Methods and compositions for treating hearing loss

Abstract

Provided herein are methods for inducing cell differentiation of undifferentiated stem cells into cells capable of functioning as sensory cells of the ear, and pharmaceutical compositions for treating hearing conditions in a subject.

Description

[Technical field]

[0001] Related Applications This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 315,830, filed March 2, 2022, and No. 63 / 322,110, filed March 21, 2022. The contents of each of the foregoing patent applications are incorporated herein by reference in their entirety.

[0002] The present invention relates generally to compositions and methods for inducing cell differentiation of pluripotent stem cells into cells capable of functioning as auditory cells of the ear, and therapeutic methods using such auditory cells for the treatment of hearing conditions in a subject. [Background technology]

[0003] More than 5% of the population in developed countries suffer from significant hearing pathology or hearing loss, the severity of which ranges from mild difficulty in understanding speech to profound hearing loss. Hearing loss is age-related, with debilitating hearing loss occurring in approximately 4% of people under 45 and 34% of people over 65. In most cases, the cause is related to the degeneration and death of hair cells and associated spiral ganglion neurons.

[0004] The ear is made up of four main parts: the outer ear, the middle ear, the inner ear, and the transmission pathway to the hearing center in the brain. The inner ear is a very dense bone capsule that contains fluid that communicates with the middle ear. The ossicles within the middle ear (malleus, incus, and stapes) transmit sound energy from the eardrum to the oval window at the entrance to the cochlea of ​​the inner ear. The action of the stapes on the oval window exerts pressure on the fluid within the cochlea. The pressure is transmitted through the cochlea and ultimately causes a second window, the round window, to vibrate. The basilar membrane that defines the fluid-filled chamber of the cochlea transmits the vibrations to the organ of Corti. Hair cells are found in the epithelial lining of the inner ear (i.e., in the organ of Corti of the cochlea) and in the vestibular sensory epithelium of the saccule macula, utricle macula, and the cristae of the semicircular canals of the labyrinth. Cochlear hair cells send signals to the cochlear spiral ganglion, and the collective neuronal bodies transmit those signals to the cochlear nucleus in the brainstem.

[0005] Mechanosensitive sensory hair cells are the basis of hearing and balance. The inner ear contains approximately 13,000-15,000 cochlear hair cells and a similar number of vestibular sensory hair cells, which are the mechanoreceptors for hearing and balance. Because of their small number, molecular studies on hair cells are limited, and as a result, the molecular basis of their function is unknown. In addition to being few in number, hair cells are also sensitive to mechanical and chemical stimuli. Acoustic overstimulation, chemotherapy, side effects of aminoglycosides, the effects of aging, and an increasingly noisy environment contribute to hearing deterioration over time. As a result, hundreds of millions of patients worldwide are permanently debilitated by hearing loss and balance disorders. The main reason for the persistence of these chronic disorders is the fact that mammalian cochlear hair cells do not regenerate naturally, and the limited regeneration observed in the vestibular system is insufficient to restore function.

[0006] Auditory neuropathy is a hearing disorder in which the inner ear detects sound fine, but has problems transmitting signals from the ear to the brain. Current, up-to-date medical knowledge suggests that auditory neuropathy plays a major role in hearing impairment and hearing loss. Hearing relies on a series of complex steps that transform sound waves in the air into electrical signals. The auditory nerve then transmits these signals to the brain. The outer hair cells help amplify the sound vibrations that enter the inner ear from the middle ear. When hearing is functioning normally, the inner ear hair cells convert these vibrations into electrical signals that are transmitted to the brain as nerve impulses, which the brain interprets as sound. Auditory neuropathy can be caused by many factors, including (i) damage to the auditory nerve cells that transmit sound information from the cochlear hair cells, which are specialized sensory cells in the inner ear, to the brain, (ii) damage to the cochlear hair cells themselves, (iii) inheritance of a gene with a mutation in the auditory system or damage to the auditory system that can cause poor connections between the cochlear hair cells and the auditory nerve that leads from the inner ear to the brain, or (iv) damage to the auditory nerve itself. Researchers are still searching for an effective treatment for people with auditory neuropathy.

[0007] Several protocols have been developed for the differentiation of human pluripotent stem cells, such as human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), into auris sensory cells that can be used in cell therapy to treat hearing loss. Although these methods have been successful in producing auris sensory cells, challenges remain regarding the quality, scalability, and cost of goods associated with translating existing protocols into a clinical commercial-scale production process for such sensory cells.

[0008] Thus, there is a need for improved methods for differentiating pluripotent stem cells into auris sensory cell populations, and compositions comprising these differentiated pluripotent stem cells. Such methods should be readily scalable to produce sufficient quantities of auris sensory cells for cell therapy applications, while consistently and reproducibly producing targeted sensory cells and pharmaceutical formulations thereof with desired sensory cell quality characteristics for the treatment of hearing conditions. Summary of the Invention

[0009] The present disclosure provides pharmaceutical compositions comprising a population of auditory cells. In some embodiments, (a) 20% or more of the cells in the population express SOX2, (b) 10% or more of the cells in the population express β-tubulin III, (c) 5% or more of the cells in the population express TrkB, and (d) 1% or less of the cells in the population express TRA-1-60 and / or SSEA5. In some embodiments, the pharmaceutical compositions comprise a population of at least 100,000 cells. Some populations of cells include between 100,000 cells and 10 million cells. In some embodiments, the pharmaceutical compositions include a pharmaceutically acceptable carrier.

[0010] In some embodiments of the pharmaceutical compositions of the present disclosure, 30% or more of the cells in the population express SOX2. In some embodiments, 30% or more of the cells in the population express beta-tubulin III. In some embodiments, 20% or more of the cells in the population express TrkB. In some embodiments, 0.1% or less of the cells in the population express TRA-1-60 and / or SSEA5. In some embodiments, 50% or more of the cells in the population express nestin. In some embodiments, 30% or more of the cells in the population express PAX2. In some embodiments, 60% or less of the cells in the population express PAX8. In some embodiments, 10% or more of the cells in the population express GluA4. In some embodiments, 40% or less of the cells in the population express Myo7A. In some embodiments of the pharmaceutical compositions of the present disclosure, 50% or more of the cells in the population express CD133.

[0011] In some embodiments of the pharmaceutical compositions of the present disclosure, (a) 30% or more of the cells in the population express SOX2, (b) 30% or more of the cells in the population express PAX2, (c) 30% or more of the cells in the population express beta-tubulin III, (d) 20% or more of the cells in the population express TrkB, (e) 30% or more of the cells in the population express GluA4, (f) 20% or less of the cells in the population express Myo7A, and (d) 0.1% or less of the cells in the population express TRA-1-60 and / or SSEA5.

[0012] In some embodiments of the pharmaceutical compositions of the present disclosure, (a) about 30% to 95% of the cells in the population express SOX2, (b) about 10% to 60% of the cells in the population express β-tubulin III, (c) about 5% to 70% of the cells in the population express TrkB, and (d) 0 to about 1% of the cells in the population express TRA-1-60 and / or SSEA5. In some embodiments, about 30% to 95% of the cells in the population express PAX2. In some embodiments, about 5% to 95% of the cells in the population express GluA4. In some embodiments, 0 to about 30% of the cells in the population express Myo7A.

[0013] In some embodiments of the pharmaceutical compositions of the present disclosure, the population of auditory cells comprises non-neural ectoderm (NNE) cells, anterior placodal ectoderm (PPE) cells, early otic neural progenitor (ONP) cells, mid-stage ONP cells, late-stage ONP cells, or any combination thereof. In some embodiments, the population of auditory cells comprises a sensory cell population of the ear. In some embodiments, the sensory cell population is selected from the group consisting of hair cells, supporting cells, otic neural progenitor cells, and sensory neural progenitor cells.

[0014] In some embodiments of the pharmaceutical compositions of the present disclosure, the composition comprises a cryopreservation medium.

[0015] In some embodiments of the pharmaceutical compositions of the present disclosure, the composition comprises aggregates of cells, single cells, or a combination thereof.

[0016] In various embodiments, methods are provided for obtaining a population of auditory cells derived from undifferentiated pluripotent stem cells.

[0017] The present disclosure provides a method for obtaining a population of auditory cells derived from undifferentiated pluripotent stem cells, the method comprising: a) obtaining a culture of pluripotent stem cells; b) culturing the pluripotent cells for an initial period of time under culture conditions sufficient to induce differentiation of the pluripotent cells into non-neural ectodermal cells; and c) culturing the non-neural ectodermal cells of b) under culture conditions sufficient to differentiate the non-neural ectodermal cells into auditory cells.

[0018] The disclosure provides a method of producing a composition comprising a population of auditory cells, comprising: (a) culturing a population of undifferentiated pluripotent stem cells in a first cell culture medium comprising bone morphogenetic protein 4 (BMP4), fibroblast growth factor 2 (FGF2), and 4-[4-(2H-1,3-benzodioxol-5-yl)-5-(pyridin-2-yl)-1H-imidazol-2-yl]benzamide (SB431542) for 1 to 9 days under conditions sufficient to produce non-neural ectodermal (NNE) cells, thereby producing a cell population comprising NNE cells; and (b) culturing the cell population comprising NNE cells produced in step (a) in a first cell culture medium comprising SB431542, FGF2, and 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-1-yl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl}quinoline (LDN193189) for 1 to 9 days under conditions sufficient to produce preplacode ectoderm (PPE) cells, thereby producing a cell population comprising PPE cells; and (c) culturing the cell population comprising PPE cells produced in step (b) in a second cell culture medium comprising 6-((2-((4-(2,(d) culturing the cells in a third cell culture medium containing 4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-yl)amino)ethyl)amino)nicotinonitrile (CHIR99021), FGF2, and insulin-like growth factor 1 (IGF-1) for 5 to 9 days under conditions sufficient to produce early otic neural progenitor (ONP) cells, thereby producing a cell population containing early ONP cells; and (d) culturing the cell population containing early ONP cells produced in step (c) in a third cell culture medium containing sonic hedgehog (SHH), retinoic acid (RA), epidermal growth factor (EGF), FGF2, and IGF-1. (e) culturing the cell population comprising mid-late ONP cells produced in step (d) in a fifth cell culture medium comprising brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3) and IGF-1 for 3-65 days under conditions sufficient to produce late ONP cells, thereby producing a cell population comprising late ONP cells; and (f) collecting the cell population, thereby producing a composition comprising a population of auditory cells. In some embodiments, the first cell culture medium does not comprise FGF2.

[0019] In some embodiments, BMP4 is present in the first cell culture medium at a concentration of about 1-25 ng / mL. In some embodiments, BMP4 is present in the first cell culture medium at a concentration of 10 ng / mL. In some embodiments, SB431542 is present in the first cell culture medium and / or the second cell culture medium at a concentration of about 0.1-10 μM. In some embodiments, SB431542 is present in the first cell culture medium and / or the second cell culture medium at a concentration of about 1 μM. In some embodiments, FGF2 is present in the first, second, third and / or fourth cell culture medium at a concentration of about 1-25 ng / mL. In some embodiments, FGF2 is present in the first, second, third and / or fourth cell culture medium at a concentration of 10 ng / mL. In some embodiments, IWP-2 is present in the second cell culture medium at a concentration of about 0.5-10 μM. In some embodiments, IWP-2 is present in the second cell culture medium at a concentration of 2 μM. In some embodiments, LDN193189 is present in the second cell culture medium at a concentration of about 20-400 nM. In some embodiments, LDN193189 is present in the second cell culture medium at a concentration of 100 nM. In some embodiments, CHIR99021 is present in the third cell culture medium at a concentration of about 1-25 μM. In some embodiments, CHIR99021 is present in the third cell culture medium at a concentration of 6 μM. In some embodiments, IGF-1 is present in the third, fourth and / or fifth cell culture medium at a concentration of about 5-100 ng / mL. In some embodiments, IGF-1 is present in the third, fourth and / or fifth cell culture medium at a concentration of 50 ng / mL. In some embodiments, SHH is present in the fourth cell culture medium at a concentration of about 50-1000 ng / mL. In some embodiments, SHH is present in the fourth cell culture medium at a concentration of 500 ng / mL.

[0020] In some embodiments, RA is present in the fourth cell culture medium at a concentration of about 0.2-2 μM. In some embodiments, RA is present in the fourth cell culture medium at a concentration of 0.5 μM. In some embodiments, EGF is present in the fourth cell culture medium at a concentration of about 5-100 ng / mL. In some embodiments, EGF is present in the fourth cell culture medium at a concentration of 20 ng / mL. In some embodiments, BDNF is present in the fifth cell culture medium at a concentration of about 5-100 ng / mL. In some embodiments, BDNF is present in the fifth cell culture medium at a concentration of 10 ng / mL. In some embodiments, NT3 is present in the fifth cell culture medium at a concentration of about 5-100 ng / mL. In some embodiments, NT3 is present in the fifth cell culture medium at a concentration of 10 ng / mL. In some embodiments, the undifferentiated pluripotent stem cells include human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs).

[0021] In some embodiments, the culture of the undifferentiated pluripotent stem cells and / or auditory cells comprises dynamic culture conditions.

[0022] In some embodiments, auditory cells are grown in static two-dimensional (2D) adherent cultures in either small-scale platforms such as tissue culture flasks, or in larger-scale platforms such as multi-layer flasks.

[0023] In some embodiments, the method includes dynamic culture conditions in any one of steps (a)-(e).

[0024] In some embodiments, auditory cells are grown in static three-dimensional (3D) culture, such as cell aggregates on a non-adherent substrate.

[0025] In some embodiments, auditory cells are grown in dynamic two-dimensional (2D) large-scale platforms, such as microcarriers suspended in a bioreactor.

[0026] In some embodiments, auditory cells are grown in dynamic three-dimensional (3D) large-scale platforms, such as aggregates suspended in bioreactors.

[0027] In some embodiments, the suspension of auditory cells comprises aggregates of auditory cells.

[0028] In some embodiments, the method includes, prior to step (a), seeding the undifferentiated pluripotent stem cells in a monolayer at a density of 1,200-20,000 viable cells / cm2 and culturing the cells until the lactate concentration in the cell culture medium is 1.5-12.5 mM and the confluence rate is 5-80%.

[0029] In some embodiments, the population of undifferentiated pluripotent stem cells is cultured in a first cell culture medium for 3-7 days. In some embodiments, the cell population comprising NNE cells is cultured in a second cell culture medium for 3-7 days. In some embodiments, the cell population comprising PPE cells is cultured in a third cell culture medium for 7 days. In some embodiments, the cell population comprising early ONP cells is cultured in a fourth cell culture medium for 7 days. In some embodiments, culturing the cell population comprising mid-late ONP cells comprises (i) harvesting the cell population comprising mid-late ONP cells, (ii) seeding the harvested cell population in a vessel comprising a fifth cell culture medium, (iii) culturing the seeded cell population for 7-35 days, (iv) harvesting the cell population, (v) seeding the cell population in a vessel comprising the fifth cell culture medium, and (vi) culturing the cell population for 7-30 days.

[0030] In some embodiments, the method includes, prior to step (e), cryopreserving the cell population comprising mid-late ONP cells, followed by thawing and culturing in a fifth cell culture medium.

[0031] In some embodiments, the method includes cryopreserving the population of auditory cells.

[0032] In some embodiments, a method for obtaining a population of auditory cells includes obtaining a culture of pluripotent stem cells, culturing the pluripotent stem cells for an initial period of time under dynamic 2D or dynamic 3D culture conditions sufficient to induce differentiation of hESCs into non-neural ectodermal cells, and culturing the non-neural ectodermal cells under culture conditions sufficient to differentiate the non-neural ectodermal cells into auditory cells.

[0033] In some embodiments, a method for obtaining a population of auditory cells includes obtaining a dynamic culture of undifferentiated hESCs, culturing the undifferentiated hESCs under culture conditions sufficient to induce differentiation of the hESCs into mid-late ONP cells, for example with a combination of cell culture media described herein, and preparing a cryopreserved mid-late ONP cell composition for generating an intermediate cell bank. In some embodiments, the method includes characterizing and releasing thawed cells from the bank, and further culturing the mid-late ONP cells under dynamic culture conditions sufficient to differentiate the mid-late ONP cells into auditory cells.

[0034] In some embodiments, the method for obtaining a population of auditory cells comprises obtaining a dynamic culture of undifferentiated hESCs, culturing the undifferentiated hESCs for an initial period of time under conditions sufficient to induce differentiation of the hESCs into non-neural ectodermal cells, culturing the non-neural ectodermal cells under culture conditions sufficient to cause the non-neural ectodermal cells to differentiate into preplacode ectodermal cells, culturing the preplacode ectodermal cells under culture conditions sufficient to cause the preplacode ectodermal cells to differentiate into early otic neural progenitor cells, culturing the early otic neural progenitor cells under culture conditions sufficient to cause the early otic neural progenitor cells to differentiate into mid-stage otic neural progenitor cells, culturing the mid-stage otic neural progenitor cells under culture conditions sufficient to cause the mid-stage otic neural progenitor cells to differentiate into late-stage otic neural progenitor cells, and using the same to prepare a cryopreserved cell composition to generate an intermediate somatic cell bank of mid-stage and late-stage otic neural progenitor cells, characterizing and releasing thawed cells from the bank and further culturing the cells under dynamic culture conditions sufficient to cause the differentiation into sensory neural progenitor cells, such as spiral ganglion cells.

[0035] The present disclosure provides methods for preparing cryopreserved auditory cell compositions for administration to a subject immediately after thawing.

[0036] In some embodiments, a method of preparing a cryopreserved auditory cell composition includes (a) suspending auditory cells in a cryopreservation medium to form a cell suspension, (b) storing the cell suspension at cryopreservation temperatures (−80° C. or below or −140° C. or below), and (c) thawing the cryopreserved suspension for administration to a subject. In embodiments, the cryopreserved auditory cell composition is prepared for long-term storage for reseeding to continue the process immediately after thawing. In embodiments, the cryopreserved auditory cell composition is prepared for administration to a subject immediately after thawing.

[0037] The present disclosure provides a method of treating a subject having an auditory condition, comprising administering a therapeutic amount of a pharmaceutical composition of the present disclosure to the subject's inner or middle ear.

[0038] The disclosure provides a method of treating a subject having an auditory condition, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising a population of auditory cells, wherein (a) more than 20% of the cells in the population express SOX2, (b) more than 10% of the cells in the population express beta-tubulin III, (c) more than 5% of the cells in the population express TrkB, and (d) less than 1% of the cells in the population express TRA-1-60 and / or SSEA5, wherein the composition is administered to the subject's inner ear or middle ear.

[0039] In some embodiments of the therapeutic methods of the present disclosure, (a) 30% or more of the cells in the population express SOX2, (b) 30% or more of the cells in the population express PAX2, (c) 30% or more of the cells in the population express beta-tubulin III, (d) 20% or more of the cells in the population express TrkB, (e) 30% or more of the cells in the population express GluA4, (f) 20% or less of the cells in the population express Myo7A, and (d) 0.1% or less of the cells in the population express TRA-1-60 and / or SSEA5.

[0040] In some embodiments, the composition is administered by injection. In some embodiments, the injection comprises administration to the scala tympani or modiolus of the subject. In some embodiments, the injection comprises cannulating through a hole in the otic capsule or cannulating through the round window. In some embodiments, about 100,000 to 1 million cells are administered to the subject.

[0041] The present disclosure provides compositions for use in treating any auditory condition in a subject, including pharmaceutical compositions comprising a population of auditory cells as described herein.

[0042] The present disclosure provides compositions for use in the manufacture of a medicament for the treatment of any auditory condition in a subject, including pharmaceutical compositions comprising a population of auditory cells as described herein.

[0043] The present disclosure provides kits that include the pharmaceutical compositions described herein.

[0044] In some embodiments, a pharmaceutical composition is provided for administration to a subject, the pharmaceutical composition comprising the auditory cells described herein and a cryopreservation medium.

[0045] In embodiments, a pharmaceutical composition is provided for administration to a subject, the pharmaceutical composition comprising auditory cells as described herein and a cryopreservation medium.

[0046] 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, or tinnitus.

[0047] In some embodiments, the pharmaceutical composition is administered to the inner ear.

[0048] In some embodiments, the pharmaceutical composition is administered to the middle ear.

[0049] In some embodiments, the present disclosure provides a method for replacing an auditory nerve in a subject in need of such replacement, comprising administering to the subject a therapeutically effective amount of any of the pharmaceutical compositions described herein.

[0050] In some embodiments, the present disclosure provides a method for enhancing an existing but damaged auditory nerve population in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any of the pharmaceutical compositions described herein. [Brief description of the drawings]

[0051] [Figure 1A] 1 shows the auditory neuron (AN) differentiation stages of an exemplary method of the present disclosure, including a scheme for directed differentiation of human embryonic stem cells (hESCs) into placode-derived spiral ganglion-like sensory neurons, including exemplary differentiation timeframes (DTFs) along the process. [Figure 1B] 1 shows growth factors at different stages of auditory neuron differentiation for an exemplary method of the present disclosure. hESCs are exposed to different combinations of growth factors for directed differentiation into otic neural progenitor cells. Differentiation factors used in each DTF are shown. [Diagram 2] 1 shows images of auditory neuron (AN) morphology at the end of each DTF described in Example 1. [Diagram 3] 1 shows images of human embryonic stem cell (hESC) morphology prior to the initiation of differentiation as described in Example 2. [Figure 4] 1 shows images of cell morphology of DTF#1 during differentiation of cells into AN as detailed in Table 10 and Example 3. [Diagram 5] 1 shows images of cell morphology of DTF#2 during differentiation of cells into AN as detailed in Table 12 and Example 4. [Figure 6] 1 shows images of cell morphology exposed to different growth factor (GF) combinations in DTF#1 as described in Table 14 and Example 5. [Figure 7]FIG. 16 shows images of the morphology of cells replated in different culture systems (static, dynamic, single cell, aggregates) during maturation of otic neural progenitor cells (ONPs) as described in Table 16 and Example 6. [Figure 8] 1 shows images of molten aggregates and the morphology of aggregates during continued culture in different culture systems (static, dynamic) as detailed in Table 17 and Example 7. [Figure 9A] FIG. 1 shows an exemplary scheme for directed differentiation of human embryonic stem cells into placode-derived spiral ganglion-like sensory neurons. [Figure 9B] FIG. 1 shows a scheme for the directed differentiation of human embryonic stem cells into otic neural progenitor cells. [Figure 9C] An exemplary cell culture scheme and protocol for directed differentiation of human embryonic stem cells into otic neural progenitor cells according to the present disclosure is shown. hESCs are cultured for 0-3 days using iMatrix-511 directly coated vessels (1-step seeding). Cell culture and differentiation may also proceed on directly coated MCs in a PB wheel bioreactor (1-step seeding). Differentiation may be continued for 3-32 days, optionally substituting SB431542 (TGF-beta inhibitor) for NIC, according to Needham and Nayagam 2014. ONP production can be achieved by neural crest induction (using only FGF and EGF), possibly combined with FBi protocols (inhibition of FGF and BMP signaling). At day 26, the cells may be cryopreserved to create an intermediate somatic cell bank (ICB, LONP stage after culture in a large bioreactor). Single cell survival and in vivo maturation for transplantation can be tested, and cellular impurities can also be characterized. Cells harvested on day 25 can be seeded for spheroid formation by seeding in a PBS wheel for 7 days as single cell culture. Spheroids can be formed in Aggrewell plates for homogenous spheroids (final product). The end result of the exemplary protocol can result in a thaw and inject (TAI) formulation (single cell / spheroid). [Figure 10]1 shows an exemplary batch release profile of sensory neural progenitor cells according to the present disclosure. [Figure 11] 1 shows an exemplary batch release marker profile of sensory neural progenitor cells according to the present disclosure. [Figure 12] 1 shows an exemplary in-process control (IPC) test scheme for directed differentiation of human embryonic stem cells into otic neural progenitor cells according to the present disclosure. [Figure 13] 1 shows an exemplary IPC and marker profile of sensory neural progenitor cells according to the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] This specification is not intended to be a detailed enumeration of all the different ways in which the disclosure may be implemented or all the features that may be added to the 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 some embodiments of the disclosure may exclude or omit any feature or combination of features described herein. In addition, numerous modifications and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the present disclosure, and do not depart from the disclosure. In other instances, well-known structures, interfaces, and processes have not been shown in detail so as not to unnecessarily obscure the invention. No part of this specification is intended to result in a denial of any portion of the entire scope of the invention. Thus, the following description is intended to illustrate certain aspects of the disclosure, but is not intended to exhaustively specify all permutations, combinations, and variations thereof.

[0053] 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 terms used in the description of the present disclosure herein are for the purpose of describing particular embodiments only and are not intended to be limiting of the present disclosure.

[0054] All publications, patent applications, patents, and other references cited herein are incorporated by reference in their entirety.

[0055] It is specifically contemplated that the various features of the present disclosure described herein can be used in any combination, unless the context indicates otherwise. Further, it is contemplated that in some embodiments of the present disclosure, the present disclosure can exclude or omit any feature or combination of features described herein.

[0056] The methods disclosed herein may include one or more steps or actions for achieving the described method. The steps and / or actions of the methods may be interchanged with one another without departing from the scope of the invention. In other words, unless a specific order of steps or actions is required for proper operation of an embodiment, the order and / or use of specific steps and / or actions may be modified without departing from the scope of the invention. [Table 1]

[0057] definition As used in the description of this 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.

[0058] As used herein, "and / or" refers to and includes every possible combination of one or more of the associated listed items, as well as the lack of combination when interpreted in the alternative ("or").

[0059] The terms "about" and "approximately" as used herein when referring to measurable values ​​such as percentages, densities, volumes, and the like, are meant to encompass variations of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount.

[0060] 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 "about X to Y" mean "about X to about Y".

[0061] "Cell" as used herein refers to a cell that performs metabolic or other functions sufficient to maintain or replicate its genomic DNA. Cells can be identified by methods well known in the art, including, for example, the presence of an intact membrane, staining with a particular dye, the ability to produce progeny, or, in the case of gametes, the ability to combine with a second gamete to produce viable progeny. Cells can 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, such as mammalian cells, insect (e.g., spodoptera) cells, and human cells. Cells can be useful when they are naturally non-adherent or have been treated to prevent them from adhering to surfaces, for example, by trypsinization.

[0062] "Comprising" or "comprising" is intended to mean that the compositions and methods include the recited elements but do not exclude other elements. When used to define compositions and methods, "consisting essentially of" shall mean excluding other elements that are essential to the combination for the stated purpose. Thus, a composition consisting essentially of the elements defined herein will not exclude other materials or steps that do not materially affect the basic and novel feature(s) of the claimed invention. "Consisting of" shall mean excluding more than trace amounts of other component elements and substantial method steps. Embodiments defined by each of these transitional terms are within the scope of this disclosure.

[0063] An "effective amount" is an amount sufficient for the composition to achieve a stated purpose (e.g., achieve the effect for which the composition is administered, treat a disease, reduce enzyme activity, increase enzyme activity, attenuate a signal transduction pathway, or alleviate one or more signs or symptoms of a disease or condition) compared to the absence of the composition. An example of an "effective amount" is an amount sufficient to contribute to the treatment, prevention, or alleviation of a symptom or symptoms of a disease, which may also be referred to as a "therapeutically effective amount." "Alleviation" of a symptom or symptoms (and grammatical equivalents of this phrase) refers to a reduction in the severity or frequency of the symptom(s), or the elimination of the symptom(s). A "prophylactically effective amount" of a drug (e.g., a cell as described herein) is an amount of drug that, when administered to a subject, will have an intended prophylactic effect, such as preventing or delaying the onset (or recurrence) of an injury, disease, condition, or condition, or reducing the likelihood of onset (or recurrence) of an injury, disease, condition, or condition, or alleviating the symptoms thereof. A complete preventive effect may not necessarily occur by administration of one dose, but may occur only after administration of a series of doses. Thus, a prophylactically effective amount can be administered in one or more doses. "Activity-reducing amount" as used herein refers to the amount of antagonist required to reduce the activity of an enzyme compared to the absence of antagonist. "Function-perturbing amount" as used herein refers to the amount of antagonist required to perturb the function of an enzyme or protein compared to the absence of antagonist. The exact amount depends on the purpose of treatment and can be ascertained by those skilled in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (vols.1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).For any composition described herein, the therapeutically effective amount can be initially determined from cell culture assays. The target concentration is the concentration (e.g., cell concentration or cell number) of the active composition(s) that can achieve the methods described herein, as measured using the methods described herein or known in the art.

[0064] "Control" or "control experiment" is used according to its plain and ordinary meaning to refer to an experiment in which an experimental subject or reagent is treated as a parallel experiment, except for the omission of an experimental procedure, reagent, or variable. In some instances, a control is used as a standard of comparison in the evaluation of experimental effects. In some embodiments, a control is a measurement of protein activity in the absence of a composition described herein (including embodiments and examples).

[0065] As used herein, "implantation" or "implantation" refers to the administration of a cell population to a target tissue using an appropriate delivery technique (eg, using an injection device).

[0066] "Pharmaceutically acceptable excipient" and "pharmaceutically acceptable carrier" refer to substances that aid in the administration and absorption of an active substance by a subject and can be included in the compositions of the present disclosure without causing significant adverse toxicological effects to the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, saline, lactated Ringer's solution, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavorings, salt solutions (such as Ringer's solution), alcohols, oils, gelatin, carbohydrates such as lactose, amylose, or starch, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidine, and colorings. Such preparations are sterilized and may be mixed with auxiliary agents, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, and / or flavorings, that do not adversely react with the compositions of the present disclosure. Those of skill in the art will appreciate that other pharmaceutical excipients are useful in the present disclosure.

[0067] As used herein, a "patient" or "subject" refers to an organism suffering from or susceptible to a disease or condition that can be treated by administration of a pharmaceutical composition or implantable biodegradable scaffold as provided herein. Non-limiting examples include humans, other mammals, cows, rats, mice, dogs, monkeys, goats, sheep, dairy cows, deer, and other non-mammals. In some embodiments, the patient is a human.

[0068] As used herein, a "subject in need thereof" refers to an animal or human having damaged tissue in the central nervous system. In one embodiment, the animal or human is experiencing reduced motor function.

[0069] As used herein, "treatment" or "treating" with respect to a condition or disease is an approach to obtain a beneficial or desired result, preferably a clinical result, etc., after a condition or disease appears in a subject. Beneficial or desired results with respect to a disease include, but are not limited to, one or more of the following: amelioration of the condition associated with the disease, cure of the disease, reduction of the severity of the disease, delay of the progression of the disease, alleviation of one or more symptoms associated with the disease, improvement of the quality of life of a person suffering from the disease, prolongation of survival, and any combination thereof. Similarly, 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: amelioration of the condition, cure of the condition, reduction of the severity of the condition, delay of the progression of the condition, alleviation of one or more symptoms associated with the condition, improvement of the quality of life of a person suffering from the condition, prolongation of survival, and any combination thereof.

[0070] As used herein, "inner ear sensory hair cells" or simply "hair cells" refer to mechanosensory hair cells of the cochlea (auditory system) and mechanosensory hair cells of the saccule, saccule, crista ampullaris, and semicircular canals (vestibular system), which contribute to the detection and amplification of sound and the maintenance of balance, respectively. Hair cells resemble columnar cells, each bearing a bundle of stereocilia on their apical surface. Bending of the stereocilia opens mechanically gated ion channels, which allow small positively charged ions (mainly potassium and calcium) to enter the hair cell. Unlike many other electrically active cells, hair cells themselves do not generate action potentials. Rather, the influx of positive ions depolarizes the cell, resulting in a receptor potential. Thus, hair cells typically exhibit graded electrical responses rather than the action potential spikes typical of other nerve cells. Hair cells may express detectable levels of one or more of the following markers: atonal homolog 1 (Atoh1 / MATH1 / HATH1), myosin VI (MYO6), myosin VIIA (MYO7A), Espin (ESPN), myosin heavy chain 3 (MYH2), cadherin 23 (CDH23), protocadherin 15 (PCDH15), otoferlin (OTOF), and prestin (SLC26A5).

[0071] As used herein, "inner ear supporting cells" or simply "supporting cells" refers to cells that contribute to the complex structural and functional properties of the cochlea, such as Deiters (phalangeal) cells, Hensen cells, Claudius cells, Boettcher cells, pillar cells, marginal cells, etc., as well as cells of the saccule, utricle, crista ampullaris, and semicircular canals. Supporting cells are identifiable by short microvilli on their apical cell surface. Furthermore, they are found in close proximity to hair cells, i.e., they are found directly adjacent to and in clusters with hair cells. Supportive cells may express detectable levels of one or more of the following markers: cyclin-dependent kinase inhibitor 1B (CDKN1B, p27(KIP1)), prospero homeobox 1 (PROX1), otoancorin (OTOA), musashi homolog 1 (MSI1), SRY-box 2 (SOX2), gap junction protein beta 2, 26 kDa (connexin 26), gap junction protein beta 6, kDa (connexin 30), gap junction protein alpha 1, 43 kDa (connexin 43), hairy / enhancer-of-split related with YRPW motif 2 (HEY2).

[0072] As used herein, "pluripotent stem cells" or "pluripotent cells" refer to cells that have the ability to differentiate into any type of cell in an organism. Pluripotent cells can form teratomas and contribute to ectodermal, mesodermal, or endodermal tissues in an organism. Examples of pluripotent stem cells are embryonic stem (ES) cells, embryonic germ stem (EG) cells, and induced pluripotent stem (iPS) cells.

[0073] As used herein, "embryonic stem cell" or "ES cell" refers to a cell that a) can self-renew, b) can differentiate to produce any type of cell of an organism, and c) is derived from the inner cell mass of the blastula of a developing organism. ES cells can be cultured for extended periods of time while maintaining the ability to differentiate into any type of cell of an organism. In culture, ES cells typically grow as flat colonies with large nuclear-cytoplasmic ratios, well-defined borders, and prominent nuclei. In addition, ES cells express stage-specific embryonic antigen (SSEA) 5 (SSEA-5), POU class 5 homeobox 1 (Oct-4), Nanog homeobox (Nanog), SSEA-3, SSEA-4, TRA-1-60 antigen (TRA-1-60), TRA-1-81 antigen (TRA-1-81), and alkaline phosphatase, but not SSEA-1. Examples of methods for generating and characterizing ES cells can be found, for example, in US Pat. Nos. 7,029,913, 5,843,780, and 6,200,806, the disclosures of which are incorporated herein by reference.

[0074] As used herein, "embryonic germ stem cells", "embryonic germ cells" or "EG cells" refer to cells that a) can self-renew, b) can differentiate to produce any type of cell in an organism, and c) are derived from germ cells and germ cell precursors, such as primordial germ cells, i.e., the cells that give rise to sperm and eggs. Embryonic germ cells (EG cells) are believed to have similar properties to embryonic stem cells, as described above. Examples of methods for generating and characterizing EG cells can be found, for example, in U.S. Patent 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.

[0075] As used herein, "induced pluripotent stem cells" or "iPS cells" refer to cells that a) can self-renew, b) can differentiate to produce any cell type of an organism, and c) are derived from somatic cells. iPS cells have an ES cell-like morphology and grow as flat colonies with large nucleus-to-cytoplasm ratios, well-defined borders, and prominent nuclei. In addition, iPS cells express one or more important pluripotency markers known to those skilled in the art, including, but not limited to, alkaline phosphatase, SSEA3, SSEA4, SRY-box transcription factor 2 (Sox2), Oct-4, Nanog, TRA-1-60, TRA-1-81, teratoma-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 (Cyp26a1), telomerase reverse transcriptase (TERT), and ZFP42 zinc finger protein (ZFP42). iPS cells can be generated by providing cells with "reprogramming factors," i.e., one or more biologically active factors, i.e., a cocktail thereof, that act on the cell to alter transcription, thereby reprogramming the cell to pluripotency. These reprogramming factors can be provided to the cells individually or as a single composition, i.e., as a premix composition of reprogramming factors. These factors can be provided in the same molar ratio or different molar ratios. These factors can be provided once or multiple times during the process of culturing the cells of the present invention. Examples of methods for generating and characterizing iPS cells can be found, for example, in application numbers US20090047263, US20090068742, US20090191159, US20090227032, US20090246875, and US20090304646, the disclosures of which are incorporated herein by reference.

[0076] It will be 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 cell registry www.grants.nih.govstem_cells / or other hESC registries. Non-limiting examples of commercially available embryonic stem cell lines include H1, 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, WIBR2, WIBR3, WIBR4, WIBRS, WIBR6, HUES 45, Shef 3, Shef 6, BINhem19, BJNhem20, SAGO 1, and SAOO1.

[0077] As used herein, "somatic cell" refers to any cell in an organism that does not normally give rise to any type of cell in the organism in the absence of experimental manipulation. In other words, a somatic cell is a cell that is sufficiently differentiated to not naturally give rise to cells of all three germ layers of the body, namely ectoderm, mesoderm, and endoderm. For example, somatic cells include both neural cells and neural progenitor cells, the latter of which can self-renew and naturally give rise to all or some cell types of the central nervous system, but cannot give rise to cells of mesodermal or endodermal lineages.

[0078] As used herein, "endoderm" refers to the germ layer formed during animal embryonic development that gives rise to the digestive tract, respiratory tract, endocrine glands and organs, certain structures of the auditory system, and certain structures of the urinary system.

[0079] As used herein, "mesoderm" refers to the germ layer formed during animal embryonic development that gives rise to muscle, cartilage, bone, dermis, the reproductive system, adipose tissue, connective tissue of the intestine, peritoneum, certain structures of the urinary system, mesothelium, notochord, and spleen.

[0080] As used herein, "ectoderm" refers to the germ layer formed during animal embryonic development that gives rise to the nervous system, tooth enamel, epidermis, hair, nails, and the lining of mucosal tissue. During embryonic development, the embryonic ectoderm is patterned into lineage progenitors of the neural plate, neural crest, placode, and epidermis. "Non-neuronal ectoderm" or "non-neural ectoderm" refers to ectodermal cells that form non-neural structures such as the epidermis.

[0081] As used herein, "anterior ectoderm" refers to the region of ectodermal germ layer at the anterior, or "rostral" end of the embryo, i.e., toward the head region. The anterior ectoderm includes the anterior placode ectoderm as well as adjacent tissues, such as presumptive early ectoderm, presumptive neural crest, and neural tissue. Ectoderm can be induced to become anterior ectoderm by contact with rostralizing factors such as IGF1 or insulin.

[0082] As used herein, "preplacode ectoderm" refers to the narrow band of cells of the anterior ectoderm that surrounds the anterior neural plate at the end of gastrulation and gives rise to the head placode, which gives rise to the paired sensory structures of the head. Preplacode ectoderm cells may express detectable levels of one or more of the following 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). Preplacodal ectodermal cells are competent to respond to otic induction, i.e., induction of otic progenitor cells by culturing in the presence of FGF, resulting in upregulation of expression of p75, Pax8, Pax2, GATA3 and Sox10. Cells expressing preplacodal ectodermal markers and having characteristics of preplacodal ectoderm can be derived from undifferentiated pluripotent stem cells using the methods described herein.

[0083] As used herein, "otic progenitor cells" or "otic neural progenitor cells" refers to somatic cells that a) can self-renew and b) can differentiate to generate 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. Additionally, 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), and claudin 14 (CLDN14). Otic neural progenitor cells can be divided into early, intermediate, and late otic progenitor cells based on marker expression, as described herein.

[0084] As used herein, "stromal cells" refers to connective tissue cells of any organ, such as fibroblasts, pericytes, endothelial cells, etc.

[0085] As used herein, the term "microcarrier" or "MC" refers to a suspending support matrix that allows adherent cells to grow in dynamic or static cell culture and can be maintained in suspension with gentle mixing. Microcarriers can be composed of, but are not limited to, polystyrene, surface-modified polystyrene, chemically modified polystyrene, cross-linked dextran, cellulose, acrylamide, collagen, alginate, gelatin, glass, DEAE-dextran, or combinations thereof. Microcarriers can be coated with biological support matrices, including but not limited to laminin, Matrigel®, collagen, polylysine, poly-L-lysine, poly-D-lysine, vitronectin, fibronectin, tenascin, dextran, peptides, or combinations thereof. Many different types of microcarriers are commercially available, including but not limited to HyQSphere (HyClone™), Hillex (SoloHill Engineering), and low-concentration Synthemax® II (Corning) brands. Microcarriers can be made from cross-linked dextran, such as Cytodex® brand (GE Healthcare). Microcarriers can be spherical and smooth, have a microporous surface, such as CYTOPORE™ brand (GE Healthcare), and / or be rod-shaped carriers, such as DE-53 (Whatman™). Microcarriers can be impregnated with magnetic particles, which can help separate cells from beads (e.g., GEM particles from Global Cell Solutions). Chip-based microcarriers, such as μHex products (Nunc), provide a flat surface for cell growth while maintaining the high surface area to volume ratio of traditional microcarriers. Microcarrier properties can greatly affect proliferation rates and cell multipotency or pluripotency.

[0086] As used herein, "dynamic culture" refers to cell culture that, unlike cell cultures performed under static conditions (e.g., Petri dishes), is performed with deliberate active movement, enhancing mass transfer and mechanotransduction effects (e.g., bioreactors), often resulting in higher functional cell numbers. For example, in dynamic differentiation processes, bioreactors directly apply mechanical forces to create physiological conditions and promote differentiation into specific cell lineages. In dynamic cultures, cells may have a more uniform environment, which cannot be provided by diffusion alone in static cultures. For example, cells growing at the periphery of the vessel and cells growing inside the vessel. In addition, static cultures may generate diverse biologically distinct niches, as microenvironments with various cell densities are maintained, which cannot be maintained in dynamic cultures.

[0087] As used herein, "dynamic two-dimensional" or "dynamic 2D" refers to cells that grow and form a monolayer on a microcarrier. For example, cells are cultured in a dynamic culture (e.g., a bioreactor) that includes a suspended adhesive agent (e.g., a microcarrier) that allows the cells to adhere to form a dynamic 2D culture.

[0088] As used herein, "dynamic three-dimensional" or "dynamic 3D" refers to cells growing as aggregates in suspension, such as cell cultures in artificially created environments that allow biological cells to grow or interact with their surroundings in all three dimensions. Unlike 2D environments, in 3D cell cultures, cells in vitro can grow in all directions, similar to in vivo. These three-dimensional cultures can be grown, for example, in bioreactors, miniature capsules where cells can grow into spheroids, or 3D cell aggregates.

[0089] As used herein, "sensory neural progenitor cells" refers to multipotent cells with the ability to self-renew that initially generate radial glial progenitor cells that generate the neurons and glia of the nervous system of all animals during embryonic development. Although sensory neural progenitor cells can occur naturally, their cellular composition differs from cells derived from undifferentiated pluripotent stem cells using the methods disclosed herein.

[0090] As used herein, "auditory neuron(s)" (abbreviated AN) or "auditory cell(s)" refers to sensory cell populations of the ear including, but not limited to, one or more of hair cells, supporting cells, otic progenitor cells, sensory neuron progenitor cells, etc. The term "auditory cell" may in some cases refer to a mixed cell population encompassing any combination of the above cell types in any ratio.

[0091] As used herein, "auditory disorder" or "auditory condition" or "hearing disorder" or "hearing condition" refers to a condition or disorder including, but not limited to, conductive hearing loss, sensorineural hearing loss, central hearing loss, mixed hearing loss, auditory neuropathy spectrum disorder, central auditory processing disorder, and tinnitus.

[0092] As used herein, "conductive hearing loss" refers to impaired transmission of sound waves from the ear canal to the bones of the middle ear.

[0093] As used herein, "sensorineural hearing loss" refers to pathological changes in the structures of the inner ear or the auditory nerve.

[0094] As used herein, "central hearing loss" refers to a pathological condition above the junction of the auditory nerve and the brainstem.

[0095] As used herein, "mixed hearing loss" refers to a subject who has both conductive and sensorineural hearing loss.

[0096] As used herein, "auditory neuropathy spectrum disorder" refers to a type of sensorineural hearing loss in which the auditory nerve fails to send coherent messages to the hearing center of the brain.

[0097] As used herein, "central auditory processing disorder" refers to a defect in the neural processing of auditory information in the central auditory nervous system.

[0098] Methods for generating populations of auditory cells The present disclosure provides a method for producing a population of auditory cells from undifferentiated pluripotent stem cells. The method includes culturing a population of undifferentiated pluripotent stem cells in different combinations of growth factors and growth factor inhibitors in a series of steps that induce the differentiation of the undifferentiated pluripotent stem cells to an auditory neuronal fate through a series of differentiation steps. In an exemplary differentiation pathway, human embryonic stem cells (hESCs) are induced to differentiate into non-neural ectoderm (NNE) cells, which are induced to differentiate into anterior placode ectoderm (PPE) cells, which are induced to differentiate into early otic neural progenitor (ONP) cells, mid-otic progenitor cells, late otic progenitor cells, and spiral ganglion neurons. The resulting population of cells may include a mixture of cell types. However, cells at later stages of the pathway may predominate, with only few or no hESCs remaining. Without wishing to be bound by theory, it is believed that compositions including mixed cell populations may be more suitable as therapeutic agents for hearing diseases and disorders than compositions including homogenous populations of cells. This is because a broader range of cell types increases the number of niches that the cells can engraft when administered to a subject, thereby increasing the number of fates that the cells can adopt following administration.

[0099] Methods for Proliferating and Maintaining Human Embryonic Stem Cells (hESCs) In one aspect, provided herein is a method for expanding and maintaining human embryonic stem cells (hESCs) in an undifferentiated, pluripotent state, the method comprising: (a) simultaneously combining human embryonic stem cells and extracellular matrix components (ECM) in growth medium in a tissue culture flask for static expansion; and (b) culturing the adherent hESCs for a period of time.

[0100] In some embodiments, statically grown cultured human embryonic stem cells are non-enzymatically harvested using ReLeSR™ and cultured in mTeSR™ plus medium on iMatrix-511 coated vessels. In some embodiments, the hESCs are further expanded by repeating steps (a) and (b).

[0101] In some embodiments, statically grown cultured human embryonic stem cells are harvested and further differentiated.

[0102] In one aspect, provided herein is a method for growing and maintaining human embryonic stem cells (hESCs) in an undifferentiated, pluripotent state, the method comprising: (a) simultaneously combining human embryonic stem cells, extracellular matrix components (ECM), and microcarriers in a growth medium to form suspension growth complexes; and (b) culturing the suspension growth complexes for a period of time.

[0103] In some embodiments, the cultured human embryonic stem cells in the suspension growth complexes are harvested and further expanded by repeating steps (a) and (b).

[0104] In some embodiments, the cultured human embryonic stem cells in the suspension growth complexes are harvested and further differentiated.

[0105] Human embryonic stem cells can be isolated from human blastocysts. Human blastocysts are typically obtained from human in vivo preimplantation embryos or in vitro fertilized (IVF) embryos. Alternatively, single-cell human embryos can be grown to the blastocyst stage. In 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 trophectoderm cells are lysed and removed from the intact ICM by gentle pipetting. The ICM is then plated in a tissue culture flask containing an appropriate medium that allows its growth. After 9-15 days, the ICM-derived outgrowths are dissociated into clumps by either mechanical dissociation or enzymatic digestion, and the cells are then replated in fresh tissue culture medium. Colonies that exhibit undifferentiated morphology are individually selected by micropipette, mechanically dissociated into clumps, and replated. The resulting ES cells are then split periodically every 4-7 days. For details on the preparation of human ES cells, see Reubinoff et al. Nat Biotechnol 2000, May:18(5):559; Thomson et al., [US 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].

[0106] Furthermore, ES cells have been used in mouse (Mills and Bradley, 2001), golden hamster [Doetschman et al., 1988, Dev Biol. 127:224-7], rat [Iannaccone 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], and several livestock species [Notarianni et al., 1991, J Reprod Fertil Suppl. 43:255-60; Wheeler 1994, Reprod Fertil Dev. 6:563-8; Mitalipova et al. al., 2001, Cloning. 3:59-67] and other species, including non-human primate species (rhesus monkeys and marmosets) [Thomson et al., 1995, Proc Natl Acad Sci US A. 92:7844-8; Thomson et al., 1996, Biol Reprod. 55:254-9].

[0107] Expanded blastocyst cells (EBCs) can be obtained from blastocysts at least 9 days post-fertilization at the pre-gastrulation stage. Prior to culturing the blastocysts, the zona pellucida is digested [e.g., by Tyrode's acid solution (Sigma Aldrich, St Louis, MO, USA)] to expose the inner cell mass. The blastocysts are then cultured in vitro as whole embryos for at least 9 to no more than 14 days post-fertilization (i.e., before the gastrulation event) using standard embryonic stem cell culture methods.

[0108] 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 involves removing a single cell from an embryo during an in vitro fertilization process. The embryo is not destroyed in the process.

[0109] EG (embryonic germ) cells are prepared from primordial germ cells obtained from fetuses of about 8-11 weeks gestation (for human fetuses) using laboratory techniques known to those skilled in the art. The genital ridges are excised, cut into small pieces, and then disaggregated into cells by mechanical dissociation. The EG cells are then grown in tissue culture flasks containing the appropriate medium. The cells are cultured with daily changes of medium until a cell morphology consistent with EG cells is observed, typically after 7-30 days or 1-4 passages. For further details of methods for the preparation of human EG cells, see Shamblott et al., [Proc. Natl. Acad. Sci. USA 95:13726, 1998] and U.S. Patent No. 6,090,622.

[0110] Yet another method for preparing ES cells is by parthenogenesis, a process that also does not destroy the embryo.

[0111] Cells can be grown in suspension with or without microcarriers or in monolayers. Growth of mixed cell populations in monolayer or suspension cultures can be adapted to large-scale growth in bioreactors or multi / hyperstacks by methods well known to those skilled in the art.

[0112] According to some embodiments, the growth phase is carried out for at least 1 week to 20 weeks, such as at least 1 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 growth phase is carried out 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 period can be any value or subrange within the recited range, including the endpoints.

[0113] According to yet other embodiments, the proliferation phase is carried out until a suitable lactate concentration in the cell culture medium and / or a confluence rate (%) is achieved. Confluence rate is the percentage of the culture vessel surface area that appears covered with a cell layer when observed under a microscope. In some embodiments, the undifferentiated pluripotent stem cells are cultured until the lactate concentration in the cell culture medium is about 1.0-13.0 mM, or about 1.5-12.5 mM. In some embodiments, the cells are cultured until the lactate concentration in the cell culture medium is about 1.68-12.29 mM. In some embodiments, the confluence rate is 5%-85%.

[0114] According to yet other embodiments, the mixed cell population is passaged at least once during the growth phase, at least twice during the growth phase, at least three times during the growth phase, at least four times during the growth phase, at least five times during the growth phase, at least six times during the growth phase, or at least seven times during the growth phase.

[0115] If the cells are enzymatically harvested, they can continue to grow for more than 8, more than 9, or even more than 10 passages (e.g., 11-15 passages). The total number of cell doublings can increase to more than 30, e.g., 31, 32, 33, 34, or more. (See International Patent Application Publication No. WO2017 / 021973, which is incorporated by reference in its entirety.)

[0116] The extracellular matrix (ECM) is a three-dimensional meshwork of extracellular polymers and minerals, such as collagen, enzymes, glycoproteins, and hydroxyapatite, that provides structural and biochemical support to surrounding cells. Because multicellularity evolved independently in different multicellular lineages, the composition of the ECM varies among multicellular structures. However, cell adhesion, cell-cell communication, and differentiation are common functions of the ECM.

[0117] The extracellular matrix of animals includes the interstitial matrix and the basement membrane. The interstitial matrix is ​​present between the various animal cells (i.e., within the intercellular spaces). A gel of polysaccharides and fibrous proteins fills the intercellular spaces and acts as a compressive cushion against stresses on the ECM. The basement membrane is a sheet-like deposit of ECM on which the various epithelial cells rest. There are different types of ECM for different types of connective tissue in animals: collagen fibers and bone minerals comprise the ECM of bone tissue, reticular fibers and ground substance comprise the ECM of loose connective tissue, and plasma is the ECM of blood.

[0118] 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, derivatives thereof, or combinations thereof. In certain embodiments, the human laminin is human laminin 511 E8 fragment.

[0119] In some embodiments, the microcarrier 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 microcarrier is comprised of polystyrene, surface-modified polystyrene, chemically modified polystyrene, cross-linked dextran, cellulose, acrylamide, collagen, alginate, gelatin, glass, DEAE-dextran, or combinations thereof. In some embodiments, the microcarrier is comprised of polystyrene. In some embodiments, the microcarrier is comprised of surface-modified polystyrene. In some embodiments, the microcarrier is comprised of chemically modified polystyrene. In some embodiments, the microcarrier is comprised of cross-linked dextran. In some embodiments, the microcarrier is comprised of cellulose. In some embodiments, the microcarrier is comprised of acrylamide. In some embodiments, the microcarrier is comprised of collagen. In some embodiments, the microcarrier is comprised of alginate. In some embodiments, the microcarrier is comprised of gelatin. In some embodiments, the microcarrier is comprised of glass. In some embodiments, the microcarrier is comprised of DEAE-dextran. In some embodiments, the microcarriers are uncoated.

[0120] In some embodiments, the microcarriers are coated. In embodiments, the microcarriers may be coated with Matrigel®, laminin, vitronectin, collagen, derivatives thereof, or combinations thereof. In embodiments, the microcarriers may be coated with polylysine, poly-L-lysine, poly-D-lysine, fibronectin, tenascin, dextran, peptides, or combinations thereof. In some embodiments, the microcarriers are coated with laminin. In some embodiments, the microcarriers are coated with Matrigel®. In some embodiments, the microcarriers are coated with collagen. In some embodiments, the microcarriers are coated with polylysine. In some embodiments, the microcarriers are coated with poly-L-lysine. In some embodiments, the microcarriers are coated with poly-D-lysine. In some embodiments, the microcarriers are coated with vitronectin. In some embodiments, the microcarriers are coated with fibronectin. In some embodiments, the microcarriers are coated with tenascin. In some embodiments, the microcarriers are coated with dextran. In some embodiments, the microcarriers are coated with peptides.

[0121] In some embodiments, the microcarriers may be spherical, smooth, macroporous, rod-shaped, or a combination thereof. In some embodiments, the microcarriers may be bound with protamine or polylysine. In some embodiments, the microcarriers are spherical. In some embodiments, the microcarriers are ellipsoidal. In some embodiments, the microcarriers are rod-shaped. In some embodiments, the microcarriers are disc-shaped. In some embodiments, the microcarriers are porous. In some embodiments, the microcarriers are non-porous. In some embodiments, the microcarriers are smooth. In some embodiments, the microcarriers are flat.

[0122] In some embodiments, the microcarriers are neutral. In some embodiments, the microcarriers are negatively charged. In some embodiments, the microcarriers are hydrophilic.

[0123] In some embodiments, the microcarriers may have a surface area 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 range, including the endpoints.

[0124] In certain embodiments, the microcarriers are surface treated to enhance cell attachment, thereby maximizing cell yield and cell viability. The microcarriers may be constructed of USP Class VI polystyrene material, which provides a consistent platform. In some embodiments, the microcarriers create a synthetic surface on the microcarrier for stem cell growth. The enhanced attachment surface treatment infuses oxygen onto the surface of the microcarrier to improve cell attachment. In some embodiments, the microcarriers are non-pyrogenic. In some embodiments, the microcarriers are optimized for mesenchymal stem cell applications. In certain embodiments, the bead size may vary between 125-212 μm. In certain embodiments, the density of the microcarriers may be 1.026±0.004. In certain embodiments, the microcarriers are 360 ​​cm 2 / gram.

[0125] In some embodiments, the method includes combining hESCs with laminin or a derivative thereof to improve cell adhesion to the carrier surface. In a particular embodiment, the laminin is human laminin 511. In alternative embodiments, several other extracellular matrices can be used for cell adhesion, including, but not limited to, vitronectin, fibronectin, collagen, Matrigel®, or derivatives thereof.

[0126] In some embodiments, the cells may be cultured for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days.

[0127] In some embodiments, the cells can be cultured in a working volume between 10 mL and 3,000 mL, e.g., 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 can be any value or subrange within the recited range, including the endpoints.

[0128] In some embodiments, the cultured cells may be further expanded.

[0129] In some embodiments, the cultured cells may remain undifferentiated. Undifferentiated cells may be identified by expression of various markers, including but not 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 (see IPC#0 in FIG. 2).

[0130] In some embodiments, the cells may be cultured in feeder cell conditioned medium. ES culture methods may include the use of a feeder cell layer that secretes factors required for the proliferation of stem cells while at the same time inhibiting their differentiation. The culture is typically carried out on a solid surface, such as a surface coated with gelatin or vimentin. Exemplary feeder layers include human embryonic fibroblasts, adult fallopian tube epithelial cells, primary mouse embryonic fibroblasts (PMEF), mouse embryonic fibroblasts (MEF), mouse fetal fibroblasts (MFF), human embryonic fibroblasts (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 bone marrow stromal cells (hMSC). 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 can be added to the medium to maintain hESCs in a naive, undifferentiated state. See, e.g., Kalkan et al., 2014, Phil. Trans. R. Soc. B, 369:20130540.

[0131] hESCs are typically plated on top of feeder cells after 1-4 days in supportive medium (e.g., NUT(+) with human serum albumin, mTeSR™plus, or mTeSR™1 StemFit™). Additional factors such as bFGF and TGFβ3 may be added to the medium to prevent ESC differentiation. 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 until the required amount of hESCs is reached. According to some embodiments, after the first round of expansion, the hESCs are removed using TrypLE™ Select, and after the second round of expansion, the hESCs are removed using collagenase A.

[0132] Feeder cell-free systems have also been used for ES cell culture, and such systems utilize a matrix supplemented with serum replacement, cytokines, and growth factors (including IL6 and soluble IL6 receptor chimeras) as an alternative to a 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 culture medium (e.g., Lonza L7™ system, mTeSR™, StemPro™, XFKSR, E8, NUTRISTEM®). Unlike feeder-based cultures, which require simultaneous growth of feeder cells and stem cells and may result in mixed cell populations, stem cells grown in feeder-free systems are easily separated from the surface. The culture medium used for stem cell growth contains factors that effectively inhibit differentiation and promote growth, such as MEF-conditioned medium and bFGF.

[0133] Also included within the scope of the present disclosure is a method 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 the population of undifferentiated hESCs with microcarriers in a growth medium, and expanding the population of cells.

[0134] 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).

[0135] According to some embodiments, when cells are cultured on a non-adherent substrate, such as a cell culture plate, the atmospheric oxygen conditions are 20%. However, it is also contemplated to manipulate the atmospheric oxygen conditions so that the atmospheric oxygen percentage is less than about 20%, less than about 15%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, or even less than about 5% (e.g., between 1%-20%, 1%-10%, or 0-5%). According to other embodiments, cells are cultured on a non-adherent substrate initially under normal atmospheric oxygen conditions, then reduced to lower than normal atmospheric oxygen conditions.

[0136] Although the above method is directed to the method of growing and maintaining hESC, similar methods directed to induced pluripotent stem cells (iPSC) are also within the scope of this disclosure. iPSC is a type of stem cell derived from somatic cells that are reprogrammed to a pluripotent state by the introduction of pluripotency-related genes, and is available from a variety of sources. Those skilled in the art will understand the necessary modifications to adapt the above hESC method for use with iPSC and the like.

[0137] Growth Composition In another aspect, provided herein is a suspension growth complex composition comprising human embryonic stem cells or IPSCs, an extracellular matrix component (ECM), and a microcarrier.

[0138] Human embryonic stem cells, extracellular matrix, and microcarriers are described in detail elsewhere herein.

[0139] The range of proliferation complexes can vary. In the table below, the range of complex components is listed in varying units of ECM components. [Table 2]

[0140] ECM components were calculated using the molecular weight of laminin-511 E8 fragment (150 KDa) in mol / cm 2 It can be expressed as: [Table 3]

[0141] ECM components are also related to Avogadro's number (6.022 × 10 23 ) multiplied by the molecular weight (150KDa) to obtain the number of molecules / cm 2 It can be expressed as: [Table 4]

[0142] In some embodiments, the following specification parameters may be expanded: Number of hESCs (cell count) - cm of microcarriers 2 4,000-600,000 cells per cm; Laminin 511 E8 fragment (µg per cm2 of microcarrier) - 0.125 µg / cm 2 The end.

[0143] In some embodiments, the composition may further comprise a growth medium. Non-limiting examples of commercially available basal media (i.e., chemically defined media or CDM) that may be utilized in accordance with the present disclosure include NUTRISTEM® (without bFGF and TGF for ESC differentiation, with bFGF and TGF for ESC proliferation), NEUROBASAL™, KO-DMEM, DMEM, DMEM / F12, CELLGRO™ Stem Cell Growth Medium, or X-VIVO™. The basal medium may be supplemented with various agents known in the art for handling cell culture. The following are non-limiting references to various additives that may be included in the cultures used in accordance with the present disclosure: serum or serum replacement containing media, such as, but not limited to, Knockout Serum Replacement (KOSR), NUTRIDOMA-CS, TCH™, N2, N2 derivatives, or B27 or combinations; extracellular matrix (ECM) components, such as, but not limited to, 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, and optionally supplemented with BrainPhys™. The ECM can then be used to deliver one or more members of the TGFI3 superfamily of growth factors; antimicrobial agents, such as, but not limited to, L-glutamine, beta-mercaptoethanol, penicillin, and streptomycin; and non-essential amino acids (NEAAs), neurotrophins known to play a role in promoting survival of SCs in culture, such as, but not limited to, BDNF, NT3, NT4, and the like.

[0144] As noted above, the microcarrier may comprise one or more of polystyrene, cross-linked dextran, magnetic particles, microchips, cellulose, hydroxylated methacrylate, collagen, gelatin, polystyrene, plastic, glass, ceramic, silicone. In some embodiments, the microcarrier is comprised of polystyrene. In some embodiments, the microcarrier is comprised of surface-modified polystyrene. In some embodiments, the microcarrier is comprised of chemically modified polystyrene. In some embodiments, the microcarrier is comprised of cross-linked dextran. In some embodiments, the microcarrier is comprised of cellulose. In some embodiments, the microcarrier is comprised of acrylamide. In some embodiments, the microcarrier is comprised of collagen. In some embodiments, the microcarrier is comprised of alginate. In some embodiments, the microcarrier is comprised of gelatin. In some embodiments, the microcarrier is comprised of glass. In some embodiments, the microcarrier is comprised of DEAE-dextran.

[0145] As noted above, microcarriers can be spherical, smooth, macroporous, rod-shaped, or combinations thereof.

[0146] In some embodiments, the microcarriers may be coated with matrigel, laminin, vitronectin, collagen, derivatives thereof, or combinations thereof. In some embodiments, the laminin is human laminin 511.

[0147] In some embodiments, the microcarriers are uncoated.

[0148] In some embodiments, the microcarriers are 25 cm 2 ~7,500cm 2 , for example, about 25 cm 2 , 50cm 2 , 75cm 2 , 100cm2 , 125cm 2 , 150cm 2 , 175cm 2 , 200cm 2 , 225cm 2 , 250cm 2 , 500cm 2 , 625cm 2 , 750cm 2 , 1,000cm 2 , 1,250cm 2 , 5,000cm 2 or 7,500 cm 2 The surface area can be any value or subrange within the recited range, including the endpoints.

[0149] In some embodiments, the microcarriers are conjugated 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.

[0150] How to make auditory cells According to the present disclosure, human pluripotent stem cells (hPSCs) can be grown in dynamic culture on microcarriers in hESC culture medium and maintained in a pluripotent state by daily replacement of the hPSC medium, as described above. hPSCs are differentiated by medium replacement to culture medium (a 1:1 mixture of DMEM / F12 and neurobasal medium) that induces non-neuronal ectoderm (NNE) formation. This medium may contain, for example, B27 and N2 supplements, TGFβ agonists such as BMP4 (1-25 ng / mL), vitamin b3 derivative nicotinamide (NIC, 1-25 mM), SB431542, and / or FGF2 (1-25 ng / mL). Dynamic or static culture is continued for 3-7 days, with complete or gradual (75-100% volume daily) replacement of medium. On days 4-8 of differentiation, replace differentiation factors with FGF2, LDN193189 (20-400 nM), IWP-2 (2 uM), SB431542 (1 μM), NIC (1-25 mM), and the Wnt inhibitor IWR-endo (1-10 μM) to generate preplacode ectoderm. Continue dynamic culture for 3-7 days, changing medium every 1-3 days. On days 8-14 of differentiation, replace differentiation factors with FGF2, CHIR99021 (6 uM), and IGF1 (50 ng / ml) to generate early ONPs. Continue dynamic culture for 7-10 days, changing medium every 2-3 days. On days 17-22, differentiation factors are replaced with FGF2, EGF, retinoic acid (RA 0.2-2 µM), SHH (500 ng / ml), and IGF1 (50 ng / ml) to generate mid-late ONPs. Dynamic culture is continued for 7-10 days, with medium changes every 1-3 days. On days 22-28, mid-late ONP cells are harvested and inoculated into static / dynamic suspension as single cells in the presence of BDNF (10 ng / mL), NT3 (10 ng / mL), and IGF-1 Rock inhibitor (e.g., Y-27632 dihydrochloride, 10 µM) for 3 days to form small aggregates. On differentiation days 22-28, cells are further expanded for final maturation (see Figures 1B and 9B).

[0151] In some embodiments, hPSCs may be differentiated by 2D culture for about 14 days, for example with 50-2000 ng / ml Noggin and 0.5-20 ng / ml FGF2, followed by medium exchange to culture medium inducing neural crest formation (1:1 mixture of DMEM / F12 and neurobasal medium). At or about day 14, the cells are transferred to culture in 3D for about 5 days, for example with 5-100 ng / ml EGF and 5-100 ng / ml FGF2. At or about day 19, the cells are transferred back to 2D culture with differentiation factors FGF2, purmorphamine (0.1-1 μM), EGF, retinoic acid (RA 0.2-2 μM) and IGF1 (50 ng / ml) to generate late ONPs. Dynamic culture continues for about 7 days, with medium exchange about every 2-3 days. At or about day 25, harvest late ONP cells and inoculate them in dynamic suspension as single cells in the presence of FGF and EGF and Rock inhibitor (2-50 µM) for approximately 3 days to form small aggregates.

[0152] hPSCs can be differentiated into different cell populations by culturing in a variety of different media containing growth factors and growth factor inhibitors. In some embodiments, the undifferentiated pluripotent stem cells are subjected to conditions sufficient for directed differentiation to produce a composition comprising a cell population comprising an auditory cell population, e.g., NNE, PPE, ONP cells, neuronal cells (e.g., spiral ganglion neurons), or any combination thereof. In some embodiments, the method comprises culturing a population of hPSCs in one, two, three, four, or five culture media, each comprising a combination of growth factors and / or growth factor inhibitors, under conditions sufficient to induce the cell population into a target cell type, thereby producing a cell population comprising the target cell type.

[0153] In some embodiments, the method comprises culturing a population of undifferentiated pluripotent stem cells at between 1,200 and 20,000 viable cells / cm. 2and culturing the cells until the lactate concentration in the cell culture medium reaches about 1.68-12.29 mM and the confluence rate reaches 5%-80%. In some embodiments, the undifferentiated pluripotent stem cells are cultured until the lactate concentration in the cell culture medium reaches about 1.0-13.0 mM, or about 1.5-12.5 mM. In some embodiments, the cells are cultured until the lactate concentration in the cell culture medium reaches about 1.68-12.29 mM. In some embodiments, the confluence rate is 5%-90%, 5%-85%, or 5%-80%.

[0154] In some embodiments, the population of undifferentiated pluripotent stem cells is cultured in a first cell culture medium comprising bone morphogenetic protein 4 (BMP4) and 4-[4-(2H-1,3-benzodioxol-5-yl)-5-(pyridin-2-yl)-1H-imidazol-2-yl]benzamide (SB431542). In some embodiments, the first cell culture medium comprises BMP4, SB431542, and fibroblast growth factor 2 (FGF2).

[0155] In some embodiments, the population of undifferentiated pluripotent stem cells (PSCs) is cultured in a 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 result in differentiation into a target cell type, e.g., non-neural ectodermal (NNE) cells. In some embodiments, the PSC population is cultured in the first cell culture medium for at least 1 day. In some embodiments, the PSC population is cultured in the first cell culture medium for at least 4 days. In some embodiments, the PSC population is cultured in the first cell culture medium for at least 5 days. In some embodiments, the PSC population is cultured in the first cell culture medium for at least 7 days. In some embodiments, the PSC population is cultured in the first cell culture medium for about 1-20 days, 1-9 days, 2-10 days, 3-7 days, or 4-6 days. In some embodiments, the PSC population is cultured in the first cell culture medium for about 1-9 days. In some embodiments, the PSC population is cultured in the first cell culture medium for about 3 to 7 days. In some embodiments, culturing the population of undifferentiated PSCs in the first cell culture medium produces a cell population that includes non-neurectodermal (NNE) cells.

[0156] In some embodiments, a cell population produced by culturing PSCs in a first cell culture medium, e.g., a cell population comprising NNE cells, is 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-1-yl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl}quinoline (LDN193189).

[0157] In some embodiments, the cell population 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 result in differentiation into a target cell type, e.g., preplacodal ectoderm (PPE) cells. In some embodiments, the cell population is cultured in the second cell culture medium for at least 1 day. In some embodiments, the cell population is cultured in the second cell culture medium for at least 4 days. In some embodiments, the cell population is cultured in the second cell culture medium for at least 5 days. In some embodiments, the cell population is cultured in the second cell culture medium for at least 6 days. In some embodiments, the cell population is cultured in the second cell culture medium for at least 7 days. In some embodiments, the cell population is cultured in the second cell culture medium for about 1-20 days, 1-9 days, 2-10 days, 3-7 days, or 4-6 days. In some embodiments, the cell population is cultured in the second cell culture medium for about 1-9 days. In some embodiments, the cell population is cultured in the second cell culture medium for about 3-7 days. In some embodiments, the cell population 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 cell population is cultured in the second cell culture medium for about 5 days. In some embodiments, the cell population is cultured in the second cell culture medium for 6 days. In some embodiments, the cell population is cultured in the second cell culture medium for 7 days. In some embodiments, culturing the cell population in the second cell culture medium produces a cell population comprising PPE cells.

[0158] In some embodiments, the cell population produced by culturing the cells in the second culture medium, e.g., a cell population comprising PPE cells, is cultured in a third cell culture medium comprising 6-((2-((4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-yl)amino)ethyl)amino)nicotinonitrile (CHIR99021), FGF2, and insulin-like growth factor 1 (IGF-1).

[0159] In some embodiments, the cell population 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 cause differentiation into a target cell type, such as an early otic neural progenitor (ONP) cell. In some embodiments, the cell population is cultured in the third cell culture medium for at least 1 day. In some embodiments, the cell population is cultured in the third cell culture medium for at least 4 days. In some embodiments, the cell population is cultured in the third cell culture medium for at least 5 days. In some embodiments, the cell population is cultured in the third cell culture medium for at least 7 days. In some embodiments, the cell population is cultured in the third cell culture medium for 5 days. In some embodiments, the cell population is cultured in the third cell culture medium for 7 days. In some embodiments, the cell population is cultured in the third cell culture medium for 9 days. In some embodiments, the cell population is cultured in the third cell culture medium for about 1-20 days, 1-10 days, 1-17 days, 2-10 days, 3-7 days, or 4-6 days. In some embodiments, the cell population is cultured in the third cell culture medium for about 3-10 days. In some embodiments, the cell population is cultured in the third cell culture medium for about 5-9 days. In some embodiments, culturing the cell population in the third cell culture medium produces a cell population comprising initial ONP cells.

[0160] In some embodiments, the cell population produced by culturing the cells in the third culture medium, e.g., the cell population comprising primary ONP cells, is cultured in a fourth cell culture medium comprising sonic hedgehog (SHH), retinoic acid (RA), epidermal growth factor (EGF), FGF2, and IGF-1.

[0161] In some embodiments, the cell population 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 result in differentiation into a target cell type, e.g., mid-late ONP cells. In some embodiments, the cell population is cultured in the fourth cell culture medium for at least 1 day. In some embodiments, the cell population is cultured in the fourth cell culture medium for at least 4 days. In some embodiments, the cell population is cultured in the fourth cell culture medium for at least 5 days. In some embodiments, the cell population is cultured in the fourth cell culture medium for at least 7 days. In some embodiments, the cell population is cultured in the fourth cell culture medium for 5 days. In some embodiments, the cell population is cultured in the fourth cell culture medium for 7 days. In some embodiments, the cell population is cultured in the fourth cell culture medium for 9 days. In some embodiments, the cell population is cultured in the fourth cell culture medium for about 1-20 days, 1-10 days, 1-17 days, 2-10 days, 3-7 days, or 4-6 days. In some embodiments, the cell population is cultured in the fourth cell culture medium for about 3-10 days. In some embodiments, the cell population is cultured in the fourth cell culture medium for about 5-9 days. In some embodiments, culturing the cell population in the fourth cell culture medium produces a cell population comprising mid-late ONP cells.

[0162] In some embodiments, a cell population produced by culturing cells in the third culture medium, e.g., a cell population comprising mid-late stage ONP cells, is cultured in a fifth cell culture medium comprising brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3), and IGF-1.

[0163] In some embodiments, the cell population 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 cause differentiation into a target cell type, e.g., late ONP cells. In some embodiments, the cell population is cultured in the fifth cell culture medium for at least 1 day. In some embodiments, the cell population is cultured in the fifth cell culture medium for at least 10 days. In some embodiments, the cell population is cultured in the fifth cell culture medium for at least 20 days. In some embodiments, the cell population is cultured in the fifth cell culture medium for at least 30 days. In some embodiments, the cell population is cultured in the fifth cell culture medium for at least 45 days. In some embodiments, the cell population is cultured in the fifth cell culture medium for at least 60 days. In some embodiments, the cell population is cultured in the fifth cell culture medium for 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 cell population is cultured in the fifth cell culture medium for 7-65 days. In some embodiments, the cell population is cultured in the fifth cell culture medium for 3-45 days. In some embodiments, the cell population is cultured in the fifth cell culture medium for 10-60 days. In some embodiments, the cell population is cultured in the fifth cell culture medium for 20-45 days. In some embodiments, culturing the cell population in the fifth cell culture medium produces a cell population comprising late ONP cells.

[0164] In some embodiments, culturing the cell population in the culture medium includes (i) harvesting a cell population produced by culturing the cells in a fourth cell culture medium, e.g., a cell population comprising mid-late ONP cells; (ii) seeding the cell population in a vessel comprising a fifth cell culture medium; (iii) culturing the cell population; (iv) harvesting the cell population; (v) seeding the cell population in a vessel comprising the fifth cell culture medium; and (vi) culturing the cell population. In some embodiments, the fifth cell culture medium further comprises a ROCK inhibitor. In some embodiments, the cells are cultured in step (iii) for 5 to 35 days, 7 to 35 days, 7 to 30 days, or 10 to 25 days. In some embodiments, the cells are cultured in step (iii) for 7 to 35 days. In some embodiments, the cells are cultured in step (vi) for 7 to 30 days, 10 to 30 days, or 15 to 25 days. In some embodiments, the cells are cultured in step (vi) for 7 to 30 days. In some embodiments, step (iii) above comprises culturing the cell population in a vessel containing 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, step (vi) above comprises culturing the cell population 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.

[0165] In some embodiments, the first cell culture medium contains 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 contains BMP4 at a concentration of 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 contains BMP4 at a concentration of 1 ng / mL to 40 ng / mL. In some embodiments, the first cell culture medium contains BMP4 at a concentration of 1 ng / mL to 25 ng / mL. In some embodiments, the first cell culture medium contains BMP4 at a concentration of 5 ng / mL to 30 ng / mL. In some embodiments, the first cell culture medium contains BMP4 at a concentration of 10 ng / mL to 15 ng / mL. In some embodiments, the first cell culture medium contains BMP4 at a concentration of about 10 ng / mL. In some embodiments, the first cell culture medium contains BMP4 at a concentration of about 5 ng / mL. In some embodiments, the first cell culture medium contains BMP4 at a concentration of about 20 ng / mL.

[0166] In some embodiments, the first and / or second cell culture medium comprises SB431542 at a concentration of about 0.1 μM, about 1 μM, about 5 μM, about 10 μM, about 15 μM, or about 20 μM. In some embodiments, the first and / or second cell culture medium comprises SB431542 at a concentration of about 0.1 μM to 20 μM, 0.1 to 10 μM, 5 μM to 15 μM, or 7 μM to 13 μM. In some embodiments, the first and / or second cell culture medium comprises SB431542 at a concentration of 1 μM to 20 μM. In some embodiments, the first and / or second cell culture medium comprises SB431542 at a concentration of 5 μM to 15 μM. In some embodiments, the first and / or second cell culture medium comprises SB431542 at a concentration of 0.1 μM to 10 μM. In some embodiments, the first and / or second cell culture medium comprises SB431542 at a concentration of about 1 μM.

[0167] In some embodiments, the first, second, third and / or fourth cell culture medium comprises 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 comprises FGF2 in an amount of about 1 ng / mL to 40 ng / mL, 1 ng / mL to 30 ng / mL, 1 ng / mL to 25 ng / mL, 5 ng / mL to 30 ng / mL, 5 ng / mL to 15 ng / mL, or 10 ng / mL to 15 ng / mL. In some embodiments, the first, second, third and / or fourth cell culture medium comprises FGF2 in an amount of 1 ng / mL to 40 ng / mL. In some embodiments, the first, second, third and / or fourth cell culture medium comprises FGF2 in an amount between 1 ng / mL and 25 ng / mL. In some embodiments, the first, second, third and / or fourth cell culture medium comprises FGF2 in an amount between 10 ng / mL and 15 ng / mL. In some embodiments, the first, second, third and / or fourth cell culture medium comprises FGF2 in an amount of about 10 ng / mL.

[0168] In some embodiments, the second cell culture medium comprises IWP-2 in an amount of about 0.5 μM, 1 μM, about 2 μM, about 3 μM, about 5 μM, or about 10 μM. In some embodiments, the second cell culture medium comprises IWP-2 in an amount of about 0.5 μM to 20 μM, 0.5 μM to 10 μM, 1 μM to 10 μM, 1 μM to 5 μM, 2 μM to 7 μM, or 3 μM to 5 μM. In some embodiments, the second cell culture medium comprises IWP-2 in an amount of about 0.5 μM to 10 μM. In some embodiments, the second cell culture medium comprises IWP-2 in an amount of about 2 μM to 4 μM. In some embodiments, the second cell culture medium comprises IWP-2 in an amount of about 2 μM.

[0169] In some embodiments, the second cell culture medium comprises 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 comprises LDN193189 in an amount of about 1 nM to 600 nM, 50 nM to 500 nM, 75 nM to 200 nM, 100 nM to 400 nM, or 200 nM to 300 nM. In some embodiments, the second cell culture medium comprises LDN193189 in an amount of 1 nM to 600 nM. In some embodiments, the second cell culture medium comprises LDN193189 in an amount of 50 nM to 500 nM. In some embodiments, the second cell culture medium comprises LDN193189 in an amount of 20 nM to 400 nM. In some embodiments, the second cell culture medium comprises LDN193189 in an amount of 75 nM to 150 nM. In some embodiments, the second cell culture medium comprises LDN193189 in an amount of about 100 nM.

[0170] In some embodiments, the third cell culture medium comprises CHIR99021 at a concentration of about 1 μM, about 5 μM, about 6 μM, about 7 μM, about 10 μM, about 20 μM, about 25 μM, about 30 μM, or about 40 μM. In some embodiments, the third cell culture medium comprises CHIR99021 at a concentration of about 1 μM to 40 μM, 1 μM to 30 μM, 1 μM to 25 μM, 5 μM to 20 μM, 2 μM to 10 μM, or 5 μM to 15 μM. In some embodiments, the third cell culture medium comprises CHIR99021 at a concentration of 1 μM to 40 μM. In some embodiments, the third cell culture medium comprises CHIR99021 at a concentration of 1 μM to 25 μM. In some embodiments, the third cell culture medium comprises CHIR99021 at a concentration of 2 μM to 8 μM. In some embodiments, the third cell culture medium comprises CHIR99021 at a concentration of about 6 μM.

[0171] In some embodiments, the third, fourth and / or fifth cell culture medium comprises 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 comprises IGF-1 in an amount of 1 ng / mL to 300 ng / mL, 20 ng / mL to 200 ng / mL, 5 ng / mL to 100 ng / mL, 25 ng / mL to 300 ng / mL, or 40 ng / mL to 100 ng / mL. In some embodiments, the third, fourth and / or fifth cell culture medium comprises IGF-1 in an amount of 1 ng / mL to 300 ng / mL. In some embodiments, the third, fourth and / or fifth cell culture medium comprises IGF-1 in an amount between 25 ng / mL and 300 ng / mL. In some embodiments, the third, fourth and / or fifth cell culture medium comprises IGF-1 in an amount between 5 ng / mL and 100 ng / mL. In some embodiments, the third, fourth and / or fifth cell culture medium comprises IGF-1 in an amount of about 50 ng / mL.

[0172] In some embodiments, the fourth cell culture medium comprises 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 comprises SHH in an amount of 10 ng / mL to 1300 ng / mL, 50 ng / mL to 1000 ng / mL, 300 ng / mL to 1000 ng / mL, or 400 ng / mL to 600 ng / mL. In some embodiments, the fourth cell culture medium comprises SHH in an amount of 10 ng / mL to 1300 ng / mL. In some embodiments, the fourth cell culture medium comprises SHH in an amount of 300 ng / mL to 1000 ng / mL. In some embodiments, the fourth cell culture medium comprises SHH in an amount of 400 ng / mL to 600 ng / mL. In some embodiments, the fourth cell culture medium comprises SHH in an amount of about 500 ng / mL.

[0173] In some embodiments, the fourth cell culture medium comprises RA at a concentration of about 0.1 μM, about 0.2 μM, about 0.3 μM, about 0.5 μM, about 1 μM, about 2 μM, about 3 μM, about 5 μM, about 10 μM, about 15 μM, or about 20 μM. In some embodiments, the fourth culture medium comprises RA at a concentration of 0.1 μM to 20 μM, 0.1 μM to 5 μM, 0.5 μM to 5 μM, 0.2 μM to 5 μM, or 0.5 μM to 2 μM. In some embodiments, the fourth cell culture medium comprises RA at a concentration of 0.1 μM to 20 μM. In some embodiments, the fourth cell culture medium comprises RA at a concentration of 0.2 μM to 5 μM. In some embodiments, the fourth cell culture medium comprises RA at a concentration of 0.2 μM to 2 μM. In some embodiments, the fourth cell culture medium comprises RA at a concentration of about 0.5 μM.

[0174] In some embodiments, the fourth cell culture medium comprises 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 comprises EGF in an amount of 1 ng / mL to 300 ng / mL, 10 ng / mL to 200 ng / mL, 20 ng / mL to 300 ng / mL, 5 ng / mL to 100 ng / mL, or 10 ng / mL to 50 ng / mL. In some embodiments, the fourth cell culture medium comprises EGF in an amount of 1 ng / mL to 300 ng / mL. In some embodiments, the fourth cell culture medium comprises EGF in an amount of 5 ng / mL to 100 ng / mL. In some embodiments, the fourth cell culture medium comprises EGF in an amount of 10 ng / mL to 50 ng / mL. In some embodiments, the fourth cell culture medium comprises EGF in an amount of about 20 ng / mL.

[0175] In some embodiments, the fifth cell culture medium comprises 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 comprises BDNF in an amount of 1 ng / mL to 300 ng / mL, 5 ng / mL to 200 ng / mL, 5 ng / mL to 100 ng / mL, or 5 ng / mL to 50 ng / mL. In some embodiments, the fifth cell culture medium comprises BDNF in an amount of 1 ng / mL to 300 ng / mL. In some embodiments, the fifth cell culture medium comprises BDNF in an amount of 5 ng / mL to 100 ng / mL. In some embodiments, the fifth cell culture medium comprises BDNF in an amount of 5 ng / mL to 30 ng / mL. In some embodiments, the fifth cell culture medium comprises BDNF in an amount of about 10 ng / mL.

[0176] In some embodiments, the fifth cell culture medium comprises neurotrophin 3 (also referred to as NT3, NT-3 and NTF3). NT3 is a member of the neurotrophin family and is involved in the survival and differentiation of mammalian neural cells. NT3 is believed to be involved in the maintenance of the adult nervous system and in the development of neural cells in embryos. In some embodiments, the fifth cell culture medium comprises 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 comprises NT3 in an amount of 1 ng / mL to 300 ng / mL, 5 ng / mL to 200 ng / mL, 5 ng / mL to 100 ng / mL, or 5 ng / mL to 50 ng / mL. In some embodiments, the fifth cell culture medium comprises NT3 in an amount of 1 ng / mL to 300 ng / mL. In some embodiments, the fifth cell culture medium comprises NT3 in an amount of 5 ng / mL to 100 ng / mL. In some embodiments, the fifth cell culture medium comprises NT3 in an amount of 5 ng / mL to 30 ng / mL. In some embodiments, the fifth cell culture medium comprises NT3 in an amount of 10 ng / mL.

[0177] 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 (trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide dihydrochloride). In some embodiments, the fifth cell culture medium comprises the Rock inhibitor in an amount of about 0.5 μM, 1.0 μM, 1.5 μM, 2.0 μM, 3.0 μM, 5 μM, 7 μM, 9 μM, 10 μM, 11 μM, 12 μM, 15 μM, 20 μM, 30 μM, 40 μM, 50 μM, or 60 μM. In some embodiments, the fifth cell culture medium comprises a Rock inhibitor at about 0.5 μM to 60 μM, 1 μM to 50 μM, 2 μM to 50 μM, 1 μM to 30 μM, 2 μM to 5 μM, 5 μM to 20 μM, 1 μM to 15 μM, or 5 μM to 15 μM. In some embodiments, the fifth cell culture medium comprises a Rock inhibitor in an amount of about 2 μM to 50 μM. In some embodiments, the fifth cell culture medium comprises a Rock inhibitor in an amount of about 10 μM.

[0178] In some embodiments, the method includes (a) culturing a population of undifferentiated pluripotent stem cells in a first cell culture medium comprising FGF2 at a concentration of 1-25 ng / mL, BMP4 at a concentration of 1-25 ng / mL, and SB431542 at a concentration of 0.1-10 μM, for 1-9 days under conditions sufficient to produce non-neural ectodermal (NNE) cells, thereby producing a cell population comprising NNE cells; and (b) culturing the cell population comprising NNE cells in a first cell culture medium comprising SB431542 at a concentration of 0.1-10 μM, FGF2 at a concentration of 1-25 ng / mL, BMP4 at a concentration of 0.5 μM, and SB431542 at a concentration of 0.1-10 μM. (c) culturing the cell population comprising PPE cells in a second cell culture medium comprising IWP-2 at a concentration of 10 μM, and LDN193189 at a concentration of 20-400 nM for 1-9 days under conditions sufficient to produce preplacodal ectoderm (PPE) cells, thereby producing a cell population comprising PPE cells; and (d) culturing the cell population comprising PPE cells in a third cell culture medium comprising CHIR99021 at a concentration of 1-25 μM, FGF2 at a concentration of 1-25 ng / mL, and IGF-1 at a concentration of 5-100 ng / mL for 5-9 days under conditions sufficient to produce early otic neural progenitor (ONP) cells, thereby producing a cell population comprising early ONP cells. (e) culturing the cell population comprising mid-late ONP cells in a fifth cell culture medium comprising BDNF at a concentration of 5-100 ng / mL, NT3 at a concentration of 5-100 ng / mL, and IGF-1 at a concentration of 5-100 ng / mL for 3-45 days under conditions sufficient to produce late ONP cells, thereby producing a cell population comprising late ONP cells; and (f) collecting the cell population comprising late ONP cells, thereby producing the composition. In an alternative embodiment, the first cell culture medium in step (a) contains BMP4 at a concentration of 1 to 25 ng / mL and SB431542 at a concentration of 0.1 to 10 μM, and does not contain FGF2.

[0179] In some embodiments, the method includes the steps of: (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-neural ectoderm (NNE) cells, thereby producing a cell population comprising NNE cells; (b) culturing the cell population 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 preplacodal ectoderm (PPE) cells, thereby producing a cell population comprising PPE cells; and (c) culturing the cell population comprising PPE cells in a third cell culture medium comprising CHIR99021, FGF2 and IGF-1 to produce early otic neural progenitor cells. (d) culturing the cell population 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 cell population comprising mid-late ONP cells, (e) culturing the cell population 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 cell population comprising late ONP cells, and (f) collecting the cell population comprising late ONP cells, thereby producing the composition. In an alternative embodiment, the first cell culture medium of step (a) comprises BMP4 and SB431542 and does not comprise FGF2.

[0180] In some embodiments, the method includes (a) culturing a population of undifferentiated pluripotent stem cells in a first cell culture medium comprising FGF2 at a concentration of 1-25 ng / mL, BMP4 at a concentration of 1-25 ng / mL, and SB431542 at a concentration of 0.1-10 μM under conditions sufficient to produce non-neural ectodermal (NNE) cells, thereby producing a cell population comprising NNE cells; and (b) culturing the cell population comprising NNE cells in a first cell culture medium comprising SB431542 at a concentration of 0.1-10 μM, BMP4 at a concentration of 1-25 ng / mL, and SB431542 at a concentration of 0.1-10 μM, thereby producing a cell population comprising NNE cells. (c) culturing the cell population comprising PPE cells in a second cell culture medium comprising FGF2, IWP-2 at a concentration of 0.5-10 μM, and LDN193189 at a concentration of 20-400 nM under conditions sufficient to produce preplacodal ectoderm (PPE) cells, thereby producing a cell population comprising PPE cells; and (c) culturing the cell population comprising PPE cells in a third cell culture medium comprising CHIR99021 at a concentration of 1-25 μM, FGF2 at a concentration of 1-25 ng / mL, and IGF-1 at a concentration of 5-100 ng / mL. (d) culturing the cell population comprising the early ONP cells in a fourth cell culture medium comprising SHH at a concentration of 50-1000 ng / mL, RA at a concentration of 0.2-2 μM, EGF at a concentration of 5-100 ng / mL, FGF2 at a concentration of 1-25 ng / mL, and IGF-1 at a concentration of 5-100 ng / mL under conditions sufficient to produce mid-to-late ONP cells, thereby producing a cell population comprising early ONP cells. (e) culturing the cell population comprising mid-late ONP cells in a fifth cell culture medium comprising BDNF at a concentration of 5-100 ng / mL, NT3 at a concentration of 5-100 ng / mL, and IGF-1 at a concentration of 5-100 ng / mL under conditions sufficient to produce late ONP cells, thereby producing a cell population comprising late ONP cells, and (f) collecting the cell population comprising late ONPs, thereby producing the composition. In an alternative embodiment, the first cell culture medium of step (a) comprises BMP4 at a concentration of 1-25 ng / mL and SB431542 at a concentration of 0.1-10 μM, and does not comprise FGF2.

[0181] In some embodiments, the method includes (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 μM under conditions sufficient to produce non-neural ectodermal (NNE) cells for 3 to 7 days, thereby producing a cell population comprising NNE cells; and (b) culturing the cell population comprising NNE cells in a first cell culture medium comprising SB431542 at a concentration of 1 μM, FGF2 at a concentration of 10 ng / mL, BMP4 at a concentration of 10 ng / mL, and SB431542 at a concentration of 1 μM for 3 to 7 days under conditions sufficient to produce non-neural ectodermal (NNE) cells, thereby producing a cell population comprising NNE cells; (c) culturing the cell population containing PPE cells in a third cell culture medium containing CHIR99021 at a concentration of 6 μM, FGF2 at a concentration of 10 ng / mL, and IGF-1 at a concentration of 50 ng / mL for 3 to 7 days under conditions sufficient to produce preplacodal ectoderm (PPE) cells, thereby producing a cell population containing PPE cells; and (b) culturing the cell population containing PPE cells in a third cell culture medium containing CHIR99021 at a concentration of 6 μM, FGF2 at a concentration of 10 ng / mL, and IGF-1 at a concentration of 50 ng / mL for 3 to 7 days under conditions sufficient to produce preplacodal ectoderm (PPE) cells. (d) culturing the cell population comprising the 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 μM, 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 cell population comprising mid-late ONP cells; (e) culturing the cell population comprising the 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 to 45 days under conditions sufficient to produce late ONP cells, thereby producing a cell population comprising late ONP cells; and (f) collecting the cell population comprising late ONPs, thereby producing the composition. In an alternative embodiment, the first cell culture medium in step (a) comprises BMP4 at a concentration of 10 ng / mL and SB431542 at a concentration of 1 μM, and does not comprise FGF2.

[0182] In some embodiments, any one of the above methods comprises, prior to step (a), culturing the undifferentiated pluripotent stem cells at between 1,200 and 20,000 viable cells / cm. 2 and culturing the cells until the lactate concentration in the cell culture medium reaches 1.68-12.29 mM and the confluency rate reaches 5-80%.

[0183] In some embodiments, any one of the above methods further comprises cryopreserving the population of cells comprising late ONPs.

[0184] Those skilled in the art will appreciate that in any of the above steps, the resulting population of cells can be cryopreserved and subsequently seeded and cultured in a culture medium appropriate for the next stage of the differentiation process.

[0185] Treatment method Methods for treating a subject having or at risk of developing a hearing disorder are also described and are within the scope of the present invention. Hearing disorders 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 cell population described herein to the ear of a subject. The administered cells may be obtained by the methods described herein, and the starting material may be tissue obtained from the subject to be treated. In other embodiments, the method includes administering a therapeutic agent (e.g., a differentiation agent described herein) that promotes expression of an auditory protein in cells in the inner ear. When used, the differentiation agent may be administered to cells in culture or to a subject alone (to stimulate differentiation of stem or progenitor cells in the inner ear of the subject) or 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 purmorphamine (e.g., Hh-Ag1.3).

[0186] Subjects with inner ear disorders or at risk of developing such disorders can be treated with auditory cells as described herein. If engraftment is successful, for example, at least some of the transplanted spiral ganglion neurons form synaptic connections with hair cells and with targets in the cochlear nucleus. To improve the ability of cells to engraft, stem cells can be modified before differentiation. For example, cells can be engineered to overexpress one or more anti-apoptotic genes in progenitor or differentiated cells. Fak tyrosine kinase or Akt genes are candidates for anti-apoptotic genes that may be useful for this purpose. Overexpression of FAK or Akt can prevent cell death in spiral ganglion cells and promote engraftment when transplanted into another tissue, such as an excised organ of Corti (see, for example, Mangi et al., Nat.Med.9:1195-201,2003). Neural precursor cells overexpressing α.sub.v.β.sub.3 integrin may have an improved ability to extend neurites in tissue explants, since this integrin has been shown to mediate neurite outgrowth 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 exogenously expressed cDNA, to modify EphA4 signaling events. Spiral ganglion neurons have been shown to be guided by signals from EphA4 mediated by cell surface expression of ephrin-B2 and -B3 (Brors et al., J.Comp. Neurol.462:90-100,2003). Inactivating this guidance signal may increase the number of neurons reaching their targets in the adult inner ear. The addition of exogenous factors, such as the neurotrophins BDNF and NT3, and LIF, to tissue grafts can promote neurite outgrowth and growth into the target tissue in vivo and in ex vivo tissue culture.Neurite outgrowth of sensory neurons can be promoted by the addition of neurotrophins (BDNF, NT3) and LIF (Gillespie et al., NeuroReport 12:275-279, 2001). Sonic hedgehog (Shh) polypeptides or polypeptide fragments (e.g., SHH-N) can also be useful as endogenous factors to promote neurite outgrowth. Shh is a regulator of inner ear development and a chemoattractant for axons (Charron et al., Cell 113:11 23, 2003).

[0187] Subjects who experience hearing loss or are at risk of developing hearing loss are candidates for the therapeutic methods described herein. For example, subjects may receive transplants of inner ear hair cells or spiral ganglion cells that are produced by exposure to differentiation agents, or subjects may be administered agents that are identified as capable of differentiating stem cells into cells of the inner ear. Subjects who have hearing loss or are at risk of developing hearing loss may hear less than the average subject, or may hear less than subjects before experiencing hearing loss. For example, hearing may be reduced by at least 5, 10, 30, 50% or more. Subjects may have sensorineural hearing loss due to damage or dysfunction of the sensory part (cochlea) or nerve part (auditory nerve) of the ear, or conductive hearing loss due to blockage or damage of the outer ear and / or middle ear, or subjects may have mixed hearing loss caused by problems in both conductive pathways (outer or middle ear) and nerve pathways (inner ear). An example of mixed hearing loss is a combination of conductive hearing loss due to otitis media and sensorineural hearing loss due to age-related damage.

[0188] A subject may be deaf or hard of hearing for any reason or as a result of any type of event. For example, a subject may be deaf due to genetic or congenital defects. For example, a subject may be deaf or hard of hearing as a result of being born deaf or having gradually lost hearing due to genetic or congenital defects. In another example, a subject may be deaf or hard of hearing as a result of a traumatic event, such as physical trauma to the ear structure or exposure to sudden loud noise or loud noise for a long period of time. For example, prolonged exposure to concert venues, airport runways, and construction sites can damage the inner ear and subsequently cause hearing loss. A subject may be experiencing chemical-induced ototoxicity, where ototoxic agents include therapeutic agents, including antineoplastic agents, salicylates, quinines, and aminoglycoside antibiotics, contaminants in food or medicine, and environmental or industrial pollutants. The subject may have age-related hearing loss or the subject may have tinnitus (characterized by a ringing sound in the ears).

[0189] Suitable subjects for the pharmaceutical compositions and methods described herein may include subjects with vestibular dysfunction, including bilateral and unilateral vestibular dysfunction.Vestibular dysfunction is an inner ear dysfunction characterized by symptoms including dizziness, balance problems, vertigo, nausea, and blurred vision, and may be accompanied by hearing problems, fatigue, and changes in cognitive function.Vestibular dysfunction may result from genetic or congenital defects, infections such as viral or bacterial infections, or injuries such as traumatic or non-traumatic injuries.Vestibular dysfunction is most commonly examined by measuring the individual symptoms of the disorder (e.g., vertigo, nausea, and blurred vision).

[0190] The compositions and methods described herein may be used to treat hearing impairment resulting from sensory hair cell loss or auditory neuropathy. Subjects suffering from auditory neuropathy experience loss of sensory neurons in the cochlea, while hair cells in the inner ear remain intact. Such subjects may particularly benefit from treatments that differentiate cells (stem or progenitor cells) into spiral ganglion cells, or from administration of spiral ganglion cells to the inner ear. Subjects with sensory hair cell loss experience degeneration of cochlear hair cells, which frequently results in loss of spiral ganglion neurons in the area of ​​hair cell loss. Such subjects may also experience loss of supporting cells in the organ of Corti, as well as degeneration of the lamina propria, spiral ligament, and stria vascularis in the temporal bone material. Such subjects may be treated with agents that differentiate cells into hair cells, or with tissue grafts that include hair cells implanted or injected into the inner ear. Subjects may further benefit from treatments that differentiate cells into spiral ganglion cells or from administration of spiral ganglion cells to the inner ear. For example, in mechanical compression-induced auditory neurodegeneration, most auditory spiral ganglion cells degenerate after sustained compression in Rosenthal's canal (causing transneuronal cell death of cochlear nucleus cells) and, together with astrocytes and Schwann cell columns, form a continuous "spontaneous autologous cellular bridge" that acts as an anatomical scaffold for the migration of transplanted cells to connect the PNS and CNS (Sekiya et al. 2021 Cell Transplantation Volume 30:1-20).

[0191] In some embodiments, the methods provided herein are for replacing auditory neurons in a subject in need of such replacement. In some embodiments, the methods provided herein are for enhancing an existing but damaged population of auditory neurons in a subject in need of such replacement.

[0192] The auditory cells produced by the methods described herein can be administered, for example in the form of a cell suspension, by injection, for example, into, on, or near the inner or middle ear, for example, through a retromastoid route into the lumen of the cochlea or the auditory nerve. Injection can be, for example, through the round window of the ear, or through the alveolar part surrounding the cochlea. The cells can be injected through the round window into the auditory nerve trunk in the internal auditory canal, or into the scala tympani, as described below. Administration of the auditory cells described herein can be accomplished using an injection needle or syringe positioning device known in the art, with the ability to control (e.g., manually or via a robotic interface) the navigation and positioning of the needle into the desired target structure, for example, in the inner or middle ear, for treatment of the hearing or hearing loss pathology described herein. Such a device includes the stabilization required to facilitate safe and effective delivery of auditory cells over a period of time, for example, to ensure delivery of the concentration or amount of auditory cells described herein. Exemplary routes of administration are described, for example, at otosurgeryatlas.stanford.edu / otologic-surgery-atlas / cochlear-implantation / cochlear-implant-surgical-variations / .

[0193] In an exemplary route of administration, for example, into the scala tympani by cochleostomy, a small hole is made in the otocyst at the base of the cochlea and a cannula is inserted. The hole is covered with a small piece of fascia. The cells are loaded into a 30G cannula, primed with saline, and aspirated with about 1 μL of air, followed by aspirating the composition. A pump is used to inject the cells into the scala tympani at a rate of about 1 μL / min. In an exemplary second route of administration, a cochleostomy is performed through the otocyst. A dental drill is used to create a hole for a 33G needle and cannula to enter the modiolus, which is then punctured through the hole to penetrate the bony wall of the modiolus. The hole is covered with a small piece of fascia. The cells are loaded into a 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, the compositions of the present disclosure can be delivered through the round window by first exposing the round window, then removing or incising the mucosa of the round window and removing the bony overhang. A cannula is passed through the round window into the scala tympani or modiolus, and cells are administered as described above.

[0194] Accordingly, the disclosure provides a method of treating an auditory condition comprising administering a therapeutically effective amount of a pharmaceutical composition comprising a population of auditory cells, wherein (a) more than 20% of the cells in the population express SOX2, (b) more than 10% of the cells in the population express beta-tubulin III, (c) more than 5% of the cells in the population express TrkB, and (d) less than 1% of the cells in the population express TRA-1-60 and / or SSEA5, wherein the composition is administered to the inner ear or middle ear of a subject. In some embodiments, the method comprises administering a pharmaceutical composition in which (a) 30% or more of the cells in the population express SOX2, (b) 30% or more of the cells in the population express PAX2, (c) 30% or more of the cells in the population express beta-tubulin III, (d) 20% or more of the cells in the population express TrkB, (e) 30% or more of the cells in the population express GluA4, (f) 20% or less of the cells in the population express Myo7A, and (d) 0.1% or less of the cells in the population express TRA-1-60 and / or SSEA5.

[0195] In some embodiments, the methods include administering to a subject about 100,000-50 million cells, about 100,000-10 million cells, about 100,000-1 million cells, about 200,000-10 million cells, about 500,000-1 million cells, or about 100,000-500,000 cells.

[0196] In some embodiments, administration of the compositions described herein alleviates the signs or symptoms of a hearing disease or condition in a subject. In some embodiments, the method improves the subject's hearing, reduces the severity of hearing loss, slows the progression of hearing loss, or alleviates one or more symptoms associated with a hearing disease or disorder.

[0197] Administration of the auditory cells described herein can be accomplished, for example, by pre-injection of a coating material, such as a matrix component, serum component, or biodegradable scaffold, that can enhance the adhesion and integration of the transplanted sensory neurons, ensuring delivery of the concentration or amount of auditory cells described herein. The cell product can be stored frozen in a cryovial made of plastic, glass, or other polymers or rubber and plastic copolymers, such as cyclic olefin copolymers. The vial can be sealed with a screw cap or stopper made of rubber and plastic copolymers, such as thermoplastic elastomers, that allows for aseptic transfer of the cell product to a delivery device. The cell product can be stored frozen pre-loaded in a syringe, syringe cartridge, or injection cannula, which is thawed prior to administration to a subject.

[0198] Pharmaceutical Compositions The present disclosure provides pharmaceutical compositions comprising a population of cells expressing the markers described herein. One skilled in the art will understand that the compositions described herein can include a mixed population of cell types, the characteristics of which are reflected in the percentage of cells in the population expressing one or more of the markers described herein. Depending on the differentiation state of the cells, individual cells within the population may express only a single marker described below, or individual cells may express a combination of the markers described below. Cells within the population may express neural progenitor markers such as nestin, ONP markers such as PAX2, PAX8, and / or SOX2, neuronal markers such as beta-tubulin III, auditory neuronal markers such as TrkB and / or GluA4, non-specific 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 beta-tubulin III, cells in the population express TrkB, and cells in the population express TRA-1-60 and / or SSEA5.

[0199] In some embodiments, cells in the population express SOX2, cells in the population express PAX2, cells in the population express beta 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.

[0200] In some embodiments, the cells in the population express nestin. Nestin is a member of the intermediate filament protein family and is expressed in neuronal cells. In some embodiments, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more of the cells in the population express nestin. In some embodiments, 40% or more of the cells in the population express nestin. In some embodiments, 50% or more of the cells in the population express nestin. In some embodiments, 60% or more of the cells in the population express nestin. In some embodiments, 70% or more of the cells in the population express nestin. In some embodiments, 80% or more of the cells in the population express nestin. In some embodiments, 90% or more of the cells in the population express nestin. In some embodiments, between 10% and 95%, between 20% and 90%, between 30% and 80%, between 40% and 70%, or between 30% and 60% of the cells in the population express nestin.

[0201] In some embodiments, the cells in the population express SOX2. SOX2 encodes a member of the SRY-related HMG-box (SOX) family of transcription factors involved in regulating embryonic development and determining cell fate. In some embodiments, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more of the cells in the population express SOX2. In some embodiments, 30% or more of the cells in the population express SOX2. In some embodiments, 40% or more of the cells in the population express SOX2. In some embodiments, 50% or more of the cells in the population express SOX2. In some embodiments, 60% or more of the cells in the population express SOX2. In some embodiments, 70% or more of the cells in the population express SOX2. In some embodiments, 80% or more of the cells in the population express SOX2. In some embodiments, 90% or more of the cells in the population express SOX2. In some embodiments, between 10% and 95%, between 20% and 90%, between 30% and 80%, between 40% and 70%, or between 30% and 60% of the cells in the population express SOX2.

[0202] In some embodiments, the cells in the population express PAX8. PAX8 encodes a member of the paired box family of transcription factors that includes 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, between 0.1% and 60%, between 1% and 50%, between 0.1% and 30%, between 1% and 20%, between 5% and 15%, or between 5% and 8% of the cells in the population express PAX8.

[0203] In some embodiments, the cells in the population express Six1. Six1 is a homeobox protein gene found in a cluster of related genes on chromosome 14 and is believed to be involved in limb development. In some embodiments, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more of the cells in the population express Six1. In some embodiments, 20% or more of the cells in the population express Six1. In some embodiments, 30% or more of the cells in the population express Six1. In some embodiments, 40% or more of the cells in the population express Six1. In some embodiments, 50% or more of the cells in the population express Six1. In some embodiments, 60% or more of the cells in the population express Six1. In some embodiments, 70% or more of the cells in the population express Six1. In some embodiments, 80% or more of the cells in the population express Six1. In some embodiments, between 10% and 95%, between 20% and 90%, between 30% and 80%, between 40% and 70%, or between 30% and 60% of the cells in the population express Six1.

[0204] In some embodiments, the cells in the population express PAX2. PAX2 encodes paired box gene 2, which is a target for transcriptional repression by the tumor suppressor gene WT1. In some embodiments, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more of the cells in the population express PAX2. In some embodiments, 20% or more of the cells in the population express PAX2. In some embodiments, 30% or more of the cells in the population express PAX2. In some embodiments, 40% or more of the cells in the population express PAX2. In some embodiments, 50% or more of the cells in the population express PAX2. In some embodiments, 60% or more of the cells in the population express PAX2. In some embodiments, 70% or more of the cells in the population express PAX2. In some embodiments, 80% or more of the cells in the population express PAX2. In some embodiments, 90% or more of the cells in the population express PAX2, hi some embodiments, 10%-99%, 20%-95%, 30%-80%, 40%-70%, or 50%-60% of the cells in the population express PAX2.

[0205] In some embodiments, the cells in the population express GluA4. GluA4 encodes a glutamate receptor expressed in excitatory neurotransmitter-secreting neurons in the brain and is activated in a variety of normal neurophysiological processes. In some embodiments, 1% or more, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more of the cells in the population express GluA4. In some embodiments, 10% or more of the cells in the population express GluA4. In some embodiments, 20% or more of the cells in the population express GluA4. In some embodiments, 30% or more of the cells in the population express GluA4. In some embodiments, 40% or more of the cells in the population express GluA4. In some embodiments, 50% or more of the cells in the population express GluA4. In some embodiments, 70% or more of the cells in the population express GluA4. In some embodiments, 90% or more of the cells in the population express GluA4. In some embodiments, 1%-99%, 10%-95%, 20%-90%, 30%-80%, 30%-60%, or 20%-50% of the cells in the population express GluA4. In some embodiments, about 10%-95% of the cells in the population express GluA4. In some embodiments, 30%-90% of the cells in the population express GluA4.

[0206] In some embodiments, the cells in the population express CD133. CD133 encodes a five-spanning membrane glycoprotein that is localized to membrane protrusions, is often expressed on adult stem cells, and functions to maintain stem cell properties by inhibiting differentiation. In some embodiments, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more of the cells in the population express CD133. In some embodiments, 50% or more of the cells in the population express CD133. In some embodiments, 60% or more of the cells in the population express CD133. In some embodiments, 70% or more of the cells in the population express CD133. In some embodiments, 80% or more of the cells in the population express CD133. In some embodiments, 90% or more of the cells in the population express CD133. In some embodiments, 95% or more of the cells in the population express CD133. In some embodiments, between 1% and 99%, between 10% and 95%, between 20% and 90%, between 30% and 80%, or between 40% and 70% of the cells in the population express CD133.

[0207] In some embodiments, the cells in the population express GATA3. GATA3 is a regulator of T cell development and plays a role in endothelial cell biology. Deficiency of GATA3 is a cause of hypoparathyroidism with sensorineural hearing loss. In some embodiments, less than 10%, less than 5%, less than 1%, or 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 GATA3. In some embodiments, less than 1% of the cells in the population express GATA3. In some embodiments, less than 0.1% of the cells in the population express GATA3. In some embodiments, 0.1%-10%, 0.1%-5%, or 0.1%-1% of the cells in the population express GATA3.

[0208] In some embodiments, the cells in the population express β-tubulin III (also referred to as βIII tubulin, beta3 tubulin, etc.). β-tubulin III encodes a member of the beta-tubulin family of proteins that heterodimerize and assemble to form microtubules. In some embodiments, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, or 80% or more of the cells in the population express β-tubulin III. In some embodiments, 10% or more of the cells in the population express β-tubulin III. In some embodiments, 20% or more of the cells in the population express β-tubulin III. In some embodiments, 30% or more of the cells in the population express β-tubulin III. In some embodiments, 40% or more of the cells in the population express β-tubulin III. In some embodiments, 50% or more of the cells in the population express β-tubulin III. In some embodiments, 60% or more of the cells in the population express β-tubulin III. In some embodiments, between 1% and 80%, between 10% and 70%, between 30% and 60%, or between 40% and 50% of the cells in the population express β-tubulin III.

[0209] In some embodiments, the cells in the population express tropomyosin-related kinase receptor B (TrkB). TrkB is involved in nervous system development and allows for brain-derived neurotrophic factor binding activity and brain-derived neurotrophic factor (BDNF) activator receptor activity. In some embodiments, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, or 80% or more of the cells in the population express TrkB. In some embodiments, 10% or more of the cells in the population express TrkB. In some embodiments, 20% or more of the cells in the population express TrkB. In some embodiments, 30% or more of the cells in the population express TrkB. In some embodiments, 40% or more of the cells in the population express TrkB. In some embodiments, 60% or more of the cells in the population express TrkB. In some embodiments, between 5% and 80%, between 10% and 70%, between 20% and 50%, or between 30% and 40% of the cells in the population express TrkB.

[0210] In some embodiments, the cells in the population express tropomyosin-related kinase receptor C (TrkC). TrkC is predicted to act upstream or within several processes including neurogenesis, propagation of neuronal action potentials, and to enable several functions such as 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%-10%, 0.01%-5%, or 0.01%-1% of the cells in the population express TrkC.

[0211] In some embodiments, the cells in the population express brain-specific homeobox / POU domain protein 3a (BRN3A). BRN3A enables several functions including DNA binding activity, DNA binding transcription activator activity, and is involved in nervous system development. In some embodiments, less than 10%, less than 5%, less than 1%, or less than 0.1% of the cells in the population express BRN3A. In some embodiments, less than 5% of the cells in the population express BRN3A. In some embodiments, less than 1% of the cells in the population express BRN3A. In some embodiments, less than 0.1% of the cells in the population express BRN3A. In some embodiments, 0.1%-10%, 0.1%-5%, or 0.1%-1% of the cells in the population express BRN3A.

[0212] In some embodiments, the cells in the population express Myo7A. Mutations in MYO7A are known to play an important role in the development of deafness and blindness. Myo7A is expressed in the nervous system, allows protein domain specific binding activity, and acts upstream 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%, 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, between 0.1% and 40%, between 1% and 30%, between 5% and 20%, or between 10% and 15% of the cells in the population express Myo7A.

[0213] In some embodiments, the cells in the population express stage-specific embryonic antigen (SSEA-5). Undifferentiated cells may be identified by expression of various markers, such as 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, 0.01%-10%, 0.1%-5%, or 0.1%-1% of the cells in the population express SSEA-5.

[0214] In some embodiments, the cells in the population express T cell receptor alpha locus (TRA-1-60). Undifferentiated cells may be identified by expression of various markers, such as TRA-1-60. 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 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, 0.01%-10%, 0.1%-5%, or 0.1%-1% of the cells in the population express TRA-1-60.

[0215] In some embodiments, (a) 20% or more of the cells in the population express SOX2, (b) 10% or more of the cells in the population express beta-tubulin III, (c) 5% or more of the cells in the population express TrkB, and (d) 1% or less of the cells in the population express TRA-1-60 and / or SSEA5.

[0216] In some embodiments, (a) 30% or more of the cells in the population express SOX2, (b) 30% or more of the cells in the population express PAX2, (c) 30% or more of the cells in the population express beta-tubulin III, (d) 20% or more of the cells in the population express TrkB, (e) 30% or more of the cells in the population express GluA4, (f) 20% or less of the cells in the population express Myo7A, and (d) 0.1% or less of the cells in the population express TRA-1-60 and / or SSEA5.

[0217] After harvesting according to the methods described herein, the expanded population of auditory cells can be prepared in a specific therapeutic dose (e.g., cell number) and cryopreserved for transport to the clinic. The ready to administer (RTA) auditory cell therapeutic composition can then be administered immediately after thawing without further processing. Examples of media suitable for cryopreservation include, but are not limited to, 90% human serum / 10% DMSO, CryoStor®, CryoStor® CS10 (10% DMSO), CryoStor® CS5 (5% DMSO), CryoStor® CS2 (2% DMSO), STEM-CELLBANKER®, PRIME XV® FREEZIS, HYPOTHERMASOL®, trehalose, and the like. In an embodiment, the cryopreservation medium contains about 0.5% to about 50% DMSO, for example, 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 an embodiment, the cryopreservation medium contains about 0.5% to about 30% DMSO. In an embodiment, the cryopreservation medium contains about 1% to about 20% DMSO.

[0218] In some embodiments, the final cell composition is a cell aggregate that has been filtered to separate the cell aggregate from the carrier, cell debris, or matrix. In other embodiments, the cells are filtered prior to cryopreservation using a single-use filter, cell strainer, or mesh with a pore size of at least 40 μm, about 50 μm, about 70 μm, about 100 μm, about 60 μm to separate single cells from the cell aggregate, carrier, cell debris, or matrix. The filter can be in a closed system. In other embodiments, the separation of single cells from the cell aggregate, carrier, cell debris, or matrix can be performed by tangential flow centrifugation. The filtration system has the capacity to safely filter single cells through pore I at volumes of 1 million cells, 10 million cells, 100 million cells, 1 billion cells, 10 billion cells, 100 billion cells. The viability (%) of post-filtration cells stored in cryopreservation medium for 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 range. In other embodiments, the recovery (%) of post-filtration cells stored in cryopreservation medium for 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 range.

[0219] In alternative embodiments, the cells in the final cell composition are single cells in suspension. For example, the single cells in the composition can be produced by dissociating the aggregates described herein by any method known in the art that results in viable single cells.

[0220] In further embodiments, the viability (%) of post-filtration cells stored in neutralizing medium for about 0 to about 8 hours followed by storage in cryopreservation medium for 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 recovery (%) of post-filtration cells stored in neutralizing medium for about 0 to about 8 hours followed by storage in cryopreservation medium for 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 range.

[0221] In yet another embodiment, the viability (%) of the cells after filtration after storage in neutralizing medium for about 0 to about 8 hours, followed by storage in cryopreservation medium for about 0 to about 8 hours, and thawing the cryopreservation composition is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In yet another embodiment, the recovery (%) of the cells after storage in neutralizing medium for about 0 to about 8 hours, followed by storage in cryopreservation medium for about 0 to about 8 hours, and thawing the cryopreservation 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 range.

[0222] In some embodiments, the viability (%) of post-filtration auditory cells stored in a neutralizing medium at room temperature for 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%. In some embodiments, the viability (%) of post-filtration auditory cells stored in a cryopreservation medium at room temperature for 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%. In further embodiments, the viability (%) of post-filtration auditory cells stored in a neutralizing solution at room temperature for about 0 to about 8 hours and then stored in a cryopreservation medium at room temperature for 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 yet further embodiments, the recovery (%) of post-filtration auditory cells stored in a neutralizing solution at room temperature for about 0 to about 8 hours and then stored in a cryopreservation medium at room temperature for about 0 to about 8 hours 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 survival rate can be any value or subrange within the recited range.

[0223] Auditory cells prepared in a cryopreservation medium suitable for ready-to-thaw (RTA) use may include adenosine, dextran-40, lactobionic acid, HEPES (N-(2-hydroxyethyl)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, dimethylsulfoxide (DMSO), and auditory cells suspended in water. An example of this cryopreservation medium is commercially available under the trade name CryoStor® and is manufactured by BioLife Solutions, Inc. In some embodiments, auditory cell aggregates are prepared in a suitable cryopreservation medium for aggregates, such as CryoStem®, as a ready-to-inject product, using an aggregate-specific delivery system such as the Sutter Xenowork system, which has been used for somatic cell nuclear transfer and intracytoplasmic sperm injection, and more recently for ONP spheroids (Heuer et al. 2020).

[0224] DMSO may be used as a cryoprotectant to prevent the formation of ice crystals that may kill cells during the cryopreservation process. In some embodiments, the cryopreservable auditory cell therapy composition comprises about 0.1% to about 2% DMSO (v / v). In some embodiments, the RTA auditory cell therapy composition comprises about 1% to about 20% DMSO. In some embodiments, the RTA auditory cell therapy composition comprises about 10% DMSO. In some embodiments, the RTA auditory cell therapy composition comprises about 5% DMSO. The concentrations may be any value or subrange within the recited ranges.

[0225] In some embodiments, auditory cell therapy compositions prepared in cryopreservation medium suitable for ready-to-thaw (RTA) applications may include auditory cells suspended in cryopreservation medium that does not contain DMSO. For example, RTA sensory therapy cell compositions may be prepared without DMSO (dimethyl sulfoxide, (CH 3 ) 2SO) or any other dipolar aprotic solvents, Trolox, Na + , K + , Ca 2+ , Mg 2+ , Cl - , H 2 PO 4 - , HEPES, lactobionate, sucrose, mannitol, glucose, dextran-40, adenosine, glutathione. An example of this cryopreservation medium is commercially available under the tradename HYPOTHERMOSOL® or HYPOTHERMOSOL®-FRS, also manufactured by BioLife Solutions, Inc. In other embodiments, the auditory cell composition prepared in a cryopreservation medium suitable for ready-to-administer applications upon thawing may include auditory cells suspended in trehalose.

[0226] The RTA auditory cell therapy composition may optionally include additional factors that support auditory cell engraftment, integration, survival, efficacy, etc. In some embodiments, the RTA auditory cell therapy composition includes activators of the function of the auditory cell preparations described herein.

[0227] In some embodiments, the RTA auditory cell therapy composition may be prepared in a medium containing components that reduce molecular and cellular stress during the freeze-thaw process by scavenging free radicals, buffering pH, supporting oncotic / osmolarity, and maintaining ionic concentration balance.

[0228] In some embodiments, the cytotherapy agent prepared in a cryopreservation medium suitable for use immediately after thawing may include one or more immunosuppressant compounds. In certain embodiments, the cytotherapy agent prepared in a cryopreservation medium suitable for use immediately after thawing may include one or more immunosuppressant compounds prepared for sustained release of one or more immunosuppressant compounds. The immunosuppressant compounds for use with the formulations described herein may belong to the following classes of immunosuppressants: glucocorticoids, cytostatics (e.g., alkylating agents or antimetabolites), antibodies (polyclonal or monoclonal), drugs acting on immunophilins (e.g., cyclosporine, tacrolimus, or sirolimus). Additional drugs include interferons, opioids, TNF-binding proteins, mycophenolates, and small molecule biological agents. Examples of immunosuppressants include mesenchymal stem cells, antilymphocyte globulin (ALG) polyclonal antibody, antithymocyte globulin (ATG) polyclonal antibody, azathioprine, BAS 1L1 X 1MABO (anti-IL-2Ra receptor antibody), cyclosporine (cyclosporine A), daclizumab (anti-IL-2Ra receptor antibody), everolimus, mycophenolic acid, rituximab (anti-CD20 antibody), sirolimus, tacrolimus, and / or mycophenolate mofetil.

[0229] In embodiments, the pharmaceutical composition may be prepared for parenteral administration by injection, for example, by bolus injection or continuous infusion. Injectable preparations may be provided in unit dosage form, for example, in ampoules or multi-dose containers, with the addition of preservatives. The composition 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 above, the composition may also be prepared as a depot preparation. Such long-acting preparations may be administered by implantation (for example, subcutaneously). Thus, for example, the composition may be prepared 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.

[0230] The nature of the pharmaceutical compositions described herein depends on the mode of administration and can be easily determined by those skilled in the art. The pharmaceutical compositions described herein can include carriers or excipients, many of which are known to those skilled in the art. Excipients that can be used include buffers (e.g., citrate buffer, phosphate buffer, acetate buffer, and bicarbonate buffer), amino acids, urea, alcohol, ascorbic acid, phospholipids, polypeptides (e.g., serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, and glycerol. The modulating compounds can be prepared in various ways according to the corresponding route of administration. For example, liquid solutions can be made for administration by drops into the ear, injection, or oral ingestion, and gels or powders can be made for oral ingestion or topical application. Methods for making such formulations are well known and can be found, for example, in "Remington's Pharmaceutical Sciences".

[0231] For example, the pharmaceutical composition can be prepared for administration by ear drops, insufflation (e.g., into the ear), topical administration, or oral administration. In another mode of administration, the pharmaceutical composition can be administered directly to the cochlea of ​​the inner ear in situ, such as through a cannula, catheter, or pump. For example, a cannula, catheter, or pump can direct the pharmaceutical composition into the cochlear cavity or the round window of the ear. In another route of administration, the pharmaceutical composition can be injected into the ear, for example, into the lumen of the cochlea (e.g., scala media, scala vestibuli, and scala tympani). Injection can be, for example, through the round window of the ear or through the cochlear bulla.

[0232] The pharmaceutical composition according to the present disclosure may further comprise a pharma- ceutically acceptable carrier. In one embodiment, the pharma- ceutically acceptable carrier may comprise dimethyl sulfoxide (DMSO). In one embodiment, the pharma- ceutically acceptable carrier does not comprise dimethyl sulfoxide. As described herein, the composition may further be adapted for cryopreservation at -80°C to -195°C or lower. In an embodiment, the composition may be prepared to be thawed and administered directly to a subject, for example, via injection, without additional manipulation prior to administration. In an embodiment, the composition may be prepared including a cryopreservation fluid, such as CryoStor® 10 (CS10), as a cryopreservation medium (an animal component-free defined cryopreservation medium containing 10% DMSO). In some embodiments, the composition is prepared in CryoStor® 10. In an embodiment, the composition may be filtered using a filter kit prior to cryopreservation to avoid blockage during administration through a thin cannula, injection needle, or catheter.

[0233] A pharmaceutical composition according to the present disclosure may be administered at a concentration of about 1 million cells per milliliter, such as about 1.5 million cells per milliliter, for example, about 2 million cells per milliliter, for example, about 5 million cells per milliliter, for example, about 10 million cells per milliliter, for example, about 20 million cells per milliliter, for example, about 25 million cells per milliliter, for example, about 30 million cells per milliliter, for example, about 40 million cells per milliliter, for example, about 60 million cells per milliliter, for example, about 70 million cells per milliliter, for example, about 80 million cells per milliliter, for example, about 90 million cells per milliliter, for example, about 100 million cells per milliliter, for example, about 110 million cells per milliliter, for example, about 120 million cells per milliliter, for example, about 130 million cells per milliliter, for example, about 140 million cells per milliliter, for example, about 150 million cells per milliliter, for example, about 160 million cells per milliliter, for example, about 170 million cells per milliliter, for example, about 180 million cells per milliliter, for example, about 190 million cells per milliliter, for example, about 200 million cells per milliliter, for example, about 210 million cells per milliliter, for example, about 220 million cells per milliliter, for example, about 230 million cells per milliliter, for example, about 240 million cells per milliliter, for example, about 250 million cells per milliliter, for example, about 300 million cells per milliliter, for example, about 350 million cells per milliliter, for example, about 360 million cells per milliliter, for example, about 370 million cells per milliliter, for example, about 380 million cells per milliliter, for example, about 390 million cells per milliliter, for example, about 400 million cells per milliliter, for example, about 410 million cells per The cell number may include about 70 million cells per milliliter, for example, about 80 million cells per milliliter, for example, about 90 million cells per milliliter, for example, about 100 million cells per milliliter, for example, about 500,000 cells per milliliter, for example, about 600,000 cells per milliliter, for example, about 800,000 cells per milliliter, for example, about 900,000 cells per milliliter, for example, about 1 million cells per milliliter, for example, about 1.5 million cells per milliliter, or for example, about 50 million cells per milliliter. The cell number may be any value or subrange within the recited range.

[0234] In yet another embodiment, the pharmaceutical composition according to the present disclosure may have an amount ranging from about 2 microliters to about 2 milliliters, 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. The amount may be any value or subrange within the recited range. In one embodiment, the pharmaceutical composition according to the present disclosure may be in a container configured for cryopreservation or for administration to a subject in need thereof. In one embodiment, the container may be a prefilled syringe.

[0235] In one embodiment, the pharmaceutical composition according to the present disclosure may be administered in an amount 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 about 20 microliters, or such as about 40 microliters. The amount may be any value or subrange within the recited range.

[0236] In some embodiments, a pharmaceutical composition according to the present disclosure comprises 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 about 50,000 cells to 50 million cells, about 100,000 cells to 20 million cells, about 100,000 cells to 10 million cells, about 100,000 cells to 1 million cells, about 500,000 cells to 10 million cells, about 500,000 cells to 1 million cells, about 1 million cells to 50 million cells, or about 10 million cells to 50 million cells. In some embodiments, the pharmaceutical composition comprises about 100,000 cells to 10 million cells. In some embodiments, the pharmaceutical composition comprises about 100,000 cells to 1 million cells. In some embodiments, the pharmaceutical composition comprises about 100,000 cells to 500,000 cells. In some embodiments, the pharmaceutical composition comprises between about 500,000 cells and 1 million cells.

[0237] Kits and manufactured products The present disclosure provides kits comprising the pharmaceutical compositions described herein and articles of manufacture such as cryovials, syringes, syringe cartridge cannulas, and the like.

[0238] In some embodiments, the kit comprises instructions for use.

[0239] In some embodiments, the pharmaceutical compositions described herein are frozen and pre-packaged in unit doses in cryovials, cannulas, syringes or syringe cartridges stored at a suitable temperature (e.g., −80° C. or below or −140° C. or below) and can be immediately administered to a subject after thawing to a suitable temperature, such as room temperature.

[0240] Having now generally described the present invention, the present invention will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to limit the disclosure. It will be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or variations will be suggested to those skilled in the art in light of them, and that such modifications and variations are intended to be included within the spirit and scope of this application and the scope of the appended claims. All publications, patents, and patent applications cited herein are incorporated herein in their entirety by reference for all purposes.

[0241] Exemplary embodiments The invention can be understood with reference to the following enumerated embodiments.

[0242] Embodiment 1. A method for obtaining a population of auditory cells derived from undifferentiated pluripotent stem cells, comprising: a) obtaining a culture of pluripotent stem cells; b) culturing the pluripotent stem cells for an initial period of time under culture conditions sufficient to induce differentiation of the pluripotent stem cells into non-neural ectodermal cells; c) culturing the non-neural ectodermal cells of b) under culture conditions sufficient to differentiate the non-neural ectodermal cells into auditory cells.

[0243] Embodiment 2. The method of embodiment 1, wherein the pluripotent stem cells are human embryonic stem cells (hESCs).

[0244] Embodiment 3. The method of embodiment 1, wherein the pluripotent stem cells are human induced pluripotent stem cells (hiPSCs).

[0245] Embodiment 4. The method of embodiment 1, wherein the auditory cells comprise aggregates of auditory cells.

[0246] Embodiment 5. The method of embodiment 1, wherein the auditory cells comprise a population of sensory cells of the ear.

[0247] Embodiment 6. The method of embodiment 5, wherein the sensory cell population is selected from the group consisting of hair cells, supporting cells, otic progenitor cells, and sensory neural progenitor cells.

[0248] Embodiment 7. The method of embodiment 6, wherein the sensory cell population is selected from the group consisting of hair cells expressing a hair cell marker selected from myosin 7a, supporting cells, otic progenitor cells expressing one or more of nestin, PAX2, PAX8, GATA3 and SOX2, and one or more sensory neural progenitor cells expressing a marker selected from peripherin, BRN3a, FOXG1, β-tubulin 3, TrkB, TrkC, / MafB and GluA4.

[0249] Embodiment 8. The method of embodiment 7, wherein the therapeutic function of sensory neural progenitor cells can be assessed by electrophysiological recording, e.g., showing increased neural activity after administration of glutamate in the culture medium.

[0250] Embodiment 9. The method of embodiment 8, wherein the increase in neuronal activity can be detected by an intracellular calcium-sensitive dye.

[0251] Embodiment 10. The method of embodiment 1, wherein the auditory cells comprise aggregates of sensory neural progenitor cells.

[0252] Embodiment 11. The method according to embodiment 1, wherein the culture of pluripotent cells in step a) is in dynamic suspension.

[0253] Embodiment 12. The method of claim 12, wherein the dynamic suspension comprises a combination of hESCs, MCs (microcarriers), and ECM (extracellular matrix).

[0254] Embodiment 13: The method according to embodiment 1, wherein the culturing of the non-neural ectodermal cells in step b) is carried out under dynamic culture conditions.

[0255] Embodiment 14. A pharmaceutical composition for administration to a subject, comprising auditory cells as described in embodiment 1 and a cryopreservation medium as a formulation ready for immediate administration to a subject immediately after thawing.

[0256] Embodiment 15. A method for treating an auditory condition in a subject, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of embodiment 14.

[0257] Embodiment 16. The method of embodiment 15, wherein the therapeutically effective amount of the pharmaceutical composition comprises 50 million AN cells / ml and 10% DMSO.

[0258] Embodiment 17 The method of embodiment 17, wherein the pharmaceutical composition is administered to the inner ear.

[0259] Embodiment 18 The method of embodiment 15, wherein the pharmaceutical composition is administered to the middle ear.

[0260] Embodiment 19. The method of embodiment 15, wherein the auditory pathology is conductive hearing loss, sensorineural hearing loss, central hearing loss, mixed hearing loss, auditory neuropathy spectrum disorder, or central auditory information processing disorder.

[0261] Embodiment 20. A method for preparing a cryopreserved intermediately differentiated auditory cell composition for long-term storage, for reseeding to continue the process immediately after thawing, comprising: (a) suspending the auditory cells of embodiment 1 in a cryopreservation medium to form a cell suspension; (b) storing the cell suspension at a cryopreservation temperature; and (c) thawing the cryopreserved suspension for further differentiation.

[0262] Embodiment 21. A method for preparing a cryopreserved auditory cell composition for administration to a subject immediately after thawing, comprising: (a) suspending the auditory cells described in embodiment 1 in a cryopreservation medium to form a cell suspension; (b) storing the cell suspension at a cryopreservation temperature; and (c) thawing the cryopreserved suspension for administration to a subject.

[0263] Embodiment 22. A method for replacing an auditory nerve in a subject in need of such replacement, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of embodiment 10.

[0264] Embodiment 23. A method for enhancing an existing but damaged auditory nerve population in a subject in need of enhancement of said auditory nerve population, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of embodiment 10. EXAMPLES

[0265] Example 1: General method for differentiating pluripotent stem cells into auditory cells

[0266] General process: The differentiation of human embryonic stem cells (hESCs) into auditory neuron-like cells was based on a stepwise process in which hESCs were differentiated through different stages of the auditory neuron (AN) lineage (Figure 1A), while relevant growth factors were added to the cells throughout each stage (Figure 1B). The process began with harvesting hESCs and dissociating the cells into single cells or small clumps and seeding them. Once the cells reached the desired density and lactate concentration (1.68-12.29 mM), differentiation was initiated.

[0267] First, cells were supplemented with medium containing growth factors that induce cells into the non-neurectodermal (NNE) lineage, such as BMP4, SB431542, and optionally FGF2. This combination of GFs was used for 3-7 days and was designated Differentiation Timeframe #1 (DTF#1).

[0268] These cells were then provided with the following combination of GFs, including LDN193189, SB431542, FGF2, and IWP-2, to induce the cells into the preplacodal ectoderm (PPE) lineage: This combination of GFs was used for 3-7 days and was designated Differentiation Timeframe Number 2 (DTF#2).

[0269] Differentiation into the early otic neural progenitor (ONP) lineage was initiated by adding medium containing the growth factors CHIR99021, FGF2, and IGF-1 to the cells for 7 days, designated Differentiation Timeframe Number 3 (DTF#3). Old medium was removed and fresh medium containing the new growth factor combination was added.

[0270] The next step in hearing development was the differentiation of late otic neural progenitor cells by adding medium containing a combination of the following GFs to the cells: Shh, RA, EGF, FGF2, and IGF-1. The duration of this step was 7 days and was designated Differentiation Time Frame #4 (DTF#4).

[0271] The final step towards mature auditory neurons involved adding IGF-1, BDNF and NT-3 to the cells for 6-40 days and was designated differentiation time frame number 5 (DTF#5) and differentiation time frame number 6 (DTF#6). At the end of each differentiation time frame (DTF), typical markers of each developmental stage were assessed by flow cytometry (FCM). The identification markers were as follows: otic neural progenitor cells (GATA3, PAX8, PAX2, nestin, SOX2; Matsouka 2017), otic neural progenitor connectivity (TrkB, TrkC) and neuronal function (GluA4, Neuro-D, Brn3A, V-Glut1, β-tubulin III, peripherin, MafB, FOXG1). Cells were stained with the various identification markers mentioned above and the results and percentage (%) of positive cells for the markers tested in each DTF are shown in Table 5.

[0272] Throughout the process, cell culture was either in 2D or 3D systems, and in some cases it was beneficial to harvest cells at any time frame and re-seed the cells to optimize the differentiation process. The data in Table 5 was obtained using a 2D system. [Table 5] [Table 6]

[0273] Differentiation process from H1 hESC to ANP: Initiation of AN differentiation began with the change of medium containing growth factors (Table 6) for the first differentiation time frame (DTF#1). Cells were cultured for 6 days (Figure 2), during which the medium was changed daily except for day 1, when cells were treated with double the volume of medium, and allowed to reach ectodermal cells undisturbed until day 3 (due to the weekend). At the end of this time frame, cells were harvested and analyzed by fluorescence activated cell sorting (FACS) for the expression of typical ectodermal markers, such as PAX6, SOX2 and NNE markers (Table 7).

[0274] Differentiation of DTF#2: The differentiation process continued into the next differentiation time frame (DTF#2). At this point, cells were treated with medium containing a new combination of GFs according to Table 6. Medium was changed every day except for day 8, when cells were treated with double the volume of medium and left undisturbed until day 10 (due to the weekend). This period lasted a total of 6 days (Figure 2). At the end of this time frame, cells were harvested and analyzed by FACS for preplacode ectoderm (PPE) markers such as P75 and GATA3 (Table 7).

[0275] Differentiation of DTF#3: The differentiation process was then continued to the next differentiation time frame (DTF#3). Cells were treated with medium containing the new GF combination according to Table 6. Medium was changed every day except for day 15, when cells were treated with double the amount of medium and left undisturbed until day 17, due to the weekend. This period lasted a total of 7 days (Figure 2). At the end of this time frame, cells were harvested and analyzed by FACS for typical non-neural preplacode ectoderm (PPE), such as p75 expressing cells, and neural progenitor markers such as PAX2 and GATA3 (Table 7).

[0276] Differentiation of DTF#4: On day 19, the process continued into the next differentiation timeframe (DTF#4), where cells were treated with medium containing a new combination of GFs according to Table 7. Medium was changed every day except day 22, where cells were treated with double the volume of medium and left untouched until day 24 (due to the weekend). This period lasted a total of 7 days (Figure 2). At the end of this timeframe, cells were harvested and analyzed by FACS for typical neural progenitor markers such as PAX2, PAX8, and additional neural markers such as β-tubulin III (Table 7).

[0277] Differentiation of DTF#5 and DTF#6: At the end of DTF#4 (day 26), cells were harvested as single cells (TrypLE™ Select) and seeded into flasks containing the GF combination from DTF#5 (Table 6) and media containing ROCK inhibitor. At this stage, media was changed every 2 days until day 35 (except for day 28, when cells were treated with double the volume of media and left undisturbed until day 31 over the weekend) (Figure 2). At the end of this timeframe, cells were harvested and assessed by FACS for additional neural markers such as nestin and PAX2 (Table 7).

[0278] The process was continued by reseeding the cells harvested at the end of DTF#5 (again as single cells) into flasks containing twice the volume of medium containing the DTF#6 GF combination (Table 6) and ROCK inhibitor. The cells were incubated for 3 days after seeding, after which the medium was changed every 2 days until day 42 (Figure 2). At day 42, the cells were harvested and analyzed by FACS for mature neuronal markers such as TrkB and GluA4 (Table 7). [Table 7] [Table 8]

[0279] FACS analysis revealed marker expression of cells across each stage of AN differentiation. At the end of DTF#1, there were high levels of SOX2 and PAX6, indicating successful differentiation into ectoderm, and some expression of AP2, indicating the onset of differentiation into the NNE lineage. At the end of DTF#2, cells expressed high levels of P75, a marker for the PPE lineage, and most of the population stained positive for P75 and negative for TRA-1-60, indicating high commitment to the PPE lineage. At this stage, cells also expressed high levels of GATA3, indicating a direction towards ONP cell differentiation.

[0280] At the end of DTF#3, cells also expressed CD133, another marker of neural progenitor cells, suggesting promotion of the neural lineage. At the end of DTF#4, there were high levels of ONP markers PAX2 and PAX8 and the neural marker β-tubulin III. At the end of DTF#5, we could detect a slight increase in PAX2 expression and high levels of nestin, another marker of neural progenitor cells. At the end of DTF#6, cells expressed the neural marker TrkB and high levels of GluA4, indicative of mature auditory neurons. Taken together, these results suggest that cells underwent distinct lineages toward AN differentiation.

[0281] Example 2: hESC culture conditions and differentiation into auditory cells Culturing hESCs under different conditions results in successful differentiation into ANs. hESCs were harvested as small clumps or single cells and cultured at different densities (1,200–20,000 viable cells / cm). 2 ). Cells were cultured in a monolayer environment in different vessels such as iMatrix-511 E8-coated T-flasks and plates and subcultured for 2-5 passages until the confluency (%) before the start of differentiation was 5-80% according to Table 8 and the desired lactate concentration for the start of differentiation (1.68-12.29 mM) was reached. These parameters at the start of differentiation ensure successful differentiation into AN cells, as FACS results show expression of markers of NNE / PPE lineage such as AP2, P75 at the first stage of differentiation, as well as high levels of neural progenitor markers such as PAX8 and β-tubulin III at more advanced stages of differentiation (Table 9). Error! Invalid bookmark self-reference. The morphology of pre-differentiated hESCs of groups 8 is shown in Figure 3. [Table 9] [Table 10]

[0282] Example 3: Timing of Differentiation Timeframe 1 Differentiation timeframe #1 (DTF#1) period results in successful differentiation into AN. The complete process presented in FIG. 1 involves differentiation from non-neural ectoderm (NNE) to anterior placode ectoderm (PPE) stage. Different periods of culture with DTF#1 were tested as presented in Table 10. Morphological evaluation of DTF#1 to AN detailed in Table 10 is presented in FIG. 4. Furthermore, AN marker expression of cells at the end of each DTF for all groups is presented in Table 11. [Table 11] [Table 12]

[0283] At the end of DTF#1, the results indicate differentiation towards ectoderm with high levels of SOX2 (56–97%) and PAX6 (2–67%) markers. At the end of DTF#2, the cells expressed high levels of P75 (85–97%), a marker of PPE lineage. At the end of DTF#3, the cells expressed CD133 (15–29%), a marker of neural progenitor cells. At the end of DTF#4, there were high levels of nestin (96%), a neural progenitor marker, and β-tubulin III (22–49%), a neural marker. Furthermore, a slight increase in the expression of CD133 and GATA3 was detected at the end of DTF#4 compared to the end of DTF#3. Taken together, these results suggest that the cells underwent a distinct lineage towards AN differentiation.

[0284] hESCs were successfully cultured from non-neural ectoderm (NNE) to preplacode ectoderm (PPE) stage (DTF#1) for all time periods (3-7 days).

[0285] Example 4: Timing of Differentiation Timeframe #2 The Differentiation Timeframe #2 (DTF#2) period results in successful differentiation into AN. The complete process presented in FIG. 1 involves differentiation from the preplacode ectoderm (PPE) to the early otic neural progenitor (ONP) stage. Different periods of culture in DTF#2 were tested. Examples of periods of several days are presented in Table 12.

[0286] The morphology evaluation of DTF#2 to AN in Table 12 is presented in Figure 5 .

[0287] Furthermore, AN marker expression of cells at the end of each DTF for all groups is presented in Table 13. [Table 13] [Table 14]

[0288] At the end of DTF#1, the results show differentiation towards ectoderm with high levels of SOX2 (97%) and PAX6 (78%) markers. At the end of DTF#2, the cells expressed high levels of P75 (89-92%), a marker of PPE lineage, and furthermore cells staining positive for P75 and negative for TRA-1-60 showed high levels of expression (63-79%). At the end of DTF#3, the cells expressed the neural progenitor marker CD133 (5-9%) and the ONP marker PAX8 (9-15%). At the end of DTF#4, the cells expressed the neural marker β-tubulin III (14-29%) and the AN marker TrkB (30-46%). High levels of nestin, a marker of neural progenitor cells, were present (79-87%). Furthermore, a slight increase in CD133 expression could be detected at the end of DTF#4 compared to the end of DTF#3. Taken together, these results suggest that cells underwent distinct lineages toward AN differentiation.

[0289] Cells were successfully cultured from the preplacodal ectoderm (PPE) to early otic neural progenitor (ONP) stage (DTF#2) for all time periods (5-7 days).

[0290] Example 5: Growth factor combinations in differentiation timeframe #1 Different GF combinations for Differentiation Time Frame #1 (DTF#1). In this time frame, cells are cultured with BMP4, SB431542, and FGF2 growth factors to induce the cells into the ectodermal lineage. While the previous example showed the duration of DTF#1, this example shows different GF combinations with and without FGF2 in DTF#1. The two different combinations are detailed in Table 14. [Table 15]

[0291] Morphological evaluation of DTF#1 with and without FGF2 in AN in Table 14 is presented in FIG.

[0292] Additionally, AN marker expression of cells at the end of each DTF for all groups is presented in Table 15. [Table 16]

[0293] At the end of DTF#1, results indicate differentiation towards ectoderm with high levels of SOX2 (84-95%) and PAX6 (36-82%) markers. At the end of DTF#2, cells expressed high levels of P75 (90-99%), a marker of PPE lineage, and furthermore cells staining positive for P75 and negative for TRA-1-60 showed high levels of expression (59-85%). At the end of DTF#3, cells expressed the neural progenitor marker CD133 (33-41%) and the ONP marker PAX8 (6-21%). At the end of DTF#4, there were high levels of the neural progenitor marker nestin (69-92%) and the neural marker β-tubulin III (37-53%). Furthermore, a slight increase in CD133 and PAX8 expression could be detected at the end of DTF#4 compared to the end of DTF#3. Taken together, these results suggest that cells underwent distinct lineages toward AN differentiation.

[0294] hESCs were successfully cultured in two different GF combinations until differentiation from the non-neural ectoderm (NNE) to the preplacode ectoderm (PPE) stage (DTF#1).

[0295] Example 6: Differentiation culture system Different culture systems for ONP maturation. During process development, several systems were tested to optimize the stage of ONP maturation during AN differentiation (Figure 7). The dynamic system was used to form aggregates in 0.1 L PBS wheels, while the static system was used for aggregate formation in 96-well plates or for monolayer single cell culture in flasks (as described above in Example 2) and 6-well plates. Prior to using these systems, cells were harvested (by TrypLE™ Select or transferred as whole hole aggregates) at the final stage of the differentiation process (DTF#5 / 6) and seeded at different densities in each culture system (Table 16).

[0296] Techniques used for ONP maturation included the transfer of aggregates (either as whole aggregates or as single cells after enzymatic and mechanical dissociation of aggregates) from dynamic culture on PBS wheels to static culture in flasks. In other groups, aggregates cultured in 96-well plates were transferred to 0.1 L PBS wheels (without dissociation) until the end of DTF#5 and then transferred again as whole aggregates to T25 flasks or harvested as single cells and reseeded in T25 flasks until the end of DTF#6. [Table 17]

[0297] Example 7: Cryopreservation of intermediate cell banks Cryopreservation of intermediate cell bank of mid-late ONPs and thawing before AN maturation step. At the end of DTF#4, cells were either continued to differentiate towards the end stage of AN differentiation (DTF#5) or harvested and cryopreserved (using CS10) to generate intermediate cell bank of mid-late ONPs (ICB). When cultured in 96-well, cells were cryopreserved as aggregates and later thawed and cultured in 1 L PBS wheels (Figure 8). FACS analysis shows similar marker expression between continued and thawed aggregates at the end of DTF#5 (Table 17). [Table 18]

[0298] FACS results indicate that during continued culture, the aggregates and thawed aggregates maintain a low pluripotency as indicated by low levels of the hESC marker SSEA-5. Both groups show similar expression of neural markers, including high levels of CD133 and low levels of GATA3, and both groups express TrkB.

[0299] Example 8: Cryopreservation of auditory cells Cryopreservation of auditory neuroprogenitor cells (ANP) in a ready-to-thaw injection (TAI) formulation. Cells were harvested at day 35 of differentiation and prepared in CryoStor® 10% to a final concentration of 50 million cells / ml for a final ready-to-administer (RTA) formulation. 0.25 ml of prepared cells were aliquoted into cryovials and frozen using a controlled freezing protocol (CryoMed, Thermo Scientific). Cryovials were transferred to long-term storage in a vapor-phase N2 tank. Several batches of vials were plated onto tissue culture plates at 500,000 cells / cm. 2 Cells were tested for viability and proliferation after thawing by seeding in 10% CryoStor® 10% for 14 days. Media was changed every 2-3 days and contained BDNF, IGF1 and NT3. On day 14, cells were harvested, counted and assessed for viability (%) and yield (cells harvested / cells seeded) as detailed in Table 18. Table 18 shows cell viability and yield of several AN batches after cryopreservation (prepared as RTA formulation in CryoStor® 10% at clinically relevant cell concentrations) and after thawing, seeding and growth for 14 days. [Table 19]

[0300] The cells were viable and grew well after thawing from the RTA formulation.

[0301] Auditory cell profile Proposed functional assays include electrical behavior, axonal connectivity and calcium influx using various configurations of single cell patch clamp or multi-electrode arrays to record every single cell of the entire cell population using extracellular electrodes in combination with pharmacological agents and calcium-dependent dyes specific for inducing electrical activity in auditory neurons. See Table 19. [Table 20]

[0302] Auditory cell stage after administration The auditory cell stage is otic neural progenitor cells (ONPs) derived from pluripotent stem cells when administered to a subject. These auditory cells are more likely to survive and integrate in the form of aggregates as opposed to single cell suspensions. The cells can be delivered as cell aggregates or as a high-density single cell suspension that can form cell aggregates after delivery.

[0303] Dosage of auditory cells An approximate dose of the pharmaceutical composition is from about 30,000 to about 100,000 auditory neurons (based on the number of neurons in the nucleus of the spiral ganglion).

[0304] injection site The injection site for the pharmaceutical composition is the nucleus of the spiral ganglion. A hole may be drilled in the otic bulla for scala tympani injection using a 30G cannula, and an additional hole may be drilled in the bony wall of the modiolus for modiolus injection using a 33G cannula.

[0305] Mature auditory cell stage When this auditory cell stage matures within the explant / in vitro, it becomes a spiral ganglion nucleus sensory afferent neuron (SGN).

[0306] Equivalent The foregoing description is provided for illustrative purposes only and is not intended to limit the disclosure to the precise form disclosed. Details of one or more embodiments of the disclosure are described 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 described herein. Other features, objects, and advantages of the present disclosure will become apparent from the specification and claims. In this specification and the appended claims, the singular forms include the plural forms unless the context clearly dictates otherwise. 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. All patents and publications cited herein are incorporated herein by reference.

Claims

1. 1. A pharmaceutical composition comprising a population of auditory cells, (a) 70% or more of the cells in the population express SOX2; (b) 70% or more of the cells in the population express nestin; (c) 80% or more of the cells in the population express β-tubulin III; (d) 5% or more of the cells in the population express TrkB; (e) 10% or more of the cells in the population express GluA4; (f) no more than 5% of the cells in the population express PAX8; and (g) 0.1% or less of the cells in the population express TRA-1-60 and / or SSEA5; The pharmaceutical composition, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier. (i) 10% or more of the cells in the population express TrkB; (ii) 1% or less of the cells in the population express PAX8, and / or 2. The pharmaceutical composition of claim 1, wherein (iii) no more than 10% of the cells in the population express Myo7A.

3. (a) 80% or more of the cells in the population express SOX2; (b) 80% or more of the cells in the population express nestin; (c) 80% or more of the cells in the population express β-tubulin III; (d) 20% or more of the cells in the population express TrkB; (e) 10% or more of the cells in the population express GluA4; (f) no more than 10% of the cells in the population express Myo7A; (g) 1% or less of the cells in the population express PAX8; and (h) the pharmaceutical composition of claim 1, wherein no more than 0.1% of the cells in the population express TRA-1-60 and / or SSEA5.

4. 2. The pharmaceutical composition of claim 1, wherein the population of auditory cells comprises intermediate otic neuron precursor (ONP) cells, late ONP cells, spiral ganglion neurons, or any combination thereof.

5. 2. The pharmaceutical composition of claim 1, wherein the population of auditory cells comprises a population of sensory cells of the ear. (i) comprising a cell aggregate, a single cell, or a combination thereof; (ii) Freezing Contains a storage medium, (iii) the population of auditory cells comprises at least 100,000 cells; and / or (vi) The pharmaceutical composition of claim 1, wherein the population of auditory cells comprises between 100,000 cells and 10 million cells.

7. A method for making the pharmaceutical composition of any one of claims 1 to 6, 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 into non-neurectodermal cells; c) culturing the cells of (b) under culture conditions sufficient to differentiate the non-neural ectodermal cells into auditory cells.

8. 1. A method of producing a composition comprising a population of auditory cells, comprising: (a) culturing a population of undifferentiated pluripotent stem cells in a first cell culture medium comprising bone morphogenetic protein 4 (BMP4) and 4-[4-(2H-1,3-benzodioxol-5-yl)-5-(pyridin-2-yl)-1H-imidazol-2-yl]benzamide (SB431542) for 1 to 9 days under conditions sufficient to produce non-neural ectodermal (NNE) cells, thereby producing a cell population comprising NNE cells; (b) culturing the cell population comprising NNE cells produced in step (a) in a second cell culture medium comprising SB431542, fibroblast growth factor 2 (FGF2), 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-1-yl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl}quinoline (LDN193189) for 1 to 9 days under conditions sufficient to produce preplacode ectoderm (PPE) cells, thereby producing a cell population comprising PPE cells; (c) culturing the cell population comprising the PPE cells produced in step (b) in a third cell culture medium comprising 6-((2-((4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-yl)amino)ethyl)amino)nicotinonitrile (CHIR99021), FGF2, and insulin-like growth factor 1 (IGF-1) for 5 to 9 days under conditions sufficient to produce early otic neural progenitor (ONP) cells, thereby producing a cell population comprising early ONP cells; (d) culturing the cell population comprising early ONP cells produced in step (c) in a fourth cell culture medium comprising sonic hedgehog (SHH), retinoic acid (RA), epidermal growth factor (EGF), FGF2, and IGF-1 for 5 to 9 days under conditions sufficient to produce intermediate-to-late ONP cells, thereby producing a cell population comprising intermediate-to-late ONP cells; (e) culturing the cell population comprising intermediate-to-late ONP cells produced in step (d) in a fifth cell culture medium comprising brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3), and IGF-1 for 3 to 45 days under conditions sufficient to produce late ONP cells, thereby producing a cell population comprising late ONP cells; (f) harvesting said cell population, thereby producing said composition comprising said population of auditory cells; The method comprising:

9. 9. The method of claim 8, wherein the first cell culture medium comprises FGF2. (i) the BMP4 is present in the first cell culture medium at a concentration of about 1 to 25 ng / mL, or the BMP4 is present in the first cell culture medium at a concentration of 10 ng / mL; (ii) the SB431542 is present in the first cell culture medium and / or the second cell culture medium at a concentration of about 0.1 to 10 μM, or the SB431542 is present in the first cell culture medium and / or the second cell culture medium at a concentration of about 1 μM; (iii) the FGF2 is present in the first, second, third, and / or fourth cell culture medium at a concentration of about 1 to 25 ng / mL, or the FGF2 is present in the first, second, third, and / or fourth cell culture medium at a concentration of 10 ng / mL; (iv) the IWP-2 is present in the second cell culture medium at a concentration of about 0.5 to 10 μM, or the IWP-2 is present in the second cell culture medium at a concentration of 2 μM; (v) the LDN193189 is present in the second cell culture medium at a concentration of about 20 to 400 nM, or the LDN193189 is present in the second cell culture medium at a concentration of 100 nM; (vi) said CHIR99021 is present in said third cell culture medium at a concentration of about 1-25 μM, or said CHIR99021 is present in said third cell culture medium at a concentration of 6 μM; (vii) the IGF-1 is present in the third, fourth, and / or fifth cell culture medium at a concentration of about 5-100 ng / mL, or the IGF-1 is present in the third, fourth, and / or fifth cell culture medium at a concentration of 50 ng / mL; (viii) the SHH is present in the fourth cell culture medium at a concentration of about 50 to 1000 ng / mL, or the SHH is present in the fourth cell culture medium at a concentration of 500 ng / mL; (ix) the RA is present in the fourth cell culture medium at a concentration of about 0.2 to 2 μM, or the RA is present in the fourth cell culture medium at a concentration of 0.5 μM; (x) the EGF is present in the fourth cell culture medium at a concentration of about 5-100 ng / mL, or the EGF is present in the fourth cell culture medium at a concentration of 20 ng / mL; (xi) the BDNF is present in the fifth cell culture medium at a concentration of about 5-100 ng / mL, or the BDNF is present in the fifth cell culture medium at a concentration of 10 ng / mL, and / or (xii) the NT3 is present in the fifth cell culture medium at a concentration of about 5 ng / mL to 100 ng / mL, or the NT3 is present in the fifth cell culture medium at a concentration of 10 ng / mL.

11. 9. The method of claim 8, wherein the undifferentiated pluripotent stem cells comprise human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs), or the population of auditory cells comprises mid-stage ONP cells, late-stage ONP cells, spiral ganglion neurons, or any combination thereof.

12. The method of claim 8 , wherein the auditory cells comprise a population of sensory cells in the ear.

13. 13. The method of claim 12, wherein the sensory cell population is selected from the group consisting of hair cells, supporting cells, otic neuron precursor cells, and sensory neuron precursor cells.

14. The method of claim 8 , wherein the culturing of the undifferentiated pluripotent stem cells comprises dynamic culture conditions.

15. Before step (a), the undifferentiated pluripotent stem cells are cultured at a density of 1,200 to 20,000 viable cells / cm ２ and culturing the cells until the lactate concentration in the cell culture medium is 1.5 to 12.5 mM and the confluence is 5 to 80%.

16. 9. The method of claim 8, wherein the population of undifferentiated pluripotent stem cells is cultured in the first cell culture medium for 3 to 7 days.

17. The method of claim 15 , wherein the undifferentiated pluripotent stem cells are cultured under dynamic culture conditions. (i) culturing a cell population containing the NNE cells in the second cell culture medium for 3 to 7 days; (ii) culturing the cell population comprising the PPE cells in the third cell culture medium for 7 days; 9. The method of claim 8, wherein (iii) the cell population comprising the primary ONP cells is cultured in the fourth cell culture medium for 7 days.

19. Culturing the cell population comprising the mid-to-late ONP cells, (i) collecting a cell population containing the mid-late ONP cells; (ii) seeding the harvested cell population into a vessel containing the fifth cell culture medium; (iii) culturing the seeded cell population for 7 to 35 days; (iv) harvesting the cell population; and (v) seeding the cell population into a vessel containing the fifth cell culture medium; (vi) culturing the cell population for 7 to 30 days; The method of claim 8, comprising:

20. 20. The method of claim 19, wherein the fifth cell culture medium comprises a ROCK inhibitor.

21. 9. The method of claim 8, further comprising, prior to step (e), cryopreserving the cell population comprising the intermediate-late stage ONP cells, followed by thawing and culturing in the fifth cell culture medium.

22. The method of claim 8, further comprising cryopreserving the population of auditory cells.

23. A pharmaceutical composition described in any one of claims 1 to 7 for use in treating an auditory condition in a subject, comprising administering a therapeutically effective amount of the pharmaceutical composition to the subject's inner ear or middle ear.

24. A pharmaceutical composition comprising a population of auditory cells for use in treating a subject having an auditory condition, comprising administering a therapeutically effective amount of the pharmaceutical composition to the subject's inner ear or middle ear, (a) 70% or more of the cells in the population express SOX2; (b) 70% or more of the cells in the population express nestin; (c) 80% or more of the cells in the population express β-tubulin III; (d) 5% or more of the cells in the population express TrkB; (e) 10% or more of the cells in the population express GluA4; (f) no more than 5% of the cells in the population express PAX8; and (g) the pharmaceutical composition, wherein 0.1% or less of the cells in the population express TRA-1-60 and / or SSEA5.

25. A kit comprising the pharmaceutical composition according to any one of claims 1 to 7.

26. 26. The kit of claim 25, wherein the pharmaceutical composition is prepared in a cryovial, syringe, syringe cartridge, or cannula.

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