Combination method including cell therapy and cochlear implant

Abstract

The present invention provides a method for treating hearing loss in a subject suffering from an auditory condition and a method for enhancing the effectiveness of a cochlear implant in a subject having an auditory condition, comprising administering a population of auditory cells obtained by inducing the differentiation of pluripotent cells presumed to have an auditory fate into the inner ear of the subject.

Description

Technology Field

[0001] Cross-reference regarding related applications

[0002] This application claims priority and interest to U.S. provisional application No. 63 / 624,451 filed January 24, 2024, 63 / 649,692 filed May 20, 2024, and 63 / 695,088 filed September 16, 2024, the contents of which are incorporated herein by reference in their entirety. Background Technology

[0003] More than 5% of the population in developed countries has significant hearing or hearing loss ranging in severity from mild difficulty understanding speech to severe hearing loss. Hearing loss is age-related, as approximately 4% of the population under 45 and about 34% of the population over 65 have debilitating hearing loss. In most cases, the cause is related to the degeneration and death of hair cells within the inner ear's cochlea and associated spiral ganglion neurons. There are currently no treatment options available to overcome auditory neuron loss.

[0004] Cochlear implants can address hearing loss in some cases. Cochlear implants stimulate the auditory nerve directly through an array of electrodes that send signals from a receiver to different regions of the auditory nerve, bypassing damaged or dysfunctional hair cells within the cochlea. However, if hearing loss involves significant loss of spiral ganglion neurons, cochlear implants may not be effective, or they may be less effective if there is neuronal dysfunction or partial but significant loss of neuronal cells. Therefore, there is a need in the relevant technical field for additional methods to address hearing loss and methods to enhance the efficacy of cochlear implants.

[0005] The present disclosure provides a method for treating a subject having an auditory condition, comprising inserting a cochlear implant and administering a therapeutically effective amount of a pharmaceutical composition comprising a population of auditory cells, wherein (a) at least 75% of the cells in the population express both Nestin and SOX2; (b) at least 80% of the cells in the population express β-tubulin III; (c) at least 5% of the cells in the population express TrkB; and (d) at least 1% of the cells in the population express TRA-1-60 and / or SSEA5. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition enhances the efficacy of the cochlear implant.

[0006] The present disclosure provides a method for enhancing the efficacy of a cochlear implant in a subject having an auditory pathology, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising a population of auditory cells, wherein (a) at least 75% of the cells in the population express both Nestin and SOX2; (b) at least 80% of the cells in the population express β-tubulin III; (c) at least 5% of the cells in the population express TrkB; and (d) at least 1% of the cells in the population express TRA-1-60 and / or SSEA5. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

[0007] In some embodiments, the pharmaceutical composition is administered before the insertion of the cochlear implant. In some embodiments, the pharmaceutical composition is administered after the insertion of the cochlear implant. In some embodiments, the pharmaceutical composition is administered simultaneously with the insertion of the cochlear implant.

[0008] In some embodiments, the pharmaceutical composition is administered to the inner or middle ear of the subject. In some embodiments, administration to the inner ear includes administration to the cochlea. In some embodiments, the pharmaceutical composition is administered to the tympanic system, the cochlear axis, or the spiral ganglion. In some embodiments, the pharmaceutical composition is administered via injection. In some embodiments, the injection includes inserting a cannula through the opening of the otosac or inserting a cannula through the round window of the subject.

[0009] In some embodiments, the auditory condition includes conductive hearing loss, sensorineural hearing loss, central hearing loss, mixed hearing loss, auditory neuropathy spectrum disorder, central auditory processing disorder, tinnitus, or hidden hearing loss. In some embodiments, the auditory condition includes loss of cochlear hair cells or cochlear neurons. In some embodiments, the auditory condition includes loss of cochlear hair cells and loss of cochlear neurons.

[0010] The present disclosure provides a method for treating a subject having an auditory condition selected from the group consisting of conductive hearing loss, sensorineural hearing loss, mixed hearing loss, auditory neuropathy spectrum disorder, and hidden hearing loss, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising a population of auditory cells, wherein (a) at least 75% of the cells in the population express both Nestin and SOX2; (b) at least 80% of the cells in the population express β-tubulin III; (c) at least 5% of the cells in the population express TrkB; and (d) at least 1% of the cells in the population express TRA-1-60 and / or SSEA5. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

[0011] The present disclosure provides a method for treating a subject having hearing loss, e.g., hidden hearing loss, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising a population of auditory cells, wherein (a) at least 75% of the cells in the population express both Nestin and SOX2; (b) at least 80% of the cells in the population express β-tubulin III; (c) at least 5% of the cells in the population express TrkB; and (d) at least 1% of the cells in the population express TRA-1-60 and / or SSEA5. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

[0012] In some embodiments, the pharmaceutical composition is administered into the inner or middle ear of a subject having hidden hearing loss. In some embodiments, the subject is further treated with a cochlear implant.

[0013] In some embodiments, the pharmaceutical composition is cryopreserved, and the method includes thawing the pharmaceutical composition before administration.

[0014] In some embodiments, 85% or more of the cells in the population express both Nestin and SOX2. In some embodiments, 85% or more of the cells in the population express β-tubulin III. In some embodiments, 15% 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, 80% or more of the cells in the population express Nestin. In some embodiments, 80% or more of the cells in the population express SOX2. In some embodiments, 20% or less of the cells in the population express PAX8 and / or PAX2. In some embodiments, the cell population does not significantly express PAX2 and / or PAX8. In some embodiments, 10% or more of the cells in the population express GluA4. In some embodiments, 10% or less of the cells in the population express Myo7A.

[0015] In some embodiments, (a) more than 85% of the cells in the population express both Nestin and SOX2; (b) more than 90% of the cells in the population express β-tubulin III; (c) more than 15% of the cells in the population express TrkB; (d) more than 10% of the cells in the population express GluA4; (e) less than 10% of the cells in the population express Myo7A; and (f) less than 0.1% of the cells in the population express TRA-1-60 and / or SSEA5.

[0016] In some embodiments, more than 50% of the cells in the population express CD133.

[0017] In some embodiments, (a) about 70% to 100% of the cells in the population express both Nestin and SOX2; (b) about 80% to 100% of the cells in the population express β-tubulin III; (c) about 5% to 80% of the cells in the population express TrkB; and (d) 0% to about 0.1% of the cells in the population express TRA-1-60 and / or SSEA5.

[0018] In some embodiments, about 10% 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.

[0019] In some embodiments, the auditory cell population comprises late ONP cells, mature auditory neurons, spiral ganglion neurons, or any combination thereof. In some embodiments, the auditory cell population 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, ear neuron progenitor cells, and sensory neuron progenitor cells.

[0020] In some embodiments, the percentage of cells expressing any of the markers described herein is determined by flow cytometry or immunofluorescence. In some embodiments, the percentage of cells expressing Nestin, SOX2, β-tubulin III, TrkB, GluA4, TRA-1-60 and / or SSEA5 is determined by flow cytometry or immunofluorescence.

[0021] In some embodiments, the population of cells includes cell aggregates, single cells, or a combination thereof.

[0022] In some embodiments, the population of auditory cells comprises at least 100,000 cells. In some embodiments, the population of auditory cells comprises 100,000 to 10 million cells.

[0023] In some embodiments, about 100,000 to 1 million cells are administered to the subject. In some embodiments, about 30 million cells per milliliter to about 700 million cells per milliliter are administered to the subject.

[0024] In some embodiments, a population of auditory cells is obtained by a method comprising the following steps: (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-neuronal ectodermal cells; and (c) culturing the cells from (b) under culture conditions sufficient to differentiate the non-neuronal ectodermal cells into auditory cells. Brief explanation of the drawing

[0025] Fig. 1a It illustrates the histology showing the Rosenthal tube of a normal guinea pig cochlea. Fig. 1b This illustrates the histology of Rosenthal's tubules in guinea pig wolves after Oubain treatment. Fig. 1c is a higher magnification image of the area marked "C" in Fig. 1b. Fig. 1d is a higher magnification image of the area marked "D" in Fig. 1b. Fig. 1e This shows a series of histological images illustrating the establishment of chemically induced otopathies in guinea pigs. A normal cochlea (left) contains Rosenthal's canal packed with spiral ganglion neurons. After ouabain treatment (center), the spiral ganglion neurons were almost completely eliminated. After neomycin treatment (right), approximately 25% of the ganglion neurons survived. Fig. 2a and Fig. 2b Figure 1 shows fluorescence microscopy images of guinea pig cochleas (from 2 specimens) 7 days after injection of LCTANP1 cells (also referred to as ANP1 cells). Red fluorescent labeling indicates transplanted cells. Scale bar = 1 mm. Fig. 2cThis shows a higher magnification of the injection site (left) and the first and second rotational sections (right) of the guinea pig cochlea (center). Scale bar = 1 mm. Fig. 2d This is a series of images showing ANP1 cells (labeled in red) at 7 days after administration to guinea pig cochleas with hearing loss induced by auabain or neomycin, administered through the tympanic cavity or cochlear axis as indicated. Fig. 3a and Fig. 3b This is a fluorescence microscopy image showing that human nucleolus-specific antibodies can distinguish between human neuronal cells and host guinea pig cells. In cell culture, LCTANP1 cells were labeled with antibodies against the human nucleolus, whereas (green, Fig. 3a ), guinea pig fibroblasts were not labeled ( Fig. 3b The neurofilament proteins were stained red (phaloidine staining). Figs. 4a-4d This is a fluorescence microscopy image showing that LCTANP1 cells (light blue) survived in a guinea pig cochlea for at least one week after transplantation. Beta-tubulin III is stained red. Fig. 5a and Fig. 5b is the process of differentiating LCTANP1 cells from human embryonic stem cells (hESCs) into late auditory neural progenitor cells (ONP) / mature and / or early auditory neurons ( Fig. 5a ), and hESCs, neural progenitor cells, ectodermal and late ear neuron progenitor cells (ONP), and markers of AN late ONP as a function of differentiation time ( Fig. 5b It shows ). Over time, the removal of hESC markers and the expression of ear neuron progenitor cell markers can be observed. Fig. 5c This is a schematic diagram and graph showing the monitoring of LTCANP1 (also referred to as ANP1) differentiation using in-process controls starting from a monopluripotent stem cell line. Fig. 6a and Fig. 6bFigure 6 is a schematic illustration of various evaluations for quality control of LCTANP1 cell therapy, including in vivo assays and functional assessments that can be used to characterize the clinical applicability of the product. Figures 6a and 6b show snapshots of the development of in vitro assay methods designed to monitor the differentiation process and the final cell therapy product. Fig. 7a This is a diagram showing the development of LCTANP1 cells from hPSC cell banking and expansion to full-scale operation execution. Fig. 7b This is a pair of images and diagrams showing the directional differentiation of LCTANP1 cells during the manufacturing process, which mimics natural ear development. Fig. 8 This is a series of fluorescence microscopy images showing the expression profiles of various markers on ANP1 cells, including markers of auricular neural progenitor cells and neural markers. For each of the two sets of nine images, the left column / blue is DAPI. The center column / red or green labels the marker of interest. The right column of each of the two sets of nine images is the result of merging the left and center columns and exemplifies co-localization. ANP1 cells express auricular neural progenitor cells and neural markers. Fig. 9 This shows the bioinformatics analysis of ANP1 cells using a Uniform Manifold Approximation and Projection (UMAP) graph based on Principal Component Analysis (PCA) containing a set of auditory neuron-related genes from a publicly available dataset. Colors indicate cells expressing the major auditory neuron-related gene set. Bioinformatics confirms that the expression of major auditory neuron-related genes is distributed among the ANP1 end products. Fig. 10 This is a series of fluorescence microscopy images of ANP1 cells whose morphology was characterized in vitro based on a combination of neuronal and synapse-related markers at 3 days of culture (top row) and 3 weeks of culture (bottom row). ANP1 cells show a significant increase in marker expression with culture time. Fig. 11Figure 1 shows the electrical activity of auditory neurons on a multi-electrode array (MEA, left column). Using a multi-electrode array, the neuronal activity of multiple ANP1 cells was simultaneously recoded and recorded in the absence (basal activity; right column, top) and presence (inductive activity; right column, bottom) of glutamate receptor agonists. Figs. 12a-12c A pair of graphs (Fig. 12a-12b) illustrating the basal neuronal activity of ANP1 cells by quantifying the active area over time based on MEA activity scans and network activity scans, and a table (Fig. 12c) showing the ANP cell response to electrical stimulation. Fig. 12a exemplifies the percentage of active area over time (x-axis, days) based on MEA active scans. Fig. 12b This illustrates network activity over time (x-axis, days), showing that neuronal network activity is formed after ~30 days and increases with culture time up to ~50 days. Fig. 12c is a quantification of the neuronal response to electrical stimulation, including the active electrode, total spike, and evoked peak, and shows an increase in response correlated with the stimulation amplitude. Figs. 13a-13c This is a series of histograms and graphs illustrating the specific glutamatergic activity of ANP1 cells using MEA. Fig. 13a is a series of three histograms summing the action potentials from all recording electrodes before and after the addition of AMPA, DNQX, and TTX. Fig. 13b and 13c is from the baseline ( Fig. 13a Percentage of active electrode before the shaded area), after AMPA addition, and after DNQX addition ( Fig. 13b ) and spike count ( Fig. 13c It is a quantification of (as an expression of action potential). Figs. 14a-14b It illustrates MEA-based axon tracing confirming the propagation of action potentials along cell processes. Fig. 14ais a reconstructed axon map of 5 ANP1 cells. Scale bar = 100 μm. Fig. 14b Based on the electrical signals of ANP1 cells after about 6 weeks in vitro, Fig. 14a This is a series of graphs quantifying axon elongation and axon length-related parameters in 5 ANP1 cells. Figs. 15a-15b is a pair of graphs that quantify the results of the calcium influx test. Fig. 15a is the quantification of the calcium influx assay in ANP1 cells showing a positive response (>100) to glutamate and AMPA stimulation. Upon addition of DNQX, calcium influx is reduced ( Fig. 15b The data is normalized with respect to the baseline value. Fig. 16 is a flow cytometry (FCM) plot showing the expression of SOX2 and Nestin in a representative population of ANP1 cells of the present disclosure. Fig. 17 is an FCM plot showing the expression of TRA-1-60 and SSEA-5 in a representative population of ANP1 cells of the present disclosure. Fig. 18 is a series of immunofluorescence images showing the expression of beta-tubulin III (beta-tubulin 3), TrKB, and GluA4 in a representative population of ANP1 cells of the present disclosure. Cells are counterstained for nuclei with DAPI. Specific details for implementing the invention

[0026] introduction

[0027] The ear consists of four main sections: the outer ear, the middle ear, the inner ear, and the transmission pathway leading to the auditory center of the brain. The inner ear is a very dense bony sac containing fluid that communicates with the middle ear. Small bones within the middle ear (malleus, incus, and stapes) transmit sound energy from the eardrum to the oval window at the entrance of the cochlea in the inner ear. The stapes acts on the oval window, applying pressure to the fluid within the cochlea. This pressure is transmitted through the cochlea, ultimately causing vibrations in the round window, the second window. Subsequently, the basilar membrane defining the fluid-filled chamber of the cochlea transmits these vibrations to the organ of Corti. Hair cells are located on the epithelial lining of the inner ear (i.e., the cochlea of ​​the organ of Corti), as well as in the vestibular sensory epithelium of the saccule, utricle, and the three semicircular canal crests of the labyrinth. Cochlear hair cells send signals to the cochlear spiral ganglion, and clustered neuronal cell bodies transmit these signals to the cochlear nucleus in the brainstem.

[0028] Mechanosensitive sensory hair cells form the basis of hearing and balance senses. The inner ear possesses approximately 13,000 to 15,000 cochlear hair cells and roughly the same number of vestibular sensory hair cells, which serve as mechanoreceptors for hearing and balance. Molecular research on hair cells has been limited due to their small numbers, and consequently, the molecular basis of their function remains unknown. In addition to their scarcity, hair cells are also sensitive to mechanical and chemical insertions. The death or damage of sensory cells accounts for 90% of hearing loss in humans. Acoustic hyperstimulation, chemotherapy, side effects of aminoglycoside drugs, the effects of aging, and increasingly noisy environments contribute to hearing decline over time. As a result, hundreds of millions of patients worldwide are permanently impaired by hearing loss and balance problems. The main reason for the permanence of these chronic disorders lies in the fact that mammalian cochlear hair cells do not regenerate spontaneously, and the limited regeneration observed in the vestibular system is inadequate for functional recovery.

[0029] Auditory neuropathy is a hearing impairment in which the inner ear successfully detects sound, but there is a problem sending signals from the ear to the brain. The current state of knowledge in the relevant medical technology field suggests that auditory neuropathy plays a substantial role in hearing impairment and hearing loss. Damage to auditory neurons is currently being reported for various auditory neuropathy spectrum disorders, including those associated with aging. Hearing relies on a series of complex steps that convert sound waves in the air into electrical signals. The auditory nerve then carries these signals to the brain. Hair cells in the outer ear help amplify sound vibrations entering the inner ear from the middle ear. When hearing is functioning normally, hair cells in the inner ear convert these vibrations into electrical signals, which travel to the brain as neural impulses, where the brain interprets these impulses as sound. Auditory neuropathy can be caused by a number of factors, including: (i) damage to auditory neurons that transmit sound information from hair cells, which are specialized sensory cells in the inner ear, to the brain; (ii) damage to the hair cells themselves; (iii) if a gene with a mutation is inherited, or if there is damage to the auditory system, either of these can result in a faulty connection between the inner ear hair cells and the auditory nerve (which extends from the inner ear to the brain); or (iv) damage to the auditory nerve itself. Researchers are still looking for effective treatments for patients with auditory neuropathy.

[0030] Several protocols have been developed to differentiate human pluripotent stem cells, such as human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), into sensory cells of the ear that can be used in cell therapy for treating hearing loss. While these methods have been successful in generating sensory cells of the ear, challenges remain regarding product quality, scalability, and cost associated with converting these existing protocols into clinical and commercial-scale production processes.

[0031] Hidden hearing loss (HHL) generally refers to a condition resulting from damage to the inner ear (i.e., the cochlea) caused by exposure to loud noise, aging, or other factors. HHL may not be detected by standard audiometry because the changes causing this condition arise from abnormal signal transmission between the cochlear nerves connecting the brain and the ear, and do not occur within the sensory hair cells that convert sound waves into electrochemical signals designed to evaluate these tests. One example of treatment for HHL is a hearing aid, such as a cochlear implant. Additional treatment options are required.

[0032] A cochlear implant is a small electronic device that can be used to provide the sensation of sound to subjects with specific types of auditory conditions. The implant consists of an outer part and an inner part. The outer part is located behind the ear, and the inner part is surgically inserted. A cochlear implant typically has the following components: (a) a microphone configured to capture sounds from the environment; (b) a processor that selectively processes sounds captured by the environment, such as speech; (c) a transmitter and receiver or stimulator that receive signals from the processor, convert them into electrical impulses, and transmit them to an electrode array; and (d) an electrode array, a group of electrodes that receive electrical impulses from the transmitter and / or stimulator and send them to different regions of the auditory nerve. The auditory nerve sends these signals to the brain, and the brain recognizes these signals as sound. The cochlear implant can stimulate the auditory nerve directly, bypassing the damaged part of the ear. However, if the hair cells and / or neurons of the cochlea are damaged, the efficacy of the cochlear implant may be reduced or inhibited. Therefore, a method is needed to enhance the efficacy of the cochlear implant. Accordingly, the present disclosure provides a composition comprising auditory cells that can enhance the efficacy of a cochlear implant when administered to a subject. It is believed that by administering such a composition to the inner ear, the cells of the composition may reconstitute damaged or defective cells in the inner ear, thereby enhancing the activity of the cochlear implant. The benefits of this cell therapy include, but are not limited to, an established delivery route to the inner ear, scalable production (i.e., modulation, substrate, and harvesting) that provides product cost reduction, and an established preclinical model for testing.

[0033] This description is not intended to be a detailed catalog of all different ways in which the present disclosure may be implemented, or of all features that may be added to the present disclosure. For example, features illustrated for one embodiment may be incorporated into another embodiment, and features illustrated for a specific embodiment may be omitted from that embodiment. Accordingly, the present disclosure envisions that in some embodiments of the present disclosure, any feature or combination of features presented herein may be excluded or omitted. Furthermore, in light of the present disclosure, numerous variations and additions to the various embodiments presented herein without departing from the present disclosure will be apparent to those skilled in the art. In other cases, widely known structures, interfaces, and processes have not been described in detail to avoid unnecessarily obscuring the present invention. Nothing in this specification is intended to be construed as a disclaimer of any part of the entire scope of the present invention. Accordingly, the following description is intended to illustrate certain aspects of the present disclosure and is not intended to fully specify all permutations, combinations, and variations thereof.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this disclosure pertains. Terms used in the description of this disclosure are intended merely to describe specific embodiments and are not intended to limit this disclosure.

[0035] All publications, patent applications, patents, and other references cited herein are included by reference in their entirety.

[0036] Unless otherwise indicated in the context, the various features of the disclosures described herein are expressly intended to be used in any combination. Furthermore, the disclosure also envisions that in some embodiments of the disclosure, any feature or combination of features presented herein may be excluded or omitted.

[0037] The method disclosed herein may include one or more steps or acts to achieve the described method. Method steps and / or acts may be interchangeable without departing from the scope of the invention. In other words, the order and / or use of specific steps and / or acts may be modified without departing from the scope of the invention, unless the specific order of steps or acts is essential to the proper operation of the aspect.

[0038] Table 1. Abbreviations

[0039]

[0040] definition

[0041] The singular form used in the description of this disclosure and the appended claims is intended to include the plural form unless the context clearly indicates otherwise.

[0042] As used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated enumerated items, as well as cases where there is no combination when interpreted as an alternative ("or").

[0043] The terms “about” and “approximately” used herein when referring to measurable values, such as percentages, densities, volumes, etc., are intended to include variations of ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified amount.

[0044] Phrases such as "X to Y" and "about X to Y" as used herein shall be interpreted as including X and Y. Phrases such as "about X to Y" as used herein mean "about X to about Y," and phrases such as "about X to Y" mean "about X to about Y."

[0045] As used herein, "cell" refers to a cell that performs metabolic or other functions sufficient to preserve or replicate its genomic DNA. Cells may be identified by methods widely known in the relevant art, including, for example, the presence of an intact membrane, staining by a specific dye, the ability to produce offspring, or, in the case of gametes, the ability to combine with a second gamete to produce viable offspring. Cells may include prokaryotic and eukaryotic cells. Prokaryotic cells include, but are not limited to, bacteria. Eukaryotic cells include, but are not limited to, yeast cells and cells derived from plants and animals, such as mammals, insects (e.g., spodoptera), and human cells. Cells may be useful if they are naturally non-adherent or if they have been treated so as not to adhere to a surface, for example by trypsinization.

[0046] "Containing" or "contains" is intended to mean that the composition and method include the listed elements but do not exclude others. When used to define the composition and method, "essentially made of" will mean excluding other elements having any essential significance to the combination for the stated purpose. Accordingly, a composition essentially made of elements as defined herein will not exclude other materials or steps that do not substantially affect the basic and novel feature(s) of the claimed invention. "Made of" will mean excluding elements and substantial method steps in excess of trace amounts of other components. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0047] "Effective dose" is an amount of the composition sufficient to achieve the stated purpose (e.g., to achieve the effect of administration, to treat a disease, to reduce or increase enzyme activity, to reduce signaling pathways, or to reduce one or more signs or symptoms of a disease or pathological condition) compared to the absence of the composition. An example of an "effective dose" is an amount sufficient to contribute to the treatment, prevention, or reduction of the symptoms or signs of a disease, which may also be referred to as a "therapeutic effective dose." "Reduction" of symptoms or signs (and grammatical equivalents of this phrase) means a reduction in the severity or frequency of the symptom(s), or the elimination of the symptom(s). For example, a therapeutic effective dose may be an amount sufficient to improve the function of a cochlear implant. A person skilled in the art will recognize that function can be improved by approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or more by any appropriate measures, and that this can be considered a therapeutically effective amount.

[0048] The “prophylactic effective dose” of a composition (e.g., a composition comprising cells described herein) is an amount of the composition that, when administered to a subject, has the effect of preventing or delaying the onset (or recurrence) of injury, disease, pathology, or condition, or reducing the likelihood of onset (or recurrence) of injury, disease, pathology, or condition, or its symptoms. A complete prophylactic effect does not necessarily occur with a single dose administration, but may occur only after a series of dose administrations. Accordingly, the prophylactic effective dose may be administered in one or more doses.

[0049] As used herein, "amount of reduced activity" refers to the amount of antagonist required to reduce the activity of an enzyme compared to the absence of the antagonist. As used herein, "amount of inhibited function" refers to the amount of antagonist required to inhibit the function of an enzyme or protein compared to the absence of the antagonist.

[0050] The exact amount will vary depending on the purpose of treatment and can be verified by a person skilled in the art using known techniques (e.g., literature [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 [Refer to , 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins]). For any composition described herein, the therapeutic effective dose may be initially determined from a cell culture assay. The target concentration will be the concentration (e.g., cell concentration or number) of the active composition(s) capable of achieving the method described herein, as measured using the method described herein or a method known in the relevant art.

[0051] "Control group" or "control experiment" is used in its obvious and ordinary sense and refers to an experiment in which the subject or reagent of an experiment is treated as a parallel experiment, except for the omission of experimental procedures, reagents, or variables. In some cases, the control group is used as a standard of comparison in evaluating experimental effects. In some embodiments, the control group is a measurement of the activity of a protein in the absence of a composition as described herein (including embodiments and examples).

[0052] As used herein, “implantation” or “implantation” refers to administering a population of cells or a device into a target tissue using a suitable delivery technique (e.g., administering a population of cells using an injection device or administering a cochlear implant using a suitable surgical technique).

[0053] “Pharmaceutical acceptable excipients” and “pharmaceutical acceptable carriers” refer to substances that may be included in the composition of the present disclosure while assisting in the administration of the active agent and absorption by the target, and not causing significant harmful toxicological effects on the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solution, lactate Ringer’s solution, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coating agents, sweeteners, flavoring agents, salt solutions (e.g., Ringer’s solution), alcohols, oils, gelatin, carbohydrates, such as lactose, amylose or starch, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidine, and colorants, etc. These formulations may be sterilized and, if desired, may be mixed with adjuvants that do not react adversely with the compositions of the present disclosure, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts affecting osmotic pressure, buffers, coloring agents, and / or flavoring substances. A person skilled in the art will recognize that other pharmaceutical excipients are also useful for the present disclosure.

[0054] As used herein, “patient” or “subject” refers to a living organism suffering from or susceptible to a disease or condition that can be treated by the administration of a pharmaceutical composition or an insertable biodegradable scaffold as provided herein. Non-limiting examples include humans, other mammals, cattle, rats, mice, dogs, monkeys, goats, sheep, cattle, deer, and other non-mammalian animals. In some embodiments, the patient is a human.

[0055] As used herein, “subjects requiring this” refers to animals or humans having damaged tissue in the central nervous system. In one embodiment, the animal or human is experiencing a loss of hearing function.

[0056] With respect to a condition or disease, as used herein, “treatment” or “treating” is preferably an approach to obtain a beneficial or desired outcome, including clinical outcomes following the onset of the condition or disease in the subject. With respect to the disease, beneficial or desired outcomes include, but are not limited to, one or more of the following: improving a condition associated with the disease; curing the disease; reducing the severity of the disease; delaying the progression of the disease; alleviating one or more symptoms associated with the disease; increasing the quality of life of a patient suffering from the disease; extending survival; and any combination thereof. Likewise, for the purposes of this disclosure, beneficial or desired outcomes with respect to the condition include, but are not limited to, one or more of the following: improving the condition; curing the condition; reducing the severity of the condition; delaying the progression of the condition; alleviating one or more symptoms associated with the condition; increasing the quality of life of a patient suffering from the condition; extending survival; and any combination thereof.

[0057] As used herein, "pluripotent stem cell" or "pluripotent cell" refers to a cell that has the ability to differentiate into any type of cell within an organism. Pluripotent cells can form teratomas and can contribute to ectoderm, mesoderm, or endoderm tissues in living organisms. Examples of pluripotent stem cells are embryonic stem (ES) cells, germline (EG) cells, and induced pluripotent stem (iPS) cells.

[0058] As used herein, “embryonic stem cells” or “ES cells” refer to cells that a) are capable of self-renewing, b) can differentiate to produce all types of cells in an organism, and c) are derived from the inner cell mass of a blastocyst of a developing organism. ES cells can be cultured over a long period while maintaining the ability to differentiate into all types of cells in an organism. When cultured, ES cells typically grow as flat colonies with a large nucleus-to-cytoplasmic ratio, clear boundaries, and distinct nuclei. Additionally, 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 do not express SSEA-1. Examples of methods for generating and characterizing ES cells can be found, for example, in U.S. Patent No. 7,029,913, U.S. Patent No. 5,843,780, and U.S. Patent No. 6,200,806, the disclosures thereof are incorporated herein by reference.

[0059] As used herein, “embryonic germline stem cell,” “embryonic germ cell,” or “EG cell” refers to a) cells that are capable of self-renewing, b) cells that can differentiate to produce all types of cells in an organism, and c) cells derived from germ cells and germ cell progenitor cells, e.g., primordial germ cells, i.e., cells that will become sperm and eggs. Embryonic germ cells (EG cells) are thought to possess characteristics similar to those of embryonic stem cells as described above. Examples of methods for generating and characterizing EG cells are, for example, U.S. Patent No. 7,153,684; references [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. It can be found in [USA, 95:13726]; and [Koshimizu, U., et al. (1996) Development, 122:1235], the disclosures of which are incorporated herein by reference.

[0060] As used herein, "induced pluripotent stem cells" or "iPS cells" refer to cells that a) can self-renewal, b) can differentiate to produce all types of cells in an organism, and c) are derived from somatic cells. iPS cells have an ES cell-like morphology and grow as flat colonies with a large nucleus-to-cytoplasm ratio, clear boundaries, and distinct nuclei. In addition, iPS cells express one or more major pluripotency markers known to a person skilled in the art, including but not limited to alkaline phosphatase, SSEA3, SSEA4, SRY (sex determination region Y)-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 transcribtase (TERT), and ZFP42 zinc finger protein (zfp42). iPS cells can be generated by providing cells with "reprogramming factors," that is, one or more biologically active factors that act on cells to alter transcription and thereby reprogram the cells to pluripotency, i.e., a cocktail thereof. These reprogramming factors may be provided to cells individually or as a single composition, i.e., as a pre-mixed composition of reprogramming factors. The factors may be provided in the same molar ratio or in different molar ratios. The factors may 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 applications US20090047263, US20090068742, US20090191159, US20090227032, US20090246875, and US20090304646, the disclosures thereof are incorporated herein by reference.

[0061] It is recognized that commercially available stem cells may 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, Includes WIBR6, HUES 45, Shef 3, Shef 6, BINhem19, BJNhem20, SAGO 1, and SAOO1.

[0062] As used herein, "somatic cell" refers to any cell of an organism that, without experimental manipulation, typically does not produce all types of cells in the organism. In other words, a somatic cell is a cell that is sufficiently differentiated not to naturally produce cells of all three germ layers of the body: the ectoderm, mesoderm, and endoderm. For example, somatic cells may include both neurons and neural progenitor cells, the latter of which can self-renewal and naturally produce all or some cell types of the central nervous system, but cannot produce cells of the mesoderm or endoderm lineage.

[0063] As used herein, "endoderm" refers to the germ layer formed during animal embryonic development that generates the gastrointestinal tract, respiratory tract, endocrine glands and trachea, specific structures of the auditory system, and specific structures of the urinary system.

[0064] The term "mesoderm" as used herein refers to the germ layer formed during animal embryonic development that produces muscle, cartilage, bone, dermis, reproductive system, adipose tissue, connective tissue of the intestines, peritoneum, specific structures of the urinary system, mesoderm, notochord, and spleen.

[0065] As used herein, "ectoderm" refers to the germ layer formed during animal embryonic development that generates the nervous system, tooth enamel, epidermis, hair, nails, and the inner lining of mucosal tissues. During embryonic development, the embryonic ectoderm patterns into lineage progenitor cells for the neural plate, neural crest, plate of origin, and epidermis. "Non-neuronal ectoderm" or "non-neuroectoderm" refers to ectoderm cells that will form non-neuronal structures, such as the epidermis.

[0066] As used herein, "anterior ectoderm" refers to the region of the ectodermal germ facing the anterior, or "snout-like" end, of the embryo, i.e., the head region. The anterior ectoderm includes the preplate-like ectoderm and adjacent tissues, such as the presumed early ectoderm, presumed neural crest, and neural tissue. The ectoderm can be induced to become the anterior ectoderm by contact with an anteriorizing factor, such as IGF1 or insulin.

[0067] As used herein, "anterior ectoderm" refers to a narrow band of cells of the anterior ectoderm that surrounds the anterior neural plate at the end of intestinal germination, generates the cranial plate, and this cranial plate, in turn, generates the paired sensory structures of the head. Anterior ectoderm cells may express one or more of the following markers at detectable levels, 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), eye absence homologue 1 (EYA1), and eye absence homologue 2 (EYA2). Propane ectodermal cells are suitable for responding to ear induction, that is, the induction of ear progenitor cells by culture in the presence of FGF, resulting in the upregulation of p75, Pax8, Pax2, GATA3, and Sox10 expression. Using the method described herein, it is possible to induce cells with propane ectodermal characteristics by expressing propane ectodermal markers from undifferentiated pluripotent stem cells.

[0068] As used herein, “ear progenitor cells” or “ear neural progenitor cells” refer to somatic cells that a) are capable of self-renewing and b) can differentiate to produce inner ear sensory hair cells, auditory neurons, and supporting cells. Ear progenitor cells grow as spheres of cells when cultured under non-adherent conditions, or as clusters of cells when cultured under adherent conditions. Furthermore, ear progenitor cells may express one or more of the following markers at detectable levels: Paired Box 2 (PAX2), Paired Box 8 (PAX8), Distal-less Homeobox 5 (DLX5), Corrector Homeobox 2 (OTX2), Eye Absence Homograph 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), YRPW motif-associated hair / splitting region enhancer 1 (HEY1), YRPW motif-associated hair / splitting region enhancer 2 (HEY2), hair and splitting region enhancer 1 (HES1), hair and splitting region enhancer 6 (HES6), activin receptor (ACTIVIN-R), H6 family homeobox 3 (NKX5.1), claudin 8 (CLDN8), and claudin 14 (CLDN14). As described herein, ear neural progenitor cells can be divided into early, mid, and late ear progenitor cells based on marker expression.

[0069] As used herein, "stromal cells" refers to connective tissue cells of any organ, such as fibroblasts, perivascular cells, endothelial cells, etc.

[0070] As used herein, the terms “microcarrier” or “MC” refer to a suspension support matrix that allows adherent cells to grow in dynamic or static cell cultures and maintains a suspension state when gently mixed. The microcarrier may be composed of materials including, but not limited to, polystyrene, surface-modified polystyrene, chemically modified polystyrene, cross-linked dextran, cellulose, acrylamide, collagen, alginate, gelatin, glass, DEAE-dextran, or combinations thereof. The microcarrier may be coated with a biological support matrix including, but not limited to, laminin, Matrigel®, collagen, poly-lysine, poly-L-lysine, poly-D-lysine, vitronectin, fibronectin, tenacine, dextran, peptides, or combinations thereof. Many different types of microcarriers are commercially available, including but not limited to brands such as HyQSphere (HyClone™), Hillex (SoloHill Engineering), and Low Concentration Synthemax® II (Corning). Microcarriers can be manufactured from cross-linked dextran, such as Cytodex® (GE Healthcare). Microcarriers can be spherical and smooth, or have a microporous surface as in the CYTOPORE™ brand (GE Healthcare), or be rod-shaped carriers, such as DE-53 (Whatman™). The microcarrier can be impregnated with magnetic particles (e.g., GEM particles from Global Cell Solutions) that can help separate cells from the beads.Chip-based microcarriers, such as the μHex product (Nunc), provide a flat surface for cell growth while maintaining the high surface-to-volume ratio of traditional microcarriers. The characteristics of the microcarrier can significantly affect the expansion rate and cell pluripotency.

[0071] As used herein, "dynamic culture" refers to cell culture performed by applying intentional active movement (e.g., in a bioreactor) to enhance mass transfer and mechano-signaling effects, unlike cell culture performed under static conditions (e.g., in a Petri dish), which often increases the number of functional cells. For example, in the dynamic differentiation process, the bioreactor directly applies mechanical force to create physiological conditions and enhance differentiation into specific cell lineages. In dynamic culture, cells can also have a more uniform environment that cannot be provided by diffusion alone in static culture. For example, compare cells grown at the periphery of the vessel with cells grown in the inner region of the vessel. Furthermore, static culture can create various biologically distinct niches because it maintains microenvironments with varying cell densities that cannot be sustained in dynamic culture.

[0072] As used herein, "dynamic 2D" or "dynamic 2D" refers to cells grown on a microcarrier to form a monolayer. For example, a suspended adhesion agent that enables cells to adhere to form a dynamic 2D culture (e.g., Microcarrier It is a cell cultured in a dynamic culture (e.g., a bioreactor) having ).

[0073] As used herein, “dynamic three-dimensional” or “dynamic 3D” refers to cells grown as aggregates in a suspended state, such as in cell culture in an artificially created environment that allows biological cells to grow in all three dimensions or interact with their surroundings. Unlike a 2D environment, 3D cell culture allows cells to grow in all directions in vitro, similar to how they grow in vivo. Such 3D culture can be carried out, for example, in a bioreactor, which is a small vesicle where cells can grow into spherical bodies or 3D cell aggregates.

[0074] As used herein, "sensory neuron progenitor cells" refer to self-renewing pluripotent cells that first generate radial glial progenitor cells, which produce neurons and glial cells of the nervous system of all animals during embryonic development. Sensory neuron progenitor cells can occur naturally, but their cellular composition differs from that of cells induced from undifferentiated pluripotent stem cells using the method disclosed herein.

[0075] The "cochlear nerve" or "auditory nerve" refers to the nerve that transmits auditory sensory information from the cochlea of ​​the inner ear to the brain. In humans, there are an average of about 30,000 nerve fibers within the cochlear nerve. The cell bodies of the neurons of the cochlear nerve are located within the cochlea and collectively form the spiral ganglion.

[0076] As used herein, "inner ear sensory hair cells" or simply "hair cells" refer to the mechanosensory hair cells of the cochlea (auditory system) and the saccule, utricle, ampulla, and semicircular canals (vestibular system), which contribute to detecting and amplifying sound and maintaining balance, respectively. Hair cells resemble columnar cells, each possessing bundles of stereocilia on its apical surface. When the stereocilia bend, mechanically gated ion channels open, allowing small, positively charged ions (primarily potassium and calcium) to enter the hair cell. Unlike many other electroactive cells, hair cells themselves do not generate action potentials. Rather, the influx of cations depolarizes the cell, generating a receptor potential. Therefore, hair cells typically exhibit a differential electrical response rather than the action potential spikes typical of other neurons. Hair cells may express one or more of the following markers at detectable levels: atonal homologue 1 (Atoh1 / MATH1 / HATH1), myosin VI (MYO6), myosin VIIA (MYO7A), espin (ESPN), myosin heavy chain 3 (MYH2), cadherin 23 (CDH23), protocadherin 15 (PCDH15), autopherin (OTOF), and prestin (SLC26A5).

[0077] As used herein, "inner ear supporting cells," or simply "supporting cells," refer to cells that contribute to the complex structural and functional characteristics of the cochlea, e.g., Datas (phalangeal) cells, Hensen cells, Claudius cells, Boetzer cells, pillar cells, marginal cells, etc., and cells of the saccule, utricle, ampulla, and semicircular canals. Supporting cells can be identified by short microvilli on the surface of the apical cells. Additionally, they are found in very close proximity to hair cells; that is, they are found immediately adjacent to hair cells, as clusters containing hair cells. Supporting cells may express one or more of the following markers at detectable levels: cyclin-dependent kinase inhibitor 1B (CDKN1B, p27 (KIP1)), prosperohomeobox 1 (PROX1), autoancorin (OTOA), Musashi homologue 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), YRPW motif-associated hairy / splitting region enhancer 2 (HEY2).

[0078] As used herein, “auditory neuron,” “auditory neurons” (abbreviated as AN), “auditory cell,” or “auditory cells” refers to a group of sensory cells of the ear that includes, but is not intended to be limited to, hair cells, supporting cells, ear progenitor cells, sensory neuron progenitor cells, etc. In some cases, the term “auditory cell” may refer to a mixed group of cells that includes any combination of the cell types described above in any ratio.

[0079] As used herein, "hearing impairment," "auditory condition," "hearing disorder," or "auditory condition" refers to a condition or disorder including, but not intended to be limited to, conductive hearing loss, sensorineural hearing loss, central hearing loss, mixed hearing loss, auditory neuropathy spectrum disorder, central auditory processing disorder, and tinnitus.

[0080] As used here, "conductive hearing loss" refers to impaired transmission of sound waves through the external auditory canal to the bones of the middle ear.

[0081] As used here, "sensorineural hearing loss" refers to pathological changes in the structure of the inner ear or the auditory nerve.

[0082] The term "central hearing loss" used at this institution refers to a pathological condition above the junction of the auditory nerve and the brainstem.

[0083] As used herein, "mixed hearing loss" refers to subjects who have both conductive hearing loss and sensorineural hearing loss.

[0084] As used herein, "hidden hearing loss" (or HHL) refers to subjects having hearing loss resulting from damage to the auditory nerve / auditory neurons.

[0085] The term "auditory neuropathy spectrum disorder" used here refers to a type of sensorineural hearing loss in which the auditory nerve fails to send consistent messages to the brain's auditory center. Auditory neuropathy is a difficult hearing disorder in which the inner ear successfully detects sound but has trouble sending signals from the ear to the brain; it currently accounts for approximately 10% of cases of sensorineural hearing loss (SNHL) in children. The current state of knowledge in the relevant medical technology field suggests that auditory neuropathy plays a substantial role in hearing impairment and hearing loss. Hearing relies on a series of complex steps to convert sound waves in the air into electrical signals. The auditory nerve then carries these signals to the brain. Auditory neuropathy can be caused by numerous factors, including damage to or loss of auditory neurons, various genetic mutations, and viral infections. Researchers are still seeking effective treatments for patients with auditory neuropathy.

[0086] As used here, "central auditory processing disorder" refers to a defect in the neural processing of auditory information in the central auditory nervous system.

[0087] Treatment methods

[0088] In some aspects, the present disclosure provides a method for replacing sensory neurons. In some embodiments, the method comprises administering a therapeutically effective amount of a pharmaceutical composition comprising a population of auditory cells, wherein (a) at least 20% of the cells in the population express SOX2; (b) at least 10% of the cells in the population express β-tubulin III; (c) at least 5% of the cells in the population express TrkB; and (d) at least 1% of the cells in the population express TRA-1-60 and / or SSEA5.

[0089] In some aspects, the present disclosure provides a method for replacing sensory neurons. In some embodiments, the method comprises administering a therapeutically effective amount of a pharmaceutical composition comprising a population of auditory cells, wherein (a) at least 75% of the cells in the population express both Nestin and SOX2; (b) at least 80% of the cells in the population express β-tubulin III; (c) at least 5% of the cells in the population express TrkB; and (d) at least 1% of the cells in the population express TRA-1-60 and / or SSEA5.

[0090] The present disclosure provides a method for treating a subject with an auditory condition or impairment who has received or is scheduled to receive a cochlear implant. The method comprises administering to the subject a pharmaceutical composition comprising a population of auditory cells as described herein. The method comprises administering the pharmaceutical composition to the inner ear. Upon implantation, the population of cells can reconstitute the cochlea with functional auditory neurons. Furthermore, the population of cells may not only constitute the implantation site but also spread to an area adjacent to the administration site. Without being bound by theory, it is believed that the function of the cochlear implant is improved by increasing the number of auditory neurons.

[0091] Accordingly, the present disclosure provides a method for treating a subject having an auditory condition, comprising inserting a cochlear implant and administering a therapeutically effective amount of a pharmaceutical composition comprising a population of auditory cells, wherein (a) at least 20% of the cells in the population express SOX2; (b) at least 10% of the cells in the population express β-tubulin III; (c) at least 5% of the cells in the population express TrkB; and (d) at least 1% of the cells in the population express TRA-1-60 and / or SSEA5.

[0092] Accordingly, the present disclosure provides a method for treating a subject having an auditory condition, comprising inserting a cochlear implant and administering a therapeutically effective amount of a pharmaceutical composition comprising a population of auditory cells, wherein (a) at least 75% of the cells in the population express both Nestin and SOX2; (b) at least 80% of the cells in the population express β-tubulin III; (c) at least 5% of the cells in the population express TrkB; and (d) at least 1% of the cells in the population express TRA-1-60 and / or SSEA5.

[0093] In some embodiments, the method comprises inserting a cochlear implant and administering a therapeutically effective amount of a pharmaceutical composition comprising a population of auditory cells, wherein (a) at least 30% of the cells in the population express SOX2; (b) at least 30% of the cells in the population express PAX2; (c) at least 30% of the cells in the population express β-tubulin III; (d) at least 20% of the cells in the population express TrkB; (e) at least 30% of the cells in the population express GluA4; (f) at least 20% of the cells in the population express Myo7A; and (g) at least 0.1% of the cells in the population express TRA-1-60 and / or SSEA5.

[0094] In some embodiments, the method comprises inserting a cochlear implant and administering a therapeutically effective amount of a pharmaceutical composition comprising a population of auditory cells, wherein (a) at least 85% of the cells in the population express both Nestin and SOX2; (b) at least 90% of the cells in the population express β-tubulin III; (c) at least 20% of the cells in the population express TrkB; (d) at least 20% of the cells in the population express GluA4; (e) at least 10% of the cells in the population express Myo7A; and (f) at least 0.1% of the cells in the population express TRA-1-60 and / or SSEA5.

[0095] Additionally, the present disclosure provides a method for enhancing the efficacy of a cochlear implant in a subject having an auditory pathology, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising a population of auditory cells, wherein (a) at least 20% of the cells in the population express SOX2; (b) at least 10% of the cells in the population express β-tubulin III; (c) at least 5% of the cells in the population express TrkB; and (d) at least 1% of the cells in the population express TRA-1-60 and / or SSEA5.

[0096] Additionally, the present disclosure provides a method for enhancing the efficacy of a cochlear implant in a subject having an auditory pathology, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising a population of auditory cells, wherein (a) at least 85% of the cells in the population express both Nestin and SOX2; (b) at least 90% of the cells in the population express β-tubulin III; (c) at least 15% of the cells in the population express TrkB; and (d) at least 0.1% of the cells in the population express TRA-1-60 and / or SSEA5.

[0097] In some embodiments, a method for enhancing the efficacy of a cochlear implant in a subject having an auditory pathology comprises administering a therapeutically effective amount of a pharmaceutical composition comprising a population of auditory cells, wherein (a) at least 30% of the cells in the population express SOX2; (b) at least 30% of the cells in the population express PAX2; (c) at least 30% of the cells in the population express beta-tubulin III; (d) at least 20% of the cells in the population express TrkB; (e) at least 30% of the cells in the population express GluA4; (f) at least 20% of the cells in the population express Myo7A; and (g) at least 0.1% of the cells in the population express TRA-1-60 and / or SSEA5.

[0098] In some embodiments, a method for enhancing the efficacy of a cochlear implant in a subject having an auditory pathology comprises administering a therapeutically effective amount of a pharmaceutical composition comprising a population of auditory cells, wherein (a) at least 85% of the cells in the population express both Nestin and SOX2; (b) at least 90% of the cells in the population express β-tubulin III; (c) at least 15% of the cells in the population express TrkB; (d) at least 10% of the cells in the population express GluA4; (e) at least 10% of the cells in the population express Myo7A; and (f) at least 0.1% of the cells in the population express TRA-1-60 and / or SSEA5. In some embodiments, (a) about 70% to 100% of the cells in the population express both Nestin and SOX2; (b) about 80% to 100% of the cells in the population express β-tubulin III; (c) about 5% to 80% of the cells in the population express TrkB; and (d) 0% to about 0.1% of the cells in the population express TRA-1-60 and / or SSEA5.

[0099] In some embodiments, the pharmaceutical composition is administered prior to the insertion of a cochlear implant. For example, the pharmaceutical composition is administered at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 1 year, at least 2 years, or at least 3 years prior to the insertion of a cochlear implant.

[0100] In some embodiments, the pharmaceutical composition is administered before or in conjunction with the insertion of the cochlear implant, for example, at the same surgery. The surgery associated with the insertion may enable the delivery of cell-based therapies as described herein.

[0101] In some embodiments, the pharmaceutical composition is administered after the insertion of a cochlear implant. For example, the pharmaceutical composition is administered at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 1 year, at least 2 years, or at least 3 years after the insertion of the cochlear implant.

[0102] Any hearing impairment suitable for the method of the present disclosure includes hearing impairment involving the loss of cochlear hair cells and / or cochlear neurons. The loss of auditory nerve cells can result in auditory neuropathy even if the hair cells and cochlear nuclei remain intact. Cell-based therapies that replace lost or dysfunctional auditory neurons can restore hearing in such cases. In more severe cases where both hair cells and many neurons are lost, the success rate of a cochlear implant procedure can be improved by reconstructing the cochlea with implanted functional auditory neurons. A person skilled in the art will recognize that the loss does not have to be complete. For example, loss of more than 30%, 40%, 50%, 60%, 70%, 80%, or 90% of cochlear hair cells and / or neurons, or complete loss, may result in hearing loss suitable for treatment using the method described herein.

[0103] Cochlear implants for use in the method described herein, and methods for inserting such implants, are known in the art. Exemplary cochlear implants include, but are not limited to, the Cochlear™ Nucleus® system (a product of Cochlear® Corporation), cochlear implants available from Advanced Bionics, CI systems from MED-EL, and Oticon. In an exemplary insertion procedure, the surgeon makes a small incision behind the ear and then drills a hole in the mastoid bone to guide an electrode from the implant to the cochlea. The electrode is placed in the cochlea, and an internal processor is placed in the pocket between the bone and muscle behind the ear.

[0104] Hearing impairments and conditions that can be treated using the methods described herein include, but are not intended to be limited to, conductive hearing loss, sensorineural hearing loss, mixed hearing loss, auditory neuropathy spectrum disorders, central hearing loss, central auditory processing disorders, and tinnitus. These methods involve administering cells or populations of cells as described herein into the ear of a subject. The cells to be administered 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 comprises the step of administering a therapeutic agent (e.g., a differentiation agent as described herein) that promotes the expression of auditory proteins within cells in the inner ear. When used, the differentiation agent may be administered to cells in a culture, or to the subject alone (to stimulate differentiation of stem cells or progenitor cells in the subject's inner ear) or together with undifferentiated cells (e.g., undifferentiated cells isolated by the methods described herein). Differentiation agents may be, for example, agonists of the Hedgehog pathway, such as Sonic Hedgehog agonists, or Purmorphamin (e.g., Hh-Ag1.3).

[0105] Subjects with inner ear disorders or at risk of developing such disorders may be treated with auditory cells as described herein in addition to the cochlear implant. Upon successful engraftment, at least some of the transplanted spiral ganglion neurons will form synaptic contacts with targets, for example, hair cells and within the cochlear nucleus. To improve the ability of the cells to engraft, stem cells can be modified prior to differentiation. For example, cells can be engineered to overexpress one or more anti-apoptotic genes in progenitor or differentiated cells. The Fak tyrosine kinase or Akt gene is a candidate anti-apoptotic gene that may be useful for this purpose; overexpression of FAK or Akt can prevent apoptosis in spiral ganglion cells and increase engraftment rates when transplanted into another tissue, such as an explanted organ of Corti (see, e.g., [Mangi et al., Nat. Med. 9:1195-201, 2003]). Neural progenitor cells overexpressing alpha.sub.v.beta.sub.3 integrins may have an enhanced ability to extend neurites into tissue explants, as integrins have been shown to mediate neurite extension from spiral ganglion neurons on laminin substrates (Aletsee et al., Audiol. Neurootol. 6:57-65, 2001). In another example, ephrin B2 and ephrin B3 expression can be altered by silencing, for instance, using RNAi, or by overexpression using exogenously expressed cDNA, thereby modifying 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). Inactivation of these guide signals can improve the number of neurons reaching targets in the adult inner ear.Exogenous factors, such as the neurotrophins BDNF and NT3, and LIFs, can be added to tissue grafts to enhance neurite elongation and growth into target tissues in in vivo and in vitro tissue cultures. Neuroite elongation of sensory neurons can be enhanced by the addition of neurotrophins (BDNF, NT3) and LIFs (Gillespie et al., NeuroReport 12:275-279, 2001). Sonic Hedgehog (Shh) polypeptides or polypeptide fragments (e.g., SHH-N) may also be useful as endogenous factors to enhance neurite elongation. Shh is a developmental regulator for the inner ear and a chemoattractor for axons (Charron et al., Cell 113:11 23, 2003).

[0106] Subjects experiencing hearing loss are candidates for the treatment methods described herein. Subjects who have hearing loss or are at risk of developing hearing loss may have hearing that is lower than that of an average subject or lower than that of a subject prior to experiencing hearing loss. For example, hearing may be reduced by at least 5, 10, 30, or 50%. The subject may have sensorineural hearing loss, which is hearing loss resulting from damage or dysfunction of the sensory part (cochlea) or the neural part (auditory nerve) of the ear, or conductive hearing loss caused by blockage or damage to the outer ear and / or middle ear, or the subject may have mixed hearing loss caused by problems in both the conduction pathway (outer ear or middle ear) and the neural pathway (inner ear). In a preferred embodiment, the subject has sensorineural or mixed hearing loss. An example of mixed hearing loss is a combination of conductive loss due to middle ear infection and sensorineural loss due to damage associated with aging.

[0107] The subject may become deaf or have hearing loss for any reason or as a result of any type of event. For example, the subject may be deaf due to a genetic or congenital defect; for example, the subject may have been deaf since birth, or may become deaf or hearing impaired as a result of progressive hearing loss due to a genetic or congenital defect. In another example, the subject may become deaf or hearing impaired as a result of a traumatic event, such as physical trauma to the structure of the ear, or sudden loud noise, or prolonged exposure to loud noise. For example, prolonged exposure to concert halls, airport runways, and construction sites may cause inner ear damage and subsequent hearing loss. The subject may experience chemical-induced ototoxicity, where ototoxics include antineoplastic agents, therapeutic drugs including salicylates, quinine, and aminoglycoside antibiotics, contaminants in food or medicine, and environmental or industrial contaminants. The subject may have hearing impairment resulting from aging, or the subject may have tinnitus (characterized by ringing in the ears).

[0108] Subjects suitable for the method as described herein may include subjects having vestibular dysfunction, including bilateral and unilateral vestibular dysfunction. Vestibular dysfunction is an inner ear dysfunction characterized by symptoms including dizziness, balance disorders, vertigo, nausea, and blurred vision, and may be accompanied by hearing problems, fatigue, and changes in cognitive function. Vestibular dysfunction may be the result of 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 assessed by measuring individual symptoms of the disorder (e.g., vertigo, nausea, and blurred vision).

[0109] Methods as described herein may be used to treat hearing impairment resulting from sensorineural hair cell loss or auditory neuropathy. Subjects suffering from auditory neuropathy experience a loss of cochlear sensory neurons while the hair cells of the inner ear remain intact. These subjects will particularly benefit from treatments that induce cells (stem cells or progenitor cells) to differentiate into spiral ganglion cells, or from the administration of spiral ganglion cells into the inner ear. Subjects with sensorineural hair cell loss experience degeneration of cochlear hair cells, which frequently results in a loss of spiral ganglion neurons in the areas where hair cells were lost. These subjects may also experience a loss of supporting cells of the organ of Corti, and degeneration of vascular stems within the periphery, spiral ligament, and temporal bone material. These subjects may receive treatment using agents that induce cells to differentiate into hair cells, or treatment involving the implantation or injection of tissue grafts containing hair cells into the inner ear. Subjects may additionally benefit from treatments that induce cell differentiation into spiral ganglion cells, or from the administration of spiral ganglion cells into the inner ear. For example, in auditory neurodegeneration from mechanical compression, most auditory spiral ganglion cells degenerate after sustained compression of the Rosenthal canal (causing transneuronal apoptosis of cochlear nuclei), forming a continuous "spontaneous autologous cell bridge" along with astrocyte and Schwann cell columns, which acts as an anatomical scaffold for transplanted cell migration to connect the PNS and CNS (Sekiya et al. 2021 Cell Transplantation Volume 30: 1-20).

[0110] In some embodiments, the hearing impairment or condition is an auditory neuropathy spectrum disorder. Auditory neuropathy spectrum disorders include various conditions arising, for example, from genetic mutations and / or viral infections, which result in dysfunction of auditory nerve function. The method of the present disclosure advantageously provides a single-cell therapy for the treatment of any condition belonging to the auditory neuropathy spectrum disorder, rather than a number of targeted therapies depending on the underlying causative agent.

[0111] In some embodiments, the hearing impairment or condition is hidden hearing loss (HHL). HHL is a condition in which hearing functions normally when exposed to a single sound or frequency, but deteriorates when exposed to multiple sounds (e.g., parties, noisy streets, restaurants). Since hair cells function normally, damaged auditory neurons are thought to be the cause of the condition. Up to 10% of patients reporting hearing loss have normal audiometric results indicating HHL. HHL can be treated with hearing aids, for example, cochlear implants. In some embodiments, a subject has HHL and a cochlear implant is inserted. In some embodiments, the cochlear implant can induce sound-mediated electrical signals in functional auditory neurons and thus replace damaged or lost hair cells, but because it cannot replace the damaged or lost auditory neurons, the subject with the cochlear implant may still suffer from HHL.

[0112] In some embodiments, the method provided herein is a method of replacing auditory neurons in a subject in need and inserting a cochlear implant. In some embodiments, the method provided herein is a method of augmenting an existing but damaged population of auditory neurons in a subject in need and inserting a cochlear implant.

[0113] Auditory cells produced by the method described herein may be administered, for example, in the form of a cell suspension, by injection into, for example, the inner or middle ear, into, above, or near, the lumen of the cochlea or auditory nerve, for example, through the postmastoid pathway. In some embodiments, the method comprises administering a pharmaceutical composition comprising a population of cells as described herein to the inner or middle ear of a subject. In some embodiments, administration to the inner ear includes administration to the cochlea. In some embodiments, the composition is administered to the tympanic system, the cochlear axis, or the spiral ganglion.

[0114] In some embodiments, a population of cells is administered via injection, for example, through a microneedle. In some embodiments, the injection involves inserting a cannula through the opening of the sac or through a round window.

[0115] Injection may be performed, for example, through the round window of the ear or through the bony sac surrounding the cochlea. Cells may be injected into the auditory nerve trunk of the internal auditory canal, the cochlear axis, or the tympanic cavity through the round window as described below. Administration of auditory cells as described herein may be achieved, for example, for the treatment of auditory or hearing loss conditions as described herein, using an injection needle or syringe positioning device known in the relevant art that can control the movement and position of the needle (e.g., manually or via a robotic interface) to a desired target anatomical structure of the inner or middle ear. Such devices include stabilization necessary to facilitate the safe and effective delivery of auditory cells over a given period to ensure the delivery of a concentration or volume of auditory cells as described herein, for example. Exemplary administration routes are described, for example, at otosurgeryatlas.stanford.edu / otologic-surgery-atlas / cochlear-implantation / cochlear-implant-surgical-variations / .

[0116] For example, in an exemplary administration route to the tympanic cavity via a cochleotomy, a small hole is made through the otosac at the base of the cochlea to insert a cannula. This hole is covered with a small piece of fascia. Cells are loaded onto a 30G cannula, primed with saline, and the composition is aspirated after aspirating with about 1 μL of air. Cells are injected into the tympanic cavity using a pump at a rate of about 1 μL / min. In a second exemplary administration route, the cochleotomy is performed through the otosac. A dental drill is used to create a hole for inserting a 33G needle and cannula into the cochlear axis, and then penetrates the bony wall of the cochlear axis through the hole perforation. This hole is covered with a small piece of fascia. Cells are loaded onto the cannula and injected into the cochlear axis as described above. The cannula is left in place for about 10 minutes to allow the fluid to equilibrate. Alternatively, the composition of the present disclosure may be delivered through the round window. First, the round window is exposed, the round window mucosa is removed or incised, and bony protrusions are removed with a drill. A cannula is directed through the round window into the tympanic cavity or cochlear axis, and cells are administered as described above.

[0117] In some embodiments, a population of cells is administered simultaneously with the insertion of the cochlear implant, for example, in the same surgical procedure.

[0118] In some embodiments, the method comprises administering about 100,000 to 50 million cells, about 100,000 to 10 million cells, about 100,000 to 1 million cells, about 200,000 to 10 million cells, about 500,000 to 1 million cells, or about 100,000 to 500,000 cells to a subject. In some embodiments, about 100,000 to about 1 million cells are administered to a subject. In some embodiments, about 30 million cells per milliliter to about 700 million cells per milliliter are administered to a subject.

[0119] In some embodiments, administration of the composition described herein alleviates signs or symptoms of a hearing disease or condition in a subject. In some embodiments, the method improves hearing in a subject, reduces the severity of hearing loss, delays the progression of hearing loss, and alleviates one or more symptoms associated with a hearing disease or disorder.

[0120] The administration of auditory cells as described herein may be achieved, for example, by the pre-injection of a coating material, such as a matrix component, a serum component, or a biodegradable scaffold, which can ensure the delivery of the concentration or volume of auditory cells as described herein by enhancing the attachment and integration of implanted sensory neurons. The cell product may be cryopreserved in a cryo-vial made of plastic, glass, or other polymers, or a rubber-plastic copolymer, such as a cyclic olefin copolymer. The vial may be sealed with a screw cap or a stopper made of a rubber-plastic copolymer, such as a thermoplastic elastomer, to enable sterile transfer of the cell product to a delivery device. The cell product may be pre-loaded and cryopreserved in a syringe, syringe cartridge, or injection cannula, which is thawed before administration to a subject. The cell product may be cryopreserved and stored as a ready-made allogeneic cell therapy bank. The cell product may be frozen at a clinical dose and administered to a subject immediately after thawing at the clinical site.

[0121] Pharmaceutical composition

[0122] The present disclosure provides a pharmaceutical composition comprising a population of cells for use in the method described herein. A person skilled in the art will recognize that the composition described herein may comprise a mixed population of cell types, and that the identity of cell types is reflected in the percentage of cells in the population expressing one or more of the markers described herein. Alternatively, the population of cells may be substantially pure as determined by the expression of any combination of the markers disclosed herein (e.g., greater than 90%, greater than 95%, greater than 97%, greater than 98%, or greater than 99% pure). Depending on the differentiation state of the cells, individual cells in the population may express only the single marker described below, or individual cells may express a combination of the markers described below. Cells within the population may express neural progenitor cell markers, e.g., Nestin; ONP markers, e.g., PAX2, PAX8, and / or SOX2; neuron markers, e.g., β-tubulin III; auditory neuron markers, e.g., TrkB and / or GluA4; non-specific neuron markers, e.g., Myo7A; or hESC markers, e.g., TRA-1-60 and / or SSEA5. In some embodiments, cells within the population express SOX2; cells within the population express β-tubulin III; cells within the population express TrkB; and cells within the population express TRA-1-60 and / or SSEA5. In some embodiments, cells within the population express both Nestin and SOX2; cells within the population express β-tubulin III; cells within the population express TrkB; and cells within the population do not express TRA-1-60 and / or SSEA5.

[0123] In some embodiments, cells in the population express SOX2; cells in the population express β-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.

[0124] In some embodiments, cells in the population express both Nestin and SOX2; cells in the population express β-tubulin III; cells in the population express TrkB; cells in the population express GluA4; cells in the population express Myo7A; and optionally, cells in the population do not express TRA-1-60 and / or SSEA5.

[0125] In some embodiments, cells in the population do not express PAX2 and / or PAX8.

[0126] In some embodiments, cells in a population express Nestin. Nestin is a member of the intermediate filament protein family and is expressed in neurons. 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 a population express Nestin. In some embodiments, 40% or more of the cells in a population express Nestin. In some embodiments, 50% or more of the cells in a population express Nestin. In some embodiments, 60% or more of the cells in a population express Nestin. In some embodiments, 70% or more of the cells in a population express Nestin. In some embodiments, 80% or more of the cells in a population express Nestin. In some embodiments, 90% or more of the cells in a population express Nestin. In some embodiments, 99% or more of the cells in a population express Nestin. In some embodiments, 10% to 95%, 20% to 90%, 30% to 80%, 40% to 70%, or 30% to 60% of the cells in the population express Nestin. In some embodiments, 10% to 99%, 20% to 95%, 30% to 90%, 40% to 85%, or 50% to 80% of the cells in the population express Nestin. In some embodiments, 70% to 90% of the cells in the population express Nestin.

[0127] In some embodiments, cells in the population express SOX2. SOX2 codes for a member of the SRY (sex-determining region Y)-associated HMG-box (SOX) family of transcription factors involved in the regulation of embryonic development and the determination of 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, 99% or more of the cells in the population express SOX2. In some embodiments, 10% to 99%, 20% to 95%, 30% to 90%, 40% to 85%, or 50% to 80% of the cells in the population express SOX2. In some embodiments, 10% to 95%, 20% to 90%, 30% to 80%, 40% to 70%, or 30% to 60% of the cells in the population express SOX2. In some embodiments, 70% to 90% of the cells in the population express SOX2.

[0128] 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 both Nestin and SOX2. In some embodiments, 30% or more of the cells in the population express both Nestin and SOX2. In some embodiments, 40% or more of the cells in the population express both Nestin and SOX2. In some embodiments, 50% or more of the cells in the population express both Nestin and SOX2. In some embodiments, 60% or more of the cells in the population express both Nestin and SOX2. In some embodiments, 70% or more of the cells in the population express both Nestin and SOX2. In some embodiments, 80% or more of the cells in the population express both Nestin and SOX2. In some embodiments, 90% or more of the cells in the population express both Nestin and SOX2. In some embodiments, more than 99% of the cells in the population express both Nestin and SOX2. In some embodiments, 10% to 99%, 20% to 95%, 30% to 90%, 40% to 85%, or 50% to 80% of the cells in the population express both Nestin and SOX2. In some embodiments, 70% to 90% of the cells in the population express both Nestin and SOX2.

[0129] In some embodiments, cells in a population express PAX8. PAX8 codes for a member of the paired box family of transcription factors containing a paired box domain, an octapeptide, and a paired-type homeodomain domain. In some embodiments, less than 70%, less than 60%, less than 50%, less than 40%, less than 20%, less than 10%, or less than 5% of the cells in a population express PAX8. In some embodiments, less than 60% of the cells in a population express PAX8. In some embodiments, less than 50% of the cells in a population express PAX8. In some embodiments, less than 40% of the cells in a population express PAX8. In some embodiments, less than 20% of the cells in a population express PAX8. In some embodiments, less than 10% of the cells in a population express PAX8. In some embodiments, less than 5% of the cells in a population express PAX8. In some embodiments, less than 1% of the cells in a population express PAX8. In some embodiments, less than 0.1% of the cells in the population express PAX8. In some embodiments, less than 0.01% of the cells in the population express PAX8. In some embodiments, 0.1% to 60%, 1% to 50%, 0.1% to 30%, 1% to 20%, 5% to 15%, or 5% to 8% of the cells in the population express PAX8. In some embodiments, the cells in the population do not express PAX8 at a detectable level, that is, the level of PAX8 is below the detection limit by a suitable assay described herein and known in the art.

[0130] In some embodiments, cells in the population express Six1. Six1 is a homeobox protein gene found in a cluster of related genes on chromosome 14 and is thought to be involved in limb development. In some embodiments, 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, 10% to 95%, 20% to 90%, 30% to 80%, 40% to 70%, or 30% to 60% of the cells in the population express Six1.

[0131] In some embodiments, cells in the population express PAX2. PAX2 codes for paired box gene 2, which is the target of transcriptional repression by the tumor suppressor gene WT1. In some embodiments, 10% or less, 20% or less, 30% or less, 40% or less, 50% or less, 60% or less, or 70% or less of the cells in the population express PAX2. In some embodiments, 20% or less of the cells in the population express PAX2. In some embodiments, 30% or less of the cells in the population express PAX2. In some embodiments, 40% or less of the cells in the population express PAX2. In some embodiments, 50% or less of the cells in the population express PAX2. In some embodiments, 60% or less of the cells in the population express PAX2. In some embodiments, 70% or less of the cells in the population express PAX2. In some embodiments, 0% to 60%, 0.01% to 50%, 0.1% to 40%, or 1% to 30% of the cells in the population express PAX2. In some embodiments, 0.01% to 50% of the cells in the population express PAX2. In some embodiments, 1% to 30% of the cells in the population express PAX2. In some embodiments, the cells in the population do not express PAX2 at a detectable level, that is, the level of PAX2 is below the detection limit by a suitable assay described herein and known in the art.

[0132] In some embodiments, cells in the population express PAX2. PAX2 codes for paired box gene 2, which is the target of 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. In some embodiments, 10% to 99%, 20% to 95%, 30% to 80%, 40% to 70%, or 50% to 60% of the cells in the population express PAX2.

[0133] In some embodiments, cells in the population express GluA4. GluA4 codes for a glutamate receptor expressed in excitatory neurotransmitter-secreting neurons of the brain and is activated in various 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, 15% 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, 25% 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% to 99%, 10% to 95%, 20% to 90%, 30% to 80%, 30% to 60%, or 20% to 50% of the cells in the population express GluA4. In some embodiments, about 10% to 95% of the cells in the population express GluA4. In some embodiments, 30% to 90% of the cells in the population express GluA4. In some embodiments, about 10% to 75% of the cells in the population express GluA4. In some embodiments, about 15% to 70% of the cells in the population express GluA4.

[0134] In some embodiments, cells in the population express CD133. CD133 codes for the pentaspan transmembrane glycoprotein, which is localized at membrane protrusions and is often expressed in adult stem cells to maintain stem cell characteristics 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, more than 95% of the cells in the population express CD133. In some embodiments, 1% to 99%, 10% to 95%, 20% to 90%, 30% to 80%, or 40% to 70% of the cells in the population express CD133.

[0135] In some embodiments, cells in the population express GATA3. GATA3 is a regulator of T-cell development and plays a role in endothelial cell biology. A defect in GATA3 is the cause of hypoparathyroidism accompanied by 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% to 10%, 0.1% to 5%, or 0.1% to 1% of the cells in the population express GATA3.

[0136] In some embodiments, cells in a population express β-tubulin III (also referred to as β-III tubulin or beta-3 tubulin, etc.). β-tubulin III codes for a member of the beta-tubulin protein family that heteromerizes and assembles 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 a population express β-tubulin III. In some embodiments, 10% or more of the cells in a population express β-tubulin III. In some embodiments, 20% or more of the cells in a population express β-tubulin III. In some embodiments, 30% or more of the cells in a population express β-tubulin III. In some embodiments, 40% or more of the cells in a 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, 1% to 80%, 10% to 70%, 30% to 60%, or 40% to 50% of the cells in the population express β-tubulin III. In some embodiments, 80% to 100% of the cells in the population express β-tubulin III. In some embodiments, 70% to 99% of the cells in the population express β-tubulin III.

[0137] In some embodiments, cells in the population express tropomyosin-associated kinase receptor B (TrkB). TrkB is involved in nervous system development and enables brain-derived neurotrophic factor binding activity and brain-derived neurotrophic factor (BDNF)-activated receptor activity. In some embodiments, 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, 5% to 80%, 10% to 70%, 20% to 50%, or 30% to 40% of the cells in the population express TrkB. In some embodiments, about 5% to about 90%, about 10% to about 80%, about 0% to about 70%, about 5% to about 50%, about 20% to about 90%, or about 30% to about 70% of the cells in the population express TrkB.

[0138] In some embodiments, the fold change in the expression of one or more markers may be compared to the starting cell population. In some embodiments, the fold change in expression may be determined using qPCR. In some embodiments, the starting cell population is a population of hESC cells. In some embodiments, the marker is TrkB. In some embodiments, the fold change in TrkB expression between cells in the population and hESC cells is 100 or more, 300 or more, 500 or more, 1,000 or more, 1,500 or more, 2,000 or more, 3,000 or more, or 5,000 or more. In some embodiments, the fold change in TrkB expression between cells in the population and hESC cells is 100 or more. In some embodiments, the fold change in TrkB expression between cells in the population and hESC cells is 300 or more. In some embodiments, the fold change in TrkB expression between cells in the population and hESC cells is 500 or more. In some embodiments, the fold change in TrkB expression between cells in the population and hESC cells is 1,000 or more. In some embodiments, the fold change in TrkB expression between cells in the population and hESC cells is 1,500 or more. In some embodiments, the fold change in TrkB expression between cells in the population and hESC cells is 2,000 or more. In some embodiments, the fold change in TrkB expression between cells in the population and hESC cells is 3,000 or more. In some embodiments, the fold change in TrkB expression between cells in the population and hESC cells is 5,000 or more. In some embodiments, the fold change in TrkB expression between cells in the population and hESC cells is about 500 to about 10,000, about 1,000 to about 10,000, about 500 to about 7,000, or about 2,000 to about 5,000.

[0139] In some embodiments, cells in the population express tropomyosin-associated kinase receptor C (TrkC). TrkC acts upstream of or within several processes, including neurogenesis and the propagation of neuronal action potentials, and is predicted to enable several functions, including GPI-linked ephrine receptor activity; neurotrophin (NT3) binding activity; and p53 binding activity. In some embodiments, less than 10%, less than 5%, less than 1%, or less than 0.1% of the cells in the population express TrkC. In some embodiments, less than 5% of the cells in the population express TrkC. In some embodiments, less than 1% of the cells in the population express TrkC. In some embodiments, less than 0.1% of the cells in the population express TrkC. In some embodiments, 0.01% to 10%, 0.01% to 5%, or 0.01% to 1% of the cells in the population express TrkC. In some embodiments, TrkC expression is greater than 2%, for example, 2% to 20% or more of the cells express TrkC. In some embodiments, 1% to 100%, 2% to 100%, 2% to 80%, 2% to 50%, or 2% to 20% of the cells in the population express TrkC.

[0140] In some embodiments, cells in the population express brain-specific homeobox / POU domain protein 3a (BRN3A). BRN3A enables various functions, including DNA binding activity and DNA-binding transcription activator activity, and is involved in nervous system development. In some embodiments, less than 10%, less than 5%, less than 1%, or less than 0.1% of the cells in the population express BRN3A. In some embodiments, less than 5% of the cells in the population express BRN3A. In some embodiments, less than 1% of the cells in the population express BRN3A. In some embodiments, less than 0.1% of the cells in the population express BRN3A. In some embodiments, 0.1% to 10%, 0.1% to 5%, or 0.1% to 1% of the cells in the population express BRN3A. In some embodiments, 1% to 20% or more of the cells in the population express BRN3A. In some embodiments, 1% to 30%, 1% to 20%, 5% to 20%, or 1% to 10% of the cells in the population express BRN3A.

[0141] In some embodiments, cells in the population express Myo7A. Mutations in Myo7A are known to play a significant role in the development of hearing loss and blindness. Myo7A is expressed in the nervous system, enables protein domain-specific binding activity, and acts upstream of or within various processes, including organ morphogenesis. In some embodiments, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 1%, or less than 0.1% of the cells in the population express Myo7A. In some embodiments, less than 30% of the cells in the population express Myo7A. In some embodiments, less than 20% of the cells in the population express Myo7A. In some embodiments, less than 10% of the cells in the population express Myo7A. In some embodiments, less than 5% of the cells in the population express Myo7A. In some embodiments, less than 1% of the cells in the population express Myo7A. In some embodiments, less than 0.1% of the cells in the population express Myo7A. In some embodiments, 0.1% to 40%, 1% to 30%, 5% to 20%, or 10% to 15% of the cells in the population express Myo7A. In some embodiments, about 0.5% to about 10% of the cells in the population express Myo7A.

[0142] In some embodiments, cells in the population express stage-specific embryonic antigen (SSEA-5). Undifferentiated cells can be identified by the expression of various markers, including SSEA-5. In some embodiments, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% of the cells in the population express SSEA-5. In some embodiments, less than 5% of the cells in the population express SSEA-5. In some embodiments, less than 1% of the cells in the population express SSEA-5. In some embodiments, less than 0.1% of the cells in the population express SSEA-5. In some embodiments, less than 0.05% of the cells in the population express SSEA-5. In some embodiments, less than 0.01% of the cells in the population express SSEA-5. In some embodiments, 0.01% to 10%, 0.1% to 5%, or 0.1% to 1% of the cells in the population express SSEA-5. In some embodiments, cells in the population do not express SSEA-5 at a detectable level, that is, the level of SSEA-5 is below the detection limit by suitable assays described herein and known in the art.

[0143] In some embodiments, cells in the population express tumor rejection antigen 1-60 (TRA-1-60). Undifferentiated cells can be identified by the expression of various markers, including TRA-1-60. In some embodiments, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% of the cells in the population express TRA-1-60. In some embodiments, less than 5% of the cells in the population express TRA-1-60. In some embodiments, less than 1% of the cells in the population express TRA-1-60. In some embodiments, less than 0.1% of the cells in the population express TRA-1-60. In some embodiments, less than 0.05% of the cells in the population express TRA-1-60. In some embodiments, less than 0.01% of the cells in the population express TRA-1-60. In some embodiments, 0.01% to 10%, 0.1% to 5%, or 0.1% to 1% of the cells in the population express TRA-1-60. In some embodiments, the cells in the population do not express TRA-1-60 at a detectable level, that is, the level of TRA-1-60 is below the detection limit by suitable assays described herein and known in the art.

[0144] In some embodiments, (a) more than 20% of the cells in the population express SOX2; (b) more than 10% of the cells in the population express β-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.

[0145] In some embodiments, (a) more than 30% of the cells in the population express SOX2; (b) more than 30% of the cells in the population express PAX2; (c) more than 30% of the cells in the population express β-tubulin III; (d) more than 20% of the cells in the population express TrkB; (e) more than 30% of the cells in the population express GluA4; (f) less than 20% of the cells in the population express Myo7A; and (g) less than 0.1% of the cells in the population express TRA-1-60 and / or SSEA5.

[0146] In some embodiments, (a) at least 30% of the cells in the population express SOX2, for example, 70% to 100% of the cells express SOX2; (b) at least 30% of the cells in the population, for example, 50% to 100% of the cells express β-tubulin III; (c) at least 10% of the cells in the population, for example, 10% to 80% of the cells express TrkB; (d) at least 10% of the cells in the population, for example, 10% to 70% of the cells express GluA4; (e) at least 20% of the cells in the population, for example, 0.1% to 10% of the cells express Myo7A; and (f) at least 0.1% of the cells in the population, for example, 0 to 0.01% of the cells express TRA-1-60 and / or SSEA5.

[0147] After harvesting according to the method described herein, the population of expanded auditory cells can be formulated into a specific therapeutic dose (e.g., number of cells) and cryopreserved for transport to a clinic. Subsequently, the immediate administration (RTA) auditory cell therapy composition can be administered directly after thawing without further processing. Examples of media suitable for cryopreservation include 90% human serum / 10% DMSO, CRYOSTOR®, CRYOSTOR® CS10 (10% DMSO), CRYOSTOR® CS5 (5% DMSO), CRYOSTOR® CS2 (2% DMSO), and STEM-CELLBANKER.® , Prime XV ® FREEZIS, HYPOTHERMASOL ® Includes, but is not limited to, trehalose, etc. In an embodiment, the cryopreservation medium comprises about 0.5% to about 50%, e.g., about 0.5%, about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, or about 50% DMSO. In an embodiment, the cryopreservation medium comprises about 0.5% to about 30% DMSO. In an embodiment, the cryopreservation medium comprises about 1% to about 20% DMSO.

[0148] In some embodiments, the final cell composition is a cell aggregate, and this cell aggregate is filtered to be separated from the carrier, cell debris, or matrix. In other embodiments, before cryopreservation, the cells are filtered using a disposable filter, cell filter, or mesh having a pore size of at least 40 μm, about 50 μm, about 70 μm, about 100 μm, or about 60 μm to separate single cells from the cell aggregate, carrier, cell debris, or matrix. The filter may be located within a closed system. In other embodiments, the separation of single cells from the cell aggregate, carrier, cell debris, or matrix may be performed by tangential flow centrifugation. The filtration system has the capacity to safely filter single cells through the pores in amounts of 1 million cells, 10 million cells, 100 million cells, 1 billion cells, 10 billion cells, or 100 billion cells. The percentage viability of cells after filtration 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 may be any value or sub-range within the listed ranges. In another embodiment, the percentage recovery rate of cells after filtration 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 rate may be any value or sub-range within the listed ranges.

[0149] In an alternative embodiment, the cell in the final cell composition is a single cell in the suspension. For example, the single cell in the composition may be produced by dissociating the aggregate described herein by any method known in the art to produce a viable single cell.

[0150] In a further embodiment, the percentage viability of cells after filtration, stored in a neutralization medium for about 0 to about 8 hours and then stored in a cryopreservation medium for about 0 to 8 hours, is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In another embodiment, the percentage recovery rate of cells after filtration, stored in a neutralization medium for about 0 to about 8 hours and then stored in a 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 survival rate may be any value or sub-range within the listed range.

[0151] In a further embodiment, the percentage viability of cells after filtration, stored in a neutralization medium for about 0 to about 8 hours and then stored in a 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% after thawing of the cryopreserved composition. In a further embodiment, the percentage recovery rate of cells after filtration, stored in a neutralization medium for about 0 to about 8 hours and then stored in a 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% after thawing of the cryopreserved composition. The viability may be any value or sub-range within the listed ranges.

[0152] In some embodiments, the percentage viability of auditory cells after filtration 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 percentage viability of auditory cells after filtration 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 a further embodiment, the percentage viability of auditory cells after filtration, 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 another additional embodiment, the percentage recovery rate of auditory cells after filtration, stored in a neutralization solution at room temperature for about 0 to 8 hours and then stored in a cryopreservation medium at room temperature for about 0 to 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 viability may be any value or sub-range within the listed ranges.

[0153] Auditory cells formulated in a cryopreservation medium suitable for immediate administration after thawing (RTA) application may comprise 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, dimethyl sulfoxide (DMSO), and auditory cells suspended in water. One example of such a cryopreservation medium is Cryostor. ® It is commercially available under the trade name and is manufactured by BioLife Solutions, Inc. In some embodiments, auditory cell aggregates are formulated as an immediate injectable product in an aggregate-compatible cryopreservation medium, such as CryoStem®, using an aggregate-specific delivery system, such as the Sutter Xenowork system, which is used for somatic cell nuclear delivery and intracytoplasmic sperm injection and more recently for ONP globules (Heuer et al. 2020).

[0154] DMSO can be used as a cryoprotectant to prevent the formation of ice crystals that could kill cells during the cryopreservation process. In some embodiments, the cryopreservable auditory cell therapy composition contains about 0.1% to about 2% DMSO (v / v). In some embodiments, the RTA auditory cell therapy composition contains about 1% to about 20% DMSO. In some embodiments, the RTA auditory cell therapy composition contains about 10% DMSO. In some embodiments, the RTA auditory cell therapy composition contains about 5% DMSO. The concentration may be any value or sub-range within the listed ranges.

[0155] In some embodiments, an auditory cell therapy composition formulated in a cryopreservation medium suitable for immediate administration after thawing (RTA) application may comprise auditory cells suspended in a cryopreservation medium that does not contain DMSO. For example, the RTA sensory therapy cell composition may comprise Trolox, Na₂SO₄, without DMSO (dimethyl sulfoxide, (CH3)2SO) or any other bipolar aprotic solvent. + , K + , Ca 2+ , Mg 2+ , Cl - , H2PO4 - It may contain auditory cells suspended in HEPES, lactobionate, sucrose, mannitol, glucose, dextran-40, adenosine, and glutathione. An example of such a cryopreservation medium is hypothermosoll ® or hypothermozol ® It is commercially available under the trade name FRS and is also manufactured by Biolife Solutions, Inc. In another embodiment, an auditory cell composition formulated in a cryopreservation medium suitable for administration immediately after thawing may comprise auditory cells suspended in trehalose.

[0156] 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 an activator of the function of the auditory cell preparations described herein.

[0157] In some embodiments, the RTA auditory cell therapy composition may be formulated in a medium containing components that reduce molecular cell stress during the freezing and thawing process by scavenging free radicals, pH buffering, colloid osmotic pressure / osmotic pressure support, and maintaining ion concentration balance.

[0158] In some embodiments, an auditory cell therapy formulated in a cryopreservation medium suitable for immediate administration after thawing may comprise one or more immunosuppressive compounds. In certain embodiments, an auditory cell therapy formulated in a cryopreservation medium suitable for immediate administration after thawing may comprise one or more immunosuppressive compounds formulated to release one or more immunosuppressive compounds slowly. The immunosuppressive compounds for use in the formulations described herein may belong to the following classes of immunosuppressants: glucocorticoids, cell arresters (e.g., alkylating agents or antimetabolites), antibodies (polyclonal or monoclonal), and drugs acting on immunophyllin (e.g. Cyclosporine, tacrolimus, or sirolimus). Additional drugs include interferon, opioid, TNF-binding protein, mycophenolate, and small biological agents. Examples of immunosuppressive drugs include mesenchymal stem cells, anti-lymphocyte globulin (ALG) polyclonal antibodies, anti-thymocyte globulin (ATG) polyclonal antibodies, 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.

[0159] In an embodiment, the pharmaceutical composition may be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. The injectable formulation may be provided in a unit dosage form, for example, contained in an ampoule or a multi-dose container, and a preservative is added. The composition may take the form of a suspension, solution, or emulsion in an oily or aqueous vehicle and may contain formulation agents, such as a suspending agent, a stabilizer, and / or a dispersant. Alternatively, the active ingredient may be in powder form to be reconstituted into a suitable vehicle, for example, sterile pyrogen-free water, before use. In addition to the formulations described previously, the composition may also be formulated as a depot formulation. Such long-acting formulations may be administered by insertion (e.g., subcutaneously). Thus, for example, the composition may be formulated as a suitable polymeric or hydrophobic material (e.g., as an emulsion in an acceptable oil) or as an ion exchange resin, or as a sparingly soluble derivative, for example, as a sparingly soluble salt.

[0160] The properties of pharmaceutical compositions as described herein vary depending on the mode of administration and can be easily determined by a person skilled in the art. Pharmaceutical compositions described herein may contain carriers or excipients, many of which are known to a person skilled in the art. Excipients that may be used include buffers (e.g., citrate buffers, phosphate buffers, acetate buffers, and bicarbonate buffers), amino acids, urea, alcohols, ascorbic acid, phospholipids, polypeptides (e.g., serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, and glycerol. The compound may be formulated in various ways depending on the corresponding route of administration. For example, liquid solutions may be prepared for administration by ear drops, injection, or ingestion; gels or powders may be prepared for ingestion or topical application. Methods for manufacturing such formulations are widely known and can be found, for example, in the literature ["Remington's Pharmaceutical Sciences"].

[0161] For example, the pharmaceutical composition may be formulated for administration by drip into the ear, inhalation (e.g., into the ear), topical, or oral administration. In another mode of administration, the pharmaceutical composition may be administered directly into the cochlea of ​​the inner ear, for example, via a cannula, catheter, or pump. The cannula, catheter, or pump may guide the pharmaceutical composition, for example, into the cochlear lumen or the round window of the ear. In another route of administration, the pharmaceutical composition may be injected into the ear, for example, into the cochlear lumen (e.g., the mesosystem, vestibular system, and tympanic system). The injection may be made, for example, through the round window or cochlear sac of the ear.

[0162] The pharmaceutical composition according to the present disclosure may further comprise a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutically acceptable carrier may comprise dimethyl sulfoxide (DMSO). In one embodiment, the pharmaceutically acceptable carrier does not comprise dimethyl sulfoxide. As described herein, the composition may be further adapted for cryopreservation at -80°C to -195°C or lower. In an embodiment, the composition may be formulated to be thawed and administered directly to a subject, for example by injection, without additional manipulation prior to administration. In an embodiment, the composition may be a cryopreservation medium, such as a cryostor, as a cryopreservation medium. ® It may be formulated to include 10 (CS10) (a defined cryopreservation medium free of animal components containing 10% DMSO). In some embodiments, the composition is crioston ® It is formulated in 10. In one embodiment, the composition may be filtered using a filter kit before cryopreservation to prevent clogging during administration through a narrow cannula, syringe needle, or catheter.

[0163] A pharmaceutical composition according to the present disclosure comprises about 1 million cells per milliliter, for example, 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, 80 million cells per milliliter, for example, about 90 million cells per milliliter, for example, about 100 million cells per milliliter, for example, about 200 million cells per milliliter, for example, about 300 million cells per milliliter, for example, about 400 million cells per milliliter, for example, about 500 million cells per milliliter, for example, about 600 million cells per milliliter, for example, about 700 million cells per milliliter, for example, about 900 million cells per milliliter, for example, about It may contain 500,000 cells, 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. In some embodiments, the pharmaceutical composition according to the present disclosure may contain about 30 million cells per milliliter to about 700 million cells per milliliter. In some embodiments, the pharmaceutical composition according to the present disclosure may contain about 50 million cells per milliliter to about 400 million cells per milliliter. The number of cells may be any value or sub-range within the listed ranges. In some embodiments, the pharmaceutical composition according to the present disclosure may contain about 50 million cells per milliliter. In some embodiments, the pharmaceutical composition according to the present disclosure may contain about 400 million cells per milliliter.

[0164] In a further embodiment, the pharmaceutical composition according to the present disclosure may have a volume in the range of about 0.01 microliters to about 2 milliliters, e.g., about 0.1 microliters, e.g., about 0.5 microliters, e.g., about 1 microliter, e.g., about 3 microliters, e.g., about 4 microliters, e.g., about 5 microliters, e.g., about 6 microliters, e.g., about 7 microliters, e.g., about 10 microliters, e.g., about 20 microliters, e.g., about 50 microliters, e.g., about 80 microliters, e.g., about 100 microliters, e.g., about 200 microliters, e.g., about 500 microliters, e.g., about 1 milliliter, e.g., about 2 milliliters. In some embodiments, the pharmaceutical composition according to the present disclosure has a volume of about 50 microliters. In some embodiments, the pharmaceutical composition according to the present disclosure has a volume of about 0.5 microliters. The volume may be any value or sub-range within the listed ranges. In one embodiment, the pharmaceutical composition according to the present disclosure may be contained in a container configured for cryopreservation or for administration to a subject requiring administration. In one embodiment, the container may be a pre-filled syringe.

[0165] In one embodiment, the pharmaceutical composition according to the present disclosure may be administered in a volume ranging from about 1 microliter to about 1,800 microliters, e.g., about 2 microliters, e.g., about 3 microliters, e.g., about 4 microliters, e.g., about 50 microliters, e.g., about 100 microliters, e.g., about 200 microliters, e.g., about 450 microliters, e.g., about 1,800 microliters, e.g., about 10 microliters, e.g., about 20 microliters, or e.g., about 40 microliters. The volume may be any value or sub-range within the listed ranges. In some embodiments, the pharmaceutical composition according to the present disclosure is administered in a volume of about 50 microliters.

[0166] In some embodiments, the pharmaceutical composition according to the present disclosure comprises at least about 50,000 cells, at least about 100,000 cells, at least about 200,000 cells, at least about 300,000 cells, at least about 400,000 cells, at least about 500,000 cells, at least about 600,000 cells, at least about 700,000 cells, at least about 800,000 cells, at least about 900,000 cells, at least about 1 million cells, at least about 1.5 million cells, at least about 2 million cells, at least about 2.5 million cells, at least about 3 million cells, at least about 4 million cells, at least about 5 million cells, at least about 10 million cells, at least about 20 million cells, at least about 30 million cells, at least about 40 million cells, or at least about 59 million cells. In some embodiments, the pharmaceutical composition comprises about 50,000 to 50 million cells, about 100,000 to 20 million cells, about 100,000 to 10 million cells, about 100,000 to 1 million cells, about 500,000 to 10 million cells, about 500,000 to 1 million cells, about 1 million to 50 million cells, or about 10 million to 50 million cells. In some embodiments, the pharmaceutical composition comprises about 100,000 to 10 million cells. In some embodiments, the pharmaceutical composition comprises about 100,000 to 1 million cells. In some embodiments, the pharmaceutical composition comprises about 50,000 to 500,000 cells. In some embodiments, the pharmaceutical composition comprises about 100,000 to 500,000 cells. In some embodiments, the pharmaceutical composition comprises about 500,000 to 1 million cells.

[0167] Method for producing a population of auditory cells

[0168] The present disclosure provides a method for producing a population of auditory cells from undifferentiated pluripotent stem cells suitable for administration to a subject to enhance the function of a cochlear implant. A method for producing a suitable population of cells is described in WO2023 / 167986 (the full contents of which are incorporated herein by reference) and is described in detail below.

[0169] The method involves culturing a population of undifferentiated pluripotent stem cells with different combinations of growth factors and growth factor inhibitors in a series of steps to induce the differentiation of undifferentiated pluripotent stem cells into the fate of auditory neurons. In an exemplary differentiation pathway, human embryonic stem cells (hESCs) are induced to differentiate into non-neuronal ectoderm (NNE) cells, which are then induced to differentiate into preplate-ectoderm (PPE) cells, which are further induced to differentiate into early ear neuron progenitor (ONP) cells, mid-ear progenitor cells, late ear neuron progenitor cells, mature auditory neurons, and spiral ganglion neurons (SGN). The resulting population of cells may contain a mixture of cell types. However, cells from the late stages of the pathway may be dominant, and residual hESCs may be minimal or absent. For example, the population may be a substantially pure population of late ONPs, spiral ganglion neurons, and mature auditory neurons. While I do not wish to be bound by theory, it is believed that compositions containing mixed cell populations may be more suitable as therapeutic agents for hearing diseases and disorders than compositions containing homogeneous cell populations, because when various cell types are administered to a subject, they increase the niches in which cells can engraft and increase the number of fates that cells can adopt upon administration.

[0170] A schematic diagram of the process of differentiating hESC into mature SGN is Fig. 5a It is provided in. In some embodiments, Fig. 5b As described in [figure], biomarkers are monitored along the differentiation process. Monitoring of biomarkers increases the purity of the cell population, ensuring a high-quality final product of LCTANP1 cells.

[0171] Figs. 6a-6b Figure [document] illustrates an exemplary assay for monitoring the quality of the LCTANP1 cell product. The cell product may be evaluated in vivo and / or functionally. In vivo evaluation may include the determination of purity-specific markers and the examination of gene expression in the cell population. Markers may be evaluated by, for example and without limitation, flow cytometry, immunofluorescence, and other methods known in the art. Gene expression (e.g., late ANP gene expression) may be evaluated by, for example, quantitative PCR (qPCR), RT-qPCR (reverse transcriptase-quantitative polymerase chain reaction), RNA sequencing, and other methods known in the art. Functional evaluation may be performed in vitro. It may include tests for synapse formation and calcium signaling. In vitro Synapse formation can be evaluated, for example, by immunofluorescence microscopy (e.g., synapse formation markers) without limitation. Calcium signaling can be evaluated, for example, by detection of calcium influx without limitation. Additionally, cell morphology can be monitored through immunofluorescence methods. For example, in vitro maturation of ANP1 cells can be characterized by a combination of cell process elongation and beta-tubulin III expression.

[0172] The final cell product may be profiled. Profiling may be performed by RNA sequencing, proteome / protein analysis, marker characterization (e.g., by flow cytometry), and other characterization methods as known in the relevant art.

[0173] This method for characterizing manufacturing (differentiation) processes and final cell products facilitates favorable culture and scalability for repeatable and reproducible differentiation processes, thereby streamlining workflows, reducing costs and waste, and providing quality control for cell products.

[0174] How to expand and maintain human embryonic stem cells (hESCs)

[0175] In one aspect, a method for expanding and maintaining undifferentiated pluripotent human embryonic stem cells (hESCs) is provided herein, comprising the steps of: (a) simultaneously combining human embryonic stem cells and an extracellular matrix component (ECM) in a growth medium of a tissue culture flask for static expansion; and (b) culturing the adherent hESCs for a predetermined period.

[0176] In some embodiments, cultured human embryonic stem cells of static expansion are non-enzymatically harvested using ReLeSR™ and cultured in mTeSR™ plus medium in iMatrix-511 coated containers. In some embodiments, hESCs are further expanded by repeating steps (a) and (b).

[0177] In some embodiments, cultured human embryonic stem cells of static expansion are harvested and further differentiated.

[0178] In one aspect, a method for expanding and maintaining undifferentiated pluripotent human embryonic stem cells (hESCs) is provided herein, comprising the steps of: (a) simultaneously combining human embryonic stem cells, an extracellular matrix component (ECM), and a microcarrier in a growth medium to form a suspendable expansion complex; and (b) culturing the suspendable expansion complex for a predetermined period.

[0179] In some embodiments, cultured human embryonic stem cells of a suspensionable expansion complex are harvested and further expanded by repeating steps (a) and (b).

[0180] In some embodiments, cultured human embryonic stem cells of a suspensionable expansion complex are harvested and further differentiated.

[0181] Human embryonic stem cells can be isolated from human blastocysts. Human blastocysts are typically obtained from in vivo pre-implantation embryos or in vitro fertilized (IVF) embryos. Alternatively, single-cell human embryos can be extended to the blastocyst stage. For the isolation of human ES cells, the ICM is isolated by a procedure in which the zona pellucida is removed from the blastocyst, trophoblast cells are lysed, and the ICM is removed from the intact inner cell mass (ICM) by gentle pipetting. The ICM is then plated in a tissue culture flask containing appropriate medium to enable budding. After 9 to 15 days, the ICM-derived buddings are dissociated into clumps by mechanical dissociation or enzymatic digestion, and the cells are then re-plated in fresh tissue culture medium. Colonies exhibiting an undifferentiated morphology are individually selected by a micropipette, mechanically dissociated into clumps, and re-plated. The resulting ES cells are then divided regularly every 4 to 7 days. For further details on the method for preparing human ES cells, refer to [Reubinoff et al. Nat Biotechnol 2000, May: 18(5): 559]; Thomson et al., U.S. Patent No. 5,843,780; [Science 282: 1145, 1998]; [Curr. Top. Dev. Biol. 38: 133, 1998]; [Proc. Natl. Acad. Sci. USA 92: 7844, 1995]; [Bongso et al., Hum Reprod 4: 706, 1989]; and [Gardner et al., Fertil. Steril. 69: 84, 1998].

[0182] In addition, ES cells were used in mice (Mills and Bradley, 2001), golden hamsters [Doetschman et al., 1988, Dev Biol. 127: 224-7], rats [Iannaccone et al., 1994, Dev Biol. 163: 288-92], rabbits [Giles et al. 1993, Mol Reprod Dev. 36: 130-8; Graves & Moreadith, 1993, Mol Reprod Dev. 1993, 30 36: 424-33], and various domestic animal species [Notarianni et al., 1991, J Reprod Fertil Suppl. 43: 255-60; Wheeler 1994, Reprod Fertil Dev. 6: 563-8; Mitalipova et al., 2001, Cloning. It can be obtained from other species including [3: 59-67] and 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].

[0183] Expanded blastocyst cells (EBCs) can be obtained from a blastocyst at least 9 days after fertilization, i.e., at the pre-embryonic stage. Before culturing the blastocyst, the zona pellucida is digested [e.g., by Tyrode acid solution (Sigma Aldrich, St. Louis, Missouri, USA)] to expose the inner cell mass. The blastocyst is then cultured as a whole embryo in vitro using standard embryonic stem cell culture methods for at least 9 and 14 days (i.e., pre-embryonic stage) after fertilization.

[0184] Another method for producing ES cells is described in the literature [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 the in vitro fertilization process. In this process, the embryo is not destroyed.

[0185] EG (embryonic germ) cells are prepared from primordial germ cells obtained from a fetus (in the case of a human fetus) at approximately 8–11 weeks of gestation using laboratory techniques known to a person skilled in the art. The germ ridge is dissociated, cut into small portions, and then deaggregated into cells by mechanical dissociation. The EG cells are then grown in a tissue culture flask containing appropriate medium. The cells are cultured with daily replacement of the medium, typically for 7–30 days or after 1–4 passages, until cell morphology consistent with EG cells is observed. For additional details regarding the method of preparing human EG cells, refer to the literature [Shamblott et al., Proc. Natl. Acad. Sci. USA 95: 13726, 1998] and U.S. Patent No. 6,090,622.

[0186] Another additional method for producing ES cells is by parthenogenesis. In this process as well, the embryo is not destroyed.

[0187] Cells can be expanded in a suspension or as a monolayer, with or without a microcarrier. The expansion of a population of mixed cells in monolayer or suspension cultures can be transformed into large-scale expansion in a bioreactor or multiple / super stack by methods widely known in the relevant art.

[0188] According to some embodiments, the extension step is performed for at least 1 to 20 weeks, e.g., 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 an embodiment, the extension step is performed for 1 to 10 weeks, e.g., 2 to 10 weeks, 3 to 10 weeks, 4 to 10 weeks, or 4 to 8 weeks. The duration may be any value or sub-range within the listed ranges and includes an endpoint.

[0189] According to additional embodiments, an extension step is performed until a suitable lactate concentration in the cell culture medium and / or a percentage overall growth rate is achieved. The percentage overall growth rate is the percentage of the surface area of ​​the culture vessel that appears to be covered by a layer of cells when observed by microscopy. In some embodiments, undifferentiated pluripotent stem cells are cultured until the lactate concentration in the cell culture medium is about 1.0 to 13.0 mM, or about 1.5 to 12.5 mM. In some embodiments, cells are cultured until the lactate concentration in the cell culture medium is about 1.68 to 12.29 mM. In some embodiments, the percentage overall growth rate is 5% to 85%.

[0190] According to additional other embodiments, the mixed cell population is subcultured at least once during the expansion phase, at least twice during the expansion phase, at least three times during the expansion phase, at least four times during the expansion phase, at least five times during the expansion phase, at least six times during the expansion phase, or at least seven times during the expansion phase.

[0191] In the case of enzymatically collecting cells, more than 8 subcultures, more than 9 subcultures, and even more than 10 subcultures (e.g., Expansion can continue for 11–15 subcultures. The total number of cell doublings exceeds 30, for example It may be increased to 31, 32, 33, 34 or more times. (See International Patent Application Publication No. WO 2017 / 021973, the full text of which is incorporated herein by reference).

[0192] The extracellular matrix (ECM) is a three-dimensional network composed of extracellular macromolecules and minerals, such as collagen, enzymes, glycoproteins, and hydroxyapatite, which provides structural and biochemical support to surrounding cells. Since multicellularity evolved independently in different multicellular lineages, the composition of the ECM varies among multicellular structures; however, cell adhesion, cell-to-cell communication, and differentiation are common functions of the ECM.

[0193] The animal extracellular matrix comprises the interstitial matrix and the basement membrane. The interstitial matrix exists between various animal cells (i.e., the intercellular space). A gel of polysaccharides and fibrous proteins fills the interstitial space and acts as a compression buffer against stress applied to the ECM. The basement membrane is a sheet-like deposit of the ECM where various epithelial cells are present. Each animal's connective tissue possesses a unique type of ECM: the ECM of bone tissue consists of collagen fibers and bone minerals; the ECM of loose connective tissue consists of reticular fibers and matrix material; and the ECM of blood is plasma.

[0194] Suitable extracellular matrix components for use within the scope of this disclosure include Matrigel®, Vitronectin, Gelatin, Collagen I, Collagen IV, Laminin (e.g. Laminin 521), fibronectin, poly-D-lysine, derivatives thereof, or combinations thereof may be included, but are not necessarily limited thereto. In a specific embodiment, human laminin is a human laminin 511 E8 fragment.

[0195] 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 silicon. In some embodiments, the microcarrier is composed of polystyrene, surface-modified polystyrene, chemically modified polystyrene, cross-linked dextran, cellulose, acrylamide, collagen, alginate, gelatin, glass, DEAE-dextran, or a combination thereof. In some embodiments, the microcarrier is composed of polystyrene. In some embodiments, the microcarrier is composed of surface-modified polystyrene. In some embodiments, the microcarrier is composed of chemically modified polystyrene. In some embodiments, the microcarrier is composed of cross-linked dextran. In some embodiments, the microcarrier is composed of cellulose. In some embodiments, the microcarrier is composed of acrylamide. In some embodiments, the microcarrier is composed of collagen. In some embodiments, the microcarrier is composed of alginate. In some embodiments, the microcarrier is composed of gelatin. In some embodiments, the microcarrier is composed of glass. In some embodiments, the microcarrier is composed of DEAE-dextran. In some embodiments, the microcarrier is not coated.

[0196] In some embodiments, the microcarrier is coated. In some embodiments, the microcarrier may be coated with Matrigel®, laminin, vitronectin, collagen, derivatives thereof, or combinations thereof. In some embodiments, the microcarrier may be coated with poly-lysine, poly-L-lysine, poly-D-lysine, fibronectin, tenacin, dextran, peptides, or combinations thereof. In some embodiments, the microcarrier is coated with laminin. In some embodiments, the microcarrier is coated with Matrigel®. In some embodiments, the microcarrier is coated with collagen. In some embodiments, the microcarrier is coated with poly-lysine. In some embodiments, the microcarrier is coated with poly-L-lysine. In some embodiments, the microcarrier is coated with poly-D-lysine. In some embodiments, the microcarrier is coated with vitronectin. In some embodiments, the microcarrier is coated with fibronectin. In some embodiments, the microcarrier is coated with tenacin. In some embodiments, the microcarrier is coated with dextran. In some embodiments, the microcarrier is coated with peptides.

[0197] In some embodiments, the microcarrier may be spherical, smooth, macroporous, rod-shaped, or a combination thereof. In some embodiments, the microcarrier may be combined with protamine or polylysine. In some embodiments, the microcarrier is spherical. In some embodiments, the microcarrier is elliptical. In some embodiments, the microcarrier is rod-shaped. In some embodiments, the microcarrier is disc-shaped. In some embodiments, the microcarrier is porous. In some embodiments, the microcarrier is non-porous. In some embodiments, the microcarrier is smooth. In some embodiments, the microcarrier is flat.

[0198] In some embodiments, the microcarrier is neutral. In some embodiments, the microcarrier is negatively charged. In some embodiments, the microcarrier is hydrophilic.

[0199] In some embodiments, the microcarrier is 25 cm 2 , 50 cm 2 , 75 cm 2 , 100 cm 2 , 125 cm 2 , 150 cm 2 , 175 cm 2 , 200 cm 2 , 225 cm 2 , 250 cm 2 , 500 cm 2 , 625 cm 2 , 750 cm 2 , 1,000 cm 2 , 1,250 cm 2 , 5,000 cm 2 , or 7,500 cm 2 It may have a surface area (per gram). The surface area may be any value or sub-range within the listed ranges and includes the endpoint.

[0200] In specific embodiments, the microcarrier is surface-treated to enhance cell adhesion and maximize cell yield and viability. The microcarrier may be composed of USP Class VI polystyrene material that provides a consistent platform. In some embodiments, the microcarrier creates a synthetic surface on the microcarrier for stem cell expansion. The enhanced adhesion surface treatment improves cell adhesion by injecting oxygen into the surface of the microcarrier. In some embodiments, the microcarrier is non-thermotropic. In some embodiments, the microcarrier is optimized for mesenchymal stem cell applications. In specific embodiments, the beads may vary in size from 125 to 212 μm. In specific embodiments, the density of the microcarrier may be 1.026 ± 0.004. In specific embodiments, the microcarrier is 360 cm² 2 It can be / gram.

[0201] In some embodiments, the method comprises combining hESC with laminin or a derivative thereof to improve cell adhesion to a carrier surface. In a specific embodiment, the laminin is human laminin 511. As an alternative embodiment, various other extracellular matrices may be used for cell adhesion, including, but not necessarily limited to, vitronectin, fibronectin, collagen, Matrigel®, or derivatives thereof.

[0202] In some embodiments, cells may be cultured for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days.

[0203] In some embodiments, cells may be cultured in a working volume of 10 mL to 3,000 mL, for example, about 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 100 mL, 250 mL, 500 mL, 750 mL, 1,000 mL, or 3,000 mL. The volume may be any value or sub-range within the listed ranges and includes an endpoint.

[0204] In some embodiments, the cultured cells may be further expanded.

[0205] In some embodiments, cultured cells may remain in an undifferentiated state. Undifferentiated cells may be identified by the expression of various markers, including but not necessarily limited to, SSEA-5, TRA-1-60, Oct-4, and Nanog. In some embodiments, undifferentiated cells express SSEA-5. In some embodiments, undifferentiated cells express TRA-1-60. In some embodiments, undifferentiated cells express Oct-4. In some embodiments, undifferentiated cells express Nanog. In some embodiments, undifferentiated cells express both SSEA-5 and TRA-1-60. In some embodiments, undifferentiated cells express both Oct-4 and Nanog. In some embodiments, undifferentiated cells express SSEA-5, TRA-1-60, Oct-4, and Nanog (IPC#0).

[0206] In some embodiments, cells may be cultured in a feeder cell-conditioning medium. The ES culture method may involve the use of a feeder cell layer that secretes factors necessary for stem cell proliferation while simultaneously inhibiting differentiation. Culture is typically performed on a solid surface, for example, a surface coated with gelatin or vimentin. An exemplary feeder layer comprises 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 derived 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 derived 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, an agent to maintain hESCs in a naive, undifferentiated state may be added to the medium (see, for example, reference [Kalkan et al., 2014, Phil. Trans. R. Soc. B, 369: 20130540]).

[0207] hESCs are typically plated on top of feeder cells in a supportive medium (e.g., NUTRISTEM®, NUT(+) with human serum albumin, mTeSR™ Plus, or mTeSR™1 StemFit®) after 1–4 days. Additional factors, such as bFGF and TGFβ3, can be added to the medium to prevent the differentiation of ESCs. Once a sufficient amount of hESCs is obtained, the cells can be mechanically lysed (e.g., using a sterile tip or a disposable sterile stem cell tool (14602, Swemed)). Alternatively, cells can be removed by enzymatic treatment (e.g., collagenase A, or TrypLE™ Select). This process can be repeated several times until the required amount of hESCs is reached. According to some embodiments, after the expansion of the first round, hESC is removed using TrypLE™ Select, and after the expansion of the second round, hESC is removed using Collagenase A.

[0208] Feeder cell-free systems have also been used for ES cell culture, and these systems utilize a matrix supplemented with serum substitutes, cytokines, and growth factors (including IL-6 and soluble IL-6 receptor chimeras) as a substitute for the feeder cell layer. Stem cells can be grown on solid surfaces, such as extracellular matrices (e.g., Matrigel®, laminin, or vitronectin), in the presence of culture media (e.g., Lonza L7™ systems, mTeSR™, StemPro™, XFKSR, E8, Nutristem®). Unlike feeder-based cultures, which require the simultaneous growth of feeder cells and stem cells and can produce mixed cell populations, stem cells grown in feeder-free systems are easily detached from the surface. The culture media used to grow stem cells contain factors that effectively inhibit differentiation and promote growth, such as MEF-conditioning media and bFGF.

[0209] Also within the scope of the present disclosure is a method for expanding and maintaining human embryonic stem cells (hESCs) in an undifferentiated state, comprising the steps of: culturing human pluripotent stem cells on a non-adherent surface to obtain a population of undifferentiated hESCs; combining the population of undifferentiated hESCs with a microcarrier in a growth medium; and expanding the population of cells.

[0210] Examples of non-adherent cell culture plates include those manufactured by Nunc (e.g., Hydrocell catalog number 174912), etc. In other embodiments, non-adherent suspension culture dishes may be used (e.g., Corning).

[0211] According to some embodiments, cells are placed on a non-adherent substrate, for example When culturing in a cell culture plate, the atmospheric oxygen condition is 20%. However, when the percentage of atmospheric oxygen is about 20%, 15%, 10%, 9%, 8%, 7%, less than 6%, or even about 5% (e.g. Manipulation of atmospheric oxygen conditions to make the oxygen level 1%-20%, 1%-10%, or 0%-5% is also conceived. According to another embodiment, cells are initially cultured in a non-adherent substrate under normal atmospheric oxygen conditions, and then cultured under atmospheric oxygen conditions lower than normal.

[0212] The method described above relates to a method for expanding and maintaining hESCs, but similar methods for induced pluripotent stem cells (iPSCs) are also within the scope of this disclosure. iPSCs are a type of stem cell derived from somatic cells that have been reprogrammed to a pluripotent state through the introduction of a pluripotency-associated gene, and are available from various sources. A person skilled in the art will recognize the modifications necessary to adapt the hESC method described above for use with iPSCs, etc.

[0213] Extended composition

[0214] In another aspect, a suspendable extended complex composition comprising human embryonic stem cells or IPSCs, an extracellular matrix component (ECM), and a microcarrier is provided herein.

[0215] Human embryonic stem cells, extracellular matrix, and microcarriers are described in detail elsewhere in this invention.

[0216] The range of the extended complex may vary. In the table below, the range of complex components is described in detail by varying the units for the ECM components.

[0217] Table 2.

[0218]

[0219] The ECM component is mol / cm² using the molecular weight (150 kDa) of the laminin 511 E8 fragment. 2 It can be presented as a unit.

[0220] Table 3.

[0221]

[0222] The ECM component also has a molecular weight (150 kDa) and Avogadro's number (6.022 × 10⁻⁶). 23 Using the result of multiplying by ) number of molecules / cm 2 It can be presented as a unit.

[0223] Table 4.

[0224]

[0225] In some embodiments, the following specification parameters may be extended: # (cell) of hESC - cm of microcarrier 2 4,000–600,000 cells per cell; laminin 511 E8 fragment (cm of microcarrier) 2 (μg per gram) - cm 2 0.125 μg or more per gram.

[0226] In some embodiments, the composition may further comprise a growth medium. Non-limiting examples of commercially available base media (i.e., chemically defined media or CDM) available for use in accordance with this disclosure include Nutristem® (absence of bFGF and TGF for ESC differentiation, presence of bFGF and TGF for ESC proliferation), Neurobasal™, KO-DMEM, DMEM, DMEM / F12, CELLGRO™ stem cell growth medium, or X-VIVO™. Base media may be supplemented with various agents as known in the art dealing with cell culture. The following is a non-limiting reference to various supplements that may be included in the culture to be used in accordance with this disclosure: media containing serum or serum substitutes, e.g., Knockout Serum Substitute (KOSR), Nutridoma-CS, TCH™, N2, N2 derivatives, or B27 or combinations (not limited thereto); Extracellular matrix (ECM) components, such as fibronectin, laminin, collagen, and gelatin (not limited thereto). In some embodiments, the cell culture medium comprises a chemically defined medium (CDM) supplemented with N2, B27, or a combination thereof, optionally supplemented with BrainPhys™. The ECM may then be used to carry one or more members of growth factors of the TGFI3 superfamily; antibacterial agents, such as L-glutamine, beta-mercaptoethanol, penicillin, and streptomycin (not limited thereto); and non-essential amino acids (NEAAs), neurotrophins known to play a role in promoting the survival of SCs in culture, such as BDNF, NT3, NT4 (not limited thereto).

[0227] As described 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, and silicon. In some embodiments, the microcarrier is composed of polystyrene. In some embodiments, the microcarrier is composed of surface-modified polystyrene. In some embodiments, the microcarrier is composed of chemically modified polystyrene. In some embodiments, the microcarrier is composed of cross-linked dextran. In some embodiments, the microcarrier is composed of cellulose. In some embodiments, the microcarrier is composed of acrylamide. In some embodiments, the microcarrier is composed of collagen. In some embodiments, the microcarrier is composed of alginate. In some embodiments, the microcarrier is composed of gelatin. In some embodiments, the microcarrier is composed of glass. In some embodiments, the microcarrier is composed of DEAE-dextran.

[0228] As described above, the microcarrier may be spherical, smooth, macroporous, rod-shaped, or a combination thereof.

[0229] In some embodiments, the microcarrier may be coated with Matrigel, laminin, vitronectin, collagen, derivatives thereof, or combinations thereof. In some embodiments, the laminin is human laminin 511.

[0230] In some embodiments, the microcarrier is not coated.

[0231] In some embodiments, the microcarrier is 25 cm 2 to 7,500 cm 2 , for example, about 25 cm 2 , 50 cm 2 , 75 cm 2 , 100 cm 2 , 125 cm 2 , 150 cm 2 , 175 cm2 , 200 cm 2 , 225 cm 2 , 250 cm 2 , 500 cm 2 , 625 cm 2 , 750 cm 2 , 1,000 cm 2 , 1,250 cm 2 , 5,000 cm 2 , or 7,500 cm 2 It has a surface area (per gram). The surface area can be any value or sub-range within the listed ranges and includes endpoints.

[0232] In some embodiments, the microcarrier is bound to protamine or polylysine. In some embodiments, the microcarrier is neutral. In some embodiments, the microcarrier is negatively charged. In some embodiments, the microcarrier is hydrophilic.

[0233] Method for manufacturing auditory cells

[0234] According to the present disclosure, human pluripotent stem cells (hPSCs) can be grown in dynamic culture on a microcarrier in hPSC culture medium as described above and maintained in a pluripotent state by replacing the hPSC medium daily. The hPSCs will be differentiated by replacing the medium with a culture medium (a 1:1 mixture of DMEM / F12 and Neurobasil medium) to induce the formation of non-neuronal ectoderm (NNE). This medium may contain, for example, B27 and N2 supplements, TGF beta agonists, such as BMP4 (1-25 ng / mL), the vitamin B3 derivative nicotinamide (NIC, 1-25 mM), SB431542, and / or FGF2 (1-25 ng / mL). Dynamic or static culture will be continued for 3-7 days, and the medium will be completely or gradually replaced (75-100% volume daily). On days 4–8 of differentiation, the differentiation factors will be replaced 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 a proplate-like ectoderm. Dynamic culture will be continued for 3–7 days, and the medium will be changed every 1–3 days. On days 8–14 of differentiation, the differentiation factors will be replaced with FGF2, CHIR99021 (6 uM), and IGF1 (50 ng / ml) to generate an initial ONP. Dynamic culture will be continued for 7–10 days, and the medium will be changed every 2–3 days. From days 17 to 22, the differentiation factors will be replaced with FGF2, EGF, retinoic acid (RA 0.2-2 μM), SHH (500 ng / ml), and IGF1 (50 ng / ml) to generate mid-to-late ONP. Dynamic culture will be continued for 7-10 days, and the medium will be replaced every 1-3 days.On days 22–28, mid-to-late ONP cells will be harvested and inoculated as single cells into a static / dynamic suspension for 3 days in the presence of BDNF (10 ng / mL), NT3 (10 ng / mL), and an IGF-1 Rock inhibitor (e.g., Y-27632 dihydrochloride, 10 μM) to form small aggregates. On days 22–28 of differentiation, the cells will be further expanded to reach final maturation.

[0235] In some embodiments, hPSCs can be differentiated by culturing in 2D with 50-2000 ng / ml of Noggin and 0.5-20 ng / ml of FGF2 for about 14 days, and by replacing the medium with a culture medium that induces neural crest formation (a 1:1 mixture of DMEM / F12 and Neurobasil medium). On the 14th day or about the 14th day, cells are transferred and 3D cultured for about 5 days with, for example, 5-100 ng / ml of EGF and 5-100 ng / ml of FGF2. On the 19th day or about the 19th day, cells are returned to 2D culture with differentiation factor FGF2, furmorphamine (0.1-1 μM), EGF, retinoic acid (RA 0.2-2 μM), and IGF1 (50 ng / ml) to induce late ONP. Dynamic culture will be continued for about 7 days, and the medium will be replaced every 2-3 days. On the 25th day or about the 25th day, late ONP cells will be harvested and inoculated into a dynamic suspension as single cells for about 3 days in the presence of FGF, EGF and Rock inhibitor (2-50 μM) to form small aggregates.

[0236] hPSCs can be differentiated into different populations of cells through culture in various different media containing growth factors and growth factor inhibitors. In some embodiments, undifferentiated pluripotent stem cells will undergo conditions sufficient for directed differentiation to produce a composition comprising a population of auditory cells, e.g., NNE, PPE, ONP cells, neurons (e.g., spiral ganglion neurons), or any combination thereof. In some embodiments, the method comprises culturing a population of hPSCs under conditions sufficient to induce a population of cells into a target cell type in 1, 2, 3, 4, or 5 culture media, each containing a combination of growth factors and / or growth factor inhibitors, thereby producing a population of cells comprising the target cell type.

[0237] In some embodiments, the method comprises a population of undifferentiated pluripotent stem cells at 1,200–20,000 live cells / cm² 2 Start by seeding in an optional monolayer at a density and culturing the cells until the lactate concentration in the cell culture medium reaches 1.68–12.29 mM and a percentage overall growth rate of 5% to 80% is achieved. In some embodiments, undifferentiated pluripotent stem cells are cultured until the lactate concentration in the cell culture medium reaches about 1.0 to 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 percentage overall growth rate is 5% to 90%, 5% to 85%, or 5% to 80%.

[0238] In some embodiments, a 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-imidazole-2-yl]benzamide (SB431542). In some embodiments, the first cell culture medium comprises BMP4, SB431542, and fibroblast growth factor 2 (FGF2).

[0239] In some embodiments, a 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 induce differentiation into a target cell type, e.g., non-neuronal ectoderm (NNE) cells. In some embodiments, a population of PSCs is cultured in the first cell culture medium for at least 1 day. In some embodiments, a population of PSCs is cultured in the first cell culture medium for at least 4 days. In some embodiments, a population of PSCs is cultured in the first cell culture medium for at least 5 days. In some embodiments, a population of PSCs is cultured in the first cell culture medium for at least 7 days. In some embodiments, a population of PSCs is cultured in a 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, a population of PSCs is cultured in a first cell culture medium for about 1-9 days. In some embodiments, a population of PSCs is cultured in a first cell culture medium for about 3-7 days. In some embodiments, culturing a population of undifferentiated PSCs in a first cell culture medium produces a population of cells containing non-neuronal ectoderm (NNE) cells.

[0240] In some embodiments, a population of cells produced by culturing PSCs in a first cell culture medium, for example, a population of cells including NNE cells, is cultured in a second cell culture medium containing 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]pyrimidine-2-yl)thio]-acetamide (IWP-2) and 4-{6-[4-(piperazine-1-yl)phenyl]pyrazolo[1,5-a]pyrimidine-3-yl}quinoline (LDN193189).

[0241] In some embodiments, a population of cells is cultured in a 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 induce differentiation into a target cell type, e.g., proplate-ectoderm (PPE) cells. In some embodiments, a population of cells is cultured in the second cell culture medium for at least 1 day. In some embodiments, a population of cells is cultured in the second cell culture medium for at least 4 days. In some embodiments, a population of cells is cultured in the second cell culture medium for at least 5 days. In some embodiments, a population of cells is cultured in the second cell culture medium for at least 6 days. In some embodiments, a population of cells is cultured in the second cell culture medium for at least 7 days. In some embodiments, a population of cells is cultured in a 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, a population of cells is cultured in a second cell culture medium for about 1-9 days. In some embodiments, a population of cells is cultured in a second cell culture medium for about 3-7 days. In some embodiments, a population of cells is cultured in a second cell culture medium for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. In some embodiments, a population of cells is cultured in a second cell culture medium for about 5 days. In some embodiments, a population of cells is cultured in a second cell culture medium for 6 days. In some embodiments, a population of cells is cultured in a second cell culture medium for 7 days. In some embodiments, culturing a population of cells in a second cell culture medium produces a population of cells containing PPE cells.

[0242] In some embodiments, a population of cells produced by culturing cells in a second culture medium, e.g., a population of PPE cells, is cultured in a third cell culture medium containing 6-((2-((4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazole-2-yl)pyrimidine-2-yl)amino)ethyl)amino)nicotinonitrile (CHIR99021), FGF2, and insulin-like growth factor 1 (IGF-1).

[0243] In some embodiments, a population of cells is cultured in a 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 induce differentiation into a target cell type, e.g., early ear neuron progenitor (ONP) cells. In some embodiments, a population of cells is cultured in the third cell culture medium for at least 1 day. In some embodiments, a population of cells is cultured in the third cell culture medium for at least 4 days. In some embodiments, a population of cells is cultured in the third cell culture medium for at least 5 days. In some embodiments, a population of cells is cultured in the third cell culture medium for at least 7 days. In some embodiments, a population of cells is cultured in the third cell culture medium for 5 days. In some embodiments, a population of cells is cultured in the third cell culture medium for 7 days. In some embodiments, a population of cells is cultured in the third cell culture medium for 9 days. In some embodiments, a population of cells is cultured in a 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, a population of cells is cultured in a third cell culture medium for about 3-10 days. In some embodiments, a population of cells is cultured in a third cell culture medium for about 5-9 days. In some embodiments, culturing a population of cells in the third cell culture medium produces a population of cells containing initial ONP cells.

[0244] In some embodiments, a population of cells produced by culturing cells in a third culture medium, for example, a population of cells including early ONP cells, is cultured in a fourth cell culture medium containing Sonic Hedgehog (SHH), retinoic acid (RA), epidermal growth factor (EGF), FGF2, and IGF-1.

[0245] In some embodiments, a population of cells is cultured in a 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 induce differentiation into a target cell type, e.g., meta-to-late ONP cells. In some embodiments, a population of cells is cultured in the fourth cell culture medium for at least 1 day. In some embodiments, a population of cells is cultured in the fourth cell culture medium for at least 4 days. In some embodiments, a population of cells is cultured in the fourth cell culture medium for at least 5 days. In some embodiments, a population of cells is cultured in the fourth cell culture medium for at least 7 days. In some embodiments, a population of cells is cultured in the fourth cell culture medium for 5 days. In some embodiments, a population of cells is cultured in the fourth cell culture medium for 7 days. In some embodiments, a population of cells is cultured in the fourth cell culture medium for 9 days. In some embodiments, a population of cells is cultured in a 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, a population of cells is cultured in a fourth cell culture medium for about 3-10 days. In some embodiments, a population of cells is cultured in a fourth cell culture medium for about 5-9 days. In some embodiments, culturing a population of cells in the fourth cell culture medium produces a population of cells containing meta-to-late ONP cells.

[0246] In some embodiments, a population of cells produced by culturing cells in a third culture medium, for example, a population of cells including meta-to-late ONP cells, is cultured in a fifth cell culture medium containing brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3), and IGF-1.

[0247] In some embodiments, a population of cells is cultured in a 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 induce differentiation into a target cell type, e.g., late ONP cells. In some embodiments, a population of cells is cultured in the fifth cell culture medium for at least 1 day. In some embodiments, a population of cells is cultured in the fifth cell culture medium for at least 10 days. In some embodiments, a population of cells is cultured in the fifth cell culture medium for at least 20 days. In some embodiments, a population of cells is cultured in the fifth cell culture medium for at least 30 days. In some embodiments, a population of cells is cultured in the fifth cell culture medium for at least 45 days. In some embodiments, a population of cells is cultured in the fifth cell culture medium for at least 60 days. In some embodiments, a population of cells is cultured in a 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, a population of cells is cultured in the fifth cell culture medium for 7-65 days. In some embodiments, a population of cells is cultured in the fifth cell culture medium for 3-45 days. In some embodiments, a population of cells is cultured in the fifth cell culture medium for 10-60 days. In some embodiments, a population of cells is cultured in the fifth cell culture medium for 20-45 days. In some embodiments, culturing a population of cells in the fifth cell culture medium produces a population of cells containing late ONP cells.

[0248] In some embodiments, culturing a population of cells in a culture medium is (i) a population of cells produced by culturing cells in a fourth cell culture medium, e.g. The method comprises the steps of: (ii) harvesting a population of cells containing mid-to-late ONP cells; (iii) seeding the population of cells into a container containing the fifth cell culture medium; (iv) culturing the population of cells; (v) harvesting the population of cells; (v) seeding the population of cells into a container containing the fifth cell culture medium; and (vi) culturing the population of cells. In some embodiments, the fifth cell culture medium further comprises a ROCK inhibitor. In some embodiments, the cells are cultured 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, the described step (iii) comprises culturing a population of cells in a container 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, the described step (vi) comprises culturing a population of cells for at least 1 day, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 10 days, at least 15 days, at least 20 days, at least 25 days, at least 30 days, at least 35 days, at least 40 days, at least 50 days, or at least 60 days.

[0249] 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.

[0250] In some embodiments, the first and / or second cell culture medium contains 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 contains SB431542 at a concentration of about 0.1 μM–20 μM, 0.1–10 μM, 5 μM–15 μM, or 7 μM–13 μM. In some embodiments, the first and / or second cell culture medium contains SB431542 at a concentration of 1 μM–20 μM. In some embodiments, the first and / or second cell culture medium contains SB431542 at a concentration of 5 μM–15 μM. In some embodiments, the first and / or second cell culture medium contains SB431542 at a concentration of 0.1 μM to 10 μM. In some embodiments, the first and / or second cell culture medium contains SB431542 at a concentration of about 1 μM.

[0251] In some embodiments, the first, second, third, and / or fourth cell culture medium contains 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 contains 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 contains FGF2 in an amount of 1 ng / mL to 40 ng / mL. In some embodiments, the first, second, third, and / or fourth cell culture media contain FGF2 in an amount of 1 ng / mL to 25 ng / mL. In some embodiments, the first, second, third, and / or fourth cell culture media contain FGF2 in an amount of 10 ng / mL to 15 ng / mL. In some embodiments, the first, second, third, and / or fourth cell culture media contain FGF2 in an amount of about 10 ng / mL.

[0252] In some embodiments, the second cell culture medium contains 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 contains IWP-2 in an amount of about 0.5 μM–20 μM, 0.5 μM–10 μM, 1 μM–10 μM, 1 μM–5 μM, 2 μM–7 μM, or 3 μM–5 μM. In some embodiments, the second cell culture medium contains IWP-2 in an amount of about 0.5 μM–10 μM. In some embodiments, the second cell culture medium contains IWP-2 in an amount of about 2 μM–4 μM. In some embodiments, the second cell culture medium contains IWP-2 in an amount of about 2 μM.

[0253] In some embodiments, the second cell culture medium contains 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 contains 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 contains LDN193189 in an amount of 1 nM to 600 nM. In some embodiments, the second cell culture medium contains LDN193189 in an amount of 50 nM to 500 nM. In some embodiments, the second cell culture medium contains LDN193189 in an amount of 20 nM to 400 nM. In some embodiments, the second cell culture medium contains LDN193189 in an amount of 75 nM to 150 nM. In some embodiments, the second cell culture medium contains LDN193189 in an amount of about 100 nM.

[0254] In some embodiments, the third cell culture medium contains 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 contains CHIR99021 at a concentration of about 1 μM–40 μM, 1 μM–30 μM, 1 μM–25 μM, 5 μM–20 μM, 2 μM–10 μM, or 5 μM–15 μM. In some embodiments, the third cell culture medium contains CHIR99021 at a concentration of 1 μM–40 μM. In some embodiments, the third cell culture medium contains CHIR99021 at a concentration of 1 μM–25 μM. In some embodiments, the third cell culture medium contains CHIR99021 at a concentration of 2 μM to 8 μM. In some embodiments, the third cell culture medium contains CHIR99021 at a concentration of about 6 μM.

[0255] In some embodiments, the third, fourth, and / or fifth cell culture medium contains 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 contains 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 contains 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 contains IGF-1 in an amount of 25 ng / mL to 300 ng / mL. In some embodiments, the third, fourth, and / or fifth cell culture medium contains IGF-1 in an amount of 5 ng / mL to 100 ng / mL. In some embodiments, the third, fourth, and / or fifth cell culture medium contains IGF-1 in an amount of about 50 ng / mL. In some embodiments, the fifth cell culture medium does not contain IGF-1.

[0256] In some embodiments, the fourth cell culture medium contains 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, 1000 ng / mL, about 1100 ng / mL, about 1200 ng / mL, or about 1300 ng / mL. In some embodiments, the fourth cell culture medium contains 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 contains SHH in an amount of 10 ng / mL to 1300 ng / mL. In some embodiments, the fourth cell culture medium contains SHH in an amount of 300 ng / mL to 1000 ng / mL. In some embodiments, the fourth cell culture medium contains SHH in an amount of 400 ng / mL to 600 ng / mL. In some embodiments, the fourth cell culture medium contains SHH in an amount of about 500 ng / mL.

[0257] In some embodiments, the fourth cell culture medium contains 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 contains RA at a concentration of 0.1 μM–20 μM, 0.1 μM–5 μM, 0.5 μM–5 μM, 0.2 μM–5 μM, or 0.5 μM–2 μM. In some embodiments, the fourth cell culture medium contains RA at a concentration of 0.1 μM–20 μM. In some embodiments, the fourth cell culture medium contains RA at a concentration of 0.2 μM–5 μM. In some embodiments, the fourth cell culture medium contains RA at a concentration of 0.2 μM to 2 μM. In some embodiments, the fourth cell culture medium contains RA at a concentration of about 0.5 μM.

[0258] In some embodiments, the fourth cell culture medium contains 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 contains 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 contains EGF in an amount of 1 ng / mL to 300 ng / mL. In some embodiments, the fourth cell culture medium contains EGF in an amount of 5 ng / mL to 100 ng / mL. In some embodiments, the fourth cell culture medium contains EGF in an amount of 10 ng / mL to 50 ng / mL. In some embodiments, the fourth cell culture medium contains EGF in an amount of about 20 ng / mL.

[0259] In some embodiments, the fifth cell culture medium contains 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 contains 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 contains BDNF in an amount of 1 ng / mL to 300 ng / mL. In some embodiments, the fifth cell culture medium contains BDNF in an amount of 5 ng / mL to 100 ng / mL. In some embodiments, the fifth cell culture medium contains BDNF in an amount of 5 ng / mL to 30 ng / mL. In some embodiments, the fifth cell culture medium contains BDNF in an amount of about 10 ng / mL.

[0260] In some embodiments, the fifth cell culture medium contains neurotrophin 3 (also referred to as NT3, NT-3, and NTF3). NT3 is a member of the neurotrophin family involved in the survival and differentiation of mammalian neurons. NT3 is thought to be involved in the maintenance of the adult nervous system and in the development of neurons in embryos. In some embodiments, the fifth cell culture medium contains 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 contains NT3 in amounts 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 contains NT3 in amounts of 1 ng / mL to 300 ng / mL. In some embodiments, the fifth cell culture medium contains NT3 in amounts of 5 ng / mL to 100 ng / mL. In some embodiments, the fifth cell culture medium contains NT3 in amounts of 5 ng / mL to 30 ng / mL. In some embodiments, the fifth cell culture medium contains NT3 in amounts of 10 ng / mL.

[0261] In some embodiments, the fifth cell culture medium comprises a Rock inhibitor, for example, a small molecule inhibitor that inhibits ROCK1 and / or ROCK2-mediated signaling. In some embodiments, the Rock inhibitor is Y-27632 dihydrochloride ( Trans -4-[(1 RIt contains )-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide dihydrochloride). In some embodiments, the fifth cell culture medium contains 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 contains Rock inhibitor in an amount of about 0.5 μM–60 μM, 1 μM–50 μM, 2 μM–50 μM, 1 μM–30 μM, 2 μM–μM, 5 μM–20 μM, 1 μM–15 μM, or 5 μM–15 μM. In some embodiments, the fifth cell culture medium contains Rock inhibitor in an amount of about 2 μM–50 μM. In some embodiments, the fifth cell culture medium contains Rock inhibitor in an amount of about 10 μM.

[0262] In some embodiments, the method comprises: (a) culturing a population of undifferentiated pluripotent stem cells for 1-9 days under conditions sufficient to produce non-neuronal ectoderm (NNE) cells in a first cell culture medium containing 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, thereby producing a population of cells containing NNE cells; (b) culturing the population of cells containing NNE cells for 1-9 days under conditions sufficient to produce preplate-like ectoderm (PPE) cells in a second cell culture medium containing SB431542 at a concentration of 0.1-10 μM, FGF2 at a concentration of 1-25 ng / mL, IWP-2 at a concentration of 0.5-10 μM, and LDN193189 at a concentration of 20-400 nM, thereby producing a population of cells containing PPE cells; (c) culturing a population of cells containing PPE cells in a third cell culture medium containing 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 ear neuron progenitor (ONP) cells, thereby producing a population of cells containing early ONP cells; (d) culturing a population of cells containing early ONP cells in SHH at a concentration of 50-1000 ng / mL, 0.(e) culturing the population of cells containing mid-to-late ONP cells for 5-9 days under conditions sufficient to produce mid-to-late ONP cells in a fourth cell culture medium containing RA at a concentration of 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, thereby producing a population of cells containing mid-to-late ONP cells; (e) culturing the population of cells containing mid-to-late ONP cells for 3-45 days under conditions sufficient to produce late ONP cells in a fifth cell culture medium containing 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, thereby producing a population of cells containing late ONP cells; and (f) collecting the population of cells containing late ONP cells and thereby producing a composition. In an alternative embodiment, the first cell culture medium in step (a) contains BMP4 at a concentration of 1-25 ng / mL and SB431542 at a concentration of 0.1-10 μM, and does not contain FGF2.

[0263] In some embodiments, the method comprises: (a) culturing a population of undifferentiated pluripotent stem cells in a first cell culture medium containing BMP4, SB431542, and FGF2 for 1-9 days under conditions sufficient to produce non-neuronal ectoderm (NNE) cells, thereby producing a population of cells containing NNE cells; (b) culturing the population of cells containing NNE cells in a second cell culture medium containing SB431542, FGF2, and IWP-2 and LDN193189 for 1-9 days under conditions sufficient to produce preplate-like ectoderm (PPE) cells, thereby producing a population of cells containing PPE cells; (c) culturing the population of cells containing PPE cells in a third cell culture medium containing CHIR99021, FGF2, and IGF-1 for 5-9 days under conditions sufficient to produce early ear neuron progenitor (ONP) cells, thereby producing a population of cells containing early ONP cells; (d) culturing a population of cells containing early ONP cells in a fourth cell culture medium containing SHH, RA, EGF, FGF2, and IGF-1 for 5-9 days under conditions sufficient to produce mid-to-late ONP cells, thereby producing a population of cells containing mid-to-late ONP cells; (e) culturing a population of cells containing mid-to-late ONP cells in a fifth cell culture medium containing BDNF, NT3, and IGF-1 for 3-45 days under conditions sufficient to produce late ONP cells, thereby producing a population of cells containing late ONP cells; and (f) collecting a population of cells containing late ONP cells, thereby producing a composition. In an alternative embodiment, the first cell culture medium in step (a) contains BMP4 and SB431542 and does not contain FGF2.

[0264] In some embodiments, the method comprises: (a) culturing a population of undifferentiated pluripotent stem cells in a first cell culture medium containing 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-neuronal ectoderm (NNE) cells, thereby producing a population of cells containing NNE cells; (b) culturing the population of cells containing NNE cells in a second cell culture medium containing SB431542 at a concentration of 0.1-10 μM, FGF2 at a concentration of 1-25 ng / mL, and IWP-2 at a concentration of 0.5-10 μM, and LDN193189 at a concentration of 20-400 nM under conditions sufficient to produce preplate-like ectoderm (PPE) cells, thereby producing a population of cells containing PPE cells; (c) culturing a population of cells containing PPE cells in a third cell culture medium containing 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 under conditions sufficient to produce early ear neuron progenitor (ONP) cells, thereby producing a population of cells containing early ONP cells; (d) culturing a population of cells containing early ONP cells in SHH at a concentration of 50-1000 ng / mL, 0.(e) culturing the population of cells containing the mid-to-late ONP cells in a fourth cell culture medium containing RA at a concentration of 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 population of cells containing the mid-to-late ONP cells; (e) culturing the population of cells containing the mid-to-late ONP cells in a fifth cell culture medium containing 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 population of cells containing the late ONP cells; and (f) collecting the population of cells containing the late ONP cells, thereby producing a composition. In an alternative embodiment, the first cell culture medium in step (a) contains BMP4 at a concentration of 1-25 ng / mL and SB431542 at a concentration of 0.1-10 μM, and does not contain FGF2.

[0265] In some embodiments, the method comprises: (a) culturing a population of undifferentiated pluripotent stem cells for 3-7 days under conditions sufficient to produce non-neuronal ectoderm (NNE) cells in a first cell culture medium containing 10 ng / mL of FGF2, 10 ng / mL of BMP4, and 1 μM of SB431542, thereby producing a population of cells containing NNE cells; (b) culturing the population of cells containing NNE cells for 3-7 days under conditions sufficient to produce preplate-like ectoderm (PPE) cells in a second cell culture medium containing 1 μM of SB431542, 10 ng / mL of FGF2, and 2 μM of IWP-2 and 100 nM of LDN193189, thereby producing a population of cells containing PPE cells; (c) culturing a population of cells 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 7 days under conditions sufficient to produce early ear neuron progenitor (ONP) cells, thereby producing a population of cells containing early ONP cells; (d) culturing a population of cells containing early ONP cells in SHH at a concentration of 500 ng / mL, 0.(e) culturing the population of cells containing mid-to-late ONP cells for 7 days under conditions sufficient to produce mid-to-late ONP cells in a fourth cell culture medium containing RA at a concentration of 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, thereby producing a population of cells containing mid-to-late ONP cells; (e) culturing the population of cells containing mid-to-late ONP cells for 3-45 days under conditions sufficient to produce late ONP cells in a fifth cell culture medium containing 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, thereby producing a population of cells containing late ONP cells; and (f) collecting the population of cells containing late ONP cells and thereby producing a composition. In an alternative embodiment, the first cell culture medium in step (a) contains BMP4 at a concentration of 10 ng / mL and SB431542 at a concentration of 1 μM, and does not contain FGF2.

[0266] In some embodiments, any of the methods described above, prior to step (a), undifferentiated pluripotent stem cells at 1,200-20,000 live cells / cm² 2 The step includes seeding in a monolayer at a density of and culturing the cells until the lactate concentration in the cell culture medium reaches 1.68-12.29 mM and a percentage full growth rate of 5-80% is achieved.

[0267] In some embodiments, any one of the methods described above further includes the step of cryopreserving a population of cells containing late ONP.

[0268] A person skilled in the art will understand that, at any of the steps described above, the resulting population of cells can be cryopreserved and then seeded into a suitable culture medium for the next stage of the differentiation process.

[0269] Kits and manufactured goods

[0270] The present disclosure provides a kit comprising the pharmaceutical composition described herein, and manufactured articles, such as a freezing vial, syringe, syringe cartridge, cannula, etc.

[0271] In some embodiments, the kit includes an instruction manual.

[0272] In some embodiments, the pharmaceutical composition described herein is cryopreserved and pre-packaged in a dosing unit within a frozen vial, cannula, syringe, or syringe cartridge that is stored at a suitable temperature (e.g., -80°C or lower, or -140°C or lower), and is administered immediately to a subject after thawing to a suitable temperature, e.g., room temperature.

[0273] Although the present invention has been generally described above, it will be more easily understood by referring to the following examples, which are provided by way of example and are not intended to limit the scope of the disclosure. The embodiments and modes of embodiment described herein are for illustrative purposes only, and it is understood that various modifications or alterations may be proposed to those skilled in the art in light of this, and that they should be included within the scope and spirit of this application and the appended claims. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety for all purposes.

[0274] Examples

[0275] Example 1: Human pluripotent-derived neurons survive in guinea pig cochlea

[0276] This embodiment describes an evaluation of a guinea pig model of removing neurons and replacing them with stem cells. This embodiment also evaluates the fate of pluripotent-derived human auditory neurons (referred to as LCTANP1 cells, or ANP1 cells) when transplanted into the cochlea of ​​a guinea pig.

[0277] method

[0278] Animals & Anesthesia

[0279] Young male Hartley albino guinea pigs were obtained from Elm Hill Breeding Labs, Chelmsford, Massachusetts, USA. For anesthesia during the procedure, a combination of xylarzine at a concentration of 10 mg / kg, ketamine at a concentration of 40 mg / kg, and lidocaine at a concentration of 4 mg / kg was administered intramuscularly to the animals. For analgesia, ketoprofen at a concentration of 1 mg / kg was administered subcutaneously to the animals before surgery and for two consecutive days after surgery.

[0280] Hearing loss induction using ouabine

[0281] 5 μl of 10 mM aubain, also known as g-strophantin, a plant-derived toxin, was injected into the tympanic cavity, and the aubain-infused Gelfoam® was placed on the round window membrane of the cochlea.

[0282] Hearing loss induction using neomycin

[0283] 10 μl of 15% neomycin was injected through the round window using a syringe pump at a rate of 1 μl per minute.

[0284] LCTANP1 cells

[0285] Human auditory neurons are derived from pluripotent cells that differentiate directly into ear neuron progenitor cells according to the method described in WO2023 / 167986. Briefly, LCTANP1 (also referred to as ANP1) cells are transplantable ear neuron progenitor cells derived from human pluripotent stem cells. ANP1 cells are manufactured through an induced differentiation process that generates a pure ANP population. Production involves in-process control and batch release using developed analytical assays. ANP1 cells are capable of further maturation into auditory neurons (AN) after transplantation. Additionally, the ANP1 product can be frozen as an allogeneic cell therapy bank and immediately thawed at the clinical site to be injected into the patient's cochlea at clinical doses.

[0286] experimental group

[0287] LCTANP1 cells were injected into 8 animals per hearing loss-inducing condition (uabain or neomycin). The hearing loss-inducing control group (n=3) was euthanized 30 days after uabain hearing loss induction. The second hearing loss-inducing group was euthanized 30 days after neomycin hearing loss induction.

[0288] For the experimental group, 5 μl of LCTANP1 cells labeled with red fluorescent protein were injected into the base of the cochlea by a syringe pump at a concentration of 1 μl / min 30 days after induced hearing loss with ouabain. Injection was either (Group i) into the tympanic cavity via a cannula (n = 4) or (Group ii) directly into the cochlear axis via a microneedle (n = 4).

[0289] histology

[0290] Seven days after cell injection, guinea pigs were euthanized to evaluate the presence and location of LCTANP1 in the cochlea. The ears were collected.

[0291] The cochlea was locally perfused with 4% paraformaldehyde, and subsequently dissected under fluorescence stereomicroscopy to locate and image the labeled cells. The cochlea was then freeze-sectioned and stained with DAPI (blue), and antibodies against human nucleoli (green) and neurofilament proteins (red).

[0292] result

[0293] Guinea pigs were divided into a control group and an experimental group as described above. Fig. 1a and Fig. 1e As shown on the left, in the cochlea of ​​a normal guinea pig, the Rosenthal canal is packed with spiral ganglion neurons. After auabain injection, Fig. 1b ( Fig. 1c and Fig. 1d (presented at a higher magnification than in) and Fig. 1eAs shown in the center, even if spiral ganglion neurons were present in Rosenthal's canal, they appeared in very small numbers. Neomycin treatment resulted in approximately 25% survival of spiral ganglion neurons in guinea pigs ( Fig. 1e , right). Ouabain treatment resulted in the depletion of endogenous neurons with minimal side effects, demonstrating the utility of auabain for modeling chemically induced auditory neuropathy in rodent models, such as guinea pigs. Ouabain was chosen to establish chemically induced auditory neuropathy because it had fewer side effects on the health of animals compared to neomycin treatment and resulted in the near-complete elimination of spiral ganglion neurons, in contrast to the 25% survival observed with neomycin.

[0294] Next, LCTANP1 cells were administered to the experimental group. Fig. 2a , Fig. 2b and Fig. 2c As shown in [the text], 7 days after the injection of LCTANP1 cells, a large area of ​​the tympanic system of the cochlea appeared red by fluorescence microscopy, indicating the presence of clusters of transplanted LCTANP1 cells.

[0295] In addition, human nucleolus-specific antibodies ( Fig. 3a (Reference, stained green) and host guinea pig cells ( Fig. 3b It was confirmed that it is possible to distinguish between (see illustration of red-stained guinea pig fibroblasts).

[0296] Many LCTANP1 cells survived in guinea pig cochleas for at least one week. Figs. 4a-4d As shown in the immunofluorescence image illustrated in, transplanted LCTANP1 cells labeled with antibodies against human nucleoli ( Fig. 4bLCTANP1 cells (green) were clearly observable in the guinea pig cochlea and appeared as patches of various sizes and shapes in the tympanic regions of many guinea pigs. LCTANP1 cells were mostly present as aggregates near the wall of the cochlear axis within the tympanic space. No mitotic phases were observed among these human cells. Importantly, fluorescence stereomicroscopy identified red-labeled LCTANP1 cells in both the initial implantation site and the lateral region. LCTANP1 cells engrafted across a wide area of ​​the auabain-traumatized cochlea and were able to spread beyond the injection site as necessary for cochlear formation.

[0297] Collectively, these data demonstrate that ouabain can be used to model hearing loss in guinea pigs by removing auditory neurons. Human-derived auditory neurons, LCTANP1 cells, can be identified and survive in the guinea pig ear after transplantation. For both injection routes—into the perilymph of the tympanic cavity and into the cochlear axis—LCTANP1 cells survived in the ouabain-traumatized cochlea for at least 7 days after administration to either the tympanic cavity or the cochlear axis. Furthermore, LCTANP1 cells migrated beyond the initial injection / transplantation site toward the second rotator cuff and engrafted across a broad area of ​​the ouabain-traumatized cochlea. The transplanted cells were able to spread beyond the injection site as required to constitute the cochlea.

[0298] Example 2: Evaluation of the feasibility of manufacturing human pluripotent-derived auditory neuron progenitor cells (LCTANP1) as a therapeutic agent for hearing impairment

[0299] This example describes the feasibility, release criteria, and evaluation of in vitro activity of LCTANP1 cells produced in large quantities.

[0300] The loss of auditory neurons can lead to auditory neuropathy even if hair cells and cochlear nuclei remain intact. Cell-based therapies that replace lost or dysfunctional auditory neurons can restore hearing in such cases. In severe cases where both hair cells and many neurons are lost, reconstructing the cochlea with transplanted functional auditory neurons can improve the success rate of cochlear implant procedures. This embodiment describes a differentiation process for producing LCTANP1, composed of auditory neuron progenitor cells (also referred to as ear neuron progenitor cells), from pluripotent human stem cells. As an integrated process, the LCTANP1 production process and the cells were characterized by a set of biologically and functionally relevant markers using different quantitative methods developed and customized, including functional in vitro assays. LCTANP1 cells were transplanted into the cochlea of ​​guinea pigs from which neurons had already been removed via exposure to ouabain. The viability of engrafted LCTANP1 was evaluated when implanted into the hearing-induced cochlea of ​​guinea pigs.

[0301] method

[0302] The LCTANP1 differentiation process involved the expansion of a clinical-grade human pluripotent stem cell line, a series of differentiation inductions delivered within a specific time frame under specific culture conditions, the harvesting of final auditory neuron progenitor cells, and finally, cryopreservation in an immediate administration form. This process Fig. 5a It is schematically illustrated in [figure]. Biomarker kinetics according to differentiation are Fig. 5b It was monitored as exemplified in.

[0303] To ensure the quality of cell products, Fig. 6a and Fig. 6bAs described in [document], various assays were developed. For example, the development of analytical biomarkers to assess the purity of cell populations included flow cytometry, specific protein marker expression by immunofluorescence, gene expression profiles including RNA sequencing, and bioinformatics analysis including computer analysis of single-cell RNA sequencing (scRNA seq) results.

[0304] A functional assay was developed to measure the neuronal characteristics of LCTANP1. The functional assay was designed to test the ability of cells to induce calcium influx in a time-dependent manner in response to glutamatergic agonists (which plays a crucial role in multiple signaling cascades within auditory neurons) and to express synapse markers that play a role in the formation of synapses between hair cells and auditory neurons.

[0305] As described in Example 1 above, 5 μL of red fluorescently labeled LCTANP1 cells (quality controlled as described in this example) were injected into the tympanic cavity via cochleotomy or by inserting a needle into the cochlear axis of eight ouabain-treated guinea pigs, both procedures were performed at the base of the cochlea. After 7 days, the animals were euthanized, and the labeled LCTANP1 cells within the cochlea were visualized using a fluorescent stereomicroscope. The cochlea was processed for immunohistochemistry, dissected, and imaged for evaluation of LCTANP1 cells.

[0306] result

[0307] LCTANP1 cells were successfully manufactured on a large scale, met pre-established release criteria, and demonstrated relevant activity in in vitro functional tests. LCTANP1 cells were cryopreserved in a form that allows for immediate administration, thawing, and injection, and were successfully thawed and successfully transplanted in an in vivo guinea pig model, surviving for at least 7 days.

[0308] LCTANP1 is a cell-based product composed of auditory neuron progenitor cells derived from clinical-grade pluripotent stem cells. LCTANP1 has completed initial chemistry, manufacturing, and quality control studies and preclinical trials, highlighting its potential as a therapeutic agent.

[0309] For cell therapy to be successful, cells must be safe and reproducible, meet standards for purity, identity, and efficacy, and production must be scalable. When manufacturing safe and reproducible cells, source line characterization, cell banking, various scaling systems, differentiation process development, culture conditions, optimization, analytical methods, in-process control, and release criteria must all be considered. In addition, clinically appropriate post-production processing, development of analytical methods for process control and product release, functionality and performance testing, cell enhancements such as genetic modification, addition of expression systems, scalability aspects, substrates, harvesting protocols, clinical and commercial throughput, and manufacturing costs all play important roles.

[0310] By performing the steps in parallel, it was possible to develop LCTANP1 cells from cell bank production to production for large-scale operation execution in 18 months ( Fig. 7a ). During manufacturing, LCTANP1 cells undergo induced differentiation ( Fig. 7b During this period, repeatable and reproducible methods for the culture, expansion, and differentiation of hESCs were established. The large-scale differentiation process involved optimizing the differentiation cocktail in process and release trials. The resulting drug products were in the form of thaw-and-injectable (TAI) cryopreservation-immediate injection.

[0311] Table 5 presents an exemplary ANP1 cell profile, as well as the range of cells expressing the indicated marker based on 10 replicates and the test method used to test the marker expression.

[0312] Table 5. Exemplary ANP1 cell profile

[0313]

[0314] FC - Flow Cytometry

[0315] qPCR - Quantitative PCR

[0316] IF - Immunofluorescence

[0317] FIO - For providing information

[0318] HR - hESC Residual Test

[0319] Example 3: Evaluation and Characterization of ANP1 Cells in Vitro

[0320] Auditory neurons are specialized cells that transmit sound information from the ear to the brain by converting mechanical sound waves into electrical signals. They transmit this data from the hair cells of the cochlea to the cochlear nucleus of the brain, where the data is further processed to detect pitch, volume, and specific location of the sound. Auditory neurons and hair cells form synapses. Hair cells stimulate auditory neurons by releasing the neurotransmitter glutamate. An action potential is generated and propagates toward the cochlear nucleus (see, for example, [Moser and Starr, Nat Rev Neurology, 2018, 12(3): doi.org / 10.1038 / nrneurol.2016.10]). The present embodiment describes the evaluation and characterization of ANP1 cells and ANP1 cell connectivity by visualizing various cellular markers, including neural markers, auditory neuron markers, synaptic markers, glutamatergic markers, and late ONP markers.

[0321] method

[0322] ANP1 cells and culture

[0323] Human auditory neurons were derived from pluripotent cells that differentiate directly into ear neuron progenitor cells according to the method described in WO2023 / 167986 and Example 1. ANP1 cells were seeded into black 24-well plates and cultured in ANDM medium supplemented with BDNF, NT3, and IGF for 3 days and up to 3 weeks. The medium was changed twice a week. Cells were fixed using 4% PFA and stained using immunofluorescence to evaluate cell markers and connectivity.

[0324] ANP1 cells were stained with DAPI nuclear stain (blue) as well as fluorescently labeled antibodies (red or green) for neuronal and synaptic markers. Cell morphology, marker expression, and cell connectivity potential were evaluated by capturing images using a confocal microscope. The late ear neuronal progenitor cell marker SOX2, the auditory neuron marker TRKB, the neuronal marker Nestin, and synaptic markers consistent with successful progression through the differentiation process were used. Additionally, Fig. 10 As exemplified by the immunofluorescence image, the combination of neuron and synapse-related markers was 3 days ( Fig. 10 , top row) to 3 weeks ( Fig. 10 It showed an increase in expression according to the culture time of the lower row.

[0325] Example 4: Evaluation of auditory neuron electrical activity on a multi-electrode array

[0326] This embodiment evaluates neuronal activity from multiple ANP1 cells using cells grown on a multi-electrode array (MEA).

[0327] Experiment 1

[0328] method

[0329] ANP1 cells and culture

[0330] Human auditory neurons were derived from pluripotent cells that differentiate directly into ear neuron progenitor cells according to the method described in WO2023 / 167986 and Example 1. Subsequently, ANP1 cells were cultured on a 60MEA200 / 10iR-ITO standard microelectrode array (MCS-multichannel system, MEA) for 3 weeks. Cells were cultured in ANDM medium supplemented with BDNF and NT3 in an incubator at 37°C. The medium was replaced twice a week for 3 weeks prior to recording. At the time of electrophysiological recording, the medium was replaced with recording buffer (HBSS containing HEPES), and the culture was equilibrated for more than 1 hour prior to recording ( Fig. 11 , left; scale bar = 500 μm).

[0331] Neuron activity test

[0332] Neuronal activity of multiple ANP1 cells grown on MEA in the absence of stimulation by the glutamate receptor agonist alpha-amino-3-hydroxy-5-methyl-4-isooxazolepropionic acid (AMPA) (basal activity; Fig. 11 , upper right) and presence (inductively active; Fig. 11 It was recorded simultaneously under the lower right side.

[0333] Experiment 2

[0334] method

[0335] ANP1 cells and culture

[0336] Human auditory neurons were derived from pluripotent cells that differentiate directly into ear neuron progenitor cells according to the method described in WO2023 / 167986 and Example 1. ANP1 cells were then cultured on a MaxOne MEA chip (Maxwell Biosystems) for up to 8 weeks. Cells were cultured supplemented with neurotrophic growth factors in a 1:1 mixture of DMEM / F12 and Neurobasil medium or Brainpiece medium (Stem Cell Technologies) in a 37°C incubator. The medium was changed three times a week. During the culture period, baseline electrophysiological activity was monitored, recorded, and quantified. Once sufficient neural network activity was reached, ANP1 cell neural activity and electrical stimulation in response to glutamatergic agonists and blockers were tested.

[0337] Neuron activity test

[0338] Neuronal activity of multiple ANP1 cells grown on MEA was tested for AMPA (glutamate agonist, TOCRIS), 6,7-dinitroquinoxalin-2,3-dione ("DNQX", AMPA receptor-specific blocker, Alomone Labs), and tetrodotoxin (IUPAC designation: 4 R ,4a R ,5 R ,6 S ,7 S ,8 S ,8a R ,10 S ,12 S )-2-azaniumylidene-4,6,8,12-tetrahydroxy-6-(hydroxymethyl) - 2,3,4,4a,5,6,7,8-octahydro-1 H -8a,10-methano-5,7-(epoxymethanooxy)quinazolin-10-oleate, "TTX", sodium channel blocker, Alomone Labs) absence (basal activity) and presence (inductive activity; Fig. 13aSimultaneously recorded under ). Single neuron and network activity were monitored and quantified over time. Subsequently, an axon tracing module was used. This module enabled the recording and reconstruction of neuronal footprints across the electrode array and the automatic verification of action potential propagation along cell processes ( Figs. 14a-14b ).

[0339] result

[0340] The results of Experiments 1 and 2 demonstrate that LCT-ANP1 cells successfully exhibited basal neural activity and responded to electrical stimulation. The ability to respond to electrical stimulation is important for cells to interact effectively with the cochlear implant. The response (active electrode, total spike, and evoked peak) increases in correlation with the amplitude of the electrical stimulation ( Fig. 12c ). Fig. 11 and Fig. 13a As shown in the graph illustrated in [figure], ANP1 cells grown on MEA exhibited auditory-specific glutamatergic neuronal responses. Single-neuron and network activities were monitored and quantified over time, and the formation of neuronal network activity was demonstrated after 30 days ( Figs. 12a-12b Neural activity was tested before and after the addition of AMPA. In LCT-ANP1 cells, the frequency of action potentials increased after the addition of AMPA ( Figs. 13a-13c Addition of DNQX reduced this activity ( Figs. 13a-13c When TTX was added, electrical activity was blocked ( Fig. 13a Additionally, quantified single-neuron and network activities indicated the formation of neuronal network activity after 30 days, and activity steadily increased with culture time (maximum ~50 days) ( Figs. 12a-12b ).

[0341] In addition, LCT-ANP1 cells exhibited axonal elongation based on electrical signal footprints ( Figs. 14a-14bUsing an axon tracking module, neuronal footprints were recorded and reconstructed across the electrode array, and action potential propagation along cell processes was automatically verified. LCT-ANP1 cells exhibit axonal elongation based on electrical signal footprints after approximately 6 weeks in vitro using an MEA system. The reconstructed axon maps of five LCT-ANP1 cells are Fig. 14a It is exemplified in, and after analysis, the axon length-related parameters are Fig. 14b It is exemplified in.

[0342] Example 5: Bioinformatics Analysis of Auditory Neuron-Related Gene Expression in ANP1 Cells

[0343] This embodiment illustrates the expression of a set of auditory neuron genes selected from publicly available databases and scientific literature using bioinformatics analysis of single-cell RNA sequencing (scRNA seq) of ANP1 cells. This computational approach can facilitate a deeper understanding of ANP1 cell function and differentiation.

[0344] method

[0345] ANP1 cells and culture

[0346] Human auditory neurons were derived from pluripotent cells that differentiate directly into ear neuron progenitor cells according to the method described in WO2023 / 167986 and Example 1. ANP1 cells were thawed and processed for scRNA.

[0347] Single-cell RNA-Seq (scRNA-Seq)

[0348] Single-cell RNA sequencing was performed on the thawed ANP1 drug product. 10X Genomics® Chromium™ 3' gene expression RNA-seq was used, with a target number of 10,000 and reads per cell of 50,000. Sequencing included basic analysis, a quality report (pdf), FASTQ raw data files, and interactive differential gene expression analysis via 10X Genomics®, Cell Ranger™, and Loupe™ Cell Browser.

[0349] Bioinformatics Analysis

[0350] Bioinformatics analysis of scRNA-Seq results was performed using Uniform Manifold Approximation and Projection (UMAP) graphs based on Principal Component Analysis (PCA), which included sets of auditory neuron-related genes from publicly available datasets (e.g., MSigDB, Panglao, and Cell Marker) and related published scientific literature. Graph ( Fig. 9 The color scale in ) represents cells expressing a set of major auditory neuron-related genes.

[0351] result

[0352] Fig. 17 As shown in the graph illustrated in, flow cytometry analysis of ANP1 cells using the combination of the hPSC-specific protein markers TRA-1-60 and SSEA5 indicated that no residual hPSC cells were detected within the ANP1 population. Gene set ( Fig. 9 Further analysis including protein markers for ) and late ONP markers, neural markers, and auditory neuron markers is consistent with successful progression through the differentiation process.

[0353] Example 6: Ouabain hearing loss-inducing paradigm in guinea pigs

[0354] Animal models of auditory neuropathy are useful for testing cell-based therapies to replace lost spiral ganglion neurons. In models where inner ear hair cells are preserved, the contribution of transplanted cells can be tested using acoustic stimulation, whereas models lacking hair cells are useful for testing the function of cochlear implants. Guinea pigs are interesting because they are commonly and successfully used in auditory research and cochlear implant studies. The goal of this project is to compare two Ouavine hearing loss induction paradigms and their effects on hair cell and auditory nerve survival in guinea pigs.

[0355] 5 μl of 7.5 mM or 10 mM aubaine was injected into Hartley albino guinea pigs through the round window membrane into the perilymph of the tympanic cavity. Subsequently, a gel foam soaked in aubaine was placed in the round window. In the low-dose group, baseline pure tone auditory brainstem response (ABR) of the left ear was measured at 4, 8, and 16 kHz. Two weeks after aubaine administration, ABR was measured after hearing loss induction with simultaneous 50 dB white noise masking applied to the opposite ear.

[0356] The high-dose aubaine group was processed for histological examination one month after ototoxic insertion. Low-dose animals were processed at 17 days. The temporal bone was fixed in paraformaldehyde, decalcified, embedded in resin, and sectioned near the center of the cochlear axis plane for light microscopy.

[0357] Animals treated with both doses of ouabain developed vestibular signs, such as transient nystagmus and torticollis, during anesthesia. In the high-dose group, histology revealed near-complete loss of all hair cells throughout the cochlea and near-complete loss of the auditory nerve. Histology in the low-dose group indicated that while many external ear hair cells were missing, inner ear hair cells were present in all cochlear spinous regions. Many spiral ganglion neurons were missing, but some cells remained in all spinous regions. These data confirm the ABR results.

[0358] Injecting 7.5 mM ouabain into the perilymph of guinea pigs is useful for studying auditory neuropathy involving the insertion of neural progenitor cells. A small number of neurons remain at the lesion site, which can provide a scaffold for discovering pathways into the cochlear nucleus and also toward surviving inner ear hair cells. For studies combining cochlear implants with cell transplantation, higher doses were found to be more suitable because inner ear hair cells do not survive.

[0359] Example 7: Evaluation of auditory neuron activity using calcium influx assay

[0360] At the excitatory synapse between a hair cell and an auditory neuron, glutamate release activates ionotropic receptors, causing a rapid influx of ions into the postsynaptic cell. Ca 2+ It acts as a secondary messenger in response to glutamate, activating intracellular signal transduction and causing the transcription of genes involved in synaptic plasticity. Calcium influx can be measured using fluorescent dyes (e.g., Fluo-8, Abcam) that are added to the cell and become brighter upon calcium binding in response to glutamatergic stimulation. Changes in fluorescence over time in response to external stimuli can be monitored and quantified using fluorescence microscopy and a fluorescence plate reader (e.g., Tecan).

[0361] method

[0362] ANP1 cells and culture

[0363] Human auditory neurons were derived from pluripotent cells that differentiate directly into ear neuron progenitor cells according to the method described in WO2023 / 167986 and Example 1. ANP1 cells were then cultured in 96 wells supplemented with neurotrophic growth factor. The medium was changed 2-3 times per week. Before testing, the medium was replaced with Hanks' equilibrium salt solution (Gibco) supplemented with HEPES in the presence of CaCl2.

[0364] Calcium Influx Test

[0365] ANP1 cells were incubated with Fluo-8 fluorescent dye (Abcam). After washing, at baseline and positive control (ionomycin, IUPAC designation: (4 R ,6 S ,8 S ,10 Z ,12 R ,14 R ,16 E ,18 R ,19 R ,20 S ,21 S )-19,21-dihydroxy-22-{(2 S ,2' R ,5 S ,5' S )-5'-[(1 R Fluorescence quantification was obtained using a Tekane plate reader upon the addition of )-1-hydroxyethyl]-2,5'-dimethyloctahydro-2,2'-bifuran-5-yl}-4,6,8,12,14,18,20-heptamethyl-11-oxydo-9-oxodocosa-10,16-dienoic acid, Sigma) and glutamatergic agonists (glutamate, AMPA, and tocris). Calcium influx induction was calculated as relative fluorescence according to Equation 1 below:

[0366]

[0367] Additionally, DNQX (Alomon Labs) was added to cells prior to AMPA addition to show that specific calcium influx was reduced upon its addition ( Figs. 15a-15b ).

[0368] result

[0369] Fig. 15a As exemplified by, ANP1 cells showed a positive response (>100) to glutamate and AMPA stimulation. Upon addition of DNQX ( Fig. 15b ), ANP1 cells show a decrease in calcium influx, which demonstrates the specificity of the assay. The results of six repeated ANP1 experiments are summarized in Table 6 below.

[0370] Table 6.

[0371]

[0372] Equivalents

[0373] The foregoing description is provided merely for illustrative purposes and is not intended to limit the present disclosure to the exact form disclosed. Details of one or more embodiments of the present disclosure are provided in the appended description. Any method and material similar or equivalent to those described herein may be used to practice or test the present disclosure, but preferred methods and materials are described herein. Other features, purposes, and advantages of the present disclosure will be apparent from this description and claims. In this specification and appended claims, the singular form includes the plural subject unless the context clearly indicates otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which the present disclosure pertains. All patents and publications cited in this specification are incorporated by reference.

Claims

Claim 1 A method for treating a subject having an auditory pathology, comprising inserting a cochlear implant and administering a therapeutically effective amount of a pharmaceutical composition comprising a population of auditory cells, wherein (a) at least 75% of the cells in the population express both Nestin and SOX2; (b) at least 80% of the cells in the population express β-tubulin III; (c) at least 5% of the cells in the population express TrkB; and (d) at least 1% of the cells in the population express TRA-1-60 and / or SSEA5. Claim 2 A method according to claim 1, wherein the pharmaceutical composition enhances the efficacy of a cochlear implant. Claim 3 A method for enhancing the efficacy of a cochlear implant in a subject having an auditory pathology, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising a population of auditory cells, wherein (a) at least 75% of the cells in the population express both Nestin and SOX2; (b) at least 80% of the cells in the population express β-tubulin III; (c) at least 5% of the cells in the population express TrkB; and (d) at least 1% of the cells in the population express TRA-1-60 and / or SSEA5. Claim 4 A method according to any one of claims 1 to 3, wherein the pharmaceutical composition is administered prior to the insertion of a cochlear implant. Claim 5 A method according to any one of claims 1 to 3, wherein the pharmaceutical composition is administered after the insertion of a cochlear implant. Claim 6 A method according to any one of claims 1 to 3, wherein the pharmaceutical composition is administered simultaneously with the insertion of a cochlear implant. Claim 7 A method according to any one of claims 1 to 6, wherein the pharmaceutical composition is administered to the inner or middle ear of a subject. Claim 8 A method according to claim 7 in which administration to the inner ear includes administration to the cochlea. Claim 9 A method according to claim 8, wherein the pharmaceutical composition is administered to the tympanic cavity, cochlear axis, or spiral ganglion. Claim 10 A method according to any one of claims 1 to 9, wherein the pharmaceutical composition is administered via injection. Claim 11 A method according to claim 10, wherein the injection involves inserting a cannula through the opening of the sac or inserting a cannula through the round window of the subject. Claim 12 A method according to any one of claims 1 to 11, wherein the auditory condition comprises conductive hearing loss, sensorineural hearing loss, central hearing loss, mixed hearing loss, auditory neuropathy spectrum disorder, central auditory processing disorder, tinnitus, or hidden hearing loss. Claim 13 A method according to any one of claims 1 to 12, wherein the auditory condition comprises the loss of cochlear hair cells or cochlear neurons. Claim 14 A method according to any one of claims 1 to 12, wherein the auditory condition comprises loss of cochlear hair cells and loss of cochlear neurons. Claim 15 A method for treating a subject having an auditory condition selected from the group consisting of conductive hearing loss, sensorineural hearing loss, mixed hearing loss, auditory neuropathy spectrum disorder and hidden hearing loss, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising a population of auditory cells, wherein (a) at least 75% of the cells in the population express both Nestin and SOX2; (b) at least 80% of the cells in the population express β-tubulin III; (c) at least 5% of the cells in the population express TrkB; and (d) at least 1% of the cells in the population express TRA-1-60 and / or SSEA5. Claim 16 In paragraph 15, a method in which the auditory condition includes hidden hearing loss. Claim 17 A method according to claim 15 or 16, wherein the pharmaceutical composition is administered to the inner or middle ear of the subject. Claim 18 A method in which, in any one of paragraphs 15 through 17, the subject receives additional treatment with a cochlear implant. Claim 19 A method according to any one of claims 1 to 18, wherein the pharmaceutical composition is cryopreserved and the method comprises thawing the composition before administration. Claim 20 A method according to any one of claims 1 to 19, wherein at least 85% of the cells in the population express both Nestin and SOX2. Claim 21 A method according to any one of claims 1 to 20, wherein 85% or more of the cells in the population express β-tubulin III. Claim 22 A method according to any one of claims 1 to 21, wherein 15% or more of the cells in the population express TrkB. Claim 23 A method according to any one of claims 1 to 22, wherein 0.1% or less of the cells in the population express TRA-1-60 and / or SSEA5. Claim 24 A method according to any one of claims 1 to 23, wherein 20% or less of the cells in the population express PAX8 and / or PAX2. Claim 25 A method according to any one of claims 1 to 23, wherein the population of cells does not significantly express PAX2 and / or PAX8. Claim 26 A method according to any one of claims 1 to 25, wherein 10% or more of the cells in the population express GluA4. Claim 27 A method according to any one of claims 1 to 26, wherein 10% or less of the cells in the population express Myo7A. Claim 28 A method according to any one of claims 1 to 27, wherein (a) 85% or more of the cells in the population express both Nestin and SOX2; (b) 90% or more of the cells in the population express β-tubulin III; (c) 15% or more of the cells in the population express TrkB; (d) 10% or more of the cells in the population express GluA4; (e) 10% or less of the cells in the population express Myo7A; and (f) 0.1% or less of the cells in the population express TRA-1-60 and / or SSEA5. Claim 29 A method according to any one of claims 1 to 28, wherein (a) about 70% to 100% of the cells in the population express both Nestin and SOX2; (b) about 80% to 100% of the cells in the population express β-tubulin III; (c) about 5% to 80% of the cells in the population express TrkB; and (d) 0% to about 0.1% of the cells in the population express TRA-1-60 and / or SSEA5. Claim 30 A method according to claim 29 in which about 10% to 95% of the cells in the population express GluA4. Claim 31 A method according to claim 29 or 30 in which 0 to about 30% of the cells in the population express Myo7A. Claim 32 A method according to any one of claims 1 to 31, wherein 50% or more of the cells in the population express CD133. Claim 33 A method according to any one of claims 1 to 32, wherein the population of auditory cells comprises: a. late ear neuron progenitor (ONP) cells; b. late and meta-ONP cells; or c. meta-ONP cells, late ONP cells, mature auditory neurons, spiral ganglion neurons, or any combination thereof. Claim 34 A method according to any one of claims 1 to 33, wherein the percentage of cells expressing Nestin, SOX2, β-tubulin III, TrkB, GluA4, TRA-1-60 and / or SSEA5 protein markers is determined by flow cytometry or immunofluorescence. Claim 35 A method according to any one of claims 1 to 34, wherein the group of auditory cells comprises a group of sensory cells of the ear. Claim 36 In paragraph 35, the sensory cell population is selected from a group consisting of hair cells, supporting cells, ear neuron progenitor cells, and sensory neuron progenitor cells. Claim 37 A method according to any one of claims 1 to 36, wherein the group of cells comprises a cell aggregate, a single cell, or a combination thereof. Claim 38 A method according to any one of claims 1 to 37, wherein the group of auditory cells comprises at least 100,000 cells. Claim 39 A method according to any one of claims 1 to 38, wherein the group of auditory cells comprises 100,000 to 10 million cells. Claim 40 A method in which, in any one of claims 1 to 39, about 100,000 to about 1 million cells are administered to a subject. Claim 41 A method according to any one of claims 1 to 40, wherein approximately 30 million cells per milliliter to approximately 700 million cells per milliliter are administered to a subject. Claim 42 A method according to any one of claims 1 to 41, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier. Claim 43 A method according to any one of claims 1 to 42, wherein a population of auditory cells is obtained by a method comprising the following steps: a) obtaining a culture of undifferentiated pluripotent cells; b) culturing the undifferentiated pluripotent cells under culture conditions sufficient to induce differentiation of the pluripotent cells into non-neuronal ectodermal cells; and c) culturing the cells from (b) under culture conditions sufficient to differentiate the non-neuronal ectodermal cells into auditory cells.

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