Combination methods comprising cell therapy and cochlear implant

Abstract

Provided herein are methods for treating hearing loss in a subject suffering from auditory conditions and for enhancing the effectiveness of cochlear implants in a subject with an auditory condition, comprising administering to the inner ear of the subject a population of auditory cells obtained by inducing differentiation of pluripotent cells to assume auditory fates.

Description

COMBINATION METHODS COMPRISING CELL THERAPY AND COCHLEAR IMPLANTCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to, and benefit of, U.S. Provisional Application Nos. 63 / 624,451, filed on January 24, 2024, 63 / 649,692, filed on May 20, 2024, and 63 / 695,088 filed on September 16, 2024, the contents of which are incorporated by reference herein in their entireties.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] Cochlear implants can address hearing loss in some cases. Cochlear implants bypass damaged or dysfunctional hair cells in the cochlea, and directly stimulate the auditory nerve via an array of electrodes that sends signals from a receiver to different regions of the auditory nerve. However, where hearing loss includes significant loss of spiral ganglia neurons, cochlear implants may not be effective, or be less effective in cases where there is neuronal disfunction or partial but significant neuronal cell loss. There thus exists a need in the art for additional methods to address hearing loss, and methods of enhancing the efficacy of cochlear implants.SUMMARY

[0004] 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 75% 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. In some embodiments, thepharmaceutical composition comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition enhances efficacy of the cochlear implant.

[0005] 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 75% 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. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

[0006] In some embodiments, the pharmaceutical composition is administered prior to implantation of the cochlear implant. In some embodiments, the pharmaceutical composition is administered after implantation of the cochlear implant. In some embodiments, the pharmaceutical composition is administered at the same time as implantation of the cochlear implant.

[0007] In some embodiments, the pharmaceutical composition is administered to an inner or middle ear of the subject. In some embodiments, administration to the inner ear comprises administration to the cochlea. In some embodiments, the pharmaceutical composition is administered to the Scala tympani, modiolus or spiral ganglion. In some embodiments, the pharmaceutical composition is administered via injection. In some embodiments, the injection comprises inserting a cannula through a hole in the otic capsule, or inserting a cannula through a round window of the subject.

[0008] In some embodiments, the auditory condition comprises conductive hearing loss, sensorineural hearing loss, central hearing loss, mixed hearing loss, auditory neuropathy spectrum disorder, central auditory processing disorder, tinnitus, or hidden hearing loss. In some embodiments, the auditory condition comprises loss of cochlear hair cells or cochlear neurons. In some embodiments, the auditory condition comprises loss of cochlear hair cells and loss of cochlear neurons.

[0009] The disclosure provides methods of treating a subject with an auditory condition selected from the group consisting of conductive hearing loss, sensorineural hearing loss, mixed hearing loss, auditory neuropathy spectrum disorder and hidden hearing loss, comprising 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 both Nestin and SOX2; (b) greater than or equal to 80% of the cellsin 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. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

[0010] The disclosure provides methods of treating a subject with hearing loss, for example hidden hearing loss, comprising 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 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. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

[0011] In some embodiments, the pharmaceutical composition is administered to the inner or middle ear of the subject with hidden hearing loss. In some embodiments, the subject is further treated with a cochlear implant.

[0012] In some embodiments, the pharmaceutical composition is cryopreserved, and the method comprises thawing the pharmaceutical composition prior to administration.

[0013] In some embodiments, greater than or equal to 85% of the cells in the population express both Nestin and SOX2. 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 15% of the cells in the population express TrkB. In 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, greater than or equal to 80% of the cells in the population express Nestin. In some embodiments, greater than or equal to 80% of the cells in the population express SOX2. In some embodiments, less than or equal to 20% of the cells in the population express PAX8 and / or PAX2. In some embodiments, 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 10% of the cells in the population express Myo7A.

[0014] In some embodiments, (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 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.

[0015] In some embodiments, greater than or equal to 50% of the cells in the population express CD133.

[0016] 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 P 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.

[0017] In some embodiments, between about 10% to 95% of the cells in the population express GluA4. In some embodiments, between 0 to about 30% of cells in the population express Myo7A.

[0018] In some embodiments, the population of auditory cells comprises late ONP cells, mature auditory neurons, spiral ganglion neurons, or any combination thereof. In some embodiments, the population of auditory cells comprises sensory cell populations of the ear. In some embodiments, the sensory cell populations are selected from the group consisting of hair cells, supporting cells, otic neuronal progenitor cells and sensory neuronal progenitor cells.

[0019] In some embodiments, the percentage of cells expressing any of the markers described herein is determined by flow cytometry or immunofluorescence. In some embodiments, the percentage of cells expressing Nestin, SOX2, P tubulin III, TrkB, GluA4, TRA-1-60 and / or SSEA5 is determined by flow cytometry or immunofluorescence.

[0020] In some embodiments, the population of cells comprises cellular aggregates, single cells, or a combination thereof.

[0021] In some embodiments, the population of auditory cells comprises at least 100,000 cells. In some embodiments, the population of auditory cells comprises between 100,000 cells and 10 million cells.

[0022] In some embodiments, between about 100,000 to 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.

[0023] In some embodiments, the population of auditory cells is obtained by a method comprising: (a) obtaining a culture of undifferentiated pluripotent stem cells; (b) culturing the undifferentiated pluripotent stem cells under culture conditions sufficient to induce differentiation of the pluripotent stem cells to non-neuronal ectoderm cells; and (c) culturing the cells from (b) under culture conditions sufficient to differentiate the non-neuronal ectoderm cells into auditory cells.BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIG. 1A depicts histology showing Rosenthal’s canal in the normal guinea pig cochlea.

[0025] FIG. IB depicts guinea pig cochlea histology of Rosenthal’s canal after ouabain treatment.

[0026] FIG. 1C is a higher magnification image of the region indicated “C” in FIG. IB.

[0027] FIG. ID is a higher magnification image of the region indicated “D” in FIG. IB.

[0028] FIG. IE depicts a series of histology images illustrating the establishment of chemically induced audiopathy in guinea pig. A normal cochlea (left) includes a Rosenthal’s canal packed with spiral ganglion neurons. After ouabain treatment (middle), there is near complete ablation of spiral ganglion neurons. After neomycin treatment (right) there is about 25% survival of ganglion neurons.

[0029] FIG. 2A and FIG. 2B show fluorescence microscopy images of guinea pig cochlea (from two specimens) seven days after injection of LCTANP1 cells (also referred to as ANP1 cells). Red fluorescent label indicates the transplanted cells. Scale bars = 1 mm.

[0030] FIG. 2C shows higher magnification of the injection site (left) and 1stand 2ndturn (right) in the guinea pig cochlea (middle). Scale bars = 1 mm.

[0031] FIG. 2D is a series of images that show ANP1 cells (labeled red) at 7 days after administration to ouabain or neomycin deafened guinea pig cochlea, administered either through the scala tympani or the modiolus, as indicated.

[0032] FIG. 3A and FIG. 3B are fluorescence microscopy images showing that a human nucleoli-specific antibody can distinguish between human neuronal cells and the host guinea pig cells. In cell culture, LCTANP1 cells were labeled with antibodies to human nucleoli (green, FIG. 3A) whereas guinea pig fibroblasts were not labeled (FIG. 3B). Neurofilament protein was stained in red (phalloidin stain).

[0033] FIGS 4A - 4D are fluorescence microscopy images showing that LCTANP1 cells (light blue) survived in the guinea pig cochleae for at least 1-week post-transplantation. Beta tubulin III is stained in red.

[0034] FIG. 5A and FIG. 5B demonstrate the differentiation process of LCTANP1 cells from Human Embryonic Stem Cells (hESCs) to late otic neural progenitors (ONP) / mature and / or early auditory neurons (FIG. 5A) and markers of hESCs, neural progenitors, ectoderm and late otic neuronal progenitors (ONP), and AN late ONP as a function of differentiation time (FIG. 5B). As time progresses, the elimination of hESC markers and the expression of otic neuronal progenitor markers can be seen.

[0035] FIG. 5C is a schematic and a graph showing monitoring of LTCANP1 (also referred to as ANP1) differentiation using in process controls starting from a single pluripotent stem cell line.

[0036] FIG. 6A and FIG. 6B are schematic depictions of various assessments for LCTANP1 cell therapy quality control, including bio-analytical and functional assessments, which can be used to characterize the clinical applicability of the product. FIGS. 6A and 6B show a snapshot of in vitro analytical method development designed to monitor the differentiation process and the final cell therapy product.

[0037] FIG. 7A is a diagram showing the development of LCTANP1 cells from hPSC cell banking and expansion to a full scale engineering run.

[0038] FIG. 7B is a pair of images and a diagram that show the directed differentiation of LCTANP1 cells during the manufacturing process, which mimics natural otic development.

[0039] FIG. 8 is a series of fluorescence microscopy images showing the expression profiles of various markers in ANP1 cells including markers of otic neural progenitors and neural markers. For each of the two sets of 9 images, the left column / blue is DAPI. The middle column / red or green label the marker of interest. The right column of each of the two sets of 9 images is the merged result of the left and middle columns illustrating colocalization. ANP1 cells express otic neural progenitor and neural markers.

[0040] FIG. 9 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. Color represents cells expressing key auditory neuron related gene sets. Bioinformatics reveals expression of key auditory neuron related genes distributed among ANP1 final product.

[0041] FIG. 10 is a series of fluorescence microscopy images of ANP1 cells whose morphology has been characterized in vitro based on a combination of neural and synapse related markers at 3 days of culture (top row) and 3 weeks of culture (bottom row). ANP1 cells show a significant increase in marker expression with culture time.

[0042] FIG. 11 depicts auditory neuron electric activity on multi electrode arrays (MEA, left column). Neuronal activity from multiple ANP1 cells was recoded recorded simultaneously in the absence (basal activity; right column, top) and presence (induced activity; right column, bottom) of a glutamate receptor agonist using a multi electrode array.

[0043] FIGS. 12A-12C are a pair of graphs illustrating the basal neuronal activity of ANP1 cells by quantifying the active area over time (FIGS. 12A-12B), based on MEA activity scans and network activity scans, and a table showing ANP cell response to electrical stimulation(FIG. 12C). FIG. 12A illustrates the percentage of active area over time (x-axis, in days) based on MEA activity scan. FIG. 12B illustrates the network activity over time (x-axis, in days) demonstrating the formation of neuronal network activity after ~30 days and increasing in activity with culture time up to ~50 days. FIG. 12C is a quantification of the neuronal response to electrical stimulation, including active electrodes, total spikes, and evoked peaks, and shows an increase in response in correlation to stimulation amplitude.

[0044] FIGS. 13A-13C are a series of histograms and graphs illustrating specific glutamatergic activity of ANP1 cells using MEA. FIG. 13A is a series of three histograms summing action potentials from all recording electrodes before and after the addition of AMP A, DNQX, and TTX. FIGS. 13B and 13C quantify the percentage of active electrodes (FIG. 13B) and the number of spikes (FIG. 13C, as a representation of action potentials) at baseline (before the shaded regions in FIG. 13A), after AMP A addition, and after the addition of DNQX.

[0045] FIGS. 14A-14B depict axon tracking based on MEA to identify action potential propagation along cellular processes. FIG.14A is a reconstructed axon map of 5 ANP1 cells. Scale Bar = 100 pm. FIG. 14B is a series of graphs quantifying the axon elongation and axon length-related parameters in the 5 ANP1 cells from FIG. 14A, based on the electrical signal in ANP1 cells after about 6 weeks in vitro.

[0046] FIGS. 15A-15B are a pair of graphs quantifying the results of a calcium influx assay. FIG. 15A is a quantification of the calcium influx assay in ANP1 cells showing a positive reaction (>100) to glutamate and AMP A stimuli. Upon the addition of DNQX, calcium influx is reduced (FIG. 15B). Data are normalized to baseline values.

[0047] FIG. 16 is a Flow cytometry (FCM) plot showing expression of SOX2 and Nestin in a representative population of ANP1 cells of the disclosure.

[0048] FIG. 17 is a FCM plot showing expression of TRA-1-60 and SSEA-5 in a representative population of ANP1 cells of the disclosure.

[0049] FIG. 18 is a series of immunofluorescence images showing expression of beta tubulin III (beta tubulin 3), TrKB and GluA4 in representative population of ANP1 cells of the disclosure. Cells are counterstained for nuclei with DAPI.DETAILED DESCRIPTIONIntroduction

[0050] 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.

[0051] 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.

[0052] 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 the 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 ofauditory 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.

[0053] 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 quality, scalability, and cost of goods associated with translating the existing protocols to a clinical commercial-scale production process of such sensory cells.

[0054] Hidden hearing loss (HHL) refers to a condition typically 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 nerve 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.

[0055] 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 following 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 theelectrode 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 auditory 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 auditory 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.

[0056] 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.

[0057] 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.

[0058] All publications, patent applications, patents and other references cited herein are incorporated by reference in their entireties.

[0059] 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.

[0060] 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 without departing from the scope of the present invention. Table 1. AbbreviationsDefinitions

[0061] 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.

[0062] 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").

[0063] 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.

[0064] 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."

[0065] A “cell” 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, ability 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 are 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.

[0066] “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 consistingessentially 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.

[0067] 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.

[0068] 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.

[0069] 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 enzyme or protein relative to the absence of the antagonist.

[0070] 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 andPractice 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.

[0071] “ 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).

[0072] 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).

[0073] “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, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, and the like. Such preparations can be sterilized and, if 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] As used herein, "embryonic stem cell" or "ES 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 the inner cell mass of the blastula of a developing organism. ES cells can be cultured over a long period of time while maintaining the ability 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, S SEA-4, TRA-1-60 antigen (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.

[0079] As used herein, "embryonic germ stem cell", “embryonic 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 embryonic 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.

[0080] 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 at 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.

[0081] 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, ESI, 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, 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, WIBR.2, WIBR.3, WIBR.4, WIBRS, WIBR6, HUES 45, Shef 3, Shef 6, BINheml9, BJNhem20, SAGO 1, and SAOOL

[0082] 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 of the central nervous system but cannot give rise to cells of the mesoderm or endoderm lineages.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] As used herein, "anterior ectoderm" refers to the region of the ectodermal germ layer at the anterior, or "rostral", end of the embryo, 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.

[0087] 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 growth factor receptor 2 (FGFR2), fibroblast growth factor receptor 3 (FGFR3), SIX homeobox 1 (SIX1), SIX homeobox 4 (SIX4), eyes absent homolog 1 (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.

[0088] 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), SIX 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), hairy and 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.

[0089] As used herein, "stromal cells" refers to connective tissue cells of any organ, e.g. fibroblasts, pericytes, endothelial cells, etc.

[0090] 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-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). Chipbased 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.

[0091] 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.

[0092] 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.

[0093] 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, forexample, grown in bioreactors, small capsules in which the cells can grow into spheroids, or 3D cell aggregates.

[0094] 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.

[0095] “ 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 nerve. The cell bodies of the neurons of the cochlear nerve lie within the cochlea, and collectively form the spiral ganglion.

[0096] 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 charged 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 / MATH 1 / HATH 1), myosin VI (MY06), myosin VIIA (MY07A), Espin (ESPN), myosin heavy chain 3 (MYH2), cadherin23 (CDH23), protocadherinl5 (PCDH15), otoferlin (OTOF), and prestin (SLC26A5).

[0097] 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 (OTOA), musashi homolog 1 (MSH), 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).

[0098] 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, sensory neuronal progenitor cells, and the like. The term “auditory cells” may, in some cases, refer to a mixed population of cells encompassing any combination of the cell types described above, in any ratio.

[0099] 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.

[0100] 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.

[0101] As used herein, “sensorineural hearing loss” refers to a pathologic change in structures with the inner ear or in the acoustic nerve.

[0102] As used herein, “central hearing loss” refers to a pathologic condition above the junction of the acoustic nerve and the brainstem.

[0103] As used herein, “mixed hearing loss” refers to a subject having both conductive hearing loss and sensorineural hearing loss.

[0104] As used herein, “hidden hearing loss” (or HHL) refers to a subject having hearing loss resulting from damage to the auditory nerve / auditory neurons.

[0105] As used herein, “auditory neuropathy spectrum disorder” refers to a type of sensorineural hearing loss where the auditory nerve 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 geneticmutations, and viral infections. Researchers are still seeking effective treatments for those affected with auditory neuropathy.

[0106] 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 Treatment

[0107] 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 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.

[0108] 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 75% 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.

[0109] 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 auditory cells 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 auditory neurons, the function of the cochlear implant is thereby improved.

[0110] 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 P tubulin III; (c) greater thanor 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.

[0111] 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 75% 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.

[0112] 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 PAX2; (c) greater than or equal to 30% of the cells in the population express P 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.

[0113] 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 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 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.

[0114] 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 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 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.

[0115] 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 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; and (d) less than or equal to 0.1% of the cells in the population express TRA-1-60 and / or SSEA5.

[0116] 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 30% of the cells in the population express SOX2; (b) greater 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 beta 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.

[0117] 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 (f) 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 P 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.

[0118] 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 2months, 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.

[0119] 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 delivery of cell-based therapies as described herein.

[0120] 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.

[0121] 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 auditory 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.

[0122] 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.

[0123] 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 maybe 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).

[0124] 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 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 alpha. sub. v. beta. sub.3 integrin 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 neurons 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 growth 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 usefulas 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).

[0125] 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, which 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.

[0126] The subject can be deaf or have a hearing loss for any reason or as a result of any type 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).

[0127] 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).

[0128] 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).

[0129] In some embodiments, the auditory disorder or condition is an auditory neuropathy spectrum disorder. Auditory neuropathy 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 spectrum disorder, rather than a multitude of targeted therapies dependent on the underlying causal agent.

[0130] In some embodiments, the auditory 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 has HHL and a cochlear implant. In some embodiments, a subject with a cochlear implant still suffers from HHL as the cochlear implant can induce sound mediatedelectrical signals in functional auditory neurons and thus replace damaged or lost hair cells, but it cannot replace damaged or lost auditory neurons.

[0131] In some embodiments, the methods provided herein are methods for replacing auditory neurons 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.

[0132] Auditory cells 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.

[0133] 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.

[0134] 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 or 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 delivery of 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- impl antati on / cochl ear-impl ant- surgi cal -vari ati ons / .

[0135] 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 ofthe 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 tympani or modiolus, and cells are administered as described above.

[0136] 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.

[0137] 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.

[0138] In some embodiments, administration of the compositions described herein alleviates a sign or symptom of the auditory disease or condition in the subject. In some 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.

[0139] 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 syringecartridge, 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

[0140] The disclosure provides pharmaceutical compositions comprising a population of cells for use in the methods described herein. The skilled artisan will appreciate that the compositions described herein can comprise mixed populations of cell types, whose identities are reflected in percentages of cells 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 markers such as PAX2, PAX8, and / or SOX2, neuronal markers such as P Tubulin III, auditory neuron markers such as TrkB and / or GluA4, nonspecific neuronal markers such as Myo7A, or hESC markers such as TRA-1-60 and / or SSEA5. In some embodiments, cells in the population express SOX2; cells in the population express P tubulin III; cells in the population express TrkB; and cells in the population express TRA-1-60 and / or SSEA5. 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; and cells in the population do not express TRA-1-60 and / or SSEA5.

[0141] In some embodiments, cells in the population express 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 express TRA-1-60 and / or SSEA5.

[0142] 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.

[0143] In some embodiments, cells in the population do not express PAX2 and / or PAX8.

[0144] 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.

[0145] 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 of transcription factors involved in the regulation of embryonic 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.

[0146] 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 the 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.

[0147] In some embodiments, cells in the population 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.

[0148] In some embodiments, cells in the population express Sixl. Sixl is a homeobox protein gene found in a cluster of related genes on chromosome 14 and is thought to be involved in limb development. 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 Sixl . In some embodiments, greater than or equal to 20% of the cells in the population express Sixl . In some embodiments, greater than or equal to 30% of the cells in the population express Sixl . In some embodiments, greater than or equal to 40% of the cells in the population express Sixl. In some embodiments, greater than or equal to 50% of the cells in the population express Sixl . In some embodiments, greater than or equal to 60% of the cells in the population express Sixl . In some embodiments, greater than or equal to 70% of the cells in the population express Sixl. In some embodiments, greater than or equal to 80% of the cells in the population express Sixl . 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 Sixl.

[0149] In some embodiments, cells in the population 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 or 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.

[0150] In some embodiments, cells in the population express PAX2. PAX2 encodes paired box gene 2 is a target of transcriptional suppression by the tumor suppressor gene WT1. 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 PAX2. In some embodiments, greater than or equal to 20% of the cells in the population express PAX2. In some embodiments, greater than or equal to 30% of the cells in the population express PAX2. In some embodiments, greater than or equal to 40% of the cells in the population express PAX2. In some embodiments, greater than or equal to 50% of the cells in the population express PAX2. In some embodiments, greater than or equal to 60% of the cells in the population express PAX2. In some embodiments, greater than or equal to 70% of the cells in the population express PAX2. In some embodiments, greater than or equal to 80% of the cells in the population express PAX2. In some embodiments, greater than or equal to 90% of the cells in the population express PAX2. In some embodiments, 10% to 99%, 20% to 95%, 30% to 80%, 40% to 70%, or 50% to 60% of the cells in the population express PAX2.

[0151] 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.

[0152] In some embodiments, cells in the population express CD 133. CD 133 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 CD133. 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 CD 133.

[0153] In some embodiments, cells in the population express GAT A3. GAT A3 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 population express GAT A3. In some embodiments, less than 1% of the cells in the population express GAT A3. In some embodiments, less than 0.1% of the cells in the population express GAT A3. In some embodiments, 0.1% to 10%, 0.1% to 5%, or 0.1% to 1% of the cells in the population express GAT A3.

[0154] In some embodiments, cells in the population express P tubulin III (also referred to as P 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, 1% to 80%, 10% to 70%, 30% to 60%, or 40% to 50% 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.

[0155] 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%, 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.

[0156] In some embodiments, the fold change in expression of one or more of the markers can be compared to the starting cell 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 someembodiments, the fold change in expression of TrkB between the cells in the population and hESC 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 hESC 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 hESC 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 hESC 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 hESC 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 hESC 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 hESC cells is greater than or equal to 2,000. In some embodiments, the fold change in expression of TrkB between the cells in the population and hESC 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 hESC 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 hESC 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.

[0157] 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.

[0158] 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 BRN3 A. In some embodiments, less than 1% of the cells in the population express BRN3 A. 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 BRN3 A. 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.

[0159] In some embodiments, cells in the population express 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.

[0160] In some embodiments, cells in the population express stage-specific embryonic antigen (SSEA-5). Undifferentiated cells may be identified by expression of various 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.

[0161] 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 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 express 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 expressTRA-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.

[0162] 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 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.

[0163] In some embodiments, (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 PAX2; (c) greater than or equal to 30% of the cells in the population express P 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.

[0164] 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. between 50% and 100%, of the cells in the population express P 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 thecells 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.

[0165] 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 directly after thawing without further processing. 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), STEMCELLBANKER®, 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 cryopreservation medium comprises between about 0.5% and about 30% DMSO. In embodiments, the cryopreservation medium comprises between about 1% and about 20% DMSO.

[0166] 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 60p.m. 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 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 recovery can be any value or subrange within the recited ranges.

[0167] 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.

[0168] In further embodiments, the percent viability of 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 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.

[0169] In yet other embodiments, the percent viability of 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, 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 of 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, post-thawing 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 within the recited ranges.

[0170] In some embodiments, the percent viability 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 viability 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 viability 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 cry opreservation 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%. In still 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 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%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 140%, 150%. The viability can be any value or subrange within the recited ranges.

[0171] Auditory cells formulated in cry opreservation 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-N'- (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 cryopreservation medium such as CryoStem®, as ready to inject product, using an aggregate specific delivery system such as the Sutter Xenowork system that is used for somatic cell nuclear transfer and intracytoplasmic sperm injection, and recently used for ONP spheroids (Heuer et al. 2020).

[0172] DMSO can be used as a cryoprotective 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 cells cell therapy composition comprises about 5% DMSO. The concentration can be any value or subrange within the recited ranges.

[0173] In some embodiments, auditory cell therapy compositions formulated in cryopreservation media appropriate for post thaw ready to administer (RTA) applications may comprise auditory cells suspended in cry opreservation media that does not contain DMSO. For example, RTA sensory therapeutic cell compositions may comprise auditory cells suspended in Trolox, Na+, K+, Ca2+, Mg2+, Cl’, H2PO4’, HEPES, lactobionate, sucrose, mannitol, glucose, dextran-40, adenosine, glutathione without DMSO (dimethyl sulfoxide, (CH 2SO) or any other dipolar aprotic solvents. An example of this cryopreservation 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 in cryopreservation media appropriate for post thaw ready to administer applications may comprise auditory cells suspended in Trehalose.

[0174] 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.

[0175] 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.

[0176] 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 cryopreservation 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 use 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.

[0177] 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 compositionscan 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.

[0178] 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 topical application. Methods for making such formulations are well known and can be found in, for example, "Remington's Pharmaceutical Sciences."

[0179] 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.

[0180] 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 cry opreservation at or below -80°C to -195°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 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 isformulated in CRYOSTOR®10. In an embodiment, a composition can be filtered using a filter kit before cry opreservation, to avoid clogging during administration through a narrow cannula, syringe needle or a catheter.

[0181] 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 90 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.

[0182] 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 inaccordance 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 cryopreservation or for administration to a subject in need thereof. In an embodiment, a container can be a prefilled syringe.

[0183] 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 4 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.

[0184] 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

[0185] The disclosure provides methods of producing populations of auditory cells from undifferentiated pluripotent stem cells suitable for administration to a subject to enhance the function of a cochlear implant. Methods of 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.

[0186] The methods comprise culturing populations of undifferentiated pluripotent stem cells in different combinations of growth factors and growth factor inhibitors, in a series of steps that induces the differentiation of the undifferentiated pluripotent stem cells towards 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, 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 mixed 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.

[0187] A schematic depiction of the process of hESC differentiation to mature SGN is provided in FIG. 5A. In some embodiments, biomarkers are monitored along the differentiation process, as depicted in FIG. 5B. Monitoring of biomarkers facilitates purity of the cell populations, to ensure a high-quality final product of LCTANP1 cells.

[0188] FIGS. 6A-6B depict exemplary assays for monitoring the quality of the LCTANP1 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. Markers can be assessed, for example and without limitation, by flow cytometry, immunofluorescence, and other methods as known in the art. Gene expression (e.g., late ANP gene expression) can be assessed, for example, by quantitativePCR (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 synapse 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.

[0189] The final cell product can be profiled. Profiling can be by RNA-sequencing, proteomic / protein analysis, marker characterization (e.g., by flow cytometry), and other methods of characterization as known in the art.

[0190] 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 Embryonic Stem Cells (hESCs)

[0191] 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.

[0192] 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).

[0193] In some embodiments, the cultured human embryonic stem cells of the static expansion are harvested and further differentiated.

[0194] 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, 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.

[0195] 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).

[0196] In some embodiments, the cultured human embryonic stem cells of the suspendable expansion complex are harvested and further differentiated.

[0197] Human embryonic 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 embryo 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 re-plated on a fresh tissue culture medium. Colonies demonstrating undifferentiated morphology are individually selected by micropipette, mechanically dissociated into clumps, and re-plated. 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],

[0198] 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, 30 36: 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],

[0199] 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 as whole embryos for at least nine and no more than fourteen days post fertilization (z.e., prior to the gastrulation event) in vitro using standard embryonic stem cell culturing methods.

[0200] 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.

[0201] 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. USA 95: 13726, 1998] and U.S. Patent No. 6,090,622.

[0202] Yet another method for preparing ES cells is by parthenogenesis. The embryo is also not destroyed in the process.

[0203] 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.

[0204] According to some embodiments, the expansion phase is effected for at least one to 20 weeks, 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 weeks, 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.

[0205] 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 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 85%.

[0206] 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.

[0207] 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).

[0208] 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.

[0209] The animal extracellular matrix includes the interstitial matrix and the basement membrane. Interstitial matrix is present between various animal cells (z.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.

[0210] 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-lysine, their derivatives, or a combination thereof. In specific embodiments, the human laminin is human laminin 511 E8 fragment.

[0211] In some embodiments, the microcarriers may comprise one or more of polystyrene, cross-linked dextran, magnetic particles, microchips, cellulose, hydroxylated methacrylate, collagen, gelatin, polystyrene, 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.

[0212] In some embodiments, the microcarriers are coated. In embodiments, the microcarriers may be coated with Matrigel®, 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.

[0213] In some embodiments, the microcarriers may be spherical, smooth, macroporous, rodshaped, 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 microcarrier is ellipsoidal. In some embodiments, the microcarrier is rodshaped. In some embodiments, the microcarrier is disc-shaped. In some embodiments, the microcarrier is porous. In some embodiments, the microcarrier is non-porous. In some embodiments, the microcarrier is smooth. In some embodiments, the microcarrier is flat.

[0214] In some embodiments, the microcarriers are neutral. In some embodiments, the microcarriers are negatively charged. In some embodiments, the microcarriers are hydrophilic.

[0215] 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.

[0216] 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 cm2 / gram.

[0217] 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.

[0218] 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.

[0219] In some embodiments, the cells may be cultured in a working volume of between 10 mL and 3,000 mL, for example about 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.

[0220] In some embodiments, the cultured cells may be expanded further.

[0221] 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).

[0222] 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.

[0223] 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™l StemFit®). Additional factors may be added to the medium to prevent differentiation of the ESCs such as bFGF and TGFP3. 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.

[0224] 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 feeder-based 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 growth such as MEF-conditioned medium and bFGF.

[0225] 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.

[0226] 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., Corning).

[0227] 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.

[0228] 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.Expansion Compositions

[0229] 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.

[0230] Human embryonic stem cells, extracellular matrices, and microcarriers are described in detail elsewhere herein.

[0231] 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 2.

[0232] The ECM component can be presented by mol / cm2by using the laminin 511 E8 fragment’s molecular weight (150 KDa).Table 3.

[0233] 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 4.

[0234] 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.

[0235] In some embodiments, the composition may further comprise a growth medium. Nonlimiting 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), NEUROB AS AL™, KO-DMEM, DMEM, DMEM / F12, CELLGRO™ Stem Cell Growth Medium, or X-VIVOTM. 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 varioussupplements 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 carry the one or more members of the TGFI3 superfamily of growth factors; an antibacterial agent, such as, without 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, NT4.

[0236] As described above, the microcarriers may comprise one or more of polystyrene, crosslinked 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.

[0237] As described above, the microcarriers may be spherical, smooth, macroporous, rodshaped, or a combination thereof.

[0238] 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.

[0239] In some embodiments, the microcarriers are not coated.

[0240] 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.

[0241] 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

[0242] In accordance with the present disclosure, human pluripotent stem cells (hPSCs) can be grown in dynamic culture on microcarriers in a hESC culture media, and maintained in a pluripotency state by daily replacement of the hPSC media, as described above. The hPSCs will be differentiated by medium replacement into a culture media (1 : 1 mixture of DMEM / F12 and Neurobasal medium) 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 of static culture will 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 will be replaced with FGF2, LDN193189 (20-400 nM), IWP-2 (2uM), SB431542 (1 pM), NIC (1-25 mM), the Wnt inhibitor IWR-endo (1-10 pM), to generate the pre-placodal ectoderm Dynamic culture will continue for 3-7 days with medium replacement every 1-3 days. At differentiation days 8-14 differentiation factors will be replaced with FGF2, CHIR99021(6uM), and IGF1 (50ng / ml), to generate the early ONP. Dynamic culture will continue for 7-10 days with medium replacement every 2-3 days. On days 17-22 differentiation factors will be replaced with FGF2, EGF, retinoic acid (RA 0.2-2 pM), SHH (500ng / ml) and IGF1 (50 ng / ml), to generate the mid-Late ONP. Dynamic culture will continue for 7-10 days with medium replacement every 1-3 days. On days 22-28, Mid-Late ONP cells will be harvested and inoculated into static / dynamic suspension as single cells for 3 days in the presence of BDNF (10 ng / mL), NT3 (10 ng / mL), and IGF-1 Rock inhibitor (e.g., Y-27632 Dihydrochloride, 10 pM) to form small aggregates. At differentiation days 22-28 the cells will be further expanded for final maturation.

[0243] 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- IpM), 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.

[0244] 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 type.

[0245] In some embodiments the method begins with seeding a population of undifferentiated pluripotent stem cells at a density of 1,200-20,000 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 between about 1.0 to 13. 0 mM, or between about 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%.

[0246] In some embodiments, the population of undifferentiated pluripotent stem cells 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).

[0247] 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, thepopulation 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.

[0248] 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 growth 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]pyrimidin-3 -yl] quinoline (LDN193189).

[0249] 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 type, for example, pre-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 culturemedium 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.

[0250] 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).

[0251] 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 1 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 cell 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.

[0252] 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.

[0253] 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 producedifferentiation 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.

[0254] In some embodiments, the population of cells produced by culturing the cells in the third culture medium, e.g. the population of cells comprising mid-late ONP cells, are cultured in fifth cell culture medium comprising Brain derived neurotrophic factor (BDNF), Neurotrophin-3 (NT3), and IGF-1.

[0255] 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 culturemedium 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.

[0256] 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, or 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.

[0257] 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 someembodiments, 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.

[0258] In some embodiments, the first and / or second cell culture medium includes SB431542 at a concentration of about 0.1 gM, about 1 gM, about 5 gM, about 10 gM, about 15 gM, or about 20 gM. In some embodiments, the first and / or second cell culture medium includes SB431542 at a concentration of between about 0.1 gM - 20 gM, 0.1 - 10 gM, 5 gM 15 gM, or 7 gM 13 gM. In some embodiments, the first and / or second cell culture medium includes SB431542 at a concentration of 1 gM - 20 gM. In some embodiments, the first and / or second cell culture medium includes SB431542 at a concentration of 5 gM - 15 gM. In some embodiments, the first and / or second cell culture medium includes SB431542 at a concentration of 0.1 gM - 10 gM. In some embodiments, the first and / or second cell culture medium includes SB431542 at a concentration of about 1 gM.

[0259] 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.

[0260] 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.

[0261] 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.

[0262] 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 20 |rM, 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 |rM, 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 gM- 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.

[0263] 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.

[0264] 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.

[0265] 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, about 2 gM, about 3 gM, about 5 gM, about 10 gM, about 15 gM, or about 20 gM. In some embodiments, the fourth culture medium includes RA at a concentration of 0.1 gM - 20 gM, 0.1 gM - 5 gM, 0.5 gM - 5 gM, 0.2 gM - 5 gM, or 0.5 gM - 2 gM. In some embodiments, the fourth cell culture medium includes RA at a concentration of 0.1 gM - 20 gM. In some embodiments, the fourth cell culture medium includes RA at a concentration of 0.2 gM - 5 gM. In some embodiments, the fourth cell culture medium includes RA at a concentration of 0.2 gM - 2 gM. In some embodiments, the fourth cell culture medium includes RA at a concentration of about 0.5 gM.

[0266] 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.

[0267] 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 - 300ng / 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.

[0268] 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 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 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.

[0269] 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 ( traw -4-[(U?)-l- Aminoethyl]-7V-4-pyridinylcyclohexanecarboxamide 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.

[0270] In some embodiments, the methods comprise (a) culturing a population of undifferentiated pluripotent stem cells in a first cell culture medium comprising FGF2 at aconcentration 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 of between 5- 100 ng / mL for 5-9 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 5-9 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 (f) collecting the population of cells comprising late ONPs, 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.

[0271] 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 ina third cell culture medium comprising CHIR99021, FGF2, and IGF-1 for 5-9 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 5-9 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 (f) collecting the population of cells comprising late ONPs, thereby producing the composition. In alternative embodiments, the first cell culture medium at step (a) comprises BMP4 and SB431542, and does not comprise FGF2.

[0272] 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 aconcentration 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, thereby producing a population of cells comprising late ONP cells; and (f) collecting the population of cells comprising late ONPs, 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.

[0273] 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 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 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 a step (f) collecting the population of cells comprising late ONPs, 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.

[0274] 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 livecells / 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.

[0275] In some embodiments, any one of the methods described above further comprises a step of cry opreserving the population of cells comprising late ONPs.

[0276] 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

[0277] The disclosure provides kits comprising the pharmaceutical compositions described herein, and articles of manufacture such as cryovials, syringes, syringe cartridges, cannula and the like.

[0278] In some embodiments, the kit comprises instructions for use.

[0279] In some embodiments, the pharmaceutical compositions described herein are prepackaged 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.

[0280] Having now generally described the invention, the same will 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: Human Pluripotent Derived Neurons Survive in the Guinea Pig Cochlea

[0281] This Example describes assessment of a guinea pig model for eliminating neurons and replacing the neurons with stem cells. The Example also assesses the fate of pluripotent derived human auditory neurons (termed LCTANP1 cells, or ANP1 cells) when transplanted into the guinea pig cochlea.MethodsAnimals & Anesthesia

[0282] Young male Hartley albino guinea pigs were obtained from Elm Hill Breeding Labs (Chelmsford, MA). For anesthesia during procedures, animals were administered intramuscularly a combination of xylazine at a concentration of 10 mg / kg, ketamine at a concentration of 40 mg / kg, and lidocaine at a concentration of 4 mg / kg. For analgesia, animals were subcutaneously administered ketoprofen at a concentration of 1 mg / kg before surgery and for two consecutive days post-surgery.Deafening with Ouabain

[0283] 5 pl of 10 mM ouabain, also known as g-strophanthin, a plant-derived toxin, was infused into the scala tympani and ouabain-infused Gelfoam® was placed on the round window membrane of the cochlea.Deafening with Neomycin

[0284] 10 pl of 15 % neomycin was infused through the round window using a syringe pump at a rate of 1 pL per minute.LCTANP1 cells

[0285] Human auditory neurons were derived from pluripotent cells with direct differentiation to otic neuronal progenitors according to the methods described in WO2023 / 167986. In brief, LCTANP1 (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 a pure ANP 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.Experimental groups

[0286] 8 animals per deafening condition (ouabain or neomycin) were injected with LCTANP1 cells. A deafened control group (n=3) was euthanized 30 days after the ouabain deafening. A second deafened group was euthanized 30 days after the neomycin deafening.

[0287] For the experimental groups, 30 days after ouabain deafening, 5 pl of LCTANP1 cells labeled with red fluorescent protein were infused by syringe pump at a concentration of 1 pl / min into the base of the cochlea. Infusion was either into (group i) the scala tympani via cannula (n = 4) or (group ii) directly into the modiolus via microneedle (n = 4).Histology

[0288] Guinea pigs were euthanized 7 days after cell infusion for assessment of LCTANP1 presence and location in the cochleae. Ears were harvested.

[0289] Cochleae were locally perfused with 4% paraformaldehyde and were subsequently dissected under a fluorescence stereo-microscope to locate and photograph labeled cells. Cochleae were then cryo-sectioned and labeled with DAPI (blue) and with antibodies to human nucleoli (green) and neurofilament protein (red).Results

[0290] Guinea pigs were divided into control and experimental groups as described above. As shown in FIG. 1A and FIG. IE (left), in the normal guinea pig cochlea, Rosenthal’s canal is packed with spiral ganglion neurons. Following ouabain infusion, few if any spiral ganglion neurons were seen in the canal as shown in FIG. IB (with higher magnification shown in FIG. 1C and FIG. ID) and FIG. IE (middle). Neomycin treatment showed about 25% survival of spiral ganglion neurons in guinea pig (FIG. IE, right). Ouabain treatment showed depletion of endogenous neurons with minimal side effects demonstrating its utility for modeling chemically induced auditory neuropathy in rodent models such as guinea pigs. Ouabain was chosen to establish chemically induced audiopathy as it had fewer side effects on animals’ health compared to neomycin treatment, and resulted in near complete ablation of spiral ganglion neurons, as opposed to the 25% survival seen with neomycin.

[0291] LCTANP1 cells were then administered to the experimental groups. As shown in FIG. 2A, FIG. 2B and FIG. 2C, seven days after injection of LCTANP1 cells, large areas of the scala tympani in the cochleae appeared red by fluorescence microscopy, indicating the presence of clusters of transplanted LCTANP1 cells.

[0292] It was further determined that a human nucleoli-specific antibody could distinguish between human neuronal cells (see FIG. 3A, staining in green) and the host guinea pig cells (see FIG. 3B, depicting guinea pig fibroblasts stained in red).

[0293] Many LCTANP1 cells survived in the guinea pig cochleae for at least 1 week. As shown in the immunofluorescence images depicted in FIGS. 4A-4D, transplanted LCTANP1 cellslabeled with antibodies to human nucleoli (FIG. 4B, green) were clearly observable in the guinea pig cochlea, and were seen in patches of various sizes and shapes in the scala tympani of multiple guinea pigs. The LCTANP1 cells were mostly present as aggregates near the modiolar wall in the scala tympani space. Mitotic figures could not be observed among these human cells. Importantly, fluorescence stereoscopy revealed red-labeled LCTANP1 cells at both the initial transplantation site and in flanking areas. LCTANP1 cells engrafted throughout a large area in the ouabain-traumatized cochlea and were able to spread beyond the injection site, as needed for populating the cochlea.

[0294] Taken together, these data demonstrate that ouabain can be used to eliminate auditory neurons in the guinea pig, modeling deafness. Human-derived auditory neurons, LCTANP1 cells, survive and can be identified in guinea pig ears following transplantation. For both injection routes, into the perilymph of the scala tympani and into the modiolus, LCTANP1 cells survived in the ouabain-traumatized cochlea for at least 7 days after administration to the scala tympani or modiolus. Further, the LCTANP1 cells migrated up the second turn beyond the initial injection / transplantation site and engrafted throughout a large area in the ouabain- traumatized cochlea. The transplanted cells were able to spread beyond the injection site, as needed for populating the cochlea.Example 2: Assessment of Manufacturability of Human Pluripotent Derived Auditory Neuron Progenitors (LCTANP1) as a Therapeutic for Auditory Disorders

[0295] This Example describes the assessment of manufacturability, release criteria, and in vitro activity of LCTANP1 cells manufactured at scale.

[0296] Loss of auditory 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 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 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 LCTANP1 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. LCTANP1 cells were transplanted via direct administration into the cochlea of guinea pigs in which neurons werepreviously eliminated via exposure to ouabain. The survivability of engrafted LCTANP1 when transplanted into the deafened cochlea of guinea pigs was assessed.Methods

[0297] The LCTANP1 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. 5A. Biomarker kinetics along the differentiation were monitored, as illustrated in FIG. 5B

[0298] To ensure the quality of the cell product, various assays were developed, as depicted in FIG. 6A and FIG. 6B. 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.

[0299] Functional assays were developed to measure neuronal properties of LCTANP1. Functional assays were designed to test 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.

[0300] As described in Example 1 above, the cochleae of eight ouabain-treated guinea pigs were infused with 5 pL of red fluorescent labeled LCTANP1 cells (quality controlled as described in the instant Example) via a cochleostomy into the scala tympani, or via a needle inserted into the modiolus, both procedures performed at the base of the cochlea. Seven days later, animals were euthanized and labeled LCTANP1 cells were visualized within the cochlea using a fluorescence stereoscope. Cochlea were processed for immunohistochemistry, dissected, and imaged for assessment of LCTANP1 cells.Results

[0301] LCTANP1 cells were successfully manufactured at scale, met pre-set release criteria, and demonstrated relevant activity in in-vitro functional tests. LCTANP1 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 7 days.

[0302] LCTANP1 is a cell-based product composed of Auditory Neuron Progenitors derived from clinical grade pluripotent stem cells. LCTANP1 completed initial chemistry, manufacturing, and control studies and preclinical testing, highlighting its potential as a therapeutic.

[0303] 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 be 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.

[0304] Working on steps in parallel, it was possible to develop LCTANP1 cells from cell bank production and production to an at scale engineering run in 18 months (FIG. 7A). During manufacturing, LCTANP1 cells undergo directed differentiation (FIG. 7B). 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.

[0305] An exemplary ANP1 cell profile is shown in Table 5, as well as the ranges of cells expressing the indicated marker based on 10 replicates, and the test method used to assay marker expression.Table 5. Exemplary ANP1 cell profileFC-Flow Cytometry qPCR-quantitative PCR IF -immunofluorescenceFIO -For information only HR-hESCs residual AssayExample 3: Assessment and Characterization of ANP1 cells in vitro

[0306] Auditory neurons are specialized cells that transmit sound information from the ear to the brain by converting mechanical sound waves into electrical signals. They transfer this data from hair cells in the cochlea to the cochlear nucleus in the brain, where it is further processed to detect pitch, volume, and special localization of sounds. The auditory neurons and hair cells form synapses. The hair cells release glutamate neurotransmitters to stimulate the auditory neuron. An action potential is generated and propagated towards the Cochlear Nucleolus (see, e.g., Moser and Starr, Nat Rev Neurology, 2018, 12(3): doi.org / 10.1038 / nmeurol.2016.10). This Example describes the assessment and characterization of ANP1 cells and ANP1 cellular connectivity by visualizing various cellular markers including neural markers, auditory neuron markers, synapse markers, glutamatergic markers, and Late ONP markers.MethodsANP1 cells and culturing

[0307] 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 were seeded on black 24 well plates and cultured for 3 days and up to 3 weeks in ANDM media supplemented with BDNF, NT3 and IGF. Media was changed twice a week. Cells were fixed using 4% PFA and stained using immunofluorescent methods to assess cellular markers and connectivity.

[0308] ANP1 cells were stained with fluorescently labeled antibodies against neural and synaptic markers (red or green) as well as DAPI nuclear (blue) staining. Images were taken using a confocal microscope to assess cell morphology, marker expression and cellular connectivity potential. -Late otic neural progenitor marker SOX2, auditory neuron markers TRKB and neural marker Nestin and synaptic markers consistent with successful progression through the differentiation process were used. Additionally, as illustrated by the immunofluorescence images in FIG. 10, the combination of neural and synapse-related markers showed an increase in expression with culture time from 3 days (FIG. 10, top row) to 3 weeks (FIG. 10, bottom row).Example 4: Assessment of Auditory Neuron Electric Activity on Multi-Electrode Arrays

[0309] This Example assesses the neuronal activity from multiple ANP1 cells using cells grown on Multi Electrode Arrays (MEAs).Experiment 1MethodsANP1 cells and culturing

[0310] 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 were then cultured for 3 weeks on 60MEA200 / 10iR-ITO Standard Microelectrode array (MCS-MULTI CHANNELS SYSTEMS, MEAs). Cells were cultured in ANDM media at 37 °C degrees in an incubator, and supplemented with BDNF and NT3. Media was changed twice a week for 3 weeks before recording. Upon electrophysiological recording, media was changed into recording buffer (HBSS with HEPES) and the culture was equilibrated for more than an hour before recording (FIG. 11, left; scale bar = 500 pm).Neuronal Activity Assay

[0311] The neuronal activity of multiple ANP1 cells grown on MEA was recorded simultaneously in the absence (basal activity; FIG. 11, top right) and presence (induced activity; FIG. 11, bottom right) of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMP A), a glutamate receptor agonist stimulation.Experiment 2MethodsANP1 cells and culturing

[0312] 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 were then cultured for up to 8 weeks on MaxOne MEA chips (Maxwell Biosystems). Cells were cultured in 1 : 1 mixture of DMEM / F12 and Neurobasal medium or BrainPhys media (Stem Cell Technologies) at 37°C in an incubator and supplemented with neurotropic growth factors. Media was changed 3 times a week. Baseline Electrophysiological activity was monitored, recorded and quantified during time of culture. Upon reaching sufficient neural network activity ANP1 cells neural activity in response to glutamatergic agonists and blockers, and electrical stimulation was tested.Neuronal Activity Assay

[0313] The neuronal activity of multiple ANP1 cells grown on MEA was recorded simultaneously in the absence (basal activity) and presence (induced activity; FIG. 13A) of AMPA (glutamatergic agonist, TOCRIS), 6, 7-dinitroquinoxaline-2, 3-dione (“DNQX”, an AMPA receptor specific blocker, Alomone labs), and Tetrodotoxin (with the IUPAC name 4A,4a7?,5A,65',75',85',8a7?,105',125)-2-azaniumylidene-4,6,8,12-tetrahydroxy-6- (hydroxymethyl)-2,3,4,4a,5,6,7,8-octahydro-U / -8a,10-methano-5,7- (epoxymethanooxy)quinazolin- 10-olate, “TTX”, Sodium Chanel blocker, Alomone labs). Single neurons and network activities were monitored and quantified overtime. Axon tracking module was then used. This module enabled recording and reconstructing neuronal footprints across the electrode array and to automatically identify the action potentials propagation along cellular processes (FIGS. 14A-14B).Results

[0314] The results of Experiments 1 and 2 demonstrate that LCT-ANP1 cells successfully demonstrated basal neural activity, and responded to electrical stimulation. The ability to respond to electrical stimulation is important for the cells to be effective in interfacing with cochlear implants. The response (active electrodes, total spikes, and evoked peak) is increased in correlation to electrical stimulation amplitude (FIG. 12C). As shown in the graphs depicted in FIG. 11 and FIG. 13A, ANP1 cells grown on MEA showed auditory specific glutamatergic neuronal response. Single neurons and network activities were monitored and quantified overtime, and demonstrated formation of neuronal network activity after 30 days (FIGS. 12A- 12B) Neural Activity was tested before and after the addition of AMPA. Action potential frequency was increased after AMPA addition in LCT-ANP1 cells (FIGS. 13A-13C). This activity was reduced upon adding DNQX (FIGS. 13A-13C). The electrical activity was blocked upon addition of TTX (FIG. 13A). Additionally, the single neuron and network activity quantified demonstrated formation of neuronal network activity after 30 days with steady increase in activity with culture time (up to ~50 days) (FIGS. 12A-12B).

[0315] Further, LCT-ANP1 cells showed axon elongation based on their electrical signal footprint (FIGS. 14A-14B). An axon tracking module was used to record and reconstruct neuronal footprints across the electrode array, and to automatically identify the action potentials propagation along cellular processes. LCT-ANP1 cells show axon elongation based on their electrical signal footprint after ~6 weeks in vitro using MEA system. A reconstructed axon map of 5 LCT-ANP1 cells is illustrated in FIG. 14A and axon length related parameters after analysis is illustrated in FIG. 14B.Example 5: Bioinformatic Analysis of Auditory Neuron Related Gene Expression in ANP1 Cells

[0316] 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.MethodsANP1 cells and culturing

[0317] 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 were thawed and processed for scRNA.Single-Cell RNA-Seq (scRNA-Seq)

[0318] Single-cell RNA sequencing was performed on the thawed ANP1 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 files, and interactive differential gene expression analysis through the 10X Genomics®, Cell Ranger™ and Loupe™ Cell Browser.Bioinformatic analysis

[0319] Bioinformatic analysis of the scRNA-Seq results was performed 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 (such as MSigDB, Panglao, and Cell Marker) and relevant published scientific literature. Color scale in graphs (FIG. 9) represents cells expressing key auditory neuron related gene sets.Results

[0320] As shown in the graph depicted in FIG. 17, 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. Further analysis including gene sets (FIG. 9) and protein markers directed to Late ONP Markers, neural markers, and auditory neuronal makers are consistent with successful progression through the differentiation process.Example 6: Ouabain Deafening Paradigms in Guinea Pigs

[0321] Animal models for auditory neuropathy are useful for testing cell-based therapies for replacing lost spiral ganglion neurons. In models where inner hair cells are preserved, the contribution of transplanted cells can be tested using acoustic stimulation, whereas models with no hair cells are useful for testing function with a cochlear implant. Guinea pigs are of interest because they are commonly and successfully used for auditory research and cochlear implant research. The goal of this project is to compare two ouabain deafening paradigms and their effect on hair cell and auditory nerve survival in guinea pigs.

[0322] Hartley albino guinea pigs were given 5 pl of 7.5 mM or 10 mM ouabain, unilaterally, infused through the round window membrane into the perilymph of the scala tympani. Ouabain-soaked gelfoam was then placed on the round window. In the low dose group, a baseline pure tone auditory brainstem response (ABR) of the left ear was measured at 4, 8 and 16 kHz. Two weeks post-ouabain, a post-deafening ABR was measured with simultaneous 50 dB white noise masking of the contralateral ear.

[0323] The high dose ouabain group was processed for histology 1 month after the ototoxic insult. The lower dose animals were processed at the 17-day timepoint. Temporal bones were fixed in paraformaldehyde, decalcified, embedded in resin and sectioned for light microscopy at a near-mid-modiolar plane.

[0324] Animals treated with both doses of ouabain developed vestibular signs such as brief nystagmus while anesthetized and torticollis. In the high dose group, histology showed a near complete loss of all hair cells and a near complete loss of auditory nerve throughout the cochlea. Histology in the lower dose group showed that many outer hair cells were missing, but inner hair cells were present in all cochlear turns. While many spiral ganglion neurons were missing, several cells remained in every turn. These data corroborate ABR results.

[0325] Infusion of ouabain into perilymph of guinea pigs at a concentration of 7.5 mM is useful for auditory neuropathy studies involving implantation of neural progenitors. The lesion leaves behind a small number of neurons that can provide a scaffold for path finding into the cochlear nucleus and also toward surviving inner hair cells. The higher dose appeared more suitable for studies where cochlear implants are combined with cell transplantation, since inner hair cells do not survive.Example 7: Assessment of Auditory Neuron Activity Using Calcium Influx Assays

[0326] At an excitatory synapse between hair cells and auditory neurons, glutamate release activates ionotropic receptors allowing rapid influx of ions into the post synaptic cell. Ca2+acts as a second messenger in response to glutamate, activating intracellular signaling leading to transcription of genes involved in synaptic plasticity. Calcium influx can be measured using fluorescent dye (for example Fluo-8, Abeam) which is added to the cells and becomes brighter upon calcium binding in response to Glutamatergic stimuli. The change in fluorescence can be monitored and quantified over time in response to external stimulation using Fluorescence microscopy and Fluorescence Plate readers (for example, Tecan).MethodsANP1 cells and culturing

[0327] 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 were then cultured in 96 wells supplemented with neurotropic growth factors. Media was changed 2-3 times a week. Before the assay, media was replaced to Hanks balanced salt solution (Gibco) supplemented with HEPES in the presence of CaCL2.Calcium Influx Assay

[0328] ANP1 cells were incubated with Fluo-8 florescent dye (Abeam). Following wash, florescence quantification was acquired using a Tecan Plate reader in the basal state and upon addition of a positive control (lonomycin, IUPAC name- (4 / ,6,S',8k, I OZ, l 2 / , l4 / , l 6E, 18A,19A,205',215)-19,21-Dihydroxy-22-{(25',2'A,55',5'5)-5'-[(lA)-l-hydroxyethyl]-2,5'- dimethyloctahydro-2,2'-bifuran-5-yl}-4,6,8,12,14,18,20-heptamethyl-l l-oxido-9-oxodocosa- 10,16-dienoic acid, Sigma) and glutamatergic agonists (Glutamate and AMP A, Tocris). Calcium influx induction was calculated as relative florescence according to the following Equation 1 :Additionally, DNQX (Alomone labs) was added to cells before AMPA to show that specific calcium influx was reduced upon its addition (FIGS. 15A-15B).Results

[0329] As illustrated by FIG. 15A, ANP1 cells showed a positive reaction (>100) to glutamate and AMPA stimuli. Upon the addition of DNQX (FIG. 15B), ANP1 cells show a reduction in calcium influx, demonstrating assay specificity. The results of six ANP1 experimental replicates are summarized in Table 6 below.Table 6.Equivalents

[0330] The foregoing description has been presented only for the purposes of illustration and is not intended to limit the disclosure to the precise form disclosed. The details of one or more embodiments of the disclosure are set forth in the accompanying description above. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. Other features, objects, and advantages of the disclosure will be apparent from the description and from the claims. In the specification and the appended claims, the singular forms include plural referents unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents and publications cited in this specification are incorporated by reference.

Claims

CLAIMSWhat is claimed is:

1. A method 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 75% 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.

2. The method of claim 1, wherein the pharmaceutical composition enhances efficacy of the cochlear implant.

3. A method 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 75% 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.

4. The method of any one of claims 1-3, wherein the pharmaceutical composition is administered prior to implantation of the cochlear implant.

5. The method of any one of claims 1-3, wherein the pharmaceutical composition is administered after implantation of the cochlear implant.

6. The method of any one of claims 1-3, wherein the pharmaceutical composition is administered at the same time as implantation of the cochlear implant.

7. The method of any one of claims 1-6, wherein the pharmaceutical composition is administered to an inner or middle ear of the subject.

8. The method of claim 7, wherein administration to the inner ear comprises administration to the cochlea.

9. The method of claim 8, wherein the pharmaceutical composition is administered to the Scala tympani, modiolus or spiral ganglion.

10. The method of any one of claims 1-9, wherein the pharmaceutical composition is administered via injection.

11. The method of claim 10, wherein the injection comprises inserting a cannula through a hole in the otic capsule, or inserting a cannula through a Round Window of the subject.

12. The method of any one of claims 1-11, wherein the auditory condition comprises conductive hearing loss, sensorineural hearing loss, central hearing loss, mixed hearing loss, auditory neuropathy spectrum disorder, central auditory processing disorder, tinnitus, or hidden hearing loss.

13. The method of any one of claims 1-12, wherein the auditory condition comprises loss of cochlear hair cells or cochlear neurons.

14. The method of any one of claims 1-12, wherein the auditory condition comprises loss of cochlear hair cells and loss of cochlear neurons.

15. A method of treating a subject with an auditory condition selected from the group consisting of conductive hearing loss, sensorineural hearing loss, mixed hearing loss, auditory neuropathy spectrum disorder and hidden hearing loss, comprising 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 both Nestin andS0X2;(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.

16. The method of claim 15, wherein the auditory condition comprises hidden hearing loss.

17. The method of claim 15 or 16, wherein the pharmaceutical composition is administered to the inner or middle ear of the subject.

18. The method of any one of claims 15-17, wherein the subject is further treated with a cochlear implant.

19. The method of any one of claims 1-18, wherein the pharmaceutical composition is cryopreserved, and the method comprises thawing the composition prior to administration.

20. The method of any one of claims 1-19, wherein greater than or equal to 85% of the cells in the population express both Nestin and SOX2.

21. The method of any one of claims 1-20, wherein greater than or equal to 85% of the cells in the population express P tubulin III.

22. The method of any one of claims 1-21, wherein greater than or equal to 15% of the cells in the population express TrkB.

23. The method of any one of claims 1-22, wherein less than or equal to 0.1% of the cells in the population express TRA-1-60 and / or SSEA5.

24. The method of any one of claims 1-23, wherein less than or equal to 20% of the cells in the population express PAX8 and / or PAX2.

25. The method of any one of claims 1-23, wherein the population of cells do not significantly express PAX2 and / or PAX8.

26. The method of any one of claims 1-25, wherein greater than or equal to 10% of the cells in the population express GluA4.

27. The method of any one of claims 1-26, wherein less than or equal to 10% of the cells in the population express Myo7A.

28. The method of any one of claims 1-27, 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 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.

29. The method of any one of claims 1-28, 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 P 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.

30. The method of claim 29, wherein between about 10% to 95% of the cells in the population express GluA4.

31. The method of claim 29 or 30, wherein between 0 to about 30% of cells in the population express Myo7A.

32. The method of any one of claims 1-31, wherein greater than or equal to 50% of the cells in the population express CD 133.

33. The method of any one of claims 1-32, wherein the population of auditory cells comprises: a. late otic neuronal progenitor (ONP) cells; b. late and mid ONP cells; or c. mid ONP cells, late ONP cells, mature auditory neurons, spiral ganglion neurons, or any combination thereof.

34. The method of any one of claims 1-33, wherein the percentage of cells expressing Nestin, SOX2, P tubulin III, TrkB, GluA4, TRA-1-60 and / or SSEA5 protein markers is determined by flow cytometry or immunofluorescence.

35. The method of any one of claims 1-34, wherein the population of auditory cells comprises sensory cell populations of the ear.

36. The method of claim 35, wherein the sensory cell populations are selected from the group consisting of hair cells, supporting cells, otic neuronal progenitor cells and sensory neuronal progenitor cells.

37. The method of any one of claims 1-36, wherein the population of cells comprises cellular aggregates, single cells, or a combination thereof.

38. The method of any one of claims 1-37, wherein the population of auditory cells comprises at least 100,000 cells.

39. The method of any one of claims 1-38, wherein the population of auditory cells comprises between 100,000 cells and 10 million cells.

40. The method of any one of claims 1-39, wherein between about 100,000 to about 1 million cells are administered to the subject.

41. The method of any one of claims 1-40, wherein between about 30 million cells per milliliter to about 700 million cells per milliliter are administered to the subject.

42. The method of any one of claims 1-41, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

43. The method of any one of claims 1-42, wherein the population of auditory cells is obtained by a method comprising: a) obtaining a culture of undifferentiated pluripotent cells; b) culturing the undifferentiated pluripotent cells under culture conditions sufficient to induce differentiation of the pluripotent cells to nonneuronal ectoderm cells; and c) culturing the cells from (b) under culture conditions sufficient to differentiate the non-neuronal ectoderm cells into auditory cells.