LCTI-005 / 001WG 43694-03305

[0121] In some embodiments, the method comprises clustering the cells using the methods described herein, and at least 50%, at least 60%, at least 70% or at least 80% of the cells in the 1-3 major clusters (i.e., the 1, 2 or 3 major clusters, wherein the major clusters are the clusters containing at least 80% of the population of cells as described herein) express a plurality7of any one of the genes set forth in Table 5 or6. In some embodiments, gene expression in the 1-3 major clusters is determined relative to gene expression of the cells in the one or more minor clusters, and at least 50%, at least 60%, at least 70%, or at least 80% of the cells in the 1-3 major clusters upregulate one or more genes disclosed in Table 5 or 6 as determined relative to the expression of the same genes by the cells in the one or more minor clusters.

[0122] In some embodiments, a plurality of cells, e.g. at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or any range therebetween) of cells in the 1-3 major clusters express one or more genes set forth in Table 5 or 6. In some embodiments, at least 60% of the cells in the 1-3 major clusters express one or more genes set forth in Table 5 or 6. In some embodiments, at least 70% of the cells in the 1-3 major clusters express one or more genes set forth Table 5 or 6. In some embodiments, at least 85% of the cells in the 1-3 major clusters express one or more genes set forth in Table 5 or 6. In some embodiments, at least 90% of the cells in the 1-3 major clusters express one or more genes set forth Table 5 or 6. In some embodiments, at least 95% of the cells in the 1-3 major clusters express one or more genes set forth Table 5 or 6. In some embodiments, at least 97% of the cells in the 1 -3 major clusters express one or more genes set forth in Table 5 or 6. In some embodiments, at least 99% of the cells in the major clusters express one or more genes set forth in Table 5 or 6.

[0123] In some embodiments, a plurality7of cells, e.g. at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or any range therebetween of cells in the 1-3 major clusters express at least 20, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600 or all of the genes set forth in Table 5. In some embodiments, a plurality of cells (e.g. at least 60%, at least 70% or at least 80%) in the 1-3 major clusters express at least 20 of the genes set forth in Table 5. In some embodiments, a plurality7of cells (e.g. at least 80%) in the 1-3 major clusters express at least 50 of the genes set forth in Table 5. In some embodiments, a plurality7of cells (e.g. at least 60%, at least 70% or at least 80%) in the 1-3 major clusters express at least 100 of the genes set forth in Table 5. In some embodiments, a plurality of cells (e.g. at least 80%) in the 1-3 major clusters express at least 200 of the genes set forth in Table 5. In some embodiments, aAttorney Docket No. LCTI-005 / 001WG 43694-03305 plurality of cells (e.g. at least 60%, at least 70% or at least 80%) in the 1-3 major clusters express at least 300 of the genes set forth in Table 5. In some embodiments, a plurality of cells (e.g. at least 80%) in the 1-3 major clusters express at least 400 of the genes set forth in Table 5. In some embodiments, a plurality of cells (e.g. at least 60%, at least 70% or at least 80%) in the 1-3 major clusters express at least 500 of the genes set forth in Table 5. In some embodiments, a plurality of cells (e.g. at least 60%, at least 70% or at least 80%) in the 1-3 major clusters express at least 600 of the genes set forth in Table 5. In some embodiments, a plurality of cells (e.g .at least 60%, at least 70% or at least 80%) in the 1-3 major clusters express all of the genes set forth in Table 5.

[0124] In some embodiments, a plurality of cells, e.g. at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or any range therebetween of cells in the 1-3 major clusters express at least 10, at least 15, at least 20, at least 25 or all of the genes selected from the group consisting of one or more of catenin alpha 2 (CTNNA2), cadherin 4 (CDH4), AC109466.1, carbohydrate sulfotransferase 11 (CHST11), TOX high mobility group box family member 3 (TOX3), ephrin A5 (EFNA5), NALCN channel auxiliary factor 1 (FAM155A), phospholipase C eta 1 (PLCHI), transmembrane 131 like (TMEM131L), AAA ATPase AFG2A (SPATA5), NCK associated protein 5 (NCKAP5), contactin associated protein 2 (CNTNAP2), retinoic acid receptor beta (RARB), calcium / calmodulin dependent protein kinase ID (CAMKID), aspartate beta-hydroxylase (ASPH), teneurin transmembrane protein 2 (TENM2), dihydropyrimidinase like 3 (DPYSL3), neural EGFL like 2 (NELL2), phytanoyl-CoA 2-hydroxylase interacting protein like (PHYHIPL), microtubule affinity regulating kinase 1 (MARK1), GLI2 GLI family zinc finger 2 (GLI2), roundabout guidance receptor 2 (ROBO2), collagen type IV alpha 2 chain (COL4A2), zinc finger and BTB domain containing 16 (ZBTB16), ring finger protein 24 (RNF24), thymocyte selection associated high mobility group box (TOX), laminin subunit alpha 1 (LAMA1), cysteine and tyrosine rich 1 (CYYR1), microtubule crosslinking factor 2 (SOGA), FYVE, RhoGEF and PH domain containing 4a (FYVE), RhoGEF and PH domain containing 4 (FGD4), neuroligin 1 (NLGN1), neuron navigator 1 (NAVI), matrix metallopeptidase 16 (MMP16), arkadia (RNF111) C-terminal like ring finger ubiquitin ligase 2C (RNF165), sodium channel and clathrin linker 1 (SCLT1), fibroblast growth factor 13 (FGF13), SOX2 overlapping transcript (SOX2-OT), leucine rich repeats and immunoglobulin like domains 1 (LRIG1), ELAV like RNA binding protein 3 (ELAVL3), fidgetin, microtubule severing factor (FIGN), erb-b2 receptor tyrosine kinase 4 (ERBB4), podoplanin (PDPN), adenosine deaminase RNA specific B2 (ADARB2), MMS22 like, DNA repair proteinAttorney Docket No. LCTI-005 / 001WG 43694-03305 (MMS22L), CUGBP Elav-like family member 2 (CELF2), neuromedin El (NMU), tetraspanin 18 (TSPAN18), chromosome 1 open reading frame 21 (Clorf21). WD repeat and HMG-box DNA binding protein 1 (WDHD1), neuronal cell adhesion molecule (NRCAM), splA / ry anodine receptor domain and SOCS box containing 4 (SPSB4), formin homology 2 domain containing 3 (FHOD3) and polypeptide N-acetylgalactosaminyltransferase 13 (GALNT13). In some embodiments, a plurality of cells (e.g. at least 60%, at least 70% or at least 80%) in the 1-3 major clusters express at least 10 of the genes selected from the group consisting of CTNNA2, CDH4, AC109466.1, CHST11, TOX3, EFNA5, FAM155A, PLCH1, TMEM131L, SPATA5, NCKAP5, CNTNAP2, RARE, CAMKID, ASPH, TENM2, DPYSL3, NELL2, PHYHIPL, MARK1, GLI2, ROBO2. COL4A2, ZBTB16, RNF24, TOX, LAMA1, CYYR1, SOGA. FGD4, NLGN1, NAVI, MMP16, RNF165, SCLT1. FGF13, SOX2-OT, LRIG1, ELAVL3, FIGN, ERBB4, PDPN, ADARB2, MMS22L, CELF2, NMU, TSPAN18, Clorf21, WDHD1, NRCAM, SPSB4, FHOD3 and GALNT13. In some embodiments, a plurality7of cells (e.g. at least 60%, at least 70% or at least 80%) in the 1-3 major clusters express at least 15 of the genes selected from the group consisting of CTNNA2, CDH4, AC109466.1, CHST11, TOX3, EFNA5. FAM155A, PLCHI. TMEM131L. SPATA5.NCKAP5, CNTNAP2, RARB, CAMKID, ASPH, TENM2, DPYSL3, NELL2, PHYHIPL, MARK1, GLI2, ROBO2, COL4A2, ZBTB16, RNF24, TOX, LAMA1, CYYR1, SOGA, FGD4, NLGN1, NAVI, MMP16, RNF165, SCLT1, FGF13, SOX2-OT, LRIG1, ELAVL3, FIGN, ERBB4, PDPN, ADARB2. MMS22L, CELF2. NMU, TSPAN18, Clorf21, WDHD1. NRCAM, SPSB4, FHOD3 and GALNT13. In some embodiments, a plurality of cells (e.g. at least 60%, at least 70% or at least 80%) in the 1-3 major clusters express at least 20 of the genes selected from the group consisting of CTNNA2, CDH4, AC109466.1, CHST11, TOX3, EFNA5, FAM155A, PLCHI, TMEM131L, SPATA5, NCKAP5, CNTNAP2, RARB, CAMKID, ASPH, TENM2, DPYSL3, NELL2, PHYHIPL, MARK!, GLI2, ROBO2, COL4A2, ZBTB16, RNF24, TOX, LAMA1, CYYR1, SOGA, FGD4, NLGN1, NAVI, MMP16, RNF165, SCLT1, FGF13, SOX2-OT, LRIG1, ELAVL3, FIGN, ERBB4, PDPN, ADARB2, MMS22L, CELF2, NMU, TSPAN18, ClorfZl, WDHD1, NRCAM. SPSB4, FHOD3 and GALNT13. In some embodiments, a plurality of cells (e.g. at least 60%, at least 70% or at least 80%) in the 1-3 major clusters express at least 25 of the genes selected from the group consisting of CTNNA2, CDH4, AC109466.1, CHST11, TOX3, EFNA5, FAM155A. PLCHI, TMEM131L, SPAFA5, NCKAP5, CNTNAP2, RARB, CAMKID, ASPH, TENM2, DPYSL3, NELL2, PHYHIPL, MARK1. GLI2, ROBO2, COL4A2, ZBTB16, RNF24, TOX, LAMA1, CYYR1, SOGA, FGD4, NLGN1, NAVI, MMP16,Attorney Docket No. LCTI-005 / 001WD 43694-03305 RNF165, SCLT1, FGF13, S0X2-0T, LRIG1, ELAVL3, FIGN, ERBB4, PDPN, ADARB2, MMS22L, CELF2. NMU, TSPAN18, Clorf21, WDHD1, NRCAM, SPSB4, FH0D3 and GALNT13. In some embodiments, a plurality of cells (e.g. at least 60%, at least 70% or at least 80%) in the 1-3 major clusters express all of CTNNA2, CDH4, AC 109466.1, CHST11, TOX3, EFNA5, FAM155A, PLCHI, TMEM131L, SPATA5, NCKAP5, CNTNAP2, RARB, CAMKID. ASPH, TENM2, DPYSL3, NELL2, PHYHIPL, MARK1. GLI2, ROBO2, COL4A2, ZBTB16, RNF24, TOX, LAMA1, CYYR1, SOGA, FGD4, NLGN1, NAVI. MMP16, RNF165, SCLT1, FGF13, SOX2-OT, LRIG1, ELAVL3, FIGN, ERBB4, PDPN, ADARB2, MMS22L, CELF2, NMU, TSPAN18, Clorf21, WDHD1, NRCAM, SPSB4, FHOD3 and GALNT13.Table 2: Genes that are upregulated in ANP1 cells (Group A)Attorney Docket No. LCTI-005 / 001WO 43694-03305Attorney Docket No. LCTI-005 / 001WO 43694-03305Attorney Docket No. LCTI-005 / 001WO 43694-03305Attorney Docket No. LCTI-005 / 001WO 43694-03305Attorney Docket No. LCTI-005 / 001WO 43694-03305Attorney Docket No. LCTI-005 / 001WO 43694-03305Attorney Docket No. LCTI-005 / 001WO 43694-03305Attorney Docket No. LCTI-005 / 001WO 43694-03305Attorney Docket No. LCTI-005 / 001WO 43694-03305Attorney Docket No. LCTI-005 / 001WO 43694-03305Attorney Docket No. LCTI-005 / 001WO 43694-03305Attorney Docket No. LCTI-005 / 001WO 43694-03305Attorney Docket No. LCTI-005 / 001WO 43694-03305Attorney Docket No. LCTI-005 / 001WO 43694-03305Attorney Docket No. LCTI-005 / 001WO 43694-03305Attorney Docket No. LCTI-005 / 001WO 43694-03305Attorney Docket No. LCTI-005 / 001WO 43694-03305Attorney Docket No. LCTI-005 / 001WO 43694-03305Attorney Docket No. LCTI-005 / 001WO 43694-03305Attorney Docket No. LCTI-005 / 001WO 43694-03305Attorney Docket No. LCTI-005 / 001WO 43694-03305Attorney Docket No. LCTI-005 / 001WO 43694-03305Attorney Docket No. LCTI-005 / 001WO 43694-03305Attorney Docket No. LCTI-005 / 001WO 43694-03305Attorney Docket No. LCTI-005 / 001WO 43694-03305Table 3: Genes that are upregulated in ANP1 cells (Group B)Attorney Docket No. LCTI-005 / 001WO 43694-03305Attorney Docket No. LCTI-005 / 001WO 43694-03305Attorney Docket No. LCTI-005 / 001WO 43694-03305Attorney Docket No. LCTI-005 / 001WO 43694-03305Attorney Docket No. LCTI-005 / 001WO 43694-03305Attorney Docket No. LCTI-005 / 001WO 43694-03305Attorney Docket No. LCTI-005 / 001WO 43694-03305Attorney Docket No. LCTI-005 / 001WO 43694-03305Attorney Docket No. LCTI-005 / 001WO 43694-03305Attorney Docket No. LCTI-005 / 001WO 43694-03305Attorney Docket No. LCTI-005 / 001WO 43694-03305Attorney Docket No. LCTI-005 / 001WO 43694-03305Attorney Docket No. LCTI-005 / 001WO 43694-03305Table 4: Genes that are upregulated in ANP1 cells (Group C)Attorney Docket No. LCTI-005 / 001WO 43694-03305Attorney Docket No. LCTI-005 / 001WO 43694-03305Attorney Docket No. LCTI-005 / 001WO 43694-03305Attorney Docket No. LCTI-005 / 001WO 43694-03305Attorney Docket No. LCTI-005 / 001WG 43694-03305Table 5: Genes that are upregulated in ANP1 cells (Group D)Attorney Docket No. LCTI-005 / 001WO 43694-03305Attorney Docket No. LCTI-005 / 001WO 43694-03305Attorney Docket No. LCTI-005 / 001WD 43694-03305

[0125] Table 6 shows 53 genes that are upregulated in at least 60%, e.g. between about 60% and about 95%, of the cells in the two main clusters produced by Uniform Manifold Approximation and Projection for Dimension Reduction (UMAP) analysis of the single cell RNA sequencing data (CO and Cl in FIGS. 7 and 8A).Table 6: Genes that are upregulated in ANP1 cells (Group E)>Attorney Docket No. LCTI-005 / 001WO 43694-03305

[0126] The methods disclosed herein can be used to identify proteins expressed on the surface of auditory cells, which can used for cell sorting. Table 7 shows 26 exemplary protein coding genes that are upregulated in ANP1 cells, and which can be used for cell sorting. Table 7: Genes that are upregulated in ANP1 cells (Group F)

[0127] In some embodiments, the methods further comprise determining the expression of one or more of Nestin. SOX2, (3 tubulin III, TrkB, TRA-1-60, SSEA5, PAX2, PAX8, GluA4, Myo7A and / or CD133, for example by flow cytometry, reverse transcription quantitative PCR (RT-qPCR), immunocytochemistry or immunohistochemistry (e.g. immunofluorescence followed by imaging) as described in further detail below.

[0128] In some embodiments, the methods comprise determining the expression of one or more of POU class 5 homeobox 1 (POU5F1), Nanog, lin-28 homolog A (LIN28A), DNA methyltransferase 3 beta (DMNT3B), cripto, EGF-CFC family member (TDGF1) and LINE1 ty pe transposase domain containing 1 (L1TD1) in cells of the population of auditory cells. The expression of POU5F1, Nanog, LIN28A, DMNT3B, TDGF1 and L1TD1 can be determined using any suitable method described herein or known in the art, including methods of determining RNA expression such as RNA sequencing, as well as methods of determining protein expression such as flow cytometry and immunohistochemistry' (e.g. immunofluorescence followed by imaging).

[0129] POU5F1. Nanog. LIN28A. DMNT3B. TDGF1 and L1TD1 are markers of pluripotency. In some embodiments, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% or no cells in the population express POU5F1, Nanog, LIN28A, DMNT3B, TDGF1 and / or L1TD1. In some embodiments, less than 5% of cells in the population express POU5F1, Nanog, LIN28A, DMNT3B, TDGF1 and / or LI TD1. In some embodiments, less than 3% of cells in the population express POU5F 1, Nanog, LIN28A, DMNT3B, TDGF1 and / or L1TD1. In some embodiments, less than 1% of cells in the population expressAttorney Docket No. LCTI-005 / 001WG 43694-03305 P0U5F1, Nanog, LIN28A, DMNT3B, TDGF1 and / or L1TD1. In some embodiments, no cells in the population express POU5F1. Nanog, LIN28A, DMNT3B, TDGF1 and / or L1TD1 (i. e. , the number of cells that express POU5F1, Nanog, LIN28A, DMNT3B, TDGF1 and / or L1TD1 is below the limit of detection).Methods of Characterizing a Population of Auditory cells using scRNA Seq uencing

[0130] The disclosure provides methods of characterizing populations of auditory cells, and identifying sets of genes for use in characterizing populations of auditor}’ cells, using single cell RNA sequencing.

[0131] “Single cell RNA sequencing” or “scRNA sequencing” and similar terms refer to a laboratory and computational technique used to measure gene expression levels in individual cells rather than in bulk tissue. scRNA sequencing can be sued to measure the transcription of thousands to millions of individual cells. In atypical scRNA sequencing workflow, individual cells are separated using methods such as microfluidics (e.g., dropletbased systems), fluorescence activated cell sorting or manual separation, followed by RNA capture and reverse transcription. Complementary cDNAs are appended with barcodes that identify an individual transcript as originating from a particular cell, so reads can be traced back to the cell of origin. The barcoded cDNA libraries are then sequenced using a high-throughput DNA sequencing platform (e.g., Illumina). A bioinformatics pipeline is used to, inter alia, map sequence reads a reference genome, and count the number of transcripts per gene per cell, and generate an expression matrix.

[0132] The disclosure provides methods of characterizing a population of auditory cells, such as the populations of cells derived by in vitro differentiation of pluripotent cells described herein, by (a) performing single cell RNA sequencing on the population of auditory cells, and (b) clustering cells in the population based on shared expression of a plurality of genes.

[0133] In some embodiments, the methods comprise (a) performing single cell RNA sequencing on the population of auditory cells, thereby determining the expression of a plurality of genes by individual cells in the population, (b) generating a two-dimensional representation of the similarity and / or difference of gene expression of individual cells of the population based on expression of at least a subset of genes in the plurality7of genes, (c) partitioning the two-dimensional representation into a plurality of clusters, wherein the plurality of clusters comprises 1-3 major clusters and one or more minor clusters, (d) identifying a set of genes that are upregulated in at least 50% (e.g., at least 60%, at least 70%,Attorney Docket No. LCTI-005 / 001WO 43694-03305 at least 80%, or at least 90%) of cells of the 1-3 major clusters, and (e) identifying a percentage of cells in the population that express the set of genes, thereby characterizing the population of auditory cells.

[0134] In some embodiments, the methods comprise (f) selecting a population of auditory cells wherein at least 50% (e.g., at least 60%, at least 70%, at least 80%, or at least 90%) of the cells in the population express the set of genes identified using the methods described herein.

[0135] In some embodiments, the 1-3 major clusters consist of 2 clusters. Exemplar}’ two-dimensional representations of scRNA sequencing data with two major clusters are shown in FIGS. 7 and 8A. The skilled artisan will appreciate that the number of primary clusters may vary depending on the statistical analysis technique used to analyze the data, and so the majority of cells may group together in 1, 2, 3, 4 or even 5 clusters, and still be indicative of the same primary cell types present in the population of cells. Major clusters are those subset of clusters in which scRNA sequencing data corresponding to at least 80% of the cells in the population (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89% or at least 90%) is grouped in the major clusters, as demonstrated in FIGS. 7 and 8A.

[0136] In some embodiments, the one or more minor clusters comprise or consist essentially of 1-4 clusters. In some embodiments, less than 10% of cells in the population are in any individual minor cluster, e.g.. less than 9%. less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, or less than 3% of scRNA sequencing data corresponding to cells in the population is grouped in any individual minor cluster. Exemplary minor clusters are shown in FIGS. 7 and 8A. In some embodiments, less than 20% of the cells in the population are in the one or more minor clusters, e.g. less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11% or less than 10% of the cells in the population are in the one or more minor clusters.

[0137] Cells in the population can be clustered based on shared gene expression using any suitable method known in the art. In some embodiments, the clustering comprises a dimensionality reduction technique, such as principal component analysis (PCA), UMAP or t-SNE, or a combination thereof (such as PCA, followed by UMAP or t-SNE).

[0138] One exemplary’ dimensionality’ reduction method that can be used to identify clusters of cells with shared gene expression is uniform manifold approximation and projection (UMAP). UMAP is a machine learning algorithm for dimensionality reduction of high-dimension datasets, such as single-cell RNA sequencing data. In brief, UMAP projectsAttorney Docket No. LCTI-005 / 001WO 43694-03305 high-dimensional data into a low-dimensional space (often 2D or 3D) while preserving both local and some global structure of the data. This facilitates visualization and downstream analysis of the data. UMAP assumes that the high-dimensional data lie on a lowerdimensional manifold embedded in high-dimensional space. It builds a weighted graph in the higher dimensional space based on nearest neighbors, capturing local relationships and connectivity. It then optimizes the layout of the data points in fewer dimensions, e.g. two dimensions, so that distances and relationships between the data points from the higherdimensional graph are preserved as much as possible. This enables the visualization of clusters of related data points, for example the two clusters of cells described herein (see FIGS. 7, 8A and 8B).

[0139] “Principal component analysis’" refers to a computational process that, given a plurality of feature vectors representing a plurality of occurrences in a dataset, for example cells and corresponding gene expression values, computes an orthogonal set of basis vectors (principal components) that are linear combinations of the original feature variables, such that the basis vectors are ordered according to the amount of variance in the underlying feature vectors. The first principal component capturing the greatest variance, and subsequent principal components capturing successively lesser variance, each orthogonal to all preceding components. The process can be used to generate a reduced-dimension representation of the dataset by selecting a subset of the principal components and projecting the feature vectors into the space defined by the subset.

[0140] An additional suitable dimensionality reduction technique is t-SNE. t-SNE (t-distributed Stochastic Neighbor Embedding) is a nonlinear dimensionality reduction algorithm commonly used for visualizing high-dimensional data in two or three dimensions. t-SNE models the similarity between data points in the high-dimensional space and seeks to preserve those relationships in a low-dimensional (e.g., 2 dimensional) embedding. t-SNE pairwise distances into probability distributions that represent neighborhood similarities, then minimizes the difference between these distributions in high and low dimensions using Kullback-Leibler divergence. The algorithm emphasizes preserving local structure, making it effective at revealing clusters or groupings within complex datasets, though it may distort global relationships.

[0141] Optionally, dimensionality reduction techniques such as UMAP and t-SNE can be combined with a community detection (clustering) algorithm, such as, but not limited to k-means (which partitions data into k pre-specified clusters), DBSCAN (a density-based thatAttorney Docket No. LCTI-005 / 001WD 43694-03305 finds arbitrarily shaped clusters), HDBSCAN (a hierarchical version of DBSCAN which automatically chooses the optimal number of clusters) or Louvain / Leiden.

[0142] Dimensionality reduction and clustering can be carried out using, for example, Seurat. Seurat a program in R that is designed for single-cell RNA sequencing analysis, and identifies cell populations based on gene expression profiles. In brief, Seurat filters low-quality cells based on, inter alia, gene count and representation of mitochondrial genes, normalizes the scRNA-seq data to account for differences in sequencing depth, identifies highly variable genes, applies principal component analysis or another dimensionality reduction algorithm, constructs a graph using k-nearest neighbors, and applies a community detection algorithm such as Louvain / Leiden to partition the graph into clusters of highly related cells. Finally, the data is projected into two dimensions, for example using UMAP or t-SNE.

[0143] In some embodiments, generating a two-dimensional representation of the similarity and / or difference of gene expression of individual cells of the population based on expression of at least a subset of genes in the plurality of genes comprises (i) providing single cell RNA sequencing data for individual cells in the population of auditory cells, wherein each cell is associated with expression values for the plurality7of genes based on read counts from the single cell RNA sequencing data, (ii) performing principal component analysis, thereby producing a first reduced-dimensionality representation of the single cell RNA sequencing data for individual cells, (iii) determining, for each cell, one or more neighboring cells based on a plurality of principal component scores for each cell, (iv) generating a weighted graph, wherein vertices correspond to individual cells in the population of cells and wherein edge weights correspond to a degree of similarity between the individual cells, and (v) embedding the weighted graph in two-dimensional space, whereby preservation of topological relationships from the weighted graph is maximized, thereby generating a second reduced-dimensionality representation of the single cell RNA sequencing data for individual cells.

[0144] In some embodiments, the principal component analysis (PCA) comprises determining at least the first 10 principal components (e.g., at least 10, at least 15, at least 20, at least 30, at least 40, or at least 50 principal components). In some embodiments, the principal component analysis comprises determining the first 10 principal components. In some embodiments, the principal component analysis comprises determining the first 20 principal components. In some embodiments, the principal component analysis comprises determining the first 30 principal components.Attorney Docket No. LCTI-005 / 001WG 43694-03305

[0145] In some embodiments, the method comprises determining the k-nearest neighbors (KNN) for each cell based on the principal components. KNN is a method for identifying the k closest data points to a given target point, based on a defined distance or similarity metric. For example, for scRNA sequencing data, each cell’s position relative to each other cell following PC A is used to find the k most similar other cells.

[0146] In exemplary embodiments, for example those embodiments in which PCAis followed by UMAP, the KNN relationships become the edges in the weighted graph used by UMAP.

[0147] In some embodiments, the at least a subset plurality of genes used to generate the tw o dimension representation of the similarity and / or difference of gene expression of individual cells in the population comprises at least 50 genes, at least 500 genes, at least 600 genes, at least 1,000 genes, at least 3,000 genes or at least 5,000 genes. In some embodiments, the at least a subset of the plurality of genes comprises at least 1,000 genes. In some embodiments, the at least a subset of the plurality of genes comprises at least 2,000 genes. In some embodiments, the at least a subset of the plurality of genes comprises about 2,000 genes. In some embodiments, the at least a subset of the plurality of genes comprises between about 50 genes and about 6,000 genes, e.g. between about 100 genes and about 5,000 genes, between about 500 and about 4,000 genes, or between about 1,000 and 3,000 genes. In some embodiments, the at least a subset of the plurality of genes comprises between about 20 and about 700 genes. In some embodiments, the at least a subset of the plurality of genes comprise genes selected from the group of genes disclosed in Tables 5 and 6.

[0148] The methods described herein can be repeated more than once on independently generated populations of auditory cells to identify sets of gene that consistently characterize the populations of auditory cells. For example, the methods can be repeated at least IX, 2X, 3X, 4X, 5X, 6X, 7X, 8X, 9X, 10X or more to identify' a set of genes consistently upregulated in at least 80% of the cells of the 1-3 major clusters.

[0149] In some embodiments, the set of genes, i.e. the set of genes upregulated in the 1-3 major clusters, comprises at least 10 genes, at least 20, genes, at least 30 genes, at least 40 genes, or at least 50 genes. In some embodiments, the set of genes comprises between about 20 genes and about 70 genes. In some embodiments, the set of genes comprises a plurality of the genes (e.g., between 20 and 700 hundred, between 50 and 500, between 100 and 400, or any range therebetween) set forth in Table 5. In some embodiments, the set of genes comprises, consists essentially of, or consists of the 53 genes set forth in Table 6.Attorney Docket No. LCTI-005 / 001WO 43694-03305

[0150] An exemplar}' set of genes that is consistently upregulated in the 1-3 major clusters is set forth in Table 6, and consists of CTNNA2, CDH4, AC109466.1, CHST11, TOX3, EFNA5, FAM155A, PLCHI, TMEM131L, SPATA5, NCKAP5, CNTNAP2, RARE, CAMKID, ASPH, TENM2, DPYSL3, NELL2, PHYHIPL, MARK1, GLI2, ROBO2, COL4A2, ZBTB16, RNF24, TOX, LAMA1, CYYR1, SOGA, FGD4, NLGN1, NAVI, MMP16, RNF165, SCLT1, FGF13, SOX2-OT, LRIG1, ELAVL3, FIGN, ERBB4, PDPN, ADARB2, MMS22L, CELF2. NMU, TSPAN18, Clorf21, WDHD1, NRCAM. SPSB4, FHOD3 and GALNT13.

[0151] In some embodiments, at least 60%, (e.g. at least 70%, at least 80%, at least 85% or at least 90%) of the cells in the 1-3 major clusters express one or more of catenin alpha 2 (CTNNA2), cadherin 4 (CDH4), AC109466.1, carbohydrate sulfotransferase 11 (CHST11), TOX high mobility group box family member 3 (TOX3), ephrin A5 (EFNA5), NALCN channel auxiliary factor 1 (FAM155A), phospholipase C eta 1 (PLCHI), transmembrane 131 like (TMEM131L), AAA ATPase AFG2A (SPATA5), NCK associated protein 5 (NCKAP5), contactin associated protein 2 (CNTNAP2), retinoic acid receptor beta (RARB), calcium / calmodulin dependent protein kinase ID (CAMKID), aspartate betahydroxylase (ASPH), teneurin transmembrane protein 2 (TENM2), dihydropyrimidinase like 3 (DPYSL3), neural EGFL like 2 (NELL2), phytanoyl-CoA 2-hydroxylase interacting protein like (PHYHIPL). microtubule affinity regulating kinase 1 (MARK1), GLI2 GLI family zinc finger 2 (GLI2), roundabout guidance receptor 2 (ROBO2). collagen type IV alpha 2 chain (COL4A2), zinc finger and BTB domain containing 1 (ZBTB16), ring finger protein 24 (RNF24), thymocyte selection associated high mobility group box (TOX), laminin subunit alpha 1 (LAMA1), cysteine and tyrosine rich 1 (CYYR1), microtubule crosslinking factor 2 (SOGA), FYVE. RhoGEF and PH domain containing 4a (FYVE), RhoGEF and PH domain containing 4 (FGD4), neurohgin 1 (NLGN1), neuron navigator 1 (NAVI), matrix metallopeptidase 16 (MMP16), arkadia (RNF111) C-terminal like ring finger ubiquitin ligase 2C (RNF165), sodium channel and clathrin linker 1 (SCLT1), fibroblast grow th factor 13 (FGF13). SOX2 overlapping transcript (SOX2-OT), leucine rich repeats and immunoglobulin like domains I (LRIG1), ELAV like RNA binding protein 3 (ELAVL3), fidgetin, microtubule severing factor (FIGN), erb-b2 receptor tyrosine kinase 4 (ERBB4), podoplanin (PDPN), adenosine deaminase RNA specific B2 (ADARB2), MMS22 like, DNA repair protein (MMS22L), CUGBP Elav-like family member 2 (CELF2), neuromedin U (NMU), tetraspanin 18 (TSPAN18), chromosome 1 open reading frame 21 (Clorf21). WD repeat and HMG-box DNA binding protein 1 (WDHD1), neuronal cell adhesion molecule (NRCAM),Attorney Docket No. LCTI-005 / 001WO 43694-03305 splA / ry anodine receptor domain and SOCS box containing 4 (SPSB4), formin homology 2 domain containing 3 (FHOD3) or polypeptide N-acetylgalactosaminyltransferase 13 (GALNT13).

[0152] In some embodiments, at least 60% (e.g. at least 70%, at least 80%, at least 85% or at least 90%) of the cells in thel-3 major clusters express at least 10 genes, at least 20 genes, at least 30 genes, at least 40 genes, at least 50 genes or all genes selected from the group consisting of CTNNA2, CDH4, AC109466.1, CHST11, TOX3, EFNA5. FAM155A, PLCHI, TMEM131L, SPATA5, NCKAP5, CNTNAP2, RARB, CAMKID, ASPH, TENM2, DPYSL3, NELL2, PHYHIPL, MARK1, GLI2, ROBO2, COL4A2, ZBTB16, RNF24, TOX, LAMA1, CYYR1, SOGA, FGD4, NLGN1, NAVI, MMP16, RNF165, SCLT1, FGF13, SOX2-OT, LRIG1, ELAVL3, FIGN, ERBB4, PDPN, ADARB2, MMS22L, CELF2, NMU, TSPAN18, Clorf21, WDHD1, NRCAM, SPSB4, FHOD3 and GALNT13.

[0153] In some embodiments, the cells in the 1-3 major clusters comprise mid otic neuronal progenitor (ONP) cells, late ONP cells, auditory neuron progenitors, mature auditory neurons, spiral ganglion neurons or a combination thereof

[0154] In some embodiments, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% or no cells in the population express RNA of one or more pluripotent stem cell markers. In some embodiments, the one or more pluripotent stem cell markers are selected from the group consisting of POU class 5 homeobox 1 (POU5F1), Nanog, lin-28 homolog A (LIN28A), DNA methyltransferase 3 beta (DMNT3B), cripto, EGF-CFC family member (TDGF1) and LINE1 type transposase domain containing 1 (L1TD1).

[0155] In some embodiments, the population of auditory cells selected by the methods disclosed herein is suitable for administration to a subject with an auditory' condition. For example, if less than 80% of the cells in the population fail to cluster together into two (or one) cluster based on shared gene expression, this is an indicia that the differentiation process has failed at some point, and that the resultant population of cells does not reach a purity sufficient for administration to a subject.

[0156] Suitable computer systems for implementing the methods of the disclosure will be known the art.

[0157] The disclosure provides computer systems that are programmed to implement methods of the disclosure. In some embodiments, computer system is programmed or otherwise configured to analyze single cell sequencing data using the methods described herein.Attorney Docket No. LCTI-005 / 001WG 43694-03305

[0158] In some embodiments, the computer system includes a central processing unit (CPU, also “processor” and “computer processor” herein), which can be a single core or multi core processor, or a plurality of processors for parallel processing.The computer system can also include memory or memory location (e.g., random-access memory, read-only memory7, flash memory ), electronic storage unit (e.g., hard disk), a communication interface (e.g.. network adapter) for communicating with one or more other systems, and peripheral devices, such as cache, other memory, data storage and / or electronic display adapters. The memory, storage unit, interface and peripheral devices can be in communication with the CPU through a communication bus, such as a motherboard.The storage unit can be a data storage unit (or data repository) for storing data.The computer system can be operatively coupled to a computer network (“network”) with the aid of the communication interface. The network can be the internet, an internet and / or extranet, or an intranet and / or extranet that is in communication with the Internet.The network in some cases is a telecommunication and / or data network. The network can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network, in some cases with the aid of the computer system, can implement a peer-to-peer network, which may enable devices coupled to the computer system to behave as a client or a server.

[0159] In some embodiments, the CPU can execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions may be stored in a memory location, such as the memory7. The instructions can be directed to the CPU which can subsequently program or otherwise configure the CPU to implement methods of the present disclosure. Examples of operations performed by the CPU can include fetch, decode, execute, and writeback.

[0160] In some embodiments, the CPU can execute a sequence of machine-readable instructions, for example machine-readable instructions comprising instructions for: (i) receiving single cell sequencing data derived from a population of auditory cells as described herein, (ii) from the single cell sequencing data, determining the expression of a plurality of genes by cells in the population, (iii) generating a low-dimensionality representation of the population of cells based on expression of the plurality of genes, (iv) executing a community7detection algorithm to identify 1-3 major clusters and one or more minor clusters in the low-dimensionality representation, and (v) identifying a set of genes that are upregulated in a plurality of cells in the major clusters (e.g., at least 80% of the cells) as compared to the one or more minor clusters. In some embodiments, the machine readable instructions compriseAttorney Docket No. LCTI-005 / 001WO 43694-03305 instructions for (i) receiving single cell sequencing data derived from a population of auditory cells as described herein, (ii) determining, for each cell, one or more neighboring cells based on a similarity metric determined from gene expression values, (iii) generating a weighted graph, wherein vertices correspond to individual cells in the population of cells and wherein edge weights correspond to a degree of similarity between the individual cells, (iv) embedding the weighted graph in two-dimensional space, whereby preservation of topological relationships from the weighted graph is maximized. In some embodiments, the machine-readable instructions comprise instructions for displaying weighted graph in two-dimensional space, for example on an electronic display.Cell Sorting Methods for Enriching Auditory Cell Populations

[0161] The disclosure provides one or more genes selected from the group consisting of CD24 molecule (CD24), transmembrane protein with EGF like and two follistatin like domains 1 (TMEFF1), junctional adhesion molecule 2 (JAM2), prominin 1 (PROMI), LDL receptor related protein 4 (LRP4), neuroligin 1 (NLGN1). glycoprotein M6A (GPM6A), podoplanin (PDPN), gamma-aminobutyric acid type A receptor subunit beta3 (GABRB3), cadherin 20 (CDH20), BOC cell adhesion associated, oncogene regulated (BOC), cadherin 4 (CDH4), protocadherin 11 X-linked (PCDH11X), dipeptidyl peptidase like 10 (DPP10), glutamate ionotropic receptor AMPA type subunit 4 (GRIA4), DCC netrin 1 receptor (DCC), leucine rich repeats and immunoglobulin like domains 1 (LRIG1), neuronal cell adhesion molecule (NRCAM), neuronal growth regulator 1 (NEGRI), ADAM metallopeptidase domain 22 (ADAM22), adhesion G protein-coupled receptor L2 (ADGRL2), protein tyrosine phosphatase receptor type N2 (PTPRN2), neurexin 1 (NRXN1), cadherin EGF LAG sevenpass G-type receptor 2 (CELSR2). , neurotrophic receptor tyrosine kinase 2 (NTRK2), and neurotrophic receptor tyrosine kinase 3 (NTRK3), which can be used in the cell sorting methods described herein.

[0162] The disclosure provides methods of isolating or enriching a (a) providing a population of cells comprising target auditory cells and at least one additional cell type; (b) contacting the cells with a detectable label that binds to a cell surface marker selected from the group consisting of CD24, TMEFF1, JAM2, PROMI, LRP4, NLGN1, GPM6A, PDPN, GABRB3, CDH20, BOC, CDH4, PCDH11X, DPP10, GRIA4, DCC, LRIG1, NRCAM, NEGRI, ADAM22, ADGRL2, PTPRN2, NRXN1, CELSR2, NTRK2 and NTRK3; and (c) sorting the cells using the detectable label.Attorney Docket No. LCTI-005 / 001WG 43694-03305

[0163] The cell sorting methods can be used to separate auditory cells, such as the mid ONP cells, late ONP cells, auditory neuron progenitors, mature auditory neurons, spiral ganglion neurons and combinations thereof described herein from undesirable cell types, such as pluripotent cells and other undifferentiated cell t pes present from the differentiation process.

[0164] Suitable methods of the labeling cells using the cell surface markers disclosed herein, and sorting the cells using these cell surface markers, will be known to persons of ordinary skill in the art, and include, inter alia, immunocytochemistry based methods for binding fluorescence labels to the cell surface markers.

[0165] Exemplary methods of cell sorting include flow cytometry. In flow cytometry, fluorescently labeled cells are passed single file through a focused laser beam while detectors measure scattered light to assess fluorescence form the label. The flow cytometer uses this optical data to separate the cells, based on label, into different containers.

[0166] Alternatively, the cells can be sorted using magnetic activated cell sorting (MACs), in which cells are sorted using antibodies, e.g. to one of the cell surface markers disclosed herein, which are conjugated to magnetic beads.

[0167] As a still further alternative, the cells can be sorted using column chromatography, in which antibodies specific to one of the cell surface markers disclosed herein are bound to a solid phase inside a column. A suspension of cells is passed through the column, and cells that express the cell surface marker are reversibly bound to the column.Methods of Treatment

[0168] The disclosure provides methods of treating auditor ' conditions using compositions comprising the populations of auditory cells characterized by the methods described herein. The auditory condition can include, but is not limited to, conductive hearing loss, sensorineural hearing loss, central hearing loss, mixed hearing loss, auditory neuropathy spectrum disorder, central auditor}' processing disorder, tinnitus, hidden hearing loss or a combination thereof. The compositions comprising populations of auditory cells can be combined with cochlear implants to enhance the effectiveness of the cochlear implant.

[0169] In some aspects, the present disclosure provides a method of replacing sensory neurons. In some embodiments, the method comprises administering a therapeutically effective amount of a pharmaceutical composition comprising a population of auditory cells, wherein: (a) greater than or equal to 20% of the cells in the population express SOX2; (b) greater than or equal to 10% of the cells in the population express ( tubulin III; (c) greaterAttorney Docket No. LCTI-005 / 001WG 43694-03305 than or equal to 5% of the cells in the population express TrkB; and (d) less than or equal to 1% of the cells in the population express TRA-1-60 and / or SSEA5.

[0170] In some aspects, the present disclosure provides a method of replacing sensory neurons. In some embodiments, the method comprises administering a therapeutically effective amount of a pharmaceutical composition comprising a population of auditory cells, wherein: (a) greater than or equal to 70% of the cells in the population express both Nestin and SOX2; (b) greater than or equal to 80% of the cells in the population express P tubulin III; (c) greater than or equal to 5% of the cells in the population express TrkB; and (d) less than or equal to 1% of the cells in the population express TRA-1-60 and / or SSEA5.

[0171] In some embodiments, the methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising a population of auditory cells, wherein (a) greater than or equal to 75% of the cells in the population express SOX2 and / or Nestin; (b) greater than or equal to 80% of the cells in the population express beta tubulin III; (c) greater than or equal to 5% of the cells in the population express TrkB; (c) less than or equal to 10% of the cells in the population express Myo7A; and (d) less than or equal to 0.1% of the cells in the population express TRA-1-60 and / or SSEA5.

[0172] In some embodiments, the methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising a population of auditory' cells, wherein (a) greater than or equal to 85% of the cells in the population express both Nestin and SOX2; (b) greater than or equal to 90% of the cells in the population express beta tubulin III; (c) greater than or equal to 15% of the cells in the population express TrkB; (d) greater than or equal to 10% of the cells in the population express GluA4; (e) less than or equal to 10% of the cells in the population express Myo7A; and (f) less than or equal to 0.1% of the cells in the population express TRA-1-60 and / or SSEA5.

[0173] In some embodiments, the methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising a population of auditory cells, wherein (a) greater than or equal to 85% of the cells in the population express both Nestin and SOX2; (b) greater than or equal to 90% of the cells in the population express (3 tubulin III; (c) greater than or equal to 15% of the cells in the population express TrkB; and (d) less than or equal to 0.1% of the cells in the population express TRA-1-60 and / or SSEA5.

[0174] In some embodiments, the methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising a population of auditory cells, wherein (a) greater than or equal to 30% of the cells in the population express SOX2 and / or Nestin; (b) greater than or equal to 30% of the cells in the population express beta tubulin III;Attorney Docket No. LCTI-005 / 001WG 43694-03305 (c) greater than or equal to 20% of the cells in the population express TrkB; (d) greater than or equal to 30% of the cells in the population express GluA4; (e) less than or equal to 20% of the cells in the population express Myo7A; and (1) less than or equal to 0.1% of the cells in the population express TRA-1-60 and / or SSEA5.

[0175] In some embodiments, the methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising a population of auditory cells, wherein (a) greater than or equal to 85% of the cells in the population express both Nestin and SOX2; (b) greater than or equal to 90% of the cells in the population express |3 tubulin III; (c) greater than or equal to 15% of the cells in the population express TrkB; (d) greater than or equal to 10% of the cells in the population express GluA4; (e) less than or equal 10% of the cells in the population express Myo7A; and (f) less than or equal to 0.1% of the cells in the population express TRA-1-60 and / or SSEA5.

[0176] In some embodiments, the methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising a population of auditory cells, wherein (a) between about 70% to 100% of the cells in the population express both Nestin and SOX2; (b) between about 80% to 100% of the cells in the population express (3 tubulin III; (c) between about 5% to 80% of the cells in the population express TrkB; and (d) between 0 to about 0.1% of the cells in the population express TRA-1-60 and / or SSEA5.

[0177] In some embodiments, the methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising a population of auditory cells, wherein (a) greater than or equal to 70% of the cells in the population express SOX2 and Nestin, (b) less than 5% of the cells in the population express Myo7A, and (c) greater than 85% of the cells in the population express (3 tubulin III.

[0178] In some embodiments, the methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising a population of auditory cells, wherein (a) greater than or equal to 80% of the cells in the population express SOX2 and Nestin, (b) less than 2% of the cells in the population express Myo7A, and (c) greater than 90% of the cells in the population express (3 tubulin III.

[0179] In some embodiments, the methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising a population of auditory cells, wherein (a) greater than or equal to 70% of the cells in the population express SOX2 and Nestin, (b) less than 5% of the cells in the population express Myo7A, (c) greater than 85% of the cells in the population express (3 tubulin III. (d) a fold change in GLUA2 mRNA expression compared to a undifferentiated population of pluripotent cells is at least 1000, andAttorney Docket No. LCTI-005 / 001WG 43694-03305 (e) a fold change in TRKB mRNA expression compared to a undifferentiated population of pluripotent cells is at least 1000.

[0180] In some embodiments, the methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising a population of auditor}7cells, wherein (a) greater than or equal to 70% of the cells in the population express SOX2 and Nestin, (b) less than 5% of the cells in the population express Myo7A, (c) greater than 85% of the cells in the population express tubulin III, (d) a fold change in GLUA2 mRNA expression compared to a undifferentiated population of pluripotent cells is between 1000 and 6000, and (e) a fold change in TRKB mRNA expression compared to a undifferentiated population of pluripotent cells is between 1000 and 8000.

[0181] In some embodiments, the methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising a population of auditory cells, wherein (a) greater than or equal to 80% of the cells in the population express SOX2 and Nestin, (b) less than 2% of the cells in the population express Myo7A, (c) greater than 90% of the cells in the population express 0 tubulin III. (d) a fold change in GLUA2 mRNA expression compared to a undifferentiated population of pluripotent cells is between 1000 and 6000, and (e) a fold change in TRKB mRNA expression compared to a undifferentiated population of pluripotent cells is betw een 1000 and 8000.

[0182] The disclosure provides methods of treating a subject with an auditory condition or disorder who has received, or will receive, a cochlear implant. The methods comprise administering to the subject a pharmaceutical composition comprising a population of auditor7cells as described herein. The methods include administering the pharmaceutical composition to the inner ear. Upon transplantation, the population of cells can repopulate the cochlea with functioning auditory neurons. Furthermore, the population of cells can populate not only the transplantation site, but spread to areas flanking the site of administration.Without wishing to be bound by theory, it is thought that by increasing the number of auditor}7neurons, the function of the cochlear implant is thereby improved.

[0183] Accordingly, the disclosure provides methods of treating a subject with an auditory condition, comprising implanting a cochlear implant and administering a therapeutically effective amount of a pharmaceutical composition comprising a population of auditory cells, wherein: (a) greater than or equal to 20% of the cells in the population express SOX2; (b) greater than or equal to 10% of the cells in the population express 0 tubulin III; (c) greater than or equal to 5% of the cells in the population express TrkB; and (d) less than or equal to 1% of the cells in the population express TRA-1-60 and / or SSEA5.Attorney Docket No. LCTI-005 / 001WG 43694-03305

[0184] In some embodiments, the methods comprise implanting a cochlear implant and administering a therapeutically effective amount of a pharmaceutical composition comprising a population of auditory cells, wherein: (a) greater than or equal to 70% of the cells in the population express both Nestin and SOX2; (b) greater than or equal to 80% of the cells in the population express 0 tubulin III; (c) greater than or equal to 5% of the cells in the population express TrkB; and (d) less than or equal to 1% of the cells in the population express TRA-1-60 and / or SSEA5.

[0185] In some embodiments, the methods comprise implanting a cochlear implant and administering a therapeutically effective amount of a pharmaceutical composition comprising a population of auditory cells, wherein (a) greater than or equal to 30% of the cells in the population express SOX2; (b) greater than or equal to 30% of the cells in the population express tubulin III; (c) greater than or equal to 20% of the cells in the population express TrkB; (d) greater than or equal to 30% of the cells in the population express GluA4; (e) less than or equal to 20% of the cells in the population express Myo7A; and (f) less than or equal to 0.1% of the cells in the population express TRA-1-60 and / or SSEA5.

[0186] In some embodiments, the methods comprise implanting a cochlear implant and administering a therapeutically effective amount of a pharmaceutical composition comprising a population of auditory7cells, wherein (a) greater than or equal to 85% of the cells in the population express both Nestin and SOX2; (b) greater than or equal to 90% of the cells in the population express tubulin III; (c) greater than or equal to 20% of the cells in the population express TrkB; (d) greater than or equal to 20% of the cells in the population express GluA4; (e) less than or equal to 10% of the cells in the population express Myo7A; and (f) less than or equal to 0.1% of the cells in the population express TRA-1-60 and / or SSEA5.

[0187] In some embodiments, the methods comprise implanting a cochlear implant and administering a therapeutically effective amount of a pharmaceutical composition comprising a population of auditory cells, wherein (a) greater than or equal to 70% of the cells in the population express SOX2 and Nestin, (b) less than 5% of the cells in the population express Myo7A, and (c) greater than 85% of the cells in the population express tubulin III.

[0188] In some embodiments, the methods comprise implanting a cochlear implant and administering a therapeutically effective amount of a pharmaceutical composition comprising a population of auditory cells, wherein (a) greater than or equal to 80% of the cells in the population express SOX2 and Nestin, (b) less than 2% of the cells in theAttorney Docket No. LCTI-005 / 001WO 43694-03305 population express Myo7A, and (c) greater than 90% of the cells in the population express (3 tubulin III.

[0189] In some embodiments, the methods comprise implanting a cochlear implant and administering a therapeutically effective amount of a pharmaceutical composition comprising a population of auditory cells, wherein (a) greater than or equal to 70% of the cells in the population express SOX2 and Nestin, (b) less than 5% of the cells in the population express Myo7A, (c) greater than 85% of the cells in the population express tubulin III, (d) a fold change in GLUA2 mRNA expression compared to a undifferentiated population of pluripotent cells is at least 1000, and (e) a fold change in TRKB mRNA expression compared to a undifferentiated population of pluripotent cells is at least 1000.

[0190] In some embodiments, the methods comprise implanting a cochlear implant and administering a therapeutically effective amount of a pharmaceutical composition comprising a population of auditory cells, wherein (a) greater than or equal to 70% of the cells in the population express SOX2 and Nestin, (b) less than 5% of the cells in the population express Myo7A, (c) greater than 85% of the cells in the population express (3 tubulin III, (d) a fold change in GLUA2 mRNA expression compared to a undifferentiated population of pluripotent cells is between 1000 and 6000, and (e) a fold change in TRKB mRNA expression compared to a undifferentiated population of pluripotent cells is between 1000 and 8000.

[0191] In some embodiments, the methods comprise implanting a cochlear implant and administering a therapeutically effective amount of a pharmaceutical composition comprising a population of auditory cells, wherein (a) greater than or equal to 80% of the cells in the population express SOX2 and Nestin, (b) less than 2% of the cells in the population express Myo7A, (c) greater than 90% of the cells in the population express (3 tubulin III, (d) a fold change in GLUA2 mRNA expression compared to a undifferentiated population of pluripotent cells is between 1000 and 6000, and (e) a fold change in TRKB mRNA expression compared to a undifferentiated population of pluripotent cells is between 1000 and 8000.

[0192] Further, the disclosure provides methods of enhancing the effectiveness of a cochlear implant in a subject with an auditory condition, comprising administering a therapeutically effective amount of a pharmaceutical composition disclosed herein.

[0193] For example, the disclosure provides methods of enhancing the effectiveness of a cochlear implant in a subject with an auditory condition, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising a population ofAttorney Docket No. LCTI-005 / 001WG 43694-03305 auditory cells, wherein: (a) greater than or equal to 20% of the cells in the population express SOX2; (b) greater than or equal to 10% of the cells in the population express P tubulin III; (c) greater than or equal to 5% of the cells in the population express TrkB; and (d) less than or equal to 1% of the cells in the population express TRA-1-60 and / or SSEA5.

[0194] As a further example, the disclosure provides methods of enhancing the effectiveness of a cochlear implant in a subject with an auditory condition, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising a population of auditory cells, wherein: (a) greater than or equal to 85% of the cells in the population express both Nestin and SOX2; (b) greater than or equal to 90% of the cells in the population express tubulin III; (c) greater than or equal to 15% of the cells in the population express TrkB; and (d) less than or equal to 0.1% of the cells in the population express TRA-1-60 and / or SSEA5.

[0195] As a further example, the methods of enhancing the effectiveness of a cochlear implant in a subject with an auditory condition comprise administering a therapeutically effective amount of a pharmaceutical composition comprising a population of auditory cells, wherein (a) greater than or equal to 30% of the cells in the population express SOX2; (b) greater than or equal to 30% of the cells in the population express beta tubulin III; (c) greater than or equal to 20% of the cells in the population express TrkB; (d) greater than or equal to 30% of the cells in the population express GluA4; (e) less than or equal to 20% of the cells in the population express Myo7A; and (I) less than or equal to 0.1% of the cells in the population express TRA-1-60 and / or SSEA5.

[0196] In some embodiments, the methods of enhancing the effectiveness of a cochlear implant in a subject with an auditory condition comprise administering a therapeutically effective amount of a pharmaceutical composition comprising a population of auditory cells, wherein (a) greater than or equal to 85% of the cells in the population express both Nestin and SOX2; (b) greater than or equal to 90% of the cells in the population express P tubulin III; (c) greater than or equal to 15% of the cells in the population express TrkB; (d) greater than or equal to 10% of the cells in the population express GluA4; (e) less than or equal 10% of the cells in the population express Myo7A; and (I) less than or equal to 0.1% of the cells in the population express TRA-1-60 and / or SSEA5.In some embodiments, (a) between about 70% to 100% of the cells in the population express both Nestin and SOX2; (b) between about 80% to 100% of the cells in the population express tubulin III; (c) between about 5% to 80% of the cells in the population express TrkB; and (d) between 0 to about 0.1% of the cells in the population express TRA-1-60 and / or SSEA5.Attorney Docket No. LCTI-005 / 001WO 43694-03305

[0197] In some embodiments, the pharmaceutical composition is administered prior to implantation of the cochlear implant. For example, the pharmaceutical composition is administered at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 1 year, at least 2 years or at least 3 years before implantation of the cochlear implant.

[0198] In some embodiments, the pharmaceutical composition is administered prior to or concurrent with implantation of the cochlear implant, for example in the same surgery. The surgery associated with implantation can allow for the deliver}’ of cell-based therapies as described herein.

[0199] In some embodiments, the pharmaceutical composition is administered after implantation of the cochlear implant. For example, the pharmaceutical composition is administered at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 1 year, at least 2 years or at least 3 years after implantation of the cochlear implant.

[0200] Any auditory disorders suitable for the methods of the disclosure include auditory disorders in which cochlear hair cells and / or cochlear neurons have been lost. Loss of auditor}' nerve cells can lead to auditory neuropathy, even when the hair cells and the cochlear nucleus remain intact. Cell-based therapy for replacing lost or dysfunctional auditory neurons may restore hearing in these cases. In more severe cases, where both hair cells and many neurons are lost, the degree of success of a cochlear implant procedure may be enhanced by repopulating the cochlea with transplanted, functional auditory neurons. The skilled artisan will appreciate that the loss need not be complete. For example, a loss of greater than 30%, 40%, 50%, 60%, 70%, 80%, 90% or complete loss of cochlear hair cells and / or neurons may result in hearing loss suitable for treatment using the methods described herein.

[0201] Cochlear implants, and methods of implanting same, for use in the methods described herein, are known in the art. Exemplary cochlear implants include, but are not limited to, the Cochlear™ Nucleus® System (from Cochlear® Corporation), cochlear implants available from Advanced Bionics, the CI System from MED-EL, and Oticon. In an exemplary implantation procedure, a surgeon makes a small incision behind the ear, and then creates an opening in the mastoid bone to guide the electrode from the implant to the cochlea. The electrode is placed in the cochlea, and the internal processor is placed in a pocket between the muscle and bone behind the ear.Attorney Docket No. LCTI-005 / 001WG 43694-03305

[0202] Auditory disorders and conditions that can be treated using the methods described herein include, but are not intended to be limited to, conductive hearing loss, sensorineural hearing loss, mixed hearing loss, auditory neuropathy spectrum disorder, central hearing loss, central auditory processing disorder and tinnitus. These methods include administering a cell or population of cells as described herein to the ear of the subject. The administered cells may be obtained by the methods described herein, and the starting material may be tissue obtained from the subject to be treated. In other embodiments, the methods include the step of administering a therapeutic agent that promotes the expression of an auditory protein within a cell within the inner ear (e.g., a differentiation agent as described herein). When used, the differentiation agent can be administered to cells in culture or can be administered to the subject either alone (to stimulate the differentiation of stem cells or progenitor cells within the subject's inner ear) or together with undifferentiated cells (e.g, undifferentiated cells isolated by the methods described herein). The differentiation agent can be, for example, an agonist of the hedgehog pathway, such as an agonist of Sonic hedgehog or Purmorphamin (e.g., Hh-Agl.3).

[0203] A subject having a disorder of the inner ear, or at risk for developing such a disorder, can be treated with the auditory cells as described herein in addition to, or instead of, a cochlear implant. In a successful engraftment, at least some transplanted spiral ganglion neurons, for example, will form synaptic contacts with hair cells and with targets in the cochlear nucleus. To improve the ability of the cells to engraft, the stem cells can be modified prior to differentiation. For example, the cells can be engineered to overexpress one or more anti-apoptotic genes in the progenitor or differentiated cells. The Fak tyrosine kinase or Akt genes are candidate anti-apoptotic genes that can be useful for this purpose; overexpression of FAK or Akt can prevent cell death in spiral ganglion cells and encourage engraftment when transplanted into another tissue, such as an explanted organ of Corti (see for example, Mangi et al., Nat. Med. 9:1195-201, 2003). Neural progenitor cells overexpressing integrin alpha-V beta-3 may have an enhanced ability to extend neurites into a tissue explant, as the integrin has been shown to mediate neurite extension from spiral ganglion neurons on laminin substrates (Aletsee et al., Audiol. Neurootol. 6:57-65, 2001). In another example, ephrinB2 and ephrinB3 expression can be altered, such as by silencing with RNAi or overexpression with an exogenously expressed cDNA, to modify EphA4 signaling events. Spiral ganglion neurons have been shown to be guided by signals from EphA4 that are mediated by cell surface expression of ephrin-B2 and -B3 (Brors et al., J. Comp. Neurol. 462:90-100, 2003). Inactivation of this guidance signal may enhance the number of neuronsAttorney Docket No. LCTI-005 / 001WO 43694-03305 that reach their target in an adult inner ear. Exogenous factors such as the neurotrophins BDNF and NT3, and LIF can be added to tissue transplants to enhance the extension of neurites and their grow th towards a target tissue in vivo and in ex vivo tissue cultures.Neurite extension of sensory neurons can be enhanced by the addition of neurotrophins (BDNF, NT3) and LIF (Gillespie et al., NeuroReport 12:275-279, 2001). A Sonic hedgehog (Shh) polypeptide or polypeptide fragment (e.g., SHH-N), can also be useful as an endogenous factor to enhance neurite extension. Shh is a developmental modulator for the inner ear and a chemoattractant for axons (Charron et al., Cell 113:11 23, 2003).

[0204] A subject experiencing a hearing loss is a candidate for the treatment methods described herein. A subject having or at risk for developing a hearing loss can hear less well than the average subject being, or less well than a subject before experiencing the hearing loss. For example, hearing can be diminished by at least 5, 10, 30, 50% or more. The subject can have sensorineural hearing loss, w hich results from damage or malfunction of the sensory part (the cochlea) or the neural part (the auditory' nerve) of the ear, or conductive hearing loss, which is caused by blockage or damage in the outer and / or middle ear, or the subject can have mixed hearing loss, which is caused by a problem in both the conductive pathway (in the outer or middle ear) and in the nerve pathway (the inner ear). In preferred embodiments, the subject has sensorineural or mixed hearing loss. An example of a mixed hearing loss is a conductive loss due to a middle-ear infection combined with a sensorineural loss due to damage associated with aging.

[0205] The subject can be deaf or have a hearing loss for any reason or as a result of any t pe of event. For example, a subject can be deaf because of a genetic or congenital defect; for example, a subject can have been deaf since birth, or can be deaf or hard-of-hearing as a result of a gradual loss of hearing due to a genetic or congenital defect. In another example, a subject can be deaf or hard-of-hearing as a result of a traumatic event, such as a physical trauma to a structure of the ear, or a sudden loud noise, or a prolonged exposure to loud noises. For example, prolonged exposure to concert venues, airport runways, and construction areas can cause inner ear damage and subsequent hearing loss. A subject can experience chemical-induced ototoxicity, wherein ototoxins include therapeutic drugs including antineoplastic agents, salicylates, quinines, and aminoglycoside antibiotics, contaminants in foods or medicinals, and environmental or industrial pollutants. A subject can have a hearing disorder that results from aging, or the subject can have tinnitus (characterized by ringing in the ears).Attorney Docket No. LCTI-005 / 001WO 43694-03305

[0206] A subject suitable for the methods as described herein can include a subject having a vestibular dysfunction, including bilateral and unilateral vestibular dysfunction. Vestibular dysfunction is an inner ear dysfunction characterized by symptoms that include dizziness, imbalance, vertigo, nausea, and fuzzy vision and may be accompanied by hearing problems, fatigue and changes in cognitive functioning. Vestibular dysfunction can be the result of a genetic or congenital defect; an infection, such as a viral or bacterial infection; or an injury, such as a traumatic or nontraumatic injury. Vestibular dysfunction is most commonly tested by measuring individual symptoms of the disorder (e.g., vertigo, nausea, and fuzzy vision).

[0207] The methods as described herein may be used for the treatment of hearing disorders resulting from sensorineural hair cell loss or auditory neuropathy. Subjects suffering from auditory neuropathy experience a loss of cochlear sensory neurons while the hair cells of the inner ear remain intact. Such subjects will benefit particularly from treatment that causes cells (stem cells or progenitor cells) to differentiate into spiral ganglion cells, or from administration of spiral ganglion cells into the inner ear. Subjects with sensorineural hair cell loss experience the degeneration of cochlear hair cells, which frequently results in the loss of spiral ganglion neurons in regions of hair cell loss. Such subjects may also experience loss of supporting cells in the organ of Corti, and degeneration of the limbus, spiral ligament, and stria vascularis in the temporal bone material. Such subjects can receive treatment with an agent that causes cells to differentiate into hair cells, or a tissue transplant containing hair cells grafted or injected into the inner ear. The subjects may additionally benefit from treatment that causes cells to differentiate into spiral ganglion cells, or from administration of spiral ganglion cells into the inner ear. For example, in auditory nerve degeneration from mechanical compression, most auditory spiral ganglion cells degenerate (causing trans neuronal death of the cochlear nucleus cells) following sustained compression in the Rosenthal’s canal, together with astrocytes and Schwann cell columns form a continuous, “naturally occurring autologous cell bridge’', which acts as an anatomical scaffold for grafted cells migration to connect between the PNS and the CNS (Sekiya et al. 2021 Cell Transplantation Volume 30: 1-20).

[0208] In some embodiments, the auditory disorder or condition is an auditory neuropathy spectrum disorder. Auditory7neuropathy spectrum disorders encompass various conditions resulting from, for example, genetic mutations and / or viral infections, that lead to dysfunctional auditory nerve function. The methods of the disclosure advantageously provide a single cell therapy for treatment of any disease on the auditory neuropathy spectrumAttorney Docket No. LCTI-005 / 001WO 43694-03305 disorder, rather than a multitude of targeted therapies dependent on the underlying causal agent.

[0209] In some embodiments, the auditor}’ disorder or condition is hidden hearing loss (HHL). HHL is a condition in which hearing functions normally when exposed to a single sound or frequency, but is impaired when exposed to multiple sounds (e.g., parties, busy streets, restaurants). Damaged auditory neurons are thought to be responsible for disease as hair cells function normally. Up to 10% of patients who report hearing loss have a normal audiogram, indicating HHL. HHL may be treated with a hearing aid, e g., a cochlear implant. In some embodiments, a subject with a cochlear implant still suffers from HHL as the cochlear implant can induce sound mediated electrical signals in functional auditory neurons and thus replace damaged or lost hair cells, but a cochlear implant cannot replace damaged or lost auditory neurons.

[0210] In some embodiments, the methods provided herein are methods for replacing auditory7neurons in a subject in need thereof, and implanting a cochlear implant. In some embodiments, the methods provided herein are methods for augmenting an existing but damaged auditory neuron population in a subject in need thereof, and implanting a cochlear implant.

[0211] Auditory7cells generated by the methods described herein can be administered, such as in the form of a cell suspension, into, on to, or near, for example, the inner ear or the middle ear. by injection, such as into the luminae of the cochlea or the auditory nerve through the retromastoid route. In some embodiments, the methods comprise administering a pharmaceutical composition comprising a population of cells as described herein to the inner or middle ear of the subject. In some embodiments, administration to the inner ear comprises administration to the cochlea. In some embodiments, the composition is administered to the Scala tympani, modiolus or spiral ganglion.

[0212] In some embodiments, the population of cells is administered via injection, for example injection via microneedle. In some embodiments, the injection comprises inserting a cannula through a hole in the otic capsule, or inserting a cannula through the round window.

[0213] Injection can be, for example, through the round window of the ear or through the bony capsule surrounding the cochlea. The cells can be injected through the round window into the auditory' nerve trunk in the internal auditory' meatus, into the modiolus, or into the Scala tympani, as described below. The administration of the auditory cells as described herein can be accomplished with, for example, injection needle or syringe positioning devices known in the art that have the ability to control (e.g., either manually orAtorney Docket No. LCTI-005 / 001WG 43694-03305 through a robotic interface) the navigation and position of a needle to the desired target anatomy of, for example, the inner ear or middle ear, for the treatment of auditory or hearing loss conditions as described herein. Such devices include, for example, the stabilization that is required to facilitate safe and effective delivery of the auditory cells over a period of time to ensure delivery7of the concentration or volume of auditory cells as described herein.Exemplary routes of administration are described at, for example, otosurgeryatlas.stanford.edu / otologic-surgery-atlas / cochlear-implantation / cochlear-implant-surgical -variations / .

[0214] In an exemplary' route of administration, for example to the Scala tympani by cochleostomy, a small hole is drilled through the Otic capsule in the base of the cochlea to accommodate a cannula. The hole is covered with a small piece of fascia. Cells are loaded into a 30G cannula, primed with saline and aspirated with about 1 pL air, followed by aspiration of the composition. The cells are injected into the Scala tympani using a pump, at a rate of about 1 pL / minute. In a second exemplary route of administration, the cochleostomy is through the otic capsule. A dental drill is used to create a hole to accommodate a 33G need and cannula into the modiolus, and then through the bony wall of the modiolus via the hole puncture. The hole is covered with a small piece of fascia. Cells are loaded into the cannula, and injected into the modiolus, as described above. The cannula is left in place for about 10 minutes to allow the fluids to equilibrate. Alternatively, compositions of the disclosure can be delivered through the round window. First, the round window is exposed, followed by removal or incision of the round window mucosa membrane and drilling away of the bony overhang. A cannula is directed through the round window into the Scala ty mpani or modiolus, and cells are administered as described above.

[0215] In some embodiments, the population of cells is administered at the same time as implantation of the cochlear implant, for example in the same surgical procedure.

[0216] In some embodiments, the method comprises administering between about 100,000 and 50 million cells, between about 100,000 and 10 million cells, between about 100,000 and 1 million cells, between about 200.000 and 10 million cells, between about 500,000 and 1 million cells, or between about 100,000 and 500,000 cells to the subject. In some embodiments, between about 100,000 to about 1 million cells are administered to the subject. In some embodiments, between about 30 million cells per milliliter to about 700 million cells per milliliter are administered to the subject.

[0217] In some embodiments, administration of the compositions described herein alleviates a sign or symptom of the auditory disease or condition in the subject. In someAttorney Docket No. LCTI-005 / 001WG 43694-03305 embodiments, the method improves hearing in the subject, lessens the severity of hearing loss, delays the progression of hearing loss, and alleviating one or more symptoms associated with the hearing disease or disorder.

[0218] The administration of the auditory cells as described herein can be accomplished with, for example, by pre-inj ection of a coating material, such as a matrix component, serum component, or biodegradable scaffold that may enhance the attachment and integration of the transplanted sensory neurons, to ensure delivery of the concentration or volume of auditory cells as described herein. The cell product can be cryopreserved in a cryovial, made of plastic, glass, or other polymers or rubber and plastic copolymers such as Cyclic Olefin Copolymer. The vials can be sealed with a screw cap or a stopper made of rubber and plastic copolymers such as Thermo® Plastic Elastomers that enable sterile transfer of the cell product into the delivery device. The cell product can be cryopreserved preloaded within a syringe, a syringe cartridge, or an injection cannula, which is thawed prior to administration to the subject. The cell product can be cryopreserved and stored as an off-the-shelf allogenic cell therapy bank. The cell product can be frozen at a clinical dose ready to be thawed at clinical sites for administration to the subject.Pharmaceutical Compositions

[0219] The disclosure provides pharmaceutical compositions comprising a population of cells for use in the methods described herein, which are characterized using the methods described herein.

[0220] The skilled artisan will appreciate that the compositions described herein can comprise mixed populations of cell types, whose identities are reflected in the percentages cells in the population expressing one or more of the markers described herein. Alternatively, the populations of cells can be substantially pure (e.g., greater than 90%, greater than 95%, greater than 97%, greater than 98% or greater than 99% pure) as determined by the expression of a combination of any of the markers disclosed herein. Individual cells in the population may express only a single marker described below, or individual cells may express combinations of markers described below, depending on the differentiation state of the cell. Cells in the population may express neural progenitor markers such as Nestin, ONP protein markers such as PAX2, PAX8, and / or SOX2, neuronal protein markers such as (3 Tubulin III, and auditory neuron protein markers such as TrkB and / or GluA4. In some embodiments, cells in the population express SOX2; cells in the population express (3 tubulin III: and cells in the population express TrkB. In some embodiments, cells in the population express both NestinAttorney Docket No. LCTI-005 / 001WG 43694-03305 and S0X2; cells in the population express (3 tubulin III; cells in the population express TrkB; and cells in the population do not express TRA-1-60 and / or SSEA5. Cells in the population may express any of the gene markers set forth in Tables 5 or 6.

[0221] In some embodiments, cells in the population express SOX2; cells in the population express tubulin III; cells in the population express TrkB; cells in the population express GluA4; and cells in the population express Myo7A.

[0222] In some embodiments, cells in the population express both Nestin and SOX2; cells in the population express P tubulin III; cells in the population express TrkB; cells in the population express GluA4; cells in the population express Myo7A; and optionally cells in the population do not express TRA-1-60 and / or SSEA5.

[0223] In some embodiments, cells in the population do not express PAX2 and / or PAX8.

[0224] In some embodiments, cells in the population express Nestin. Nestin is a member of the intermediate filament protein family and is expressed in neurons. In some embodiments, greater than or equal to 10%, greater than or equal to 20%, greater than or equal to 30%, greater than or equal to 40%, greater than or equal to 50%, greater than or equal to 60%, greater than or equal to 70%, greater than or equal to 80%, greater than or equal to 90%, greater than or equal to 95%, or greater than or equal to 99% of the cells in the population express Nestin. In some embodiments, greater than or equal to 40% of the cells in the population express Nestin. In some embodiments, greater than or equal to 50% of the cells in the population express Nestin. In some embodiments, greater than or equal to 60% of the cells in the population express Nestin. In some embodiments, greater than or equal to 70% of the cells in the population express Nestin. In some embodiments, greater than or equal to 80% of the cells in the population express Nestin. In some embodiments, greater than or equal to 90% of the cells in the population express Nestin. In some embodiments, greater than or equal to 99% of the cells in the population express Nestin. In some embodiments, 10% to 95%, 20% to 90%, 30% to 80%, 40% to 70%, or 30% to 60% of the cells in the population express Nestin. In some embodiments, 10% to 99%, 20% to 95%, 30% to 90%, 40% to 85%, or 50% to 80% of the cells in the population express Nestin. In some embodiments, 70% to 90% of the cells in the population express Nestin. In some embodiments, at least 75% of the cells in the population express Nestin. In some embodiments, at least 80% of the cells in the population express Nestin.

[0225] In some embodiments, cells in the population express SOX2. SOX2 encodes a member of the SRY (The Sex-determining Region Y)-related HMG-box (SOX) family ofAttorney Docket No. LCTI-005 / 001WG 43694-03305 transcription factors involved in the regulation of embry onic development and in the determination of cell fate. In some embodiments, greater than or equal to 10%. greater than or equal to 20%, greater than or equal to 30%, greater than or equal to 40%, greater than or equal to 50%, greater than or equal to 60%, greater than or equal to 70%, greater than or equal to 80%, greater than or equal to 90%, greater than or equal to 95%, or greater than or equal to 99% of the cells in the population express SOX2. In some embodiments, greater than or equal to 30% of the cells in the population express SOX2. In some embodiments, greater than or equal to 40% of the cells in the population express SOX2. In some embodiments, greater than or equal to 50% of the cells in the population express SOX2. In some embodiments, greater than or equal to 60% of the cells in the population express SOX2. In some embodiments, greater than or equal to 70% of the cells in the population express SOX2. In some embodiments, greater than or equal to 80% of the cells in the population express SOX2. In some embodiments, greater than or equal to 90% of the cells in the population express SOX2. In some embodiments, greater than or equal to 99% of the cells in the population express SOX2. In some embodiments, 10% to 99%, 20% to 95%, 30% to 90%, 40% to 85%, or 50% to 80% of the cells in the population express SOX2. In some embodiments, 10% to 95%, 20% to 90%, 30% to 80%, 40% to 70%, or 30% to 60% of the cells in the population express SOX2. In some embodiments, 70% to 90% of the cells in the population express SOX2. In some embodiments, at least 75% of the cells in the population express SOX2. In some embodiments, at least 80% of the cells in the population express SOX2.

[0226] In some embodiments, greater than or equal to 10%, greater than or equal to 20%, greater than or equal to 30%, greater than or equal to 40%, greater than or equal to 50%. greater than or equal to 60%, greater than or equal to 70%, greater than or equal to 80%, greater than or equal to 90%, greater than or equal to 95%, or greater than or equal to 99% of the cells in the population express both Nestin and SOX2. In some embodiments, greater than or equal to 30% of the cells in the population express both Nestin and SOX2. In some embodiments, greater than or equal to 40% of the cells in the population express both Nestin and SOX2. In some embodiments, greater than or equal to 50% of the cells in the population express both Nestin and SOX2. In some embodiments, greater than or equal to 60% of the cells in the population express both Nestin and SOX2. In some embodiments, greater than or equal to 70% of the cells in the population express both Nestin and SOX2. In some embodiments, greater than or equal to 80% of the cells in the population express both Nestin and SOX2. In some embodiments, greater than or equal to 90% of the cells in theAttorney Docket No. LCTI-005 / 001WG 43694-03305 population express both Nestin and SOX2. In some embodiments, greater than or equal to 99% of the cells in the population express both Nestin and SOX2. In some embodiments, 10% to 99%, 20% to 95%, 30% to 90%, 40% to 85%, or 50% to 80% of the cells in the population express both Nestin and SOX2. In some embodiments, 70% to 90% of the cells in the population express both Nestin and SOX2. In some embodiments, between 70% and 99% of the cells in the population express both Nestin and SOX2. In some embodiments, between 75% and 99% of the cells in the population express both Nestin and SOX2. In some embodiments, between 85% and 99% of the cells in the population express both Nestin and SOX2.

[0227] In some embodiments, cells in the population express PAX8. In some embodiments, cells in the population do not express PAX8. PAX8 encodes a member of the paired box family of transcription factors containing a paired box domain, an octapeptide, and a paired-type homeodomain domain. In some embodiments, less than 70%, less than 60%, less than 50%, less than 40%, %, less than 20%, less than 10%, or less than 5%, of the cells in the population express PAX8. In some embodiments, less than 60% of the cells in the population express PAX8. In some embodiments, less than 50% of the cells in the population express PAX8. In some embodiments, less than 40% of the cells in the population express PAX8. In some embodiments, less than 20% of the cells in the population express PAX8. In some embodiments, less than 10% of the cells in the population express PAX8. In some embodiments, less than 5% of the cells in the population express PAX8. In some embodiments, less than 1% of the cells in the population express PAX8. In some embodiments, less than 0.1% of cells in the population express PAX8. In some embodiments, less than 0.01% of cells in the population express PAX8. In some embodiments, 0.1% to 60%. 1% to 50%, 0.1% to 30%, 1% to 20%. 5% to 15%, or 5% to 8% of the cells in the population express PAX8. In some embodiments, cells in the population do not detectably express PAX8, i.e. the level of PAX8 is below the limit of detection by suitable assays described herein and known in the art.

[0228] In some embodiments, cells in the population express PAX2. In some embodiments, cells in the population do not express PAX2. PAX2 encodes paired box gene 2 is a target of transcriptional suppression by the tumor suppressor gene WT1. In some embodiments, less than or equal to 10%, less than or equal to 20%, less than or equal to 30%, less than or equal to 40%, less than or equal to 50%, less than or equal to 60%, or less than or equal to 70% of the cells in the population express PAX2. In some embodiments, less than or equal to 20% of the cells in the population express PAX2. In some embodiments, less than orAttorney Docket No. LCTI-005 / 001WG 43694-03305 equal to 30% of the cells in the population express PAX2. In some embodiments, less than or equal to 40% of the cells in the population express PAX2. In some embodiments, less than or equal to 50% of the cells in the population express PAX2. In some embodiments, less than or equal to 60% of the cells in the population express PAX2. In some embodiments, less than or equal to 70% of the cells in the population express PAX2. In some embodiments, 0% to 60%, 0.01% to 50%, 0.1% to 40%, or 1% to 30% of the cells in the population express PAX2. In some embodiments, 0.01% to 50% of cells express PAX2. In some embodiments, 1% to 30% of cells in the population express PAX2. In some embodiments, cells in the population do not detectably express PAX2, i.e. the level of PAX2 is below the limit of detection by suitable assays described herein and known in the art.

[0229] In some embodiments, cells in the population express GluA4. GluA4 encodes a glutamate receptor expressed in excitatory neurotransmitter secreting neurons in the brain and are activated in a variety’ of normal neurophysiologic processes. In some embodiments, greater than or equal to 1%, greater than or equal to 5%, greater than or equal to 10%, greater than or equal to 20%, greater than or equal to 30%, greater than or equal to 40%, greater than or equal to 50%, greater than or equal to 60%, greater than or equal to 70%, greater than or equal to 80%, greater than or equal to 90%, greater than or equal to 95%, or greater than or equal to 99% of the cells in the population express GluA4. In some embodiments, greater than or equal to 10% of the cells in the population express GluA4. In some embodiments, greater than or equal to 15% of the cells in the population express GluA4. In some embodiments, greater than or equal to 20% of the cells in the population express GluA4. In some embodiments, greater than or equal to 25% of the cells in the population express GluA4. In some embodiments, greater than or equal to 30% of the cells in the population express GluA4. In some embodiments, greater than or equal to 40% of the cells in the population express GluA4. In some embodiments, greater than or equal to 50% of the cells in the population express GluA4. In some embodiments, greater than or equal to 70% of the cells in the population express GluA4. In some embodiments, greater than or equal to 90% of the cells in the population express GluA4. In some embodiments, 1% to 99%, 10% to 95%, 20% to 90%, 30% to 80%, 30% to 60%, or 20% to 50% of the cells in the population express GluA4. In some embodiments, between about 10% to 95% of the cells in the population express GluA4. In some embodiments, 30% to 90% of the cells in the population express GluA4. In some embodiments, between about 10% and 75% of cells in the population express GluA4. In some embodiments, between about 15% and 70% of cells in the population express GluA4.Attorney Docket No. LCTI-005 / 001WG 43694-03305

[0230] In some embodiments, cells in the population express glutamate receptor A2 (GluA2). GluA2 is involved in auditory system signaling and enables neuronal activity in response to glutamate secreted by sound-stimulated cochlear hair cells. In some embodiments greater than or equal to 5%, greater than or equal to 10%, greater than or equal to 20%, greater than or equal to 30%, greater than or equal to 40%, greater than or equal to 50%, greater than or equal to 60%, greater than or equal to 70%, or greater than or equal to 80% of the cells in the population express GluA2. In some embodiments, greater than or equal to 10% of the cells in the population express GluA2. In some embodiments, greater than or equal to 20% of the cells in the population express GluA2. In some embodiments, greater than or equal to 30% of the cells in the population express GluA2. In some embodiments, greater than or equal to 40% of the cells in the population express GluA2. In some embodiments, greater than or equal to 60% of the cells in the population express GluA2. In some embodiments, 5% to 80%, 10% to 70%, 20% to 50%, or 30% to 40% of the cells in the population express GluA2. In some embodiments, between about 5% and about 90%, between about 10% and about 80%, between about 0% and about 70%. between about 5% and about 50%, between about 20% and about 90%, or between about 30% and about 70% of cells in the population express GluA2.

[0231] In some embodiments, the fold change in expression of GLUA2, for example fold change of mRNA expression relative to an undifferentiated population of pluripotent cells such as hESCs. can be determined using qPCR. In some embodiments, the undifferentiated population of pluripotent cells is a population of hESC cells. The undifferentiated population of pluripotent cells can be differentiated into the population of cells described herein. In some embodiments, the fold change in expression of GLUA2 between the cells in the population and the undifferentiated population of pluripotent cells is greater than or equal to 100, greater than or equal to 300, greater than or equal to 500, greater than or equal to 1,000, greater than or equal to 1,500, greater than or equal to 2,000, greater than or equal to 3,000, or greater than or equal to 5,000. In some embodiments, the fold change in expression of GLUA2 between the cells in the population and the undifferentiated population of pluripotent cells is greater than or equal to 100. In some embodiments, the fold change in expression of GLUA2 between the cells in the population and the undifferentiated population of pluripotent cells is greater than or equal to 300. In some embodiments, the fold change in expression of GLUA2 between the cells in the population and the undifferentiated population of pluripotent cells is greater than or equal to 500. In some embodiments, the fold change in expression of GLUA2 between the cells in the population and the undifferentiatedAttorney Docket No. LCTI-005 / 001WD 43694-03305 population of pluripotent cells is greater than or equal to 1,000. In some embodiments, the fold change in expression of GLUA2 between the cells in the population and the undifferentiated population of pluripotent cells is greater than or equal to 1,500. In some embodiments, the fold change in expression of GLUA2 between the cells in the population and the undifferentiated population of pluripotent cells is greater than or equal to 2,000. In some embodiments, the fold change in expression of GLUA2 between the cells in the population and the undifferentiated population of pluripotent cells is greater than or equal to 3,000. In some embodiments, the fold change in expression of GLUA2 between the cells in the population and the undifferentiated population of pluripotent cells is greater than or equal to 5,000. In some embodiments, the fold change in expression of GLUA2 between the cells in the population and the undifferentiated population of pluripotent cells is between about 500 and about 10,000, between about 1,000 and about 10,000, between about 500 and about 7,000, or between about 2,000 and about 5,000.

[0232] In some embodiments, cells in the population express CD133. CD133 encodes a pentaspan transmembrane glycoprotein that localizes to membrane protrusions and is often expressed on adult stem cells where it functions in maintaining stem cell properties by suppressing differentiation. In some embodiments, greater than or equal to 10%, greater than or equal to 20%, greater than or equal to 30%, greater than or equal to 40%, greater than or equal to 50%, greater than or equal to 60%, greater than or equal to 70%, greater than or equal to 80%, greater than or equal to 90%, greater than or equal to greater than or equal to 95%, or greater than or equal to 99% of the cells in the population express CD133. In some embodiments, greater than or equal to 50% of the cells in the population express CD 133. In some embodiments, greater than or equal to 60% of the cells in the population express CD133. In some embodiments, greater than or equal to 70% of the cells in the population express CD133. In some embodiments, greater than or equal to 80% of the cells in the population express CD133. In some embodiments, greater than or equal to 90% of the cells in the population express CD133. In some embodiments, greater than or equal to 95% of the cells in the population express CD 133. In some embodiments, 1% to 99% 10% to 95%, 20% to 90%, 30% to 80%, or 40% to 70%, of the cells in the population express CDI33.

[0233] In some embodiments, cells in the population express GATA3. GATA3 is a regulator of T-cell development and plays a role in endothelial cell biology. Defects in GATA3 are the cause of hypoparathyroidism with sensorineural deafness. In some embodiments, less than 10%, less than 5%, less than 1%, less than 0.1% of the cells in the population express GATA3. In some embodiments, less than 5% of the cells in the populationAttorney Docket No. LCTI-005 / 001WG 43694-03305 express GATA3. In some embodiments, less than 1% of the cells in the population express GATA3. In some embodiments, less than 0.1% of the cells in the population express GATA3. In some embodiments, 0.1% to 10%, 0.1% to 5%, or 0.1% to 1% of the cells in the population express GATA3.

[0234] In some embodiments, cells in the population express P tubulin III (also referred to as III tubulin or Beta 3 tubulin and the like). P tubulin III encodes a member of the beta tubulin protein family that heterodimerizes and assembles to form microtubules. In some embodiments, greater than or equal to greater than or equal to 5%, greater than or equal to 10%, greater than or equal to 20%, greater than or equal to 30%, greater than or equal to 40%, greater than or equal to 50%, greater than or equal to 60%, greater than or equal to 70%. or greater than or equal to 80% of the cells in the population express P tubulin III. In some embodiments, greater than or equal to 10% of the cells in the population express P tubulin III. In some embodiments, greater than or equal to 20% of the cells in the population express P tubulin III. In some embodiments, greater than or equal to 30% of the cells in the population express P tubulin III. In some embodiments, greater than or equal to 40% of the cells in the population express P tubulin III. In some embodiments, greater than or equal to 50% of the cells in the population express P tubulin III. In some embodiments, greater than or equal to 60% of the cells in the population express P tubulin III. In some embodiments, greater than or equal to 70% of the cells in the population express P tubulin III. In some embodiments, greater than or equal to 80% of the cells in the population express P tubulin III. In some embodiments, greater than or equal to 85% of the cells in the population express P tubulin III. In some embodiments, greater than or equal to 90% of the cells in the population express P tubulin III. In some embodiments, greater than or equal to 95% of the cells in the population express P tubulin III. In some embodiments, 1% to 80%, 10% to 70%, 30% to 60%, 40% to 50%, 80% to 100%, 85% to 95%, 85% to 100%, 90% to 100% or 95% to 99% of the cells in the population express P tubulin III. In some embodiments, 80% to 100% of the cells in the population express P tubulin III. In some embodiments, 70% to 99% of the cells in the population express P tubulin III.

[0235] In some embodiments, cells in the population express tropomyosin-related kinase receptor B (TrkB). TrkB is involved in nervous system development and enables brain-derived neurotrophic factor binding activity and brain-derived neurotrophic factor (BDNF)-activated receptor activity. In some embodiments greater than or equal to 5%, greater than or equal to 10%, greater than or equal to 20%, greater than or equal to 30%, greater than or equal to 40%, greater than or equal to 50%, greater than or equal to 60%,Attorney Docket No. LCTI-005 / 001WG 43694-03305 greater than or equal to 70%, or greater than or equal to 80% of the cells in the population express TrkB. In some embodiments, greater than or equal to 10% of the cells in the population express TrkB. In some embodiments, greater than or equal to 20% of the cells in the population express TrkB. In some embodiments, greater than or equal to 30% of the cells in the population express TrkB. In some embodiments, greater than or equal to 40% of the cells in the population express TrkB. In some embodiments, greater than or equal to 60% of the cells in the population express TrkB. In some embodiments. 5% to 80%, 10% to 70%, 20% to 50%, or 30% to 40% of the cells in the population express TrkB. In some embodiments, between about 5% and about 90%, between about 10% and about 80%, between about 0% and about 70%, between about 5% and about 50%, between about 20% and about 90%, or between about 30% and about 70% of cells in the population express TrkB.

[0236] In some embodiments, the fold change in mRNA expression of one or more of the markers can be compared to the mRNA expression in the starting cell population, e.g. the undifferentiated population of pluripotent cells such as hESC which are differentiated into the cells in the population. In some embodiments, the fold change in expression can be determined using qPCR. In some embodiments, the starting cell population is a population of hESC cells. In some embodiments, the marker is TRKB. In some embodiments, the fold change in expression of TRKB between the cells in the population and the undifferentiated population of pluripotent cells is greater than or equal to 100. greater than or equal to 300, greater than or equal to 500, greater than or equal to 1 ,000, greater than or equal to 1 ,500, greater than or equal to 2,000, greater than or equal to 3,000, or greater than or equal to 5,000. In some embodiments, the fold change in expression of TRKB between the cells in the population and the undifferentiated population of pluripotent cells is greater than or equal to 100. In some embodiments, the fold change in expression of TRKB between the cells in the population and the undifferentiated population of pluripotent cells is greater than or equal to 300. In some embodiments, the fold change in expression of TRKB between the cells in the population and the undifferentiated population of pluripotent cells is greater than or equal to 500. In some embodiments, the fold change in expression of TRKB between the cells in the population and the undifferentiated population of pluripotent cells is greater than or equal to 1,000. In some embodiments, the fold change in expression of TRKB between the cells in the population and the undifferentiated population of pluripotent cells is greater than or equal to 1,500. In some embodiments, the fold change in expression of TRKB between the cells in the population and the undifferentiated population of pluripotent cells is greater than or equal toAttorney Docket No. LCTI-005 / 001WG 43694-03305 2,000. In some embodiments, the fold change in expression of TRKB between the cells in the population and the undifferentiated population of pluripotent cells is greater than or equal to 3,000. In some embodiments, the fold change in expression of TRKB between the cells in the population and the undifferentiated population of pluripotent cells is greater than or equal to 5,000. In some embodiments, the fold change in expression of TRKB between the cells in the population and the undifferentiated population of pluripotent cells is between about 500 and about 10,000, between about 1,000 and about 10,000, between about 500 and about 7,000, or between about 2,000 and about 5,000.

[0237] In some embodiments, cells in the population express tropomyosin-related kinase receptor C (TrkC). TrkC acts upstream of or within several processes including neurogenesis, neuronal action potential propagation, and is predicted to enable several functions, including GPI-linked ephrin receptor activity; neurotrophin (NT3) binding activity; and p53 binding activity. In some embodiments, less than 10%, less than 5%, less than 1%, or less than 0.1% of the cells in the population express TrkC. In some embodiments, less than 5% of the cells in the population express TrkC. In some embodiments, less than 1% of the cells in the population express TrkC. In some embodiments, less than 0.1% of the cells in the population express TrkC. In some embodiments, 0.01% to 10%, 0.01% to 5%, or 0.01% to 1% of the cells in the population express TrkC. In some embodiments, expression of TrkC is greater than 2%, e.g. between 2% and 20% or more of the cells express TrkC. In some embodiments, between 1% and 100%. between 2% and 100%, between 2% and 80%, between 2% and 50% or between 2% and 20% of the cells in the population express TrkC.

[0238] In some embodiments, cells in the population express Brain specific homeobox / POU domain protein 3a (BRN3A). BRN3A enables several functions, including DNA binding activity; DNA-binding transcription activator activity and is involved in nervous system development. In some embodiments, less than 10%, less than 5%, less than 1%, or less than 0.1% of the cells in the population express BRN3A. In some embodiments, less than 5% of the cells in the population express BRN3A. In some embodiments, less than 1% of the cells in the population express BRN3A. In some embodiments, less than 0.1% of the cells in the population express BRN3A. In some embodiments, 0.1% to 10%, 0.1% to 5%, or 0.1% to 1% of the cells in the population express BRN3A. In some embodiments, between 1% and 20% or more of the cells express BRN3A. In some embodiments, between 1% and 30%, between 1% and 20%, between 5% and 20%, or between 1% and 10% of the cells express BRN3A.Attorney Docket No. LCTI-005 / 001WG 43694-03305

[0239] In some embodiments, cells in the population express the hair cell protein marker Myo7A. Mutations in MY07A are known to play a significant role in the development of deafness and blindness. Myo7A is expressed in the nervous system, enables protein domain specific binding activity, and acts upstream of or within several processes, including organ morphogenesis. In some embodiments, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 1%, or less than 0.1% of the cells in the population express Myo7A. In some embodiments, less than 30% of the cells in the population express Myo7A. In some embodiments, less than 20% of the cells in the population express Myo7A. In some embodiments, less than 10% of the cells in the population express Myo7A. In some embodiments, less than 5% of the cells in the population express Myo7A. In some embodiments, less than 1% of the cells in the population express Myo7A. In some embodiments, less than 0.1% of the cells in the population express Myo7A. In some embodiments, 0.1% to 40%, 1% to 30%, 5% to 20%, or 10% to 15% of the cells in the population express Myo7A. In some embodiments, between about 0.5% to about 10% of cells in the population express Myo7A. In some embodiments, between about 0.1% and about 2% of cells in the population express Myo7A.

[0240] In some embodiments, cells in the population express stage-specific embryonic antigen (S SEA-5). Undifferentiated cells may be identified by expression of various protein markers including SSEA-5. In some embodiments, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% of the cells in the population express SSEA-5. In some embodiments, less than 5% of the cells in the population express SSEA-5. In some embodiments, less than 1% of the cells in the population express SSEA-5. In some embodiments, less than 0.1% of the cells in the population express SSEA-5. In some embodiments, less than 0.05% of the cells in the population express SSEA-5. In some embodiments, less than 0.01% of the cells in the population express SSEA-5. In some embodiments, 0.01% to 10%, 0.1% to 5%, or 0.1% to 1% of the cells in the population express SSEA-5. In some embodiments, cells in the population do not detectably express SSEA-5, i.e. the level of SSEA-5 is below the limit of detection by suitable assays described herein and known in the art.

[0241] In some embodiments, cells in the population express a Tumour Rejection Antigen 1-60 (TRA-1-60). Undifferentiated cells may be identified by the expression of various protein markers including TRA-1-60. In some embodiments, less than 10%, less than 5%, less than 1%, less than 0.1% of the cells, or less than 0.01% of the cells in the population express TRA-1-60. In some embodiments, less than 5% of the cells in the population expressAttorney Docket No. LCTI-005 / 001WG 43694-03305 TRA-1-60. In some embodiments, less than 1% of the cells in the population express TRA-1-60. In some embodiments, less than 0.1% of the cells in the population express TRA-1-60. In some embodiments, less than 0.05% of the cells in the population express TRA-1-60. In some embodiments, less than 0.01% of the cells in the population express TRA-1-60. In some embodiments, 0.01% to 10%, 0.1% to 5%, or 0.1% to 1% of the cells in the population express TRA-1-60. In some embodiments, cells in the population do not detectably express TRA-1-60, i.e. the level of TRA-1-60 is below the limit of detection by suitable assays described herein and known in the art.

[0242] In some embodiments, (a) greater than or equal to 20% of the cells in the population express SOX2; (b) greater than or equal to 10% of the cells in the population express tubulin III: (c) greater than or equal to 5% of the cells in the population express TrkB; and (d) less than or equal to 1% of the cells in the population express TRA-1-60 and / or SSEA5.

[0243] In some embodiments, (a) greater than or equal to 30% of the cells in the population express SOX2; (b) less than or equal to 30% of the cells in the population express PAX2; (c) greater than or equal to 30% of the cells in the population express 0 tubulin III; (d) greater than or equal to 20% of the cells in the population express TrkB; (e) greater than or equal to 30% of the cells in the population express GluA4; (f) less than or equal to 20% of the cells in the population express Myo7A; and (g) less than or equal to 0.1% of the cells in the population express TRA-1-60 and / or SSEA5.

[0244] In some embodiments, (a) greater than or equal to 30% of the cells in the population express SOX2, e.g. between 70% and 100% of the cells express SOX2; (b) greater than or equal to 30%, e.g. betw een 50% and 100%, of the cells in the population express 0 tubulin III; (c) greater than or equal to 10%, e.g. between 10% and 80%, of the cells in the population express TrkB; (d) greater than or equal to 10% , e.g., between 10% and 70%, of the cells in the population express GluA4; (e) less than or equal to 20%, e.g. between 0.1% and 10%, of the cells in the population express Myo7A; and (f) less than or equal to 0.1%, e.g. between 0 and 0.01%, of the cells in the population express TRA-1-60 and / or SSEA5.

[0245] In some embodiments, (a) greater than or equal to 70% of the cells in the population express SOX2 and Nestin, (b) less than 5% of the cells in the population express Myo7A, and (c) greater than 85% of the cells in the population express 0 tubulin III.

[0246] In some embodiments, (a) greater than or equal to 80% of the cells in the population express SOX2 and Nestin, (b) less than 2% of the cells in the population express Myo7A, and (c) greater than 90% of the cells in the population express 0 tubulin III.Attorney Docket No. LCTI-005 / 001WG 43694-03305

[0247] In some embodiments, (a) greater than or equal to 70% of the cells in the population express SOX2 and Nestin, (b) less than 5% of the cells in the population express Myo7A, (c) greater than 85% of the cells in the population express (3 tubulin III, (d) a fold change in GLUA2 mRNA expression compared to a undifferentiated population of pluripotent cells is at least 1000, and (e) a fold change in TRKB mRNA expression compared to a undifferentiated population of pluripotent cells is at least 1000.

[0248] In some embodiments, (a) greater than or equal to 70% of the cells in the population express SOX2 and Nestin, (b) less than 5% of the cells in the population express Myo7A, (c) greater than 85% of the cells in the population express (3 tubulin III, (d) a fold change in GLUA2 mRNA expression compared to a undifferentiated population of pluripotent cells is between 1000 and 6000, and (e) a fold change in TRKB mRNA expression compared to a undifferentiated population of pluripotent cells is between 1000 and 8000.

[0249] In some embodiments, (a) greater than or equal to 80% of the cells in the population express SOX2 and Nestin, (b) less than 2% of the cells in the population express Myo7A, (c) greater than 90% of the cells in the population express (3 tubulin III, (d) a fold change in GLUA2 mRNA expression compared to a undifferentiated population of pluripotent cells is between 1000 and 6000, and (e) a fold change in TRKB mRNA expression compared to a undifferentiated population of pluripotent cells is between 1000 and 8000.

[0250] Following harvesting in accordance with the methods described herein, the expanded population of auditory cells can be formulated at a specific therapeutic dose (e.g., number of cells) and cryopreserved for shipping to the clinic. The ready to administer (RTA) auditory cell therapy composition can then be administered to patients directly after thawing without further washing or other processing steps. Examples of media suitable for cry opreservation include but are not limited to 90% Human Serum / 10% DMSO, CRYOSTOR®, CRYOSTOR® CS10 (10% DMSO), CRYOSTOR® CS5 (5% DMSO), CRYOSTOR® CS2 (2% DMSO), STEM-CELLBANKER®, PRIME XV® FREEZIS, HYPOTHERMASOL®, Trehalose, etc. In embodiments, the cryopreservation medium comprises between about 0.5% and about 50% DMSO, e.g., about 0.5%, about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, or about 50%. In embodiments, the cry opreservation medium comprises between about 0.5% and about 30% DMSO. In embodiments, the cryopreservation medium comprises between about 1% and about 20% DMSO.Attorney Docket No. LCTI-005 / 001WG 43694-03305

[0251] In some embodiments, the final cell composition are cell aggregates, filtered to separate the cell aggregates from carriers, cellular debris or matrix. In other embodiments, the cells are filtered before cryopreservation, to separate the single cells from cell aggregates, carriers, cellular debris or matrix using a single-use filter, cell strainer or mesh with pore sizes of at least 40pm, about 50pm, about 70pm, about 100pm, about 60pm. The filter can be within a closed system. In other embodiments, the separation of single cells from cell aggregates, carriers, cellular debris or matrix can be done by tangential flow centrifugation. The filtration system has the capacity to safely filter single cells through the pores in amounts of 1 million cells, 10 million cells, 100 million cells, 1 billion cells, 10 billion cells, or 100 billion cells. The percent viability of post-filtered cells stored in a cryopreservation medium for between about 0 to about 8 hours is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. The viability can be any value or subrange within the recited ranges. In other embodiments, the percent recovery of post-filtered cells stored in a cry opreservation medium for between about 0 to about 8 hours is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%. 94%, 95%. 96%. 97%. 98%. 99%. or 100%. The recovery can be any value or subrange within the recited ranges.

[0252] In alternative embodiments, cells in the final cell compositions are single cells in a suspension. For example, single cells in a composition can be generated by dissociating the aggregates described herein by any methods known in the art that result in viable single cells.

[0253] In further embodiments, the percent viability of post-filtered cells stored in a neutralization medium for between about 0 to about 8 hours follow ed by storage in cry opreservation medium for between about 0 to about 8 hours is at least about 75%, 80%, 85%, 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or 100%. In other embodiments, the percent recovery of post-filtered cells stored in a neutralization medium for between about 0 to about 8 hours followed by storage in cry opreservation medium for between about 0 to about 8 hours is at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. The viability can be any value or subrange within the recited ranges.

[0254] In yet other embodiments, the percent viability of post-filtered cells stored in a neutralization medium for between about 0 to about 8 hours follow ed by storage in cry opreservation medium for between about 0 to about 8 hours, post-thawing of the cryopreserved composition, is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In still other embodiments, the percent recovery ofAttorney Docket No. LCTI-005 / 001WG 43694-03305 post-filtered cells stored in a neutralization medium for between about 0 to about 8 hours followed by storage in cryopreservation medium for between about 0 to about 8 hours, postthawing of the cryopreserved composition, is at least about, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. The viability can be any value or subrange w ithin the recited ranges.

[0255] In some embodiments, the percent recovery of post-filtered auditory cells stored in a neutralization medium for between about 0 to about 8 hours at room temperature is at least about, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the percent recovery of post-filtered auditory cells stored in a cryopreservation medium for between about 0 to about 8 hours at room temperature is at least about. 70%. 75%. 80%. 85%. 90%. 91%. 92%. 93%. 94%. 95%. 96%, 97%, 98%, 99%, or 100%. In further embodiments, the percent recovery of post-filtered auditory cells stored in a neutralization solution at room temperature for between about 0 to about 8 hours followed by storage in cryopreservation medium for betw een about 0 to about 8 hours at room temperature is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In still further embodiments, the percent recovery of post-filtered auditor}' cells stored in a neutralization solution at room temperature for betw een about 0 to about 8 hours follow ed by storage in cryoprcservation medium for between about 0 to about 8 hours at room temperature is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%. 95%. 96%. 97%. 98%. 99% or 100%. The recovery can be any value or subrange within the recited ranges.

[0256] Auditory cells formulated in cryopreservation media appropriate for post thaw ready to administer (RTA) applications may comprise auditory' cells suspended in adenosine, dextran-40, lactobionic acid, HEPES (N-(2-Hydroxy ethyl) piperazine-N1- (2- ethanesulfonic acid)), sodium hydroxide, L-glutathione, potassium chloride, potassium bicarbonate, potassium phosphate, dextrose, sucrose, mannitol, calcium chloride, magnesium chloride, potassium hydroxide, sodium hydroxide, dimethyl sulfoxide (DMSO), and water. An example of this cryopreservation media is available commercially under the tradename, CryoStor® and is manufactured by BioLife Solutions, Inc. In some embodiments, auditory cells aggregates formulated in aggregate suitable cry opreservation medium such as CryoStem®, as ready to inject product, using an aggregate specific delivery system such as the Sutter Xeno work system that is used for somatic cell nuclear transfer and intracytoplasmic sperm injection, and recently used for ONP spheroids (Heuer et al. 2020).Attorney Docket No. LCTI-005 / 001WO 43694-03305

[0257] DMSO can be used as a cr oprotecti ve agent to prevent the formation of ice crystals, which can kill cells during the cryopreservation process. In some embodiments, the cryopreservable auditory cells therapy composition comprises between about 0.1% and about 2% DMSO (v / v). In some embodiments, the RTA Auditory cells therapy composition comprises between about 1% and about 20% DMSO. In some embodiments, the RTA auditory' cells therapy composition comprises about 10% DMSO. In some embodiments, the RTA auditory cell progenitors cell therapy composition comprises about 5% DMSO. The concentration can be any value or subrange within the recited ranges.

[0258] In some embodiments, auditory cell therapy compositions formulated in cry opreservation media appropriate for post thaw ready to administer (RTA) applications may comprise auditory cells suspended in cryopreservation media that does not contain DMSO. For example, RTA sensory therapeutic cell compositions may comprise auditory cell progenitors suspended in Trolox, Na+, K+, Ca2+, Mg2+, Cl", H2PO4, HEPES, lactobionate, sucrose, mannitol, glucose, dextran-40, adenosine, glutathione without DMSO (dimethyl sulfoxide, (CH3)2SO) or any other dipolar aprotic solvents. An example of thiscry opreservation media is available commercially under the tradename, HYPOTHERMOSOL® or HYPOTHERMOSOL®-FRS and is also manufactured by BioLife Solutions, Inc. In other embodiments, auditory cells compositions formulated incry opreservation media appropriate for post thaw ready to administer applications may comprise auditory cells suspended in Trehalose.

[0259] The RTA auditory cell therapy compositions may optionally comprise additional factors that support auditory cell engraftment, integration, survival, potency, etc. In some embodiments, the RTA auditory cell therapy composition comprises activators of a function of the auditory cell preparations described herein.

[0260] In some embodiments, the RTA auditory cell therapy compositions may be formulated in a medium comprising components that decrease the molecular cell stress during freezing and thawing processes by scavenging of free radicals, pH buffering, oncotic / osmotic support, and maintenance of the ionic concentration balance.

[0261] In some embodiments, auditory cell therapies formulated in cryopreservation media appropriate for post thaw ready to administer applications may comprise one or more immunosuppressive compounds. In certain embodiments, auditory cell therapies formulated in cry opreservation media appropriate for post thaw ready to administer applications may comprise one or more immunosuppressive compounds that are formulated for slow release of the one or more immunosuppressive compounds. Immunosuppressive compounds for useAttorney Docket No. LCTI-005 / 001WO 43694-03305 with the formulations described herein may belong to the following classes of immunosuppressive drugs: Glucocorticoids. Cytostatics (e.g. alkylating agent or antimetabolite), antibodies (polyclonal or monoclonal), drugs acting on immunophilins (e.g. cyclosporin, Tacrolimus or Sirolimus). Additional drugs include interferons, opioids, TNF binding proteins, mycophenolate and small biological agents. Examples of immunosuppressive drugs include: mesenchymal stem cells, anti-lymphocyte globulin (ALG) polyclonal antibody, anti-thymocyte globulin (ATG) polyclonal antibody, azathioprine. BAS 1L1 X 1MAB0 (anti-I L-2Ra receptor antibody), cyclosporin (cyclosporin A), daclizumab (anti-I L-2Ra receptor antibody), everolimus, mycophenolic acid, rituximab (anti-CD20 antibody), sirolimus, tacrolimus, and / or Mycophenolate mofetil.

[0262] In embodiments, the pharmaceutical compositions can be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, for example, in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, for example, sterile pyrogen-free water, before use. In addition to the formulations described previously, the compositions can also be formulated as a depot preparation. Such long-acting formulations can be administered by implantation (e.g.. subcutaneously). Thus, for example, the compositions can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

[0263] The nature of pharmaceutical compositions as described herein is dependent on the mode of administration and can readily be determined by one of ordinary skill in the art. The pharmaceutical compositions described herein can contain carriers or excipients, many of which are known to skilled artisans. Excipients that can be used include buffers (for example, citrate buffer, phosphate buffer, acetate buffer, and bicarbonate buffer), amino acids, urea, alcohols, ascorbic acid, phospholipids, polypeptides (for example, serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, and glycerol. A modulatory compound can be formulated in various ways, according to the corresponding route of administration. For example, liquid solutions can be made for administration by drops into the ear. for injection, or for ingestion; gels or powders can be made for ingestion or topicalAttorney Docket No. LCTI-005 / 001WD 43694-03305 application. Methods for making such formulations are well known and can be found in, for example. "Remington's Pharmaceutical Sciences."

[0264] For example, a pharmaceutical composition can be formulated for administration by drops into the ear, insufflation (such as into the ear), topical, or oral administration. In another mode of administration, the pharmaceutical composition can be directly administered in situ to the cochlea of the inner ear, such as via a cannula, catheter or pump. A cannula, catheter or pump can, for example, direct the pharmaceutical composition into the cochlear luminae or the round window of the ear. In another route of administration, the pharmaceutical composition can be injected into the ear, such as into the luminae of the cochlea (e.g.. the Scala media, Sc vestibuli, and Sc tympani). Injection can be, for example, through the round window of the ear or through the cochlear capsule.

[0265] Pharmaceutical compositions in accordance with the present disclosure can further comprise a pharmaceutically -acceptable carrier. In an embodiment, a pharmaceutically-acceptable carrier can comprise dimethyl sulfoxide (DMSO). In an embodiment, a pharmaceutically-acceptable carrier does not comprise dimethyl sulfoxide. As described herein, a composition can be further adapted for cryopreservation at or below -80°C to -I95°C. In embodiments, a composition can be formulated to thaw and administered directly into a subject, e.g. via injection, without additional manipulation prior to administration. In some embodiments, a composition can be formulated including a cryosolution such as CRYOSTOR®10 (CS10) as a cryopreservation media (an animal component free defined cry opreservation medium with 10% DMSO). In some embodiments, the composition is formulated in CRYOSTOR®10. In some embodiments, a composition can be formulated including a cryosolution such as CRYOSTOR®5 (CS5) as a cryopreservation media (an animal component free defined cryopreservation medium with 5% DMSO). In some embodiments, the composition is formulated in CRYOSTOR®5. In an embodiment, a composition can be filtered using a filter kit before cryopreservation, to avoid clogging during administration through a narrow7cannula, syringe needle or a catheter.

[0266] A pharmaceutical composition in accordance with the present disclosure can comprise from about 1 million cells per milliliter, such as about 1.5 million cells per milliliter, such as about 2 million cells per milliliter, such as about 5 million cells per milliliter, such as about 10 million cells per milliliter, such as about 20 million cells per milliliter, such as about 25 million cells per milliliter, such as about 30 million cells per milliliter, such as about 40 million cells per milliliter, such as about 60 million cells per milliliter, such as about 70 million cells per milliliter, such as about 80 million cells per milliliter, such as about 90Attorney Docket No. LCTI-005 / 001WG 43694-03305 million cells per milliliter, such as about 100 million cells per milliliter, such as about 200 million cells per milliliter, such as about 300 million cells per milliliter, such as about 400 million cells per milliliter, such as about 500 million cells per milliliter, such as about 600 million cells per milliliter, such as about 700 million cells per milliliter, such as about 900 million cells per milliliter, such as about 0.5 million cells per milliliter, such as about 0.6 million cells per milliliter, such as about 0.8 million cells per milliliter, such as about 0.9 million cells per milliliter, such as about 1 million cells per milliliter, such as about 1.5 million cells per milliliter, or such as about 50 million cells per milliliter. In some embodiments, a pharmaceutical composition in accordance with the present disclosure can comprise between about 30 million cells per milliliter to about 700 million cells per milliliter. In some embodiments, a pharmaceutical composition in accordance with the present disclosure can comprise between about 50 million cells per milliliter to about 400 million cells per milliliter. The number of cells can be any value or subrange within the recited ranges. In some embodiments, a pharmaceutical composition in accordance with the present disclosure can comprise about 50 million cells per milliliter. In some embodiments, a pharmaceutical composition in accordance with the present disclosure can comprise about 400 million cells per milliliter.

[0267] In yet another embodiment, a pharmaceutical composition in accordance with the present disclosure can have a volume ranging from about 0.01 microliters to about 2 milliliters, such as about 0.1 microliters, such as about 0.5 microliters, such as about 1 microliter, such as about 3 microliters, such as about 4 microliters, such as about 5 microliters, such as about 6 microliters, such as about 7 microliters, such as about 10 microliters, such as about 20 microliters, such as about 50 microliters, such as about 80 microliters, such as about 100 microliters, such as about 200 microliters, such as about 500 microliters, such as about 1 milliliter or such as about 2 milliliters. In some embodiments, a pharmaceutical composition in accordance with the present disclosure has a volume of about 50 microliters. In some embodiments, a pharmaceutical composition in accordance with the present disclosure has a volume of about 0.5 microliters. The volume can be any value or subrange within the recited ranges. In an embodiment, a pharmaceutical composition in accordance with the present disclosure can be in a container configured for cry opreservation or for administration to a subject in need thereof. In an embodiment, a container can be a prefilled syringe.

[0268] In an embodiment, a pharmaceutical composition in accordance with the present disclosure can be administered at a volume ranging from about 1 microliter to about 1,800 microliters, such as about 2 microliters, such as about 3 microliters, such as about 4Attorney Docket No. LCTI-005 / 001WO 43694-03305 microliters, such as about 50 microliters, such as about 100 microliters, such as about 200 microliters, such as about 450 microliters, such as about 1800 microliters, such as about 10 microliters, such as bout 20 microliters, or such as about 40 microliters. The volume can be any value or subrange within the recited ranges. In some embodiments, a pharmaceutical composition in accordance with the present disclosure is administered at a volume of about 50 microliters.

[0269] In some embodiments, a pharmaceutical composition in accordance with the present disclosure comprises at least about 50,000 cells, at least about 100,000 cells, at least about 200,000 cells, at least about 300,000 cells, at least about 400,000 cells, at least about 500,000 cells, at least about 600,000 cells, at least about 700,000 cells, at least about 800,000 cells, at least about 900,000 cells, at least about 1 million cells, at least about 1.5 million cells, at least about 2 million cells, at least about 2.5 million cells, at least about 3 million cells, at least about 4 million cells, at least about 5 million cells, at least about 10 million cells, at least about 20 million cells, at least about 30 million cells, at least about 40 million cells, or at least about 59 million cells. In some embodiments, the pharmaceutical composition comprises between about 50,000 cells and 50 million cells, between about 100,000 cells and 20 million cells, between about 100,000 cells and 10 million cells, between about 100,000 cells and 1 million cells, between about 500,000 cells and 10 million cells, between about 500,000 cells and 1 million cells, between about 1 million cells and 50 million cells, or between about 10 million cells and 50 million cells. In some embodiments, the pharmaceutical composition comprises between about 100,000 cells and 10 million cells. In some embodiments, the pharmaceutical composition comprises between about 100,000 cells and 1 million cells. In some embodiments, the pharmaceutical composition comprises between about 50,000 cells and 500.000 cells. In some embodiments, the pharmaceutical composition comprises between about 100,000 cells and 500,000 cells. In some embodiments, the pharmaceutical composition comprises between about 500,000 cells and 1 million cells.Methods of Producing Populations of Auditory Cells

[0270] The disclosure provides methods of producing populations of auditory cells from undifferentiated pluripotent stem cells suitable for administration to a subject. The populations of cells can be characterized by the methods described herein, e.g. by determining the expression of any of the genes set forth in Tables 5 and 6. Methods ofAttorney Docket No. LCTI-005 / 001WO 43694-03305 producing suitable populations of cells are described in WO2023 / 167986, the contents of which are incorporated by reference in their entirety herein, and are described in detail below.

[0271] The methods comprise culturing populations of undifferentiated pluripotent stem cells in different combinations of grow th factors and growth factor inhibitors, in a series of steps that induces the differentiation of the undifferentiated pluripotent stem cells tow ards auditory neuronal fates through a series of differentiation steps. In an exemplary differentiation pathway, human embryonic stem cells (hESCs) are induced to differentiate into non-neuronal ectoderm (NNE) cells, which are induced to differentiate into pre-placodal ectoderm (PPE) cells, which in turn are induced to different into early otic neuronal progenitor (ONP) cells, mid otic progenitor cells, late otic neuronal progenitor cells, auditory' neuron progenitors, mature auditory neurons and spiral ganglion neurons (SGN). The resulting population of cells may contain a mixture of cell types. However, cells from the later stages of the pathway may predominate, and residual hESCs may be minimal or absent. For example, the population may be a substantially pure population of late ONP, spiral ganglion neuron and mature auditory’ neurons. Without wishing to be bound by theory, it is thought that a composition comprising a nuxed cell population may be better suited as a therapeutic agent for auditory diseases and disorders than a composition comprising a homogenous population of cells, as the range of cell types increases the niches into which the cells can engraft when administered to a subject, and the increases the number of fates that the cells can adopt upon administration.

[0272] A schematic depiction of the process of hESC differentiation auditory neuron progenitor and further culturing to mature SGN is provided in FIG. 1A. In some embodiments, protein markers are monitored along the differentiation process, as depicted in FIGS. 1B-1C. Monitoring of protein markers facilitates purity of the cell populations, to ensure a high-quality final product of ANP1 cells.

[0273] FIG. 2 depicts exemplary assays for monitoring the quality of the ANP1 cell product. Cell products may be assessed bio-analytically and / or functionally. Bioanalytical assessments may include determining purity specific markers and examination of gene expression for the cell populations. Protein markers can be assessed, for example and without limitation, by flow cytometry, immunofluorescence, and other methods as known in the art. Gene expression (<?.g., late ANP gene expression) can be assessed, for example, by quantitative PCR (qPCR), RT-qPCR (reverse transcriptase quantitative polymerase chain reaction), RNA sequencing, and other methods as known in the art. Functional assessments may include assaying for in vitro synapse formation and calcium signaling. In vitro synapseAttorney Docket No. LCTI-005 / 001WO 43694-03305 formation can be assessed, for example and without limitation, by immunofluorescence microscopy (e.g., for synapse formation markers). Calcium signaling can be assessed, for example and without limitation, by calcium influx detection. In addition, cell morphology can be monitored via immunofluorescence methods. For example, maturation of ANP1 cells in vitro can be characterized by elongation of cell processes combined with beta tubulin III expression.

[0274] The final cell product can be profiled. Profiling can be by RNA-sequencing, proteomic / protein analysis, marker characterization (e.g., by flow- cytometry), immunofluorescence and other methods of characterization as know n in the art.

[0275] These methods of characterization of the manufacturing (differentiation) process and final cell product can facilitate advantageous culturing and expansion for a repeatable and reproducible differentiation process, streamlining workflow, reducing costs and waste, and providing quality control of the cell product.Methods for Expanding and Maintaining Human Embry onic Stem Cells (hESCs)

[0276] In an aspect, provided herein are methods for expanding and maintaining human embryonic stem cells (hESCs) in an undifferentiated, pluripotent state, the method comprising the steps of (a) simultaneously combining human embryonic stem cells and an extracellular matrix component (ECM) in growth media in tissue culture flasks for static expansion, and (b) culturing the adherent hESCs for a period of time.

[0277] In some embodiments, the cultured human embryonic stem cells of the static expansion are harvested non-enzymatically using ReLeSR™ and cultured in mTeSR™ plus media on iMatrix-511 coated vessels. In some embodiments, hESCs are expanded further by repeating steps (a) and (b).

[0278] In some embodiments, the cultured human embryonic stem cells of the static expansion are harvested and further differentiated.

[0279] In an aspect, provided herein are methods for expanding and maintaining human embryonic stem cells (hESCs) in an undifferentiated, pluripotent state, the method comprising the steps of (a) simultaneously combining human embry onic stem cells, an extracellular matrix component (ECM), and a microcarrier in growth media to form a suspendable expansion complex, and (b) culturing the suspendable expansion complex for a period of time.Attorney Docket No. LCTI-005 / 001WO 43694-03305

[0280] In some embodiments, the cultured human embryonic stem cells of the suspendable expansion complex are harvested and expanded further by repeating steps (a) and (b).

[0281] In some embodiments, the cultured human embryonic stem cells of the suspendable expansion complex are harvested and further differentiated.

[0282] Human embry onic stem cells can be isolated from human blastocysts. Human blastocysts are typically obtained from human in vivo preimplantation embryos or from in vitro fertilized (IVF) embryos. Alternatively, a single cell human embry o can be expanded to the blastocyst stage. For the isolation of human ES cells the zona pellucida is removed from the blastocyst and the inner cell mass (ICM) is isolated by a procedure in which the trophectoderm cells are lysed and removed from the intact ICM by gentle pipetting. The ICM is then plated in a tissue culture flask containing the appropriate medium which enables its outgrowth. Following 9 to 15 days, the ICM derived outgrowth is dissociated into clumps either by a mechanical dissociation or by an enzymatic degradation and the cells are then replated on a fresh tissue culture medium. Colonies demonstrating undifferentiated morphology are individually selected by micropipette, mechanically dissociated into clumps, and replated. Resulting ES cells are then routinely split every 4-7 days. For further details on methods of preparation human ES cells, see Reubinoff et al. Nat Biotechnol 2000, May: 18(5): 559; Thomson et al., [U.S. Patent No. 5,843,780; Science 282: 1145, 1998; Curr. Top. Dev. Biol. 38: 133. 1998; Proc. Natl. Acad. Sci. USA 92: 7844, 1995]; Bongso et al.. [Hum Reprod 4: 706, 1989]; and Gardner et al., [Fertil. Steril. 69: 84, 1998],

[0283] In addition, ES cells can be obtained from other species, including mouse (Mills and Bradley, 2001), golden hamster [Doetschman et al., 1988, Dev Biol. 127: 224-7], rat [lannaccone et al., 1994, Dev Biol. 163: 288-92], rabbit [Giles et al. 1993, Mol Reprod Dev. 36: 130-8; Graves & Moreadith, 1993, Mol Reprod Dev. 1993, 3036: 424-33], several domestic animal species [Notarianni et al., 1991, J Reprod Fertil Suppl. 43: 255-60; Wheeler 1994, Reprod Fertil Dev. 6: 563-8; Mitalipova et al., 2001, Cloning. 3: 59-67] and non-human primate species (Rhesus monkey and marmoset) [Thomson et al.. 1995, Proc Natl Acad Sci U S A. 92: 7844-8; Thomson et al.. 1996, Biol Reprod. 55: 254-9],

[0284] Extended blastocyst cells (EBCs) can be obtained from a blastocyst of at least nine days post fertilization at a stage prior to gastrulation. Prior to culturing the blastocyst, the zona pellucida is digested [for example by Tyrode’s acidic solution (Sigma Aldrich, St Louis, MO, USA)] so as to expose the inner cell mass. The blastocysts are then cultured asAttorney Docket No. LCTI-005 / 001WO 43694-03305 whole embryos for at least nine and no more than fourteen days post fertilization (i.e., prior to the gastrulation event) in vitro using standard embryonic stem cell culturing methods.

[0285] Another method for preparing ES cells is described in Chung et al.. Cell Stem Cell, Volume 2, Issue 2, 113-117, 7 February 2008. This method comprises removing a single cell from an embryo during an in vitro fertilization process. The embryo is not destroyed in this process.

[0286] EG (embryonic germ) cells are prepared from the primordial germ cells obtained from fetuses of about 8-11 weeks of gestation (in the case of a human fetus) using laboratory techniques known to anyone skilled in the arts. The genital ridges are dissociated and cut into small portions which are thereafter disaggregated into cells by mechanical dissociation. The EG cells are then grown in tissue culture flasks with the appropriate medium. The cells are cultured with daily replacement of medium until a cell morphology consistent with EG cells is observed, typically after 7-30 days or 1-4 passages. For additional details on methods of preparation human EG cells see Shamblott et al., [Proc. Natl. Acad. Sci. USA95: 13726, 1998] and U.S. Patent No. 6,090.622.

[0287] Yet another method for preparing ES cells is by parthenogenesis. The embryo is also not destroyed in the process.

[0288] The cells may be expanded in suspension, with or without a microcarrier, or in a monolayer. The expansion of the mixed population of cells in monolayer cultures or in suspension culture may be modified to large scale expansion in bioreactors or multi / hyper stacks by methods well known to those versed in the art.

[0289] According to some embodiments, the expansion phase is effected for at least one to 20 w eeks, for example at least one week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks or even 10 weeks. In embodiments, the expansion phase is effected for 1 week to 10 weeks, such as 2 weeks to 10 weeks, 3 weeks to 10 w eeks, 4 weeks to 10 weeks, or 4 weeks to 8 weeks. The time period may be any value or subrange within the recited ranges, including endpoints.

[0290] According to still other embodiments, the expansion phase is effected until a suitable lactate concentration in the cell culture medium, and / or percent confluence is achieved. Percent confluence is the percentage of the culture vessel surface area that appears covered by a layer of cells when observed by microscopy. In some embodiments, the undifferentiated pluripotent stem cells are cultured until the lactate concentration in the cell culture medium is between about 1.0 to 13. 0 mM, or between about 1.5-12.5 mM. In someAttorney Docket No. LCTI-005 / 001WO 43694-03305 embodiments, cells are cultured until the lactate concentration in the cell culture medium is between about 1.68-12.29 mM. In some embodiments, the percent confluence is between 5% and 85%.

[0291] According to still other embodiments, the mixed population of cells is passaged at least one time during the expansion phase, at least twice during the expansion phase, at least three times during the expansion phase, at least four times during the expansion phase, at least five times during the expansion phase, at least six times during the expansion phase, or at least seven times during the expansion phase.

[0292] When cells are collected enzymatically, it is possible to continue the expansion for more than 8 passages, more than 9 passages and even more than 10 passages (e.g. 11-15 passages). The number of total cell doublings can be increased to greater than 30, e.g. 31, 32, 33, 34 or more. (See international patent application publication number WO 2017 / 021973, incorporated herein by reference in its entirety).

[0293] An extracellular matrix (ECM) is a three-dimensional network consisting of extracellular macromolecules and minerals, such as collagen, enzymes, glycoproteins and hydroxyapatite that provide structural and biochemical support to surrounding cells. Because multicellularity evolved independently in different multicellular lineages, the composition of ECM varies between multicellular structures; however, cell adhesion, cell-to-cell communication and differentiation are common functions of the ECM.

[0294] The animal extracellular matrix includes the interstitial matrix and the basement membrane. Interstitial matrix is present between various animal cells (i.e., in the intercellular spaces). Gels of polysaccharides and fibrous proteins fill the interstitial space and act as a compression buffer against the stress placed on the ECM. Basement membranes are sheet-like depositions of ECM on which various epithelial cells rest. Each type of connective tissue in animals has a type of ECM: collagen fibers and bone mineral comprise the ECM of bone tissue; reticular fibers and ground substance comprise the ECM of loose connective tissue; and blood plasma is the ECM of blood.

[0295] Suitable extracellular matrix components for use within the scope of the present disclosure may include, but are not necessarily limited to, Matrigel®. vitronectin, gelatin, collagen I, collagen IV, laminin (e.g. laminin 521), fibronectin poly -D-ly sine, their derivatives, or a combination thereof. In specific embodiments, the human laminin is human laminin 511 E8 fragment.

[0296] In some embodiments, the microcarriers may comprise one or more of polystyrene, cross-linked dextran, magnetic particles, microchips, cellulose, hydroxylatedAttorney Docket No. LCTI-005 / 001WO 43694-03305 methacrylate, collagen, gelatin, polysty rene, plastic, glass, ceramic, or silicone. In some embodiments, the microcarriers are composed of polystyrene, surface-modified polystyrene, chemically modified polystyrene, cross-linked dextran, cellulose, acrylamide, collagen, alginate, gelatin, glass, DEAE-dextran, or a combination thereof. In some embodiments, the microcarrier is composed of polystyrene. In some embodiments, the microcarrier is composed of surface-modified polystyrene. In some embodiments, the microcarrier is composed of chemically modified polystyrene. In some embodiments, the microcarrier is composed of cross-linked dextran. In some embodiments, the microcarrier is composed of cellulose. In some embodiments, the microcarrier is composed of acrylamide. In some embodiments, the microcarrier is composed of collagen. In some embodiments, the microcarrier is composed of alginate. In some embodiments, the microcarrier is composed of gelatin. In some embodiments, the microcarrier is composed of glass. In some embodiments, the microcarrier is composed of DEAE-dextran. In some embodiments, the microcarriers are not coated.

[0297] In some embodiments, the microcarriers are coated. In embodiments, the microcarriers may be coated with Matngel®, laminin, vitronectin, collagen, their derivatives, or a combination thereof. In embodiments, the microcarriers may be coated by poly-lysine, poly-L-lysine, poly-D-lysine, fibronectin, tenascin, dextran, a peptide, or a combination thereof. In some embodiments, the microcarrier is coated with laminin. In some embodiments, the microcarrier is coated with Matrigel®. In some embodiments, the microcarrier is coated with collagen. In some embodiments, the s microcarrier is coated with poly-lysine. In some embodiments, the microcarrier is coated with poly-L-lysine. In some embodiments, the microcarrier is coated with poly-D-lysine. In some embodiments, the microcarrier is coated with vitronectin. In some embodiments, the microcarrier is coated with fibronectin. In some embodiments, the microcarrier is coated with tenascin. In some embodiments, the microcarrier is coated with dextran. In some embodiments, the microcarrier is coated with a peptide.

[0298] In some embodiments, the microcarriers may be spherical, smooth, macroporous, rod-shaped, or a combination thereof. In some embodiments, the microcarriers may be coupled with protamine or polylysine. In some embodiments, the microcarrier is spherical. In some embodiments, the s microcarrier is ellipsoidal. In some embodiments, the microcarrier is rod-shaped. In some embodiments, the microcarrier is disc-shaped. In some embodiments, the microcarrier is porous. In some embodiments, the microcarrier is nonAttorney Docket No. LCTI-005 / 001WG 43694-03305 porous. In some embodiments, the microcarrier is smooth. In some embodiments, the microcarrier is flat.

[0299] In some embodiments, the microcarriers are neutral. In some embodiments, the microcarriers are negatively charged. In some embodiments, the microcarriers are hydrophilic.

[0300] In some embodiments, the microcarriers may have a surface area (per gram) of 25 cm2, 50 cm2, 75 cm2, 100 cm2, 125 cm2, 150 cm2, 175 cm2, 200 cm2, 225 cm2, 250 cm2, 500 cm2, 625 cm2, 750 cm2, 1,000 cm2, 1,250 cm2, 5,000 cm2, or 7,500 cm2. The surface area may be any value or subrange within the recited ranges, including endpoints.

[0301] In specific embodiments, the microcarriers are surface treated to enhance cell attachment, maximizing cell yield and viability. The microcarriers may be comprised of USP Class VI polystyrene material, which provides a consistent platform. In some embodiments, the microcarriers create a synthetic surface on the microcarriers for stem cell expansion. An enhanced attachment surface treatment infuses the surface of the microcarriers with oxygen to improve cell attachment. In some embodiments, the microcarriers are nonpyrogenic. In some embodiments, the microcarriers are optimized for mesenchymal stem cell applications. In specific embodiments, the beads may vary in size from 125-212 pm. In specific embodiments, the density of the microcarriers may be 1.026 ± 0.004. In specific embodiments, the microcarriers may be 360 crn gram.

[0302] In some embodiments, the method comprises combining the hESCs with laminin or a derivative thereof to improve the cell attachment to the carrier surface. In specific embodiments, the laminin is human laminin 511. As alternative embodiments, several other extracellular matrices may be used for cell attachment, such as including, but not necessarily limited to, vitronectin, fibronectin, collagen, Matrigel®, or derivatives thereof.

[0303] In some embodiments, the cells may be cultured for one day, two days, three days, four days, five days, six days, seven days, eight days, nine days, ten days, eleven days, twelve days, thirteen days, or fourteen days.

[0304] In some embodiments, the cells may be cultured in a working volume of between 4 mL and 3,000 mL, for example about 4 mL, 5 mL, 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 100 mL, 250 mL, 500 mL, 750 mL, 1,000 mL, or 3,000 mL. The volume may be any value or subrange within the recited ranges, including endpoints.

[0305] In some embodiments, the cultured cells may be expanded further.Attorney Docket No. LCTI-005 / 001WO 43694-03305

[0306] In some embodiments, the cultured cells may remain undifferentiated.Undifferentiated cells may be identified by expression of various markers, such as including, but not necessarily limited to, SSEA-5, TRA-1-60, Oct-4, and Nanog. In some embodiments, undifferentiated cells express SSEA-5. In some embodiments, undifferentiated cells express TRA-1-60. In some embodiments, undifferentiated cells express Oct-4. In some embodiments, undifferentiated cells express Nanog. In some embodiments, undifferentiated cells express both SSEA-5 and TRA-1-60. In some embodiments, undifferentiated cells express both Oct-4 and Nanog. In some embodiments, undifferentiated cells express SSEA-5, TRA-1-60, Oct-4, and Nanog (IPC#0).

[0307] In some embodiments, the cells may be cultured in a feeder cell-conditioned medium. ES culturing methods may include the use of feeder cell layers which secrete factors needed for stem cell proliferation, while at the same time, inhibiting their differentiation. The culturing is typically effected on a solid surface, for example, a surface coated with gelatin or vimentin. Exemplary feeder layers include human embryonic fibroblasts, adult fallopian epithelial cells, primary mouse embryonic fibroblasts (PMEF), mouse embryonic fibroblasts (MEF), murine fetal fibroblasts (MFF), human embryonic fibroblast (HEF), human fibroblasts obtained from the differentiation of human embryonic stem cells, human fetal muscle cells (HFM), human fetal skin cells (HFS), human adult skin cells, human foreskin fibroblasts (HFF), human umbilical cord fibroblasts, human cells obtained from the umbilical cord or placenta, and human marrow stromal cells (hMSCs). Growth factors may be added to the medium to maintain the ESCs in an undifferentiated state. Such growth factors include bFGF and / or TGF. In another embodiment, agents may be added to the medium to maintain the hESCs in a naive undifferentiated state - see for example Kalkan et al., 2014, Phil. Trans. R. Soc. B, 369: 20130540.

[0308] hESCs are typically plated on top of the feeder cells 1-4 days later in a supportive medium (e.g. NUTRISTEM®, NUT(+) with human serum albumin, mTeSR™ plus, or mTeSR™! StemFit®). Additional factors may be added to the medium to prevent differentiation of the ESCs such as bFGF and TGF 3. Once a sufficient amount of hESCs is obtained, the cells may be mechanically disrupted (e.g. by using a sterile tip or a disposable sterile stem cell tool; 14602 Swemed). Alternatively, the cells may be removed by enzymatic treatment (e.g. collagenase A, or TrypLE™ Select). This process may be repeated several times to reach the necessary amount of hESC. According to some embodiments, following the first round of expansion, the hESCs are removed using TrypLE™ Select and following the second round of expansion, the hESCs are removed using collagenase A.Attorney Docket No. LCTI-005 / 001WO 43694-03305

[0309] Feeder cell free systems have also been used in ES cell culturing, such systems utilize matrices supplemented with serum replacement, cytokines and growth factors (including IL6 and soluble IL6 receptor chimera) as a replacement for the feeder cell layer. Stem cells can be grown on a solid surface such as an extracellular matrix (e.g., MATRIGEL®, laminin or vitronectin) in the presence of a culture medium - for example, the Lonza L7™ system, mTeSR™, StemPro™, XFKSR, E8, NUTRISTEM®). Unlike feederbased cultures which require the simultaneous growth of feeder cells and stem cells and which may result in mixed cell populations, stem cells grown on feeder-free systems are easily separated from the surface. The culture medium used for growing the stem cells contains factors that effectively inhibit differentiation and promote their grow th such as MEF-conditioned medium and bFGF.

[0310] Also within the scope of the present disclosure are methods for expanding and maintaining human embryonic stem cells (hESCs) in an undifferentiated state, comprising culturing human pluripotent stem cells on a non-adherent surface to obtain a population of undifferentiated hESCs, combining said population of undifferentiated hESCs with microcarriers in growth media, and expanding said population of cells.

[0311] Examples of non-adherent cell culture plates include those manufactured by Nunc (e.g. Hydrocell Cat No. 174912), etc. In other embodiments, non-adherent suspension culture dishes may be used (e.g.. Coming).

[0312] According to some embodiments, when the cells are cultured on the non-adherent substrate, e.g. cell culture plates, the atmospheric oxygen conditions are 20%.However, manipulation of the atmospheric oxygen conditions is also contemplated such that the atmospheric oxygen percent is less than about 20%, 15%, 10%, 9%, 8%, 7%, 6% or even less than about 5% (e.g. between 1% - 20%. 1%-10% or 0-5 %). According to other embodiments, the cells are cultured on the non-adherent substrate initially under normal atmospheric oxygen conditions and then lowered to less than normal atmospheric oxygen conditions.

[0313] While methods described above are directed to methods of expanding and maintaining hESCs, analogous methods directed to induced pluripotent stem cells (iPSCs) are also within the scope of the present disclosure. iPSCs are a type of stem cell derived from somatic cells which have been reprogrammed back into a pluripotent state through the introduction of pluripotency associated genes, and are available from a variety of sources. The person of ordinary skill in the art will appreciate the changes necessary’ to adapt the hESC methods described above for use with iPSCs and the like.Attorney Docket No. LCTI-005 / 001WD 43694-03305Expansion Compositions

[0314] In another aspect, provided herein are suspendable expansion complex compositions comprising human embryonic stem cells or IPSCs, an extracellular matrix component (ECM), and a microcarrier.

[0315] Human embry onic stem cells, extracellular matrices, and microcarriers are described in detail elsewhere herein.

[0316] Expansion complex ranges may vary. In the following tables, the range of the complex components is detailed in different units for the ECM component.Table 8.

[0317] The ECM component can be presented by mol / cm2by using the laminin 511 E8 fragment's molecular weight (150 KDa).Table 9.

[0318] The ECM component can also be presented by the number of molecules / cm2by using molecular weight (150 KDa) multiplied by Avogadro’s number (6.022xl023).Table 10.Attorney Docket No. LCTI-005 / 001WO 43694-03305

[0319] In some embodiments, the following specification parameters may be expanded: # Of hESCs (cells) - 4,000 - 600.000 cells per cm2of microcarriers; Laminin 511 E8 fragment (pg per cm2 of microcarriers) - 0.125 pg per cm2or higher.

[0320] In some embodiments, the composition may further comprise a growth medium. Non-limiting examples of commercially available basic media (i.e. a chemically defined medium or CDM) that may be utilized in accordance with this disclosure comprise NUTRISTEM® (without bFGF and TGF for ESC differentiation, with bFGF and TGF for ESC expansion), NEUROBASAL™, KO-DMEM, DMEM, DMEM / F12, CELLGRO™ Stem Cell Growth Medium, or X-VIVO™ The basic medium may be supplemented with a variety of agents as known in the art dealing with cell cultures. The following is a non-limiting reference to various supplements that may be included in the culture to be used in accordance with the present disclosure: serum or with a serum replacement containing medium, such as, without being limited thereto, knock out serum replacement (KOSR), NUTRIDOMA-CS, TCH™, N2, N2 derivative, or B27 or a combination; an extracellular matrix (ECM) component, such as, without being limited thereto, fibronectin, laminin, collagen and gelatin. In some embodiments, the cell culture medium comprises a chemically defined medium (CDM) supplemented with N2, B27, or a combination thereof, optionally supplemented (with BrainPhys™, The ECM may then be used to cany' the one or more members of the TGF0 superfamily of growth factors; an antibacterial agent, such as. w ithout being limited thereto, L-glutamine. beta mercaptoethanol, penicillin and streptomycin; and non-essential amino acids (NEAA), neurotrophins which are known to play a role in promoting the survival of SCs in culture, such as, without being limited thereto, BDNF, NT3 and NT4.

[0321] As described above, the microcarriers may comprise one or more of polystyrene, cross-linked dextran, magnetic particles, microchips, cellulose, hydroxylated methacrylate, collagen, gelatin, polystyrene, plastic, glass, ceramic, silicone. In some embodiments, the microcarrier is composed of polystyrene. In some embodiments, the microcarrier is composed of surface-modified polystyrene. In some embodiments, the microcarrier is composed of chemically modified polystyrene. In some embodiments, the microcarrier is composed of cross-linked dextran. In some embodiments, the microcarrier is composed of cellulose. In some embodiments, the microcarrier is composed of acrylamide. In some embodiments, the microcarrier is composed of collagen. In some embodiments, the microcarrier is composed of alginate. In some embodiments, the microcarrier is composed of gelatin. In some embodiments, the microcarrier is composed of glass. In some embodiments, the microcarrier is composed of DEAE-dextran.Attorney Docket No. LCTI-005 / 001WG 43694-03305

[0322] As described above, the microcarriers may be spherical, smooth, macroporous, rod-shaped, or a combination thereof.

[0323] In some embodiments, the microcarriers may be coated with matrigel, laminin, vitronectin, collagen, their derivatives, or a combination thereof. In some embodiments, the laminin is human laminin 511.

[0324] In some embodiments, the microcarriers are not coated.

[0325] In some embodiments, the microcarriers have a surface area (per gram): of 25 cm2to 7,500 cm2, e.g., about 25 cm2, 50 cm2, 75 cm2, 100 cm2, 125 cm2, 150 cm2, 175 cm2, 200 cm2, 225 cm2, 250 cm2, 500 cm2, 625 cm2, 750 cm2, 1,000 cm2, 1,250 cm2, 5,000 cm2, or 7,500 cm2. The surface area may be any value or subrange within the recited ranges, including endpoints.

[0326] In some embodiments, the microcarriers are coupled with protamine or polylysine. In some embodiments, the microcarriers are neutral. In some embodiments, the microcarriers are negatively charged. In some embodiments, the microcarriers are hydrophilic.Methods of Making Auditory Cells

[0327] In accordance with the present disclosure, human pluripotent stem cells (hPSCs) can be grown in static or dynamic culture on microcarriers in ahESC culture media, and maintained in a pluripotency state by daily replacement of the hPSC media, as described above. The hPSCs can be differentiated by medium replacement into a culture media (1 : 1 mixture of DMEM / F12 and Neurobasal medium with added GlutaMax™, an L-alanyl-L-glutamine dipeptide that stabilizes the L-glutamine) that will induce non neuronal ectoderm (NNE) formation. This media can include, for example. B27 and N2 supplements, TGF beta agonists such as BMP4 (1-25 ng / mL), vitamin b3 derivative nicotine amide (NIC, 1-25 mM), SB431542, and / or FGF2 (1-25 ng / mL). The dynamic or static culture can continue for 3-7 days, and medium will be replaced either fully or gradually (75-100% of volume each day). At differentiation days 4-8 differentiation factors are replaced with FGF2, LDN193189 (20-400 nM). IWP-2 (2uM), SB431542 (1 pM). and the Wnt inhibitor IWR-endo (1-10 pM). to generate the pre-placodal ectoderm. Static or dynamic culture continues for 5-7 days with medium replacement every 1-3 days. At differentiation days 10-14 differentiation factors can be replaced with FGF2, CHIR99021 (6 pM), and IGF1 (50 ng / ml), to generate the early ONP. Static or dynamic culture will continue for 7-10 days with medium replacement every 2-3 days. On days 15-25 differentiation factors can be replaced with FGF2, EGF, retinoic acidAttorney Docket No. LCTI-005 / 001WD 43694-03305 (RA 0.2-2 pM), SHH (500 ng / ml) and IGF1 (50 ng / ml), to generate the mid-Late ONP. Static or dynamic culture can continue for 7-9 days with medium replacement every 1-3 days. On days 22-35, Mid-Late ONP cells are harvested and cultured as single cells for 2-3 days in the presence of BDNF (10 ng / mL), NT3 (10 ng / mL), IGF-1, and Rock inhibitor (e.g., Y-27632 Dihydrochloride, 10 pM) to form small aggregates, and for an additional two weeks without Rock inhibitor. At differentiation days 42-55 the cells are further expanded for final maturation.

[0328] In some embodiments, the hPSCs can be, for example, differentiated by medium replacement into a culture media (1 : 1 mixture of DMEM / F12 and Neurobasal medium) that will induce Neural Crest formation, by culturing in 2D with 50-2000 ng / ml Noggin and 0.5-20 ng / ml FGF2 for about 14 days. On or about day 14, cells are transferred to culturing in 3D with, for example, 5-100 ng / ml EGF and 5-100 ng / ml FGF2 for about 5 days. On or about day 19, cells are returned to a 2D culturing with differentiation factors FGF2, Purmorphamine (0.1-lpM), EGF, retinoic acid (RA 0.2-2 pM) and IGF1 (50 ng / ml), to generate the Late ONP. Dynamic culture will continue for about 7 days with medium replacement about every 2-3 days. On or about day 25, Late ONP cells will be harvested and inoculated into dynamic suspension as single cells for about 3 days in the presence of FGF and EGF and Rock inhibitor (2-50 pM) to form small aggregates.

[0329] hPSCs can be differentiated into different populations of cells through culture in a variety of different mediums comprising growth factors and growth factor inhibitors. In some embodiments, undifferentiated pluripotent stem cells are subjected to conditions sufficient for directed differentiation to produce a composition comprising a population of auditory' cells, for example, a population of cells comprising NNE, PPE, ONP cells, neurons (e.g., spiral ganglion neurons) or any combination thereof. In some embodiments, the method comprises culturing a population of hPSCs in 1, 2, 3, 4, or 5 culture media, each comprising a combination of growth factors and / or growth factor inhibitors, under conditions sufficient to drive the population of cells towards a target cell type, thereby producing a population of cells comprising the target cell t pe.

[0330] In some embodiments the method begins with seeding a population of undifferentiated pluripotent stem cells at a density’ of 1,400-20,000 live cells / cm2, optionally in a monolayer, and culturing the cells until a lactate concentration in the cell culture medium reaches 1.68-12.29 mM and a percent confluency of 5% to 80% is achieved. In some embodiments, the undifferentiated pluripotent stem cells are cultured until the lactate concentration in the cell culture medium is betw een about 1.0 to 13. 0 mM, or between aboutAttorney Docket No. LCTI-005 / 001WG 43694-03305 1.5-12.5 mM. In some embodiments, cells are cultured until the lactate concentration in the cell culture medium is between about 1.68-12.29 mM. In some embodiments, the percent confluence is between 5% and 90%, between 5% and 85%, or between 5% and 80%.

[0331] In some embodiments, the population of undifferentiated pluripotent stem cells (PSCs) is cultured a first cell culture medium comprising Bone morphogenetic protein 4 (BMP4) and 4-[4-(2H-l,3-Benzodioxol-5-yl)-5-(pyridin-2-yl)-lH-imidazol-2-yl]benzamide (SB431542). In some embodiments, the first cell culture medium comprises BMP4, SB431542, and Fibroblast growth factor 2 (FGF2).

[0332] In some embodiments, the population of undifferentiated pluripotent stem cells (PSCs) are cultured in the first cell culture medium for at least 1 day, at least 2 days, at least 3 days, at least 5 days, at least 6 days, at least 7 days, at least 9 days, at least 11 days, at least 15 days, or at least 20 days, under conditions sufficient to produce differentiation into a target cell type, for example non-neuronal ectodermal (NNE) cells. In some embodiments, the population of PSCs is cultured in the first cell culture medium for at least 1 day. In some embodiments, the population of PSCs is cultured in the first cell culture medium for at least 4 days. In some embodiments, the population of PSCs is cultured in the first cell culture medium for at least 5 days. In some embodiments, the population of PSCs is cultured in the first cell culture medium for at least 7 days. In some embodiments, the population of PSCs is cultured in the first cell culture medium for between about 1-20 days. 1-9 days, 2-10 days, 3-7 days, or 4-6 days. In some embodiments, the population of PSCs is cultured in the first cell culture medium for between about 1-9 days. In some embodiments, the population of PSCs is cultured in the first cell culture medium for between about 3-7 days. In some embodiments, culturing the population of undifferentiated PSCs in the first cell culture medium produces a population of cells comprising non-neuronal ectodermal (NNE) cells.

[0333] In some embodiments, the population of cells produced by culturing the PSCs in the first cell culture medium, e.g., a population of cells comprising NNE cells, are cultured in a second cell culture medium comprising SB431542, Fibroblast grow th factor 2 (FGF2), and N-(6-Methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidin-2-yl)thio] -acetamide (IWP-2) and 4-{6-[4-(Piperazin-l-yl)phenyl]pyrazolo[l,5-a] pyrimi din-3 -yl } quinoline (LDN 193189).

[0334] In some embodiments, the population of cells is cultured in the second cell culture medium for at least 1 day, at least 2 days, at least 3 days, at least 5 days, at least 6 days, at least 7 days, at least 9 days, at least 11 days, at least 15 days, or at least 20 days under conditions sufficient to produce differentiation into a target cell t pe, for example, pre-Attorney Docket No. LCTI-005 / 001WO 43694-03305 placodal ectodermal (PPE) cells. In some embodiments, the population of cells is cultured in the second cell culture medium for at least 1 day. In some embodiments, the population of cells is cultured in the second cell culture medium for at least 4 days. In some embodiments, the population of cells is cultured in the second cell culture medium for at least 5 days. In some embodiments, the population of cells is cultured in the second cell culture medium for at least 6 days. In some embodiments, the population of cells is cultured in the second cell culture medium for at least 7 days. In some embodiments, the population of cells is cultured in the second cell culture medium for between about 1-20 days, 1-9 days, 2-10 days, 3-7 days, or 4-6 days. In some embodiments, the population of cells is cultured in the second cell culture medium for between about 1-9 days. In some embodiments, the population of cells is cultured in the second cell culture medium for between about 3-7 days. In some embodiments, the population of cells is cultured in the second cell culture medium for 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days. In some embodiments, the population of cells is cultured in the second cell culture medium for about 5 days. In some embodiments, the population of cells is cultured in the second cell culture medium for 6 days. In some embodiments, the population of cells is cultured in the second cell culture medium for 7 days. In some embodiments, culturing the population of cells in the second cell culture medium produces a population of cells comprising PPE cells.

[0335] In some embodiments, the population of cells produced by culturing the cells in the second culture medium, e.g. the population of cells comprising PPE cells, are cultured in a third cell culture medium comprising 6-((2-((4-(2,4-Dichlorophenyl)-5-(4-methyl-lH-imidazol-2-yl)pyrimidin-2-yl)amino)ethyl)amino)nicotinonitrile (CHIR99021), FGF2, and Insulin-like growth factor 1 (IGF-1).

[0336] In some embodiments, the population of cells is cultured in the third cell culture medium for at least 1 day, at least 3 days, at least 5 days, at least 6 days, at least 7 days, at least 9 days, at least 11 days, at least 15 days, or at least 20 days, under conditions sufficient to produce differentiation into a target cell type, for example, early otic neuronal progenitor (ONP) cells. In some embodiments, the population of cells is cultured in the third cell culture medium for at least I day. In some embodiments, the population of cells is cultured in the third cell culture medium for at least 4 days. In some embodiments, the population of cells is cultured in the third cell culture medium for at least 5 days. In some embodiments, the population of cells is cultured in the third cell culture medium for at least 7 days. In some embodiments, the population of cells is cultured in the third cell culture medium for 5 days. In some embodiments, the population of cells is cultured in the third cellAttorney Docket No. LCTI-005 / 001WO 43694-03305 culture medium for 7 days. In some embodiments, the population of cells is cultured in the third cell culture medium for 9 days. In some embodiments, the population of cells is cultured in the third cell culture medium for between about 1-20 days, 1-10 days, 1-17 days, 2-10 days, 3-7 days, or 4-6 days. In some embodiments, the population of cells is cultured in the third cell culture medium for between about 3-10 days. In some embodiments, the population of cells is cultured in the third cell culture medium for between about 5-9 days. In some embodiments, culturing the population of cells in the third cell culture medium produces a population of cells comprising early ONP cells.

[0337] In some embodiments, the population of cells produced by culturing the cells in the third culture medium, e.g. the population of cells comprising early ONP cells, are cultured in a fourth cell culture medium comprising Sonic Hedgehog (SHH). retinoic acid (RA), Epidermal growth factor (EGF), FGF2 and IGF-1.

[0338] In some embodiments, the population of cells is cultured in the fourth cell culture medium for at least 1 day, at least 3 days, at least 5 days, at least 7, days at least, 9 days, at least 11 days, or at least 15 days, or at least 20 days, under conditions sufficient to produce differentiation into a target cell type, for example, mid-late ONP cells. In some embodiments, the population of cells is cultured in the fourth cell culture medium for at least 1 day. In some embodiments, the population of cells is cultured in the fourth cell culture medium for at least 4 days. In some embodiments, the population of cells is cultured in the fourth cell culture medium for at least 5 days. In some embodiments, the population of cells is cultured in the fourth cell culture medium for at least 7 days. In some embodiments, the population of cells are cultured in the fourth cell culture medium for 5 days. In some embodiments, the population of cells is cultured in the fourth cell culture medium for 7 days. In some embodiments, the population of cells is cultured in the fourth cell culture medium for 9 days. In some embodiments, the population of cells is cultured in the fourth cell culture medium for between about 1-20 days, 1-10 days, 1-17 days, 2-10 days, 3-7 days, or 4-6 days. In some embodiments, the population of cells is cultured in the fourth cell culture medium for between about 3-10 days. In some embodiments, the population of cells is cultured in the fourth cell culture medium for between about 5-9 days. In some embodiments, culturing the population of cells in the fourth cell culture medium produces a population of cells comprising mid-late ONP cells.

[0339] In some embodiments, the population of cells produced by culturing the cells in the fourth culture medium, e.g. the population of cells comprising mid-late ONP cells, areAttorney Docket No. LCTI-005 / 001WG 43694-03305 cultured in fifth cell culture medium comprising Brain derived neurotrophic factor (BDNF), Neurotrophin-3 (NT3), and IGF-1.

[0340] In some embodiments, the population of cells is cultured in the fifth cell culture medium for at least 1 day, at least 5 days, at least 10 days, at least 20 days, at least 40 days, at least 45 days, at least 50 days, at least 60 days, at least 70 days, at least 80 days, at least 90 days, or at least 100 days, under conditions sufficient to produce differentiation into a target cell type, for example late ONP cells. In some embodiments, the population of cells are cultured in the fifth cell culture medium for at least 1 day. In some embodiments, the population of cells is cultured in the fifth cell culture medium for at least 10 days. In some embodiments, the population of cells are cultured in the fifth cell culture medium for at least 20 days. In some embodiments, the population of cells is cultured in the fifth cell culture medium for at least 30 days. In some embodiments, the population of cells is cultured in the fifth cell culture medium for at least 45 days. In some embodiments, the population of cells is cultured in the fifth cell culture medium for at least 60 days. In some embodiments, the population of cells is cultured in the fifth cell culture medium for between about 1-65 days, 1-60 days, 1-50 days, 1-40 days, 1-20 days, 7-65 days, 5-50 days, 10-40 days, 10-30 days, 10-20 days, 20-60 days, 20-50 days, 20-45 days or 30-45 days. In some embodiments, the population of cells is cultured in the fifth cell culture medium for 7-65 days. In some embodiments, the population of cells is cultured in the fifth cell culture medium for 3-45 days. In some embodiments, the population of cells is cultured in the fifth cell culture medium for 10-60 days. In some embodiments, the population of cells is cultured in the fifth cell culture medium for 20-45 days. In some embodiments, culturing the population of cells in the fifth cell culture medium produces a population of cells comprising late ONP cells.

[0341] In some embodiments, culturing the population of cells in the culture medium comprises (i) harvesting the population of cells produced by culturing the cells in the fourth cell culture medium, e.g. a population of cells comprising mid-late ONP cells; (ii) seeding the population of cells in containers comprising the fifth cell culture medium; (iii) culturing the population of cells; (iv) harvesting the population of cells; (v) seeding the population of cells in containers comprising the fifth cell culture medium; and (vi) culturing the population of cells. In some embodiments the fifth cell culture medium further comprises a ROCK inhibitor. In some embodiments, the cells are cultured for between 5 and 35 days, between 7 and 35 days, between 7 and 30 days, or between 10 and 25 days at step (iii). In some embodiments, the cells are cultured for between 7 and 35 days at step (iii). In some embodiments, the cells are cultured for between 7 and 30 days, between 10 and 30 days, orAttorney Docket No. LCTI-005 / 001WG 43694-03305 between 15 and 25 days at step (vi). In some embodiments, the cells are cultured for between 7 and 30 days at step (vi). In some embodiments, the step (iii) described above comprises culturing the population of cells in containers comprising the fifth cell culture medium for at least 1 day, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 10 days, at least 15 days, at least 20 days, at least 25 days, at least 30 days, at least 35 days, at least 40 days, at least 50 days, at least 60 days, or at least 70 days. In some embodiments, the step (vi) described above comprises culturing the population of cells for at least 1 day, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 10 days, at least 15 days, at least 20 days, at least 25 days, at least 30 days, at least 35 days, at least 40 days, at least 50 days, or at least 60 days.

[0342] In some embodiments, the first cell culture medium includes BMP4 at a concentration of about 1 ng / mL, about 10 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, or about 40 ng / mL. In some embodiments, the first cell culture medium includes BMP4 at a concentration of between about 1 ng / mL to 40 ng / mL, 1 ng / mL to 25 ng / mL, 5 ng / mL to 30 ng / mL, or 10 ng / mL to 15 ng / mL. In some embodiments, the first cell culture medium includes BMP4 at a concentration of 1 ng / mL to 40 ng / mL. In some embodiments, the first cell culture medium includes BMP4 at a concentration of 1 ng / mL to 25 ng / mL. In some embodiments, the first cell culture medium includes BMP4 at a concentration of 5 ng / mL to 30 ng / mL. In some embodiments, the first cell culture medium includes BMP4 at a concentration of 10 ng / mL to 15 ng / mL. In some embodiments, the first cell culture medium includes BMP4 at a concentration of about 10 ng / mL. In some embodiments, the first cell culture medium includes BMP4 at a concentration of about 5 ng / mL. In some embodiments, the first cell culture medium includes BMP4 at a concentration of about 20 ng / mL.

[0343] In some embodiments, the first and / or second cell culture medium includes SB431542 at a concentration of about 0.1 pM, about 1 pM, about 5 pM, about 10 pM, about 15 pM, or about 20 pM. In some embodiments, the first and / or second cell culture medium includes SB431542 at a concentration of between about 0.1 pM - 20 pM, 0.1 - 10 pM, 5 pM 15 pM. or 7 pM 13 pM. In some embodiments, the first and / or second cell culture medium includes SB431542 at a concentration of 1 pM - 20 pM. In some embodiments, the first and / or second cell culture medium includes SB431542 at a concentration of 5 pM - 15 pM. In some embodiments, the first and / or second cell culture medium includes SB431542 at a concentration of 0.1 pM - 10 pM. In some embodiments, the first and / or second cell culture medium includes SB431542 at a concentration of about 1 pM.Attorney Docket No. LCTI-005 / 001WG 43694-03305

[0344] In some embodiments, the first, second, third and / or fourth cell culture medium includes FGF2 in an amount of about 1 ng / mL, about 10 ng / mL. about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, or about 40 ng / mL. In some embodiments, the first, second, third and / or fourth cell culture medium includes FGF2 in an amount of between about 1 ng / mL - 40 ng / mL, 1 ng / mL - 30 ng / mL, 1 ng / mL - 25 ng / mL, 5 ng / mL-30 ng / mL, 5 ng / mL - 15 ng / mL, or 10 ng / mL- 15 ng / mL. In some embodiments, the first, second, third and / or fourth cell culture medium includes FGF2 in an amount of 1 ng / mL- 40 ng / mL. In some embodiments, the first, second, third and / or fourth cell culture medium includes FGF2 in an amount of 1 ng / mL- 25 ng / mL. In some embodiments, the first, second, third and / or fourth cell culture medium includes FGF2 in an amount of 10 ng / mL- 15 ng / mL. In some embodiments, the first, second, third and / or fourth cell culture medium includes FGF2 in an amount of about 10 ng / mL.

[0345] In some embodiments, the second cell culture medium includes IWP-2 in an amount of about 0.5 gM, 1 gM, about 2 gM, about 3 gM, about 5 gM, or about 10 gM. In some embodiments, the second cell culture medium includes IWP-2 in an amount of between about 0.5 gM - 20 gM, 0.5 gM - 10 gM, 1 gM - 10 gM, 1 gM - 5 gM, 2 gM - 7 gM, or 3 gM-5 gM. In some embodiments, the second cell culture medium includes IWP-2 in an amount of between about 0.5 gM - 10 gM. In some embodiments, the second cell culture medium includes IWP-2 in an amount of about 2 gM - 4 gM. In some embodiments, the second cell culture medium includes IWP-2 in an amount of about 2 gM.

[0346] In some embodiments, the second cell culture medium includes LDN193189 in an amount of about 10 nM, about 50 nM, about 100 nM, about 150 nM, about 200 nM, about 250 nM, about 300 nM, about 350 nM, about 400 nM, about 450 nM, about 500 nM, about 550 nM, or about 600 nM. In some embodiments, the second cell culture medium includes LDN193189 in an amount of between about 1 nM - 600 nM, 50 nM - 500 nM, 75 nM - 200 nM,l 00 nM - 400 nM, or 200 nM-300 nM. In some embodiments, the second cell culture medium includes LDN193189 in an amount of 1 nM- 600 nM. In some embodiments, the second cell culture medium includes LDN193189 in an amount of 50 nM- 500 nM. In some embodiments, the second cell culture medium includes LDN193189 in an amount of 20 nM- 400 nM. In some embodiments, the second cell culture medium includes LDN193189 in an amount of 75 nM- 150 nM. In some embodiments, the second cell culture medium includes LDN193189 in an amount of about 100 nM.

[0347] In some embodiments, the third cell culture medium includes CHIR99021 at a concentration of about 1 gM, about 5 gM, about 6 gM, about 7 gM, about 10 gM, about 20Attorney Docket No. LCTI-005 / 001WG 43694-03305 gM, about 25 gM, about 30 gM. or about 40 gM. In some embodiments, the third cell culture medium includes CHIR99021 at a concentration of between about 1 gM - 40 gM, 1 gM - 30 gM, 1 gM - 25 gM, 5 gM -20 gM, 2 gM -10 gM, or 5 gM - 15 gM. In some embodiments, the third cell culture medium includes CHIR99021 at a concentration of 1 |iM- 40 gM. In some embodiments, the third cell culture medium includes CHIR99021 at a concentration of 1 gM - 25 gM. In some embodiments, the third cell culture medium includes CHIR99021 at a concentration of 2 gM - 8 gM. In some embodiments, the third cell culture medium includes CHIR99021 at a concentration of about 6 gM.

[0348] In some embodiments, the third, fourth and / or fifth cell culture medium includes IGF-1 in an amount of about 1 ng / mL, about 10 ng / mL, about 25 ng / mL, about 40 ng / mL. about 50 ng / mL. about 60 ng / mL, about 100 ng / mL, about 150 ng / mL, about 200 ng / mL, about 250 ng / mL, or about 300 ng / mL. In some embodiments, the third, fourth and / or fifth cell culture medium includes IGF-1 in an amount of 1 ng / mL - 300 ng / mL, 20 ng / mL-200 ng / mL, 5 ng / mL - 100 ng / mL, 25 ng / mL-300 ng / mL, or 40 ng / mL-100 ng / mL. In some embodiments, the third, fourth and / or fifth cell culture medium includes IGF-1 in an amount of 1 ng / mL - 300 ng / mL. In some embodiments, the third, fourth and / or fifth cell culture medium includes IGF-1 in an amount of 25 ng / mL- 300 ng / mL. In some embodiments, the third, fourth and / or fifth cell culture medium includes IGF-1 in an amount of 5 ng / mL- 100 ng / mL. In some embodiments, the third, fourth and / or fifth cell culture medium includes IGF-1 in an amount of about 50 ng / mL. In some embodiments, the fifth cell culture medium does not include IGF-1.

[0349] In some embodiments, the fourth cell culture medium includes SHH in an amount of about 10 ng / mL, 30 ng / mL, about 50 ng / mL, about 100 ng / mL, about 300 ng / mL, about 500 ng / mL, about 600 ng / mL. about 700 ng / mL, about 800 ng / mL. about 900 ng / mL, about 1000 ng / mL, about 1100 ng / mL, about 1200 ng / mL, or about 1300 ng / mL. In some embodiments, the fourth cell culture medium includes SHH in an amount of 10 ng / mL - 1300 ng / mL, 50 ng / mL - 1000 ng / mL, 300 ng / mL - 1000 ng / mL, or 400 ng / mL - 600 ng / mL. In some embodiments, the fourth cell culture medium includes SHH in an amount of 10 ng / mL -1300 ng / mL. In some embodiments, the fourth cell culture medium includes SHH in an amount of 300 ng / mL - 1000 ng / mL. In some embodiments, the fourth cell culture medium includes SHH in an amount of 400 ng / mL - 600 ng / mL. In some embodiments, the fourth cell culture medium includes SHH in an amount of about 500 ng / mL.

[0350] In some embodiments, the fourth cell culture medium includes RA at a concentration of about 0.1 gM, about 0.2 gM, about 0.3 gM, about 0.5 gM, about 1 gM,Attorney Docket No. LCTI-005 / 001WD 43694-03305 about 2 pM, about 3 pM, about 5 pM, about 10 pM, about 15 pM, or about 20 pM. In some embodiments, the fourth culture medium includes RA at a concentration of 0.1 pM - 20 pM.0.1 pM - 5 pM, 0.5 pM - 5 pM, 0.2 pM - 5 pM, or 0.5 pM - 2 pM. In some embodiments, the fourth cell culture medium includes RA at a concentration of 0.1 pM - 20 pM. In some embodiments, the fourth cell culture medium includes RA at a concentration of 0.2 pM - 5 pM. In some embodiments, the fourth cell culture medium includes RA at a concentration of 0.2 pM - 2 pM. In some embodiments, the fourth cell culture medium includes RA at a concentration of about 0.5 pM.

[0351] In some embodiments, the fourth cell culture medium includes EGF in an amount of about 5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL. about 50 ng / mL. about 100 ng / mL, about 150 ng / mL. about 200 ng / mL, about 250 ng / mL, or about 300 ng / mL. In some embodiments, the fourth cell culture medium includes EGF in an amount of 1 ng / mL - 300 ng / mL, 10 ng / mL - 200 ng / mL, 20 ng / mL - 300 ng / mL, 5 ng / mL - 100 ng / mL, or 10 ng / mL -50 ng / mL. In some embodiments, the fourth cell culture medium includes EGF in an amount of 1 ng / mL - 300 ng / mL. In some embodiments, the fourth cell culture medium includes EGF in an amount of 5 ng / mL - 100 ng / mL. In some embodiments, the fourth cell culture medium includes EGF in an amount of 10 ng / mL - 50 ng / mL. In some embodiments, the fourth cell culture medium includes EGF in an amount of about 20 ng / mL.

[0352] In some embodiments, the fifth cell culture medium includes BDNF in an amount of about 5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 25 ng / mL, about 50 ng / mL, about 100 ng / mL, about 150 ng / mL, about 200 ng / mL, about 250 ng / mL, or about 300 ng / mL. In some embodiments, the fifth cell culture medium includes BDNF in an amount of 1 ng / mL - 300 ng / mL, 5 ng / mL-200 ng / mL, 5 ng / mL-100 ng / mL, or 5 ng / mL-50 ng / mL. In some embodiments, the fifth cell culture medium includes BDNF in an amount of 1 ng / mL - 300 ng / mL. In some embodiments, the fifth cell culture medium includes BDNF in an amount of 5 ng / mL - 100 ng / mL. In some embodiments, the fifth cell culture medium includes BDNF in an amount of 5 ng / mL- 30 ng / mL. In some embodiments, the fifth cell culture medium includes BDNF in an amount of about 10 ng / mL.

[0353] In some embodiments, the fifth cell culture medium comprises neurotrophin 3 (NT3, also referred to as NT-3, and NTF3). NT3 is a member of the neurotrophin family, which is involved in the survival and differentiation of mammalian neurons. NT3 is thought to be involved in the maintenance of the adult nervous system, and the development of neurons in the embryo. In some embodiments, the fifth cell culture medium includes NT3 inAttorney Docket No. LCTI-005 / 001WO 43694-03305 an amount of about 5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 25 ng / mL, about 50 ng / mL. about 100 ng / mL, about 150 ng / mL. about 200 ng / mL, about 250 ng / mL, or about 300 ng / mL. In some embodiments, the fifth cell culture medium includes NT3 in an amount of 1 ng / mL - 300 ng / mL, 5 ng / mL-200 ng / mL, 5 ng / mL-100 ng / mL, or 5 ng / mL-50 ng / mL. In some embodiments, the fifth cell culture medium includes NT3 in an amount of 1 ng / mL -300 ng / mL. In some embodiments, the fifth cell culture medium includes NT3 in an amount of 5 ng / mL - 100 ng / mL. In some embodiments, the fifth cell culture medium includes NT3 in an amount of 5 ng / mL- 30 ng / mL. In some embodiments, the fifth cell culture medium comprises NT3 in an amount of 10 ng / mL.

[0354] In some embodiments, the fifth cell culture medium comprises a Rock inhibitor, e.g. a small molecule inhibitor that inhibits ROCK1 and / or ROCK2 mediated signaling. In some embodiments, the Rock inhibitor comprises Y-27632 dihydrochloride ( / ro -4-|(l / ?)-l-Ammoethyl |-N-4-pyridmylc clohexanecarboxarriide dihydrochloride). In some embodiments, the fifth cell culture medium comprises a Rock inhibitor in an amount of about 0.5 pM, 1.0 pM. 1.5 pM, 2.0 pM. 3.0 pM, 5 pM. 7 pM, 9 pM. 10 pM. 11 pM, 12 pM, 15 pM, 20 pM, 30 pM, 40 pM, 50 pM, or 60 pM. In some embodiments, the fifth cell culture medium comprises a Rock inhibitor in an amount of between about 0.5 pM - 60 pM, 1 pM - 50 pM, 2 pM - 50 pM, 1 pM - 30 pM, 2 - pM, 5 pM - 20 pM, 1 pM - 15 pM or 5 pM - 15 pM. In some embodiments, the fifth cell culture medium comprises a Rock inhibitor in an amount of between about 2 pM- 50 pM. In some embodiments, the fifth cell culture medium comprises a Rock inhibitor in an amount of about 10 pM.

[0355] In some embodiments, the methods comprise (a) culturing a population of undifferentiated pluripotent stem cells in a first cell culture medium comprising FGF2 at a concentration of between 1-25 ng / mL. BMP4 at a concentration of between 1-25 ng / mL and SB431542 at a concentration of between 0.1-10 pM for 1-9 days under conditions sufficient to produce non-neuronal ectodermal (NNE) cells, thereby producing a population of cells comprising NNE cells; (b) culturing the population of cells comprising NNE cells in a second cell culture medium comprising SB431542 at a concentration of between 0.1-10 pM, FGF2 at a concentration of between 1-25 ng / mL, IWP-2 at a concentration of between 0.5 10 pM and LDN193189 at a concentration of between 20-400 nM for 1-9 days under conditions sufficient to produce pre-placodal ectodermal (PPE) cells, thereby producing a population of cells comprising PPE cells; a step (c) culturing the population of cells comprising PPE cells in a third cell culture medium comprising CHIR99021 at a concentration of between 1-25 pM, FGF2 at a concentration of between 1-25 ng / mL, and IGF-1 at a concentration ofAttorney Docket No. LCTI-005 / 001WO 43694-03305 between 5-100 ng / mL for 5-11 days under conditions sufficient to produce early Otic Neuronal progenitor (ONP) cells, thereby producing a population of cells comprising early ONP cells; (d) culturing the population of cells comprising early ONP cells in a fourth cell culture medium comprising SHH at a concentration of between 50-1000 ng / mL, RA at a concentration of between 0.2-2 pM, EGF at a concentration of between 5-100 ng / mL, FGF2 at a concentration of between 1-25 ng / mL and IGF-1 at a concentration of between 5-100 ng / mL for 5-11 days under conditions sufficient to produce mid-late ONP cells, thereby producing a population of cells comprising mid-late ONP cells; (e) culturing the population of cells comprising mid-late ONP cells in a fifth cell culture medium comprising BDNF at a concentration of between 5-100 ng / mL, NT3 at a concentration of between 5-100 ng / mL, and IGF-1 at a concentration of between 5-100 ng / mL for 3-45 days under conditions sufficient to produce late ONP cells, thereby producing a population of cells comprising late ONP cells and auditory neuron progenitors; and (f) collecting the population of cells comprising late ONPs and auditory neuron progenitors, thereby producing the composition. In alternative embodiments, the first cell culture medium at step (a) comprises BMP4 at a concentration of between 1-25 ng / mL and SB431542 at a concentration of between 0.1-10 pM, and does not comprise FGF2.

[0356] In some embodiments, the methods comprise (a) culturing a population of undifferentiated pluripotent stem cells in a first cell culture medium comprising BMP4 and SB431542 and FGF2 for 1-9 days under conditions sufficient to produce non-neuronal ectodermal (NNE) cells, thereby producing a population of cells comprising NNE cells; (b) culturing the population of cells comprising NNE cells in a second cell culture medium comprising SB431542, FGF2, and IWP-2 and LDN193189 for 1-9 days under conditions sufficient to produce pre-placodal ectodermal (PPE) cells, thereby producing a population of cells comprising PPE cells; a step (c) culturing the population of cells comprising PPE cells in athird cell culture medium comprising CHIR99021, FGF2, and IGF-1 for 5-11 days under conditions sufficient to produce early Otic Neuronal progenitor (ONP) cells, thereby- producing a population of cells comprising early ONP cells; (d) culturing the population of cells comprising early ONP cells in a fourth cell culture medium comprising SHH, RA, EGF, FGF2 and IGF-1 for 5-11 days under conditions sufficient to produce mid-late ONP cells, thereby producing a population of cells comprising mid-late ONP cells; (e) culturing the population of cells comprising mid-late ONP cells in a fifth cell culture medium comprising BDNF. NT3. and IGF-1 for 3-45 days under conditions sufficient to produce late ONP cells, thereby producing a population of cells comprising late ONP cells and auditor}’ neuronAttorney Docket No. LCTI-005 / 001WG 43694-03305 progenitors; and (f) collecting the population of cells comprising late ONPs and auditory neuron progenitors, thereby producing the composition. In alternative embodiments, the first cell culture medium at step (a) comprises BMP4 and SB431542, and does not comprise FGF2.

[0357] In some embodiments, the methods comprise (a) culturing a population of undifferentiated pluripotent stem cells in a first cell culture medium comprising FGF2 at a concentration of between 1-25 ng / mL, BMP4 at a concentration of between 1-25 ng / mL and SB431542 at a concentration of between 0.1-10 pM under conditions sufficient to produce non-neuronal ectodermal (NNE) cells, thereby producing a population of cells comprising NNE cells; (b) culturing the population of cells comprising NNE cells in a second cell culture medium comprising SB431542 at a concentration of between 0.1-10 pM. FGF2 at a concentration of between 1-25 ng / mL, and IWP-2 at a concentration of between 0.5- 10 pM and LDN193189 at a concentration of between 20-400 nM under conditions sufficient to produce pre-placodal ectodermal (PPE) cells, thereby producing a population of cells comprising PPE cells; (c) culturing the population of cells comprising PPE cells in a third cell culture medium comprising CHIR99021 at a concentration of between 1-25 pM, FGF2 at a concentration of between 1-25 ng / mL, and IGF-1 is at a concentration of between 5-100 ng / mL under conditions sufficient to produce early Otic Neuronal progenitor (ONP) cells, thereby producing a population of cells comprising early ONP cells; (d) culturing the population of cells comprising early ONP cells in a fourth cell culture medium comprising SHH at a concentration of between 50-1000 ng / mL, RA at a concentration of between 0.2-2 pM, EGF at a concentration of between 5-100 ng / mL, FGF2 at a concentration of between 1-25 ng / mL and IGF-1 at a concentration of between 5-100 ng / mL under conditions sufficient to produce mid-late ONP cells, thereby producing a population of cells comprising mid-late ONP cells; a step (e) culturing the population of cells comprising mid-late ONP cells in a fifth cell culture medium comprising BDNF at a concentration of between 5-100 ng / mL, NT3 at a concentration of between 5-100 ng / mL, and IGF-1 at a concentration of between 5-100 ng / mL under conditions sufficient to produce late ONP cells and auditory neuron progenitors, thereby producing a population of cells comprising late ONP cells and auditory neuron progenitors; and (f) collecting the population of cells comprising late ONPs and auditory neuron progenitors, thereby producing the composition. In alternative embodiments, the first cell culture medium at step (a) comprises BMP4 at a concentration of between 1-25 ng / mL and SB431542 at a concentration of between 0.1-10 pM, and does not comprise FGF2.Attorney Docket No. LCTI-005 / 001WG 43694-03305

[0358] In some embodiments, the method comprises (a) culturing a population of undifferentiated pluripotent stem cells in a first cell culture medium comprising FGF2 at a concentration of 10 ng / mL, BMP4 at a concentration of 10 ng / mL and SB431542 at a concentration of 1 pM for 3-7 days under conditions sufficient to produce non-neuronal ectodermal (NNE) cells, thereby producing a population of cells comprising NNE cells; (b) culturing the population of cells comprising NNE cells in a second cell culture medium comprising SB431542 at a concentration of 1 pM, FGF2 at a concentration of 10 ng / mL, and IWP-2 at a concentration of between 2 pM and LDN193189 at a concentration of 100 nM for 3-7 days under conditions sufficient to produce pre-placodal ectodermal (PPE) cells, thereby producing a population of cells comprising PPE cells; (c) culturing the population of cells comprising PPE cells in a third cell culture medium comprising CHIR99021 at a concentration of 6 pM, FGF2 at a concentration of 10 ng / mL, and IGF-1 is at a concentration of 50 ng / mL for 7-10 days under conditions sufficient to produce early Otic Neuronal progenitor (ONP) cells, thereby producing a population of cells comprising early ONP cells; (d) culturing the population of cells comprising early ONP cells in a fourth cell culture medium comprising SHH at a concentration of 500 ng / mL, RA at a concentration of 0.5 pM, EGF at a concentration of 20 ng / mL, FGF2 at a concentration of 10 ng / mL and IGF-1 at a concentration of 50 ng / mL for 7-10 days under conditions sufficient to produce mid-late ONP cells, thereby producing a population of cells comprising mid-late ONP cells; (e) culturing the population of cells comprising mid-late ONP cells in a fifth cell culture medium comprising BDNF at a concentration of 10 ng / mL, NT3 at a concentration of 10 ng / mL, and IGF-1 at a concentration of 50 ng / mL for 3-45 days under conditions sufficient to produce late ONP cells, thereby producing a population of cells comprising late ONP cells and auditory neuron progenitors; and a step (f) collecting the population of cells comprising late ONPs and auditory neuron progenitors, thereby producing the composition. In alternative embodiments, the first cell culture medium at step (a) comprises BMP4 at a concentration of 10 ng / mL and SB431542 at a concentration of 1 pM, and does not comprise FGF2.

[0359] In some embodiments, any one of the methods described above comprises, prior to step (a), seeding the undifferentiated pluripotent stem cells at a density of 1,200-20,000 live cells / cm2in a monolayer, and culturing the cells until a lactate concentration in the cell culture medium reached 1.68-12.29 mM and a percent confluency of 5-80% was achieved.Attorney Docket No. LCTI-005 / 001WO 43694-03305

[0360] In some embodiments, any one of the methods described above further comprises a step of cryopreserving the population of cells comprising late ONPs and auditory neuron progenitors.

[0361] The skilled artisan will understand that at any of the steps described above, the resultant population of cells can be cryopreserved, followed by seeding and culture in the culture medium appropriate for the next stage of the differentiation process.Kits and Articles of Manufacture

[0362] The disclosure provides kits comprising the pharmaceutical compositions described herein, and articles of manufacture such as cryovials, syringes, syringe cartridges, cannula and the like. In some embodiments, the kit comprises reagents for determining the expression of one or more of the genes set forth in Tables 5 and / or 6.

[0363] In some embodiments, the kit comprises instructions for use.

[0364] In some embodiments, the pharmaceutical compositions described herein are pre-packaged in a dosage unit in a cryovial, cannula, syringe or syringe cartridge, that has been cryopreserved, stored at a suitable temperature (e.g. less than or equal to - 80 °C, or less than or equal to -140 °C), which is ready to administer to a subject after it has been thawed to a suitable temperature, such as room temperature.

[0365] Having now generally described the invention, the same w ill be more readily understood through reference to the following examples that are provided by way of illustration, and are not intended to be limiting of the present disclosure. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.EXAMPLESExample 1: Making Auditory Cell Compositions

[0366] Human auditory neuron progenitors were derived from pluripotent cells with direct differentiation to otic neuronal progenitors according to the methods described in WO2023 / 167986 and as set forth in this example below. In brief, ANP1 (also referred to as ANP1) cells are transplantable otic neuronal progenitors derived from human pluripotent stem cells. ANP1 cells are manufactured via a directed differentiation process that generates aAttorney Docket No. LCTI-005 / 001WD 43694-03305 pure population. Production is accompanied by in-process controls and batch release using developed analytical assays. ANP1 cells are permissible to further post-transplantation maturation into auditory neurons (AN). In addition, the ANP1 product can be frozen as an allogenic cell therapy bank, and at a clinical dose ready to be thawed at clinical sites and injected into a patient's cochlea.

[0367] The ranges listed below are based on process development studies that tested production success with minimal waste of resources by analyzing more than 15 biological repeats with a less than 20% failure rate.

[0368] At first, the cells were supplemented with media containing grow th factors such as BMP4, SB431542, and optionally FGF2, that drive the cells towards the Non-Neural Ectoderm (NNE) lineage. This combination of GFs was used for a duration of 3-7 days and was referred to as differentiation time frame number 1 (DTF#1).

[0369] Then the cells were provided with the next combination of GFs which included LDN193189, SB431542, FGF2 and IWP-2 to drive the cells tow ards the Pre-Placodal Ectoderm (PPE) lineage. This combination of GFs was used for a duration of 5-7 days and was referred to as differentiation time frame number 2 (DTF#2).

[0370] Production runs were monitored for the normal differentiation process.

[0371] Differentiation towards the early Otic Neuronal Progenitor (ONP) lineage w as initiated by supplementing the cells with media containing the growth factors CHIR99021, FGF2, IGF-1 for 7 days and was referred to as differentiation time frame number 3 (DTF#3). The old medium w as removed, a fresh medium with the new combination of growth factors was added.

[0372] The next stage in the auditory' development was the differentiation of the late Otic Neuronal Progenitors by supplementing the cells with media containing the GFs combination of Shh, RA, EGF, FGF2 and IGF-1. The duration of this step was 7-9 days, and is referred to as differentiation time frame number 4 (DTF#4).

[0373] The final step tow ards the mature auditory neurons included supplementing the cells with IGF-1, BDNF and NT-3 for 6-40 days and referred to as differentiation time frame number 5 (DTF#5). The cells were then harvested and cryopreserved. To assess cellular viability, cells were thawed and assayed for SOX2 and Nestin. If at least 70% of cells in the population expressed both SOX2 and Nestin, the cells were considered suitable for therapeutic use including injection into a patient’s cochlea.Attorney Docket No. LCTI-005 / 001WO 43694-03305Table 1-1. Differentiation Marker Expression.>Example 2: Assessment of Manufacturability of Human Pluripotent Derived Auditory Neuron Progenitors (ANP1) as a Therapeutic for Auditory Disorders

[0374] This Example describes the assessment of manufacturability, release criteria, and in vitro activity of ANP1 cells manufactured at scale.Attorney Docket No. LCTI-005 / 001WD 43694-03305

[0375] The present Example describes a differentiation process to manufacture LCTANP1 composed of Auditory Neuron Progenitors (also termed otic neuronal progenitors) from pluripotent human stem cells. As an integral process, the ANP1 manufacturing process and cells were characterized by biological and functionally relevant sets of markers, using different quantitative methods developed and customized including functional in vitro assays. Methods

[0376] The ANP1 differentiation process included the expansion of a clinical grade line of human pluripotent stem cells, a series of differentiation cues that are delivered under specific culture conditions, in specific time frames, harvesting the final Auditory Neuron Progenitors cells and lastly, cry opreservation in a ready to administer format. This process is schematically depicted in FIG. 1A. Biomarker kinetics along the differentiation were monitored, as illustrated in FIG. IB.

[0377] To ensure the quality of the cell product, various assays were developed, as depicted in FIG.2A and FIG. 2B. Development of analytical biomarkers, such as for assessing purity of the cell population, included the analysis of specific protein marker expression by flow cytometry, and immunofluorescence, gene expression profiles, including RNA sequencing, and bioinformatics including computational analysis of single cell RNA sequencing (scRNA seq) results.

[0378] Functional assays were developed to measure neuronal properties of ANP1. Functional assays were designed such as testing cells’ ability to elicit calcium influx (which plays an important role in multiple signaling cascades within auditory neurons), in response to glutamatergic agonists in a time-dependent manner, and to express synaptic markers which plays a role in synapse formation of auditory neurons with hair cells.Results

[0379] ANP1 cells were successfully manufactured at scale, met pre-set release criteria, and demonstrated relevant activity in in-vitro functional tests. ANP1 cells were cryopreserved in a ready-to-administer, thaw and inject format and were successfully thawed, successfully transplanted and survived in an in-vivo Guinea pig model for at least 53 days.

[0380] ANP1 is a cell-based product composed of Auditory Neuron Progenitors derived from clinical grade pluripotent stem cells. ANP1 completed initial chemistry, manufacturing, and control studies and preclinical testing, highlighting its potential as a therapeutic.

[0381] In order for a cell therapy to be successful, the cells must be safe and reproducible, meet purity, identity and potency standards, and their production must beAttorney Docket No. LCTI-005 / 001WD 43694-03305 scalable. Source line characterization, cell banking, versatile expansion systems, differentiation process development: culture conditions, optimization, analytical methods, in-process controls, and release criteria must all be considered when making safe and reproducible cells. In addition, clinically compatible post-production processing, analytical method development for process control and product release, functionality and performance testing, enhancements to cells such as genetic modification the addition of expression systems, scale up modalities, substrates, harvesting protocols, clinical and commercial throughputs and cost of manufacture all play a role.

[0382] Working on steps in parallel, it was possible to develop ANP1 cells from cell bank production and production to an at scale engineering run in 18 months. During manufacturing. ANP1 cells undergo directed differentiation (FIG. 5). During this time, repeatable and reproducible methods for culturing, expansion and differentiation of hESCs were established. The differentiation process at scale included optimizing the differentiation cocktails in process and release tests. The resulting drug product was in a thaw and inject (TAI) cry opreservation-ready to inject format.

[0383] An exemplary ANP1 cell profile is shown in Table 2-1, as well as the ranges of cells expressing the indicated marker based on 10 replicates, and the test method used to assay marker expression. Exemplary' flow cytometry' plots showing expression of SOX2, Nestin, TRA-1-60 and SSEA-5 are shown in FIG. 3 and FIG. 4.Table 2-1. Exemplary ANP1 cell profile>Attorney Docket No. LCTI-005 / 001WO 43694-03305<FC-Flow CytometryqPCR-quantitative PCRIF -immunofluorescenceFIO -For information onlyHR-hESCs residual AssayExample 3: Bioinformatic Analysis of Auditory Neuron Related Gene Expression in ANP1 Cells

[0384] This Example illustrates the expression of auditory neuron gene sets selected from publicly available databases and scientific literature using bioinformatic analysis of single cell RNA sequencing (scRNA seq) of ANP1 cells. This computational approach can facilitate a deeper understanding of ANP1 cell function and differentiation.MethodsANP 1 cells and culturing

[0385] Human auditory' neuron progenitors were derived from pluripotent cells with direct differentiation to otic neuronal progenitors according to the methods described in WO2023 / 167986 and Example 1. ANP1 cells were thawed and processed for scRNA.Single-Cell RNA-Seq (scRNA-Seq)

[0386] Single-cell RNA sequencing was performed on the thawed ANPf drug product as described in Example 4 below.Bioinformatic analysis

[0387] Bioinformatic analysis of the scRNA-Seq results was performed using Uniform Manifold Approximation and Projection (UMAP) graphs based on PrincipalAttorney Docket No. LCTI-005 / 001WO 43694-03305 Component Analysis (PCA) including auditory neuron related gene sets from publicly available data sets (such as MSigDB, Panglao, and Cell Marker) and relevant published scientific literature. Gray-scale scale in graphs (FIG.6) represents cells expressing key auditory neuron related gene sets.Results

[0388] As shown in the graph depicted in FIG.4, the flow cytometry analysis of the ANP1 cells using an hPSC specific protein markers TRA-1-60 and SSEA5 combination showed no detection of residual hPSC cells within the ANP1 population. As shown in the graphs depicted in FIG.6, the bioinformatic analysis of the ANP1 cells using ANP specific gene sets directed to Late ONP Markers, neural markers, and auditory neuronal makers is consistent with successful progression through the differentiation process.Example 4: Cluster Analysis of Auditory Cells Using Single-Cell RNA Sequencing

[0389] This example illustrates using single-cell RNA sequencing to identify additional genetic markers, and combinations of markers that identify auditory cells in the ANP1 cell population. The methods can also be used to characterize the population with respect to types and quantities of cells that are present within the sample.

[0390] Single-cell RNA sequencing (scRNA-seq) is a powerful technique that enables the analysis of gene expression profiles at the individual cell level. This method involves isolating single cells, typically through microfluidic devices or droplet-based systems, followed by RNA extraction, reverse transcription, amplification, and sequencing. scRNA-seq offers several advantages, including the ability to reveal cellular heterogeneity within tissues, identify' rare cell populations, and provide insights into developmental trajectories and gene regulatory networksMethodsANP 1 cells and culturing

[0391] Human auditory neurons were derived from pluripotent cells with direct differentiation to otic neuronal progenitors according to the methods described in WO2023 / 167986 and Example 1. ANP1 cells of multiple production batches were thawed and processed for scRNA-Seq.Attorney Docket No. LCTI-005 / 001WG 43694-03305 Single-Cell RNA-Seq (scRNA-Seq)

[0392] Single-cell RNA sequencing was performed on the thawed ANP 1 drug product. 10X Genomics® Chromium™ 3' gene expression RNA-seq was used, with target numbers of 10,000 cells and 50,000 reads per cell. Sequencing included basic analysis, quality report (pdf), FASTQ raw data fdes, and interactive differential gene expression analysis through the 10X Genomics®. Cell Ranger™ and Loupe™ Cell Browser.Bioinformatics analysis

[0393] Quality control of scRNA sequencing data: As part of quality control, only genes that were expressed in a minimum of 3 cells and cells with more than 200 genes detected were included for analysis. Cells that exhibited a unique gene count over 8,000 or less than 500 -1,000 and > 5%-l 0% mitochondrial counts per sample were removed from the analysis. To eliminate probable doublets from the data set, bioinformatical fdtration was applied on the cells using the DoubletFinder package and gold standard parameter calibration techniques for removing all events where two or more cells were captured by a single reaction volume, and thus sequenced as a single-cell artifact. After merging the biological replicates, the remaining cells were used for further analysis.

[0394] 2,000 highly variable genes were input for principal component analysis (PCA analysis), and the 20 statistically significant principal components (PCs) were used for clustering and Uniform Manifold Approximation and Projection (UMAP). The PCA and UMAP analyses were performed using the R package, Seurat v5.

[0395] Single-cell gene signature scoring was performed using the UCell package.

[0396] To identify differentially expressed genes for each cluster, genes that were expressed in less than 1% in either group or had a log fold change of <1 were filtered. Genes with an adjusted P-value below 0.05 were then used.

[0397] Enrichment tests were performed on the upregulated protein coding genes of the two main clusters vs the secondary clusters using the clusterProfiler R package. The annotation package used was org.Hs.eg.db v3.18.

[0398] Final Sample Processing included standard single-cell processing steps: the feature (gene) expression measurements for each cell were normalized by the total expression. The normalized value was then multiplied by a scale factor (10,000 by default), and the result was Log-transformed (i.e. Logiol 0,000=4). Highly variable features [variancestabilizing transformation (VST) selection method / 2,000 features] were identified, forming a list of the 2,000 most variable features. A subset of features that exhibit high cell-to-cellAttorney Docket No. LCTI-005 / 001WG 43694-03305 variation in the dataset (i.e, they are highly expressed in some cells, and lowly expressed in others) was then calculated.

[0399] For scaling the data [all the genes, not only top 2,000 from previous step] a linear transformation was applied, were mean expression across cells = 0 & variance across cells = 1. This step provided equal weight in downstream analyses, so that highly expressed genes did not dominate.

[0400] Linear dimensional reduction was performed on the scaled data (Principal Component Analysis; PC A). Only the 2,000 variable features (genes) determined in the previous step were used. Each PC essentially represents a ‘meta-feature’ that combines information across a correlated feature set.

[0401] The ‘dimensionality’ of the dataset [use first 20 PCA dimensions] was determined. To overcome the extensive technical noise in any single feature for scRNA-seq data, cells were clustered based on their PCA scores. The top 20 principal components represent a robust compression of the dataset.

[0402] Anon-linear dimensional reduction was executed for visualization using UMAP algorithm & first 20 PCA dimensions. The goal was to learn underlying structure in the dataset, in order to place similar cells together in low-dimensional space. In the following steps, cells grouped together within graph-based clusters should co-localize on these dimension reduction plots. Visualization of the cells global signatures was done using UMAP #1 & #2 (covers most of the characteristics of each cell).

[0403] Clustering analysis was done Seurat, R package with clustering resolution of 0.085 - 0.2 resulting in 5 clusters, as seen in FIG. 7.Table 4-1. Common Abbreviations.Attorney Docket No. LCTI-005 / 001WO 43694-03305<Results

[0404] The results of the cluster analysis are shown in FIG. 7. Clustering analysis in bioinformatics is a potent nonbiased technique used to group similar biological entities into distinct populations based on their shared characteristics, enabling to identify population structures. Clusters can be identified based on gene expression using statistic tests. Clustering the RNA-Seq data for the populations of ANP1 cells showed that the majority of cells (> 87% of the population) fell into two main clusters (termed C#0 and 1 in FIG. 7). These clusters were surrounded by smaller clusters. The number of cells in these clusters is summarized in Table 4-2 below:Table 4-2. Cells present in UMAP clusters<* In some production batches, this non-target cell population was not detected.Example 5: Identification of Auditory Cell Markers from Single-Cell RNA Sequencing

[0405] The cluster analysis described in Example 4 was used to identify genes that can be used to verify the composition populations of ANP1 cells. A schematic of this approach is shown in FIG. 9.

[0406] In brief, cluster analysis was used to identify differentially expressed genes. The top differentially expressed genes w ere extracted from a number of ANP1 production runs. Different cut-offs were used to generate the different lists of genes in Tables 2-5 of theAttorney Docket No. LCTI-005 / 001WG 43694-03305 application, and Tables 5-1 and 5-2 below. The number of upregulated genes in each of the three production batches presented in this example are depicted in the Venn diagram FIG. 10, showing the number of shared genes between the three production batches and between each pair of these production batches. For the lists of genes in Tables 2-4, genes were considered to be upregulated in the major clusters if at least 1% of the cells in the major clusters expressed the gene more than the cells in the minor clusters.

[0407] Table 2 shows 5,432 genes that are upregulated in ANP1 cells from production batch #1 (“Group A” genes).

[0408] Table 3 shows 3,032 genes that are upregulated in ANP1 cells from production batch #2 (“Group B’" genes).

[0409] Table 4 shows 1,236 genes that are upregulated in ANP1 cells from production batch #3 (“Group C” genes).

[0410] Genes that were considered to be upregulated in all three production batches (Tables 2-4) are shown in Table 5.

[0411] Table 5 shows 696 genes that are upregulated in ANP1 cells that are shared between the production batches (“Group D” genes).

[0412] Of the genes shown in Table 5, 53 genes were found to be expressed in at least 60% of the cells in the major clusters. These 53 genes are shown in Table 5-1 below

[0413] Table 5-1 shows 53 upregulated genes that are shared bet een ANP1 cells generated in production batches #1, #2 and #3. Ta...

Claims

Attorney Docket No. LCTI-005 / 001WG 43694-03305CLAIMSWhat is claimed is:

1. A method of characterizing a population of auditory cells derived by in vitro differentiation of pluripotent cells, the method comprising:(a) performing single cell RNA sequencing on the population of auditory cells, thereby determining the expression of a plurality of genes by individual cells in the population,(b) generating a two-dimensional representation of the si mi lari ty and / or difference of gene expression of individual cells of the population based on expression of at least a subset of genes in the plurality of genes,(c) partitioning the two-dimensional representation into a plurality of clusters, wherein the plurality of clusters comprises 1-3 major clusters and one or more minor clusters, (d) identifying a set of genes that are upregulated in at least 50% of cells of the 1-3 major clusters, and(e) identifying a percentage of cells in the population that express the set of genes, thereby characterizing the population of auditory cells.

2. The method of claim 1, wherein step (d) comprises identifying a set of genes that are upregulated in at least 60% of cells of the 1-3 major clusters.

3. The method of claim 1, comprising:(f) selecting a population of auditory cells wherein at least 50% of the cells in the population express the set of genes identified at (d).

4. The method of claim 3, wherein at least 60% of the cells in the population express the set of genes identified at (d).

5. The method of any one of claims 1-4, wherein step (b) comprises:Attorney Docket No. LCTI-005 / 001WG 43694-03305 (i) providing single cell RNA sequencing data for individual cells in the population of auditory cells, wherein each cell is associated with expression values for the plurality of genes based on read counts from the single cell RNA sequencing data,(ii) performing principal component analysis, thereby producing a first reduced-dimensionality representation of the single cell RNA sequencing data for individual cells, (iii) determining, for each cell, one or more neighboring cells based on a plurality of principal component scores for each cell,(iv) generating a weighted graph, wherein vertices correspond to individual cells in the population of cells and wherein edge weights correspond to a degree of similarity between the individual cells, and(v) embedding the weighted graph in two-dimensional space, whereby preservation of topological relationships from the weighted graph is maximized, thereby generating a second reduced-dimensionality representation of the single cell RNA sequencing data for individual cells.

6. The method of any one of claims 1-5, wherein step (b) comprises uniform manifold approximation and projection (UMAP).

7. The method of any one of claims 1-6, wherein step (c) comprises a community detection algorithm.

8. The method of any one of claims 1-7, wherein expression of the set of genes identified at (d) is upregulated in cells of the 1-3 major clusters compared to cells of the one more minor clusters.

9. The method of any one of claims 1-8, wherein the 1-3 major clusters consists of two clusters.

10. The method of any one of claims 1-9, wherein the one or more minor clusters comprise 1-4 clusters.

11. The method of any one of claims 1-10, wherein less than 10% of cells in the population are in any individual minor cluster, and / or wherein 20% or less of cells in the population are in the one or more minor clusters.Attorney Docket No. LCTI-005 / 001WO 43694-0330512. The method of any one of claims 1-11, wherein at least 80% of cells in the population are in the 1-3 major clusters.

13. The method of any one of claims 1-12, comprising repeating steps (a)-(d) at least once using a second population of auditory cells, and wherein the set of genes identified at step (d) for the population of auditory cells and the second population of auditory cells are the same.

14. The method of claim 13, wherein the population of auditory' cells and the second population of auditory cells are independently derived by in vitro differentiation of pluripotent cells.

15. The method of any one of claims 1-14, wherein the set of genes comprises between 30 and 70 genes, optionally wherein the set of genes comprises about 50 genes.

16. The method of any one of claims 1-15, wherein the set of genes comprise genes selected from the group of genes disclosed in Tables 5-6.

17. The method of any one of claims 1-15, wherein the set of genes comprises one or more of catenin alpha 2 (CTNNA2). cadherin 4 (CDH4), AC 109466.

1. carbohydrate sulfotransferase 11 (CHST11), TOX high mobility group box family member 3 (TOX3), ephrin A5 (EFNA5), NALCN channel auxiliary' factor 1 (FAM155A), phospholipase C eta 1 (PLCHI), transmembrane 131 like (TMEM131L), AAA ATPase AFG2A (SPATA5), NCK associated protein 5 (NCKAP5). contactin associated protein 2 (CNTNAP2). retinoic acid receptor beta (RARB), calcium / calmodulin dependent protein kinase ID (CAMKID), aspartate beta-hydroxylase (ASPH), teneurin transmembrane protein 2 (TENM2), dihydropyrimidinase like 3 (DPYSL3), neural EGFL like 2 (NELL2), phytanoyl-CoA 2-hydroxylase interacting protein like (PHYHIPL), microtubule affinity’ regulating kinase 1 (MARK!), GLI2 GLI family zinc finger 2 (GLI2), roundabout guidance receptor 2 (ROBO2), collagen type IV alpha 2 chain (COL4A2), zinc finger and BTB domain containing 16 (ZBTB16), ring finger protein 24 (RNF24), thymocyte selection associated high mobility' group box (TOX), laminin subunit alpha 1 (LAMA1), cysteine and ty rosine rich 1 (CYYR1). microtubule crosslinking factor 2 (SOGA), FYVE, RhoGEF and PH domain containing 4a (FYVE), RhoGEF and PH domain containing 4 (FGD4), neuroligin 1Attorney Docket No. LCTI-005 / 001WG 43694-03305 (NLGN1), neuron navigator 1 (NAVI), matrix metallopeptidase 16 (MMP16), arkadia (RNF111) C-terminal like ring finger ubiquitin ligase 2C (RNF165), sodium channel and clathrin linker 1 (SCLT1), fibroblast growth factor 13 (FGF13), SOX2 overlapping transcript (SOX2-OT), leucine rich repeats and immunoglobulin like domains 1 (LRIG1), ELAV like RNA binding protein 3 (ELAVL3), fidgetin, microtubule severing factor (FIGN), erb-b2 receptor tyrosine kinase 4 (ERBB4). podoplanin (PDPN), adenosine deaminase RNA specific B2 (ADARB2), MMS22 like, DNA repair protein (MMS22L), CUGBP Elav-like family member 2 (CELF2), neuromedin U (NMU), tetraspanin 18 (TSPAN18), chromosome 1 open reading frame 21 (Clorf21), WD repeat and HMG-box DNA binding protein 1 (WDHD1), neuronal cell adhesion molecule (NRCAM), splA / ryanodine receptor domain and SOCS box containing 4 (SPSB4), formin homology 2 domain containing 3 (FHOD3) or polypeptide N-acetylgalactosaminyltransferase 13 (GALNT13).

18. The method of any one of claims 1-15, wherein the set of genes comprises at least 10 genes, at least 20 genes, at least 30 genes, at least 40 genes, at least 50 genes or all genes selected from the group consisting of CTNNA2, CDH4, AC 109466.1, CHST11, TOX3, EFNA5, FAM155A, PLCHI, TMEM131L, SPATA5, NCKAP5, CNTNAP2, RARB, CAMKID, ASPH, TENM2, DPYSL3, NELL2, PHYHIPL, MARK1, GLI2, ROBO2, COL4A2, ZBTB16, RNF24. TOX, LAMA1, CYYR1, SOGA, FGD4, NLGN1, NAVI, MMP16. RNF165. SCLT1, FGF13, SOX2-OT, LRIG1. ELAVL3, FIGN, ERBB4. PDPN, ADARB2, MMS22L, CELF2, NMU, TSPAN18, Clorf21, WDHD1, NRCAM, SPSB4, FHOD3 and GALNT13.

19. The method of any one of claims 1-18, wherein cells in the 1-3 major clusters comprise mid otic neuronal progenitor (ONP) cells, late ONP cells, auditory neuron progenitors, mature auditory neurons, spiral ganglion neurons or a combination thereof.

20. The method of any one of claims 1-19, wherein less than 5%, less than 4%, less than 3%, less than 2%, less than 1% or no cells in the population express RNA of one or more pluripotent stem cell markers.

21. The method of claim 20, wherein the one or more pluripotent stem cell markers are selected from the group consisting of POU class 5 homeobox 1 (POU5F1), Nanog, lin-28Attorney Docket No. LCTI-005 / 001WG 43694-03305 homolog A (LIN28A), DNA methyltransferase 3 beta (DMNT3B), cripto, EGF-CFC family member (TDGF1) and LINE1 type transposase domain containing 1 (L1TD1).

22. The method of any one of claims 3-21, wherein the population of auditory cells selected at step (f) is suitable for administration to a subj ect with an auditory condition.

23. A population of auditory cells characterized by the method of any one of claims 1-22.

24. A pharmaceutical composition, comprising the population of auditory’ cells of claim 23, and a pharmaceutically acceptable carrier, diluent or excipient.

25. A method of treating an auditory condition in a subject, comprising administering the pharmaceutical composition of claim 24.

26. Use of the pharmaceutical composition of claim 24, for the treatment of an auditory’ condition in a subject.

27. A method of characterizing a population of auditory' cells comprising:(a) performing single-cell RNA sequencing on a plurality of cells in the population of auditory cells; and(b) determining a percentage of cells in the population that express one or more genes set forth in Table 5 or 6.

28. The method of claim 27, wherein at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% of the cells in the population express one or more genes set forth in Table 5 or 6.

29. The method of claim 27 or 28, wherein at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% of the cells in the population express at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600 or all of the genes set forth in Table 5.

30. The method of claim 27 or 28, wherein at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% of the cells inAttorney Docket No. LCTI-005 / 001WO 43694-03305 the population express at least 15, at least 20, at least 25, at least 30, at least 40, at least 50 or all of the genes selected from the group consisting of CTNNA2, CDH4, AC 109466.1, CHST11, TOX3, EFNA5, FAM155A, PLCHI, TMEM131L, SPATA5, NCKAP5, CNTNAP2, RARE, CAMKID, ASPH, TENM2, DPYSL3, NELL2, PHYHIPL, MARK1, GLI2, ROBO2, COL4A2, ZBTB16, RNF24, TOX, LAMA1, CYYR1, SOGA, FGD4, NLGN1, NAVI, MMP16, RNF165, SCLT1, FGF13, SOX2-OT, LRIG1, ELAVL3, FIGN, ERBB4, PDPN, ADARB2, MMS22L. CELF2, NMU, TSPAN18, Clorf21, WDHD1, NRCAM, SPSB4, FHOD3 and GALNT13.

31. The method of any one of claims 27-30, wherein the expression of the one or more genes is determined by measuring RNA or protein encoded by the one or more genes.

32. The method of claim 31, wherein the RNA is measured by single-cell RNA sequencing or RT-qPCR.

33. The method of claim 31, wherein the protein is measured by flow cytometry or immunocytochemistry.

34. The method of any one of claims 27-33, wherein the population of cells comprises mid otic neuronal progenitor (ONP) cells, late ONP cells, auditory neuron progenitors, mature auditory neurons, spiral ganglion neurons or any combination thereof.

35. The method of any one of claims 27-34, wherein less than 5%, less than 4%, less than 3%, less than 2%, less than 1% or no cells in the population express one or more pluripotent stem cell markers.

36. The method of claim 35, wherein the one or more pluripotent stem cell markers are selected from the group consisting of POU class 5 homeobox 1 (POU5F1), Nanog. lin-28 homolog A (LIN28A), DNA methyltransferase 3 beta (DMNT3B), cripto, EGF-CFC family member (TDGF1) and LINE1 type transposase domain containing 1 (L1TD1).

37. The method of claim 35 or 36, wherein expression of the one or more pluripotent stem cells markers is determined by RNA expression, optionally wherein determining RNA expression comprises single-cell RNA sequencing or RT-qPCR.Attorney Docket No. LCTI-005 / 001WG 43694-0330538. The method of any one of claims 27-37, wherein:(a) greater than or equal to 70% of the cells in the population express both Nestin and SOX2;(b) greater than or equal to 80% of the cells in the population express P tubulin III; (c) greater than or equal to 5% of the cells in the population express TrkB; and (d) less than or equal to 1% of the cells in the population express TRA-1-60 and / or SSEA5.

39. The method of any one of claims 27-37, wherein:(a) greater than or equal to 70% of the cells in the population express SOX2 and Nestin, (b) less than 5% of the cells in the population express Myo7A, and(c) greater than 80% of the cells in the population express tubulin III.

40. The method of claim 39, wherein:(d) a fold change in GLUA2 mRNA expression compared to a undifferentiated population of pluripotent cells is between 1000 and 6000, and(e) a fold change in TRKB mRNA expression compared to a undifferentiated population of pluripotent cells is between 1000 and 8000.

41. The method of any one of claims 27-40, wherein(i) greater than or equal to 15% of the cells in the population express TrkB, and / or (ii) a fold change in TRKB mRNA expression compared to a undifferentiated population of pluripotent cells is between 1000 and 8000.

42. The method of any one of claims 27-41, wherein less than or equal to 0.1% of the cells in the population express TRA-1-60 and / or SSEA5.

43. The method of any one of claims 27-42, wherein:(i) less than or equal to 20% of the cells in the population express PAX8 and / or PAX2, or wherein the population of cells do not significantly express PAX2 and / or PAX8, (ii) greater than or equal to 10% of the cells in the population express GluA4.(iii) less than or equal to 5% of the cells in the population express Myo7A, and / orAttorney Docket No. LCTI-005 / 001WG 43694-03305 (iv) greater than or equal to 50% of the cells in the population express CD133.

44. The method of any one of claims 27-43, wherein:(a) greater than or equal to 70% of the cells in the population express both Nestin and SOX2;(b) greater than or equal to 80% of the cells in the population express P tubulin III; (c) greater than or equal to 15% of the cells in the population express TrkB;(d) less than or equal to 5% of the cells in the population express Myo7A; and (e) less than or equal to 0.1% of the cells in the population express TRA-1-60 and / or SSEA5.

45. The method of any one of claims 27-43, wherein:(a) between about 70% to 100% of the cells in the population express both Nestin and SOX2;(b) between about 80% to 100% of the cells in the population express tubulin III; (c) between about 5% to 80% of the cells in the population express TrkB; and (d) between 0 to about 0.1% of the cells in the population express TRA-1-60 and / or SSEA5.

46. The method of claim 45. wherein between about 10% to 95% of the cells in the population express GluA4.

47. The method of claim 45 or 46, wherein between 0% to about 30%, optionally wherein less than 5% of cells in the population express Myo7A.

48. The method of any one of claims 27-47, wherein the population of auditor}' cells is produced by a method comprising:(a) obtaining a culture of undifferentiated pluripotent cells; and(b) culturing the undifferentiated pluripotent cells under culture conditions sufficient to induce differentiation of the pluripotent cells into the population of auditory cells.

49. A population of auditory cells characterized by the method of any one of claims 27-48, wherein at least 50% of the cells in the population express one or more genes set forth in Table 5 or 6.Attorney Docket No. LCTI-005 / 001WO 43694-0330550. The population of auditory cells of claim 49, wherein at least 60% of the cells are mid ONP cells, late ONP cells, auditory neuron progenitors, mature auditory neurons, spiral ganglion neurons any combination thereof.

51. A pharmaceutical composition, comprising the population of auditory cells of claim 49 or 50, and a pharmaceutically acceptable carrier, diluent or excipient.

52. A method of treating an auditory condition in a subject, comprising administering the pharmaceutical composition of claim 51.

53. Use of the pharmaceutical composition of claim 51 for the treatment of an auditory condition in a subject.

54. Use of the pharmaceutical composition of claim 52 in the manufacture of a medicament for the treatment of an auditory condition in a subject.

55. A method of isolating or enriching a population of target auditory' cells comprising:(a) providing a population of cells comprising target auditory cells and at least one additional cell type;(b) contacting the cells with a detectable label that binds to a cell surface marker selected from the group consisting of CD24, TMEFF1, JAM2, PROMI, LRP4, NLGN1, GPM6A, PDPN, GABRB3, CDH20, BOC, CDH4, PCDH11X, DPP10, GRIA4, DCC, LRIG1, NRCAM, NEGRI, ADAM22, ADGRL2. PTPRN2, NRXN1, CELSR2, NTRK2 and NTRK3; and(c) sorting the cells using the detectable label.

56. The method of claim 55, wherein the sorting comprises flow cytometry.

57. The method of claim 55 or 56, wherein the at least one additional cell type comprises pluripotent cells.

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