Methods for generating functional spiral ganglion neurons

Abstract

Compositions and methods are provided for generating spiral ganglion neuron-like cells from stem cells. Methods of using such spiral ganglion neuron-like cells in auditory research, disease modeling of sensorineural hearing loss, drug screening, and cell therapy are also provided.

Description

METHODS FOR GENERATING FUNCTIONAL SPIRAL GANGLION NEURONSCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims benefit under 35 U.S.C. § 119(e) of provisional application 63 / 771,540, filed March 13, 2025, which application is hereby incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED ESEARCH OR DEVELOPMENT

[0002] This invention was made with Government support under contract DC015824 awarded by the National Institutes of Health. The Government has certain rights in the invention.BACKGROUND OF THE INVENTION

[0003] Although hearing loss disables over 5% of the world’s population (who.int / news-room / fact- sheets / detail / deafness-and-hearing-loss), there are currently no effective pharmaceutical therapies for sensorineural hearing loss. The auditory neurons in the human inner ear that project to the central nervous system, the spiral ganglion neurons (SGNs), are affected in many pathologies contributing to hearing loss. However, a fundamental obstacle in the study of human SGNs is the inability to obtain routine tissue biopsies due to the small size of the cochlea, its complex three-dimensional anatomy, and encasement in dense bone. This limits the ability to develop in vitro models of human SGNs.SUMMARY OF THE INVENTION

[0004] Compositions and methods are provided for generating spiral ganglion neuron-like cells from stem cells. Methods of using such spiral ganglion neuron-like cells in auditory research, disease modeling of sensorineural hearing loss, drug screening, and cell therapy are also provided.

[0005] In one aspect, a method of producing spiral ganglion neuron (SGN)-like cells from stem cells is provided, the method comprising: (a) culturing the stem cells in a first medium comprising bone morphogenetic protein 4 (BMP4), a transforming growth factor-p (TGF-p) type I receptor inhibitor, and basic fibroblast growth factor 2 (FGF2); (b) culturing cells, produced from the stem cells in the first medium, in a second medium comprising the FGF2, the TGF-p type I receptor inhibitor, a Wnt inhibitor, and an inhibitor of ALK2 and ALK3, wherein the cells differentiate into pre-placodal ectoderm and neural crest precursors; (c) isolating the pre-placodal ectoderm and neural crest precursors; (d) culturing the isolated pre-placodal ectoderm and neural crest precursors in a third medium comprising Wnt family member 3A (WNT3A), the FGF2, insulin-like growth factor-1 (IGF-1 ),and a non-selective Rho-associated protein kinase (ROCK) inhibitor; (e) culturing the cells, produced from the pre-placodal ectoderm and neural crest precursors in the third medium, in a fourth medium comprising sonic hedgehog (SHH), retinoic acid (RA), epidermal growth factor (EGF), the FGF2, and the IGF- 1 , wherein the cells differentiate into otic neural progenitor cells; and (f) culturing the otic neural progenitor cells in a fifth medium comprising brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), the IGF-1, cyclic adenosine monophosphate (cAMP), and a selective ROCK inhibitor to induce neuronal maturation of the otic neural progenitor cells into the SGN-like cells.

[0006] In certain embodiments, the stem cells are embryonic stem cells, adult stem cells, or induced pluripotent stem cells (iPSCs). In some embodiments, the iPSCs are human induced pluripotent stem cells (hiPSCs). Exemplary hiPSC cell lines that can be used to produce SGN-like cells are SK8- A and UCSD112i-2-11.

[0007] In certain embodiments, the Wnt inhibitor is / \ / -(6-methyl-1 ,3-benzothiazol-2-yl)-2-[(4-oxo-3- phenyl-6,7-dihydrothieno[3,2-d]pyrimidin-2-yl)sulfanyl]acetamide (IWP-2).

[0008] In certain embodiments, the TGF-p type I receptor inhibitor is 4-[4-(1 ,3-benzodioxol-5-yl)-5- pyridin-2-yl-1H-imidazol-2-yl]benzamide (SB-431542).

[0009] In certain embodiments, the inhibitor of ALK2 and ALK3 is 4-[6-(4-piperazin-1- ylphenyl)pyrazolo[1 ,5-a]pyrim idin-3-yl]qu incline (LDN-193189).

[0010] In certain embodiments, the non-selective ROCK inhibitor is 4-[(1 R)-1 -aminoethyl]-N-pyridin- 4-ylcyclohexane-1 -carboxamide (Y-27632).

[0011] In certain embodiments, the selective ROCK inhibitor is 4-methyl-5-[[(2S)-2-methyl-1 ,4- diazepan-1-yl]sulfonyl]isoquinoline (H1152).

[0012] In certain embodiments, isolating the pre-placodal ectoderm and neural crest precursors (which include TRA-1-60" NGFR+cells) comprises using fluorescence-activated cell sorting or magnetic-activated cell sorting. For example, magnetic-activated cell sorting can be performed using magnetic microbeads conjugated to anti-TRA-1-60 antibodies and anti-NGFR antibodies. In some embodiments, TRA-1-60" NGFR+cells are isolated.

[0013] In certain embodiments, step (d) comprises initially seeding the third medium with about 60,000 TRA-1 -60 NGFR+cells / cm2for the culturing.

[0014] In certain embodiments, step (a) comprises culturing the stem cells in the first medium for about 3 days.

[0015] In certain embodiments, step (b) comprises culturing the stem cells in the second medium for about 5 days.

[0016] In certain embodiments, step (d) comprises culturing the cells in the third medium for about 7 days.

[0017] In certain embodiments, step (e) comprises culturing the TRA-1 -60" NGFR+cells in the fourth medium for about 7 days.

[0018] In certain embodiments, step (f) comprises culturing the otic neural progenitor cells in the fifth medium for about 6 days.

[0019] In certain embodiments, the first medium comprises 10 ng / ml BMP4, 1 pmol / L SB-431542, and 10 ng / ml FGF2.

[0020] In certain embodiments, the second medium comprises 100 nmol / L LDN-193189, 1 pmol / L SB-431542, 2 mmol / L IWP-2, and 10 ng / ml FGF2.

[0021] In certain embodiments, the third medium comprises 100 ng / ml WNT3A, 10 ng / ml FGF2, 50 ng / ml IGF-1, and 10 pmol / L Y-27632.

[0022] In certain embodiments, the fourth medium comprises 500 ng / ml SHH, 0.5 pmol / L RA, 20 ng / ml EGF, 10 ng / ml FGF2, and 50 ng / ml IGF-1.

[0023] In certain embodiments, the fifth medium comprises 10 ng / ml BDNF, 10 ng / ml NT-3, 10 ng / ml IGF-1, and 10 ng / ml H1152.

[0024] In certain embodiments, the method further comprises isolating the SGN-like cells.

[0025] In certain embodiments, the method further comprises characterizing the SGN-like cells by performing single-cell RNA sequencing, immunocytochemistry, whole-cell patch-clamping, gene expression profiling, calcium ion (Ca2+) imaging, or any combination thereof.

[0026] In certain embodiments, the method further comprises co-culturing the SGN-like cells with hair cells, glial cells, cochlear nucleus neurons, or any combination thereof.

[0027] In certain embodiments, the SGN-like cells comprise type I SGN-like cells and type II SGN- like cells. In some embodiments, the method further comprises isolating the type I SGN-like cells or the type II SGN-like cells.

[0028] In certain embodiments, the method further comprises detecting one or more cellular markers to identify the SGN-like cells, wherein the one or more cellular markers are selected from tubulin beta 3 class III (TUBB3), neurofilament light polypeptide (NEFL), neurofilament heavy polypeptide (NEFH), POU class 4 homeobox 1 (POU4F1), neuronal differentiation 1 transcription factor (NEUROD1), SRY-box transcription factor 2 (SOX2), prospero homeobox 1 (PROX1), sodium / potassium-transporting ATPase subunit alpha-3 (NKAa3), calbindin-2 (CALB2), and Ly6 / PLAUR domain-containing protein 1 (LYPD1). peripherin (PRPH), tachykinin precursor 1 (TAC1), MAF bZIP transcription factor (MAFB), and GATA binding protein 3 (GATA3).

[0029] In certain embodiments, the stem cells comprise a genetic mutation associated with an SGN- associated disorder. In some embodiments, the hiPSCs are derived from a somatic cell from a patient who has a genetic mutation associated with an SGN-associated disorder such as a disordercausing hearing loss or deafness. In some embodiments, the somatic cell is obtained from a biopsy of the inner ear of the patient.

[0030] In certain embodiments, the SGN-like cells comprise a genetic mutation associated with hearing loss or deafness.

[0031] In certain embodiments, the genetic mutation associated with hearing loss or deafness is introduced into the stem cells or the SGN-like cells by gene editing.

[0032] In certain embodiments, the genetic mutation associated with hearing loss or deafness is in a gene selected from TMPRSS3, NLRP3, FGF13, TBC1D24, GATA2, GATA3, DFNB59, GRM7, and ATP6V1B2.

[0033] In another aspect, a composition comprising SGN-like cells, produced by a method described herein, is provided.

[0034] In certain embodiments, the composition further comprises glial cells, hair cells, cochlear nucleus neurons, or any combination thereof.

[0035] In certain embodiments, the composition further comprises a pharmaceutically acceptable excipient.

[0036] In another aspect, a method of screening a candidate agent to determine its effects on SGN- like cells is provided, the method comprising: contacting the SGN-like cells, produced by a method described herein, with the candidate agent; and determining the effects of the candidate agent on morphological, electrophysiological, genetic, or functional parameters of the SGN-like cells.

[0037] In certain embodiments, the SGN-like cells comprise a genetic mutation associated with hearing loss or deafness.

[0038] In certain embodiments, determining the effects of the candidate agent comprises performing single-cell RNA sequencing, immunocytochemistry, whole-cell patch-clamping, gene expression profiling, calcium ion (Ca2+) imaging, confocal microscopy, atomic force microscopy, super-resolution microcopy, light-sheet microscopy, two-photon microscopy, fluorescence microscopy, migration assays, axonal growth and pathfinding assays, or any combination thereof.

[0039] In certain embodiments, the method further comprises contacting the SGN-like cells with glial cells, hair cells, cochlear nucleus neurons, or any combination thereof.

[0040] In certain embodiments, the method further comprises detecting the effects of the candidate agent on synapses between the SGN-like cells and the cochlear nucleus neurons or the hair cells. In some embodiments, the synapses are glutamatergic synapses.

[0041] In certain embodiments, the method further comprises detecting the effects of the candidate agent on growth of neurites of the SGN-like cells toward the hair cells.

[0042] In certain embodiments, the method further comprises detecting the effects of the candidate agent on interactions of the glial cells and the SGN-like cells.

[0043] In certain embodiments, the SGN-like cells are cultured in a two-dimensional (2D) culture system.

[0044] In another aspect, a method of treating a spiral ganglion neuron-associated disorder in a subject is provided, the method comprising administering a therapeutically effective amount of a composition comprising SGN-like cells, produced by a method described herein, locally to the ear of the subject.

[0045] In another aspect, a composition comprising SGN-like cells, produced by a method described herein, for use in treating a spiral ganglion neuron-associated disorder is provided.

[0046] In another aspect, use of a composition comprising SGN-like cells, produced by a method described herein, in the manufacture of a medicament for treating a spiral ganglion neuron- associated disorder in a subject is provided.BRIEF DESCRIPTION OF THE DRAWINGS

[0047] FIGS. 1A-1G. Stepwise differentiation of hiPSCs into human ONPs. FIG. 1A. Overview and timeline of human SGN-like neuron differentiation protocol from hiPSCs and matching bright-field images. Scale bar = 100 pm. FIG. 1B. Comparison of cells at D25 and D63 following seeding at different cell densities (25,000 or 60,000 cells / cm2) on D11 after sorting with NGFR beads. Scale bar = 100 pm. FIG. 1C. Immunostaining for NGFR+NESTIN+precursors of PPE and neural crest cells at D12. Scale bar = 100 pm. FIGS. 1D-1F. Immunostained D25 cells. Antibodies highlight neuroblast cells (NEUROD ) and otic lineage cells (PAX2+, PAX8+, and SOX2+), but no expression of neuronal markers (TUBB3) at this stage. Scale bar = 100 pm. FIG. 1G. Bar graphs showing the average proportion (%) of positive cells on D25 cells stained with NEUROD1, PAX2, PAX8, SOX2, and TUBB3. Error bars represent SD. hiPSCs human induced pluripotent stem cells, ONPs otic neural progenitors, BMP4 bone morphogenetic protein 4, LDN LDN-193189, IWP2 inhibitor of Wnt production-2, FGF2 basic fibroblast growth factor 2, NGFR nerve growth factor receptor, Wnt3 Wnt family member 3, IGF-1 insulin-like growth factor 1 , SHH sonic hedgehog, RA retinoic acid, EGF epidermal growth factor, NEUROD1 neurogenic differentiation 1, cAMP cyclic adenosine monophosphate, NT3 neurotrophin 3, BDNF brain-derived neurotrophic factor, TUBB3 tubulin beta 3 class III, PRPH peripheral neuronal marker peripherin, MPZ myelin protein zero, PPE pre-placodal ectoderm, SGC satellite glial cells, D day, SD standard deviation, GFAP glial fibrillary acidic protein, PAX2 paired box gene 2, SOX2 SRY-box transcription factor 2, DAPI 4',6-diamidino-2-phenylindole.

[0048] FIGS. 2A-2G. Differentiation of human SGN-like neurons in vitro mimics cochlear neuron development in vivo. FIG. 2A. Comparison of human SGN development in vivo (as described by Locher et al. (49)) with corresponding stages of in vitro differentiation from hiPSCs. At W12 of in vivo development, the first inner hair cells appear and are contacted by multiple neurites co-expressing TUBB3 and PRPH, with staining showing co-localization. A comparable stage is observed around D40 of in vitro differentiation. By W20 in vivo, both inner and outer hair cells are present, and PRPH expression distinguishes type I (PRPH ) from type II (PRPH+) neurites, paralleling the differentiation state observed around D90 in vitro. Scale bar = 100 pm. FIG.2B. Bar graphs and pie charts showing the average proportion (%) of TUBB3+and / or PRPH+somata in human SGN-like neurons at D35 - 90. FIG. 2C. Overall view of the morphology of human SGN-like neurons, depicted by microtubule- associated protein 2 (MAP2) expression along with glial cell marker GFAP on D90. Scale bar = 100 pm. FIG. 2D. GFAP+cell surrounding the cell body of a type I SGN-like neuron (TUBB3+) on D484. Scale bar = 5 pm. FIG. 2E. Expression of MPZ in cultures at D204. Lower panels show three- dimensional (3D) and cross-sectional view. Scale bar = 20 pm. FIGS. 2F-2G. Surgical specimens of human vestibular end organs from the inner ear. MYO7A+hair cells are innervated by TUBB3+neurons (FIG. 2F) and TUBB3+neurons are enveloped by MBP+myelinating Schwann cells (FIG.2G). Scale bar = 20 pm. SGN spiral ganglion neuron, hiPSCs human induced pluripotent stem cells, W week, TUBB3 tubulin beta 3 class III, PRPH peripheral neuronal marker peripherin, D day, GFAP glial fibrillary acidic protein, MPZ myelin protein zero, DAPI 4',6-diamidino-2-phenylindole, MBP myelin basic protein, SGC satellite glial cells.

[0049] FIGS. 3A-3H. Origin of human SGN-like neurons revealed by scRNA-seq. FIG. 3A.Schematic illustration of the developmental origins of auditory neurons in the human inner ear in vivo. FIG. 3B. UMAP plots of SK8-A D25 cells, color-coded as in FIG. 3A. FIG. 3C. Feature plots showing key gene markers for the classification of cell subtypes and determination of neuronal origins. FIG. 3D. Re-clustering of isolated otic neural primordium cells from FIG. 3B, separating into three populations: EMT, delaminating neuroblasts, and neurogenesis. FIG. 3E. Heatmap showing the top 100 differentially expressed genes across the three distinctive clusters: EMT, NB, and NG. The X and Y axes represent differentially expressed genes and single cells, respectively. FIG. 3F.Violin plots displaying marker gene expression in a log-transformed scale among the three different clusters of otic neural primordium. FIG. 3G. Feature plot visualizing expression of SGN and VGN markers in the otic neural primordium cluster, based on scRNA-seq data from previous studies (55, 59). Numbers in the lower left corners indicate the percentage of cells expressing each marker. FIG.3H. Dot plots illustrating minimal expression of hindbrain, GG, and EPI markers, and high expression of otic neuroblast and neuronal markers. To support data sharing, visualization, and analysis, theprocessed scRNA-seq dataset for FIG. 3 is available on the gEAR portal (umgear.org / p?l=sk8aNeuron). SGN spiral ganglion neuron, scRNA-seq single-cell RNA sequencing, UMAP uniform manifold approximation and projection, EMT epithelial-mesenchymal transition, NB neuroblasts, NG neurogenesis, VGN vestibular ganglion neurons, GG geniculate ganglion, EPI epibranchial placode, PLP1 proteolipid protein 1 , TWIST1 twist family bHLH transcription factor 1, TOP2A DNA topoisomerase II alpha, MEUROD1 neuronal differentiation 1, SOX2 SRY-box transcription factor 2, WNT4 Wnt family member 4, FGF8 fibroblast growth factor 8, DCX doublecortin, MEIS2 meis homeobox 2, PROX1 prospero homeobox 1 , TLX3 T cell leukemia homeobox 3, SALL3 spalt like transcription factor 3, PVALB parvalbumin, CNC cranial neural crest, ZBTB16 zinc finger and BTB domain containing 16, PAX6 paired box 6, HOXA2 homeobox A2, FOXI1 forkhead box 11, PHOX2A paired mesoderm homeobox protein 2A, FOXI2 forkhead box I2, NEUROG1 neurogenin 1, INSM1 Insulinoma-associated 1, HE56 hes family BHLH transcription factor 6, MAP2 microtubule-associated protein 2, TUBB3 tubulin beta 3 class III.

[0050] FIGS.4A-4I. Diversity of human SGN-like neurons revealed by scRNA-seq. FIG.4A. Feature plot visualizing expression of neuronal markers TUBB3 and ATP1B1 in D25, D60, D90, and D120 scRNA-seq data. Neuronal clusters indicated by red dotted circles were isolated for a downstream analysis. FIG. 4B. UMAP visualization of neurons isolated from each time point. Purple dots represent D25 neurons, which did not clearly segregate into type II populations. FIG. 4C. UMAP plots revealing 6 distinctive clusters of D60 human SGN-like neurons. FIG. 4D. Identification of type I and II human SGN-like neuron clusters based on CAMK2N1 and PRPH expression, respectively.FIG. 4E. Comparison of D60 human SGN-like neuron subtypes with developmental stages of SGNs in E14 - P1 mice (48) projected onto UMAP dimensions. FIG. 4F. Violin plots displaying log- transformed expression levels of marker genes across 4 distinct human SGN-like neuron populations. FIG. 4G. Expression profiles of selected genes related to glutamate and GABA receptors, synaptic vesicles, neurotransmitter transporters, and Ca2+binding proteins among D25, D60, D90, and D120 human SGN-like neurons. FIG. 4H. Differential expression patterns of K+and Na+channels among D25, D60, D90, and D120 human SGN-like neurons. FIG. 4I. UMAP plots depicting S100B^ peripheral glial cell cluster in the D60 sample and expression of their gene markers. To support data sharing, visualization, and analysis, the processed scRNA-seq dataset for FIG. 4 is available on the gEAR portal (umgear.org / p?l=sk8aNeuron). SGN spiral ganglion neuron, scRNA- seq single-cell RNA sequencing, D day, UMAP uniform manifold approximation and projection, SGC satellite glial cell, Non-my SC non-myelinating Schwann cells, My SC myelinating Schwann cells, PRPH peripheral neuronal marker peripherin, Ca2+calcium ion, TUBB3 tubulin beta 3 class III, ATP1B1 sodium / potassium-transporting ATPase subunit beta-1, CAMK2N1 calcium / calmodulindependent protein kinase II inhibitor 1, E embryonic day, P postnatal day, GABA gamma- aminobutyric acid, GRIN2B glutamate ionotropic receptor NMDA type subunit 2B, GLRA3 glycine receptor alpha 3, ROBO2 roundabout guidance receptor 2, LHX9 LIM homeobox 9, LYPD1 LY6 / PLAUR domain containing protein 1, SEMA5A Semaphorin 5A, POU4F1 POU class 4 homeobox 1, RPH3A Rabphilin 3A, PRPH peripherin, ETV1 ETS variant transcription factor 1, GRIA2 glutamate ionotropic receptor AMPA type subunit 2, GRIA4 glutamate ionotropic receptor AMPA type subunit 4, GABBR1 gamma-aminobutyric acid type B receptor subunit 1, GABBR2 gamma-aminobutyric acid type B receptor subunit 2, GABRB3 gamma-aminobutyric acid type A receptor subunit beta 3, CALM1 calmodulin 1, CALM3 calmodulin 3, SYT11 Synaptotagmin 11, SYN1 Synapsin I, SYNGR1 synaptogyrin 1, SYP synaptophysin, SLC17A6 solute carrier family 17 member 6, KCNMA1 potassium calcium-activated channel subfamily M alpha 1, KCNN4 potassium calcium-activated channel subfamily N member 4, KCNU1 Potassium Calcium-Activated Channel Subfamily U Member 1 , KCNJ2 potassium inwardly rectifying channel subfamily J member 2, KCNJ3 potassium inwardly rectifying channel subfamily J member 3, KCNJ11 potassium inwardly rectifying channel subfamily J member 11, KCNA1 potassium voltage-gated channel subfamily A member 1, KCNA4 potassium voltage-gated channel subfamily A member 4, KCNA5 potassium voltage-gated channel subfamily A member 5, KCNC2 potassium voltage-gated channel subfamily C member 2, KCNC4 potassium voltage-gated channel subfamily C member 4, KCNG4 potassium voltage-gated channel modifier subfamily G member 4, KCNH2 potassium voltage-gated channel subfamily H member 2, KCNH3 potassium voltage-gated channel subfamily H member 3, KCNH5 potassium voltage-gated channel subfamily H member 5, KCNH6 potassium voltage-gated channel subfamily H member 6, KCNQ3 potassium voltage-gated channel subfamily Q member 3, KCNQ4 potassium voltage-gated channel subfamily Q member 4, KCNQ5 potassium voltage-gated channel subfamily Q member 5, KCNV1 potassium voltage-gated channel modifier subfamily V member 1, KCNK7 potassium two pore domain channel subfamily K member 7, KCNK9 potassium two pore domain channel subfamily K member 9, KCNK13 potassium two pore domain channel subfamily K member 13, SCN1A sodium voltage-gated channel alpha subunit 1, SCN4A sodium voltage-gated channel alpha subunit 4, SCN8A sodium voltage-gated channel alpha subunit 8, SCN2B sodium voltagegated channel beta subunit 2, SCN4B sodium voltage-gated channel beta subunit 4, SOX10 SRY- box transcription factor 10, S100B S100 calcium binding protein B, ERBB3 erb-b2 receptor tyrosine kinase 3, NGFR nerve growth factor receptor, MPZ myelin protein zero, PLP1 proteolipid protein 1, PMP22 peripheral myelin protein 22, CRYAB crystallin alpha B.

[0051] FIGS. 5A-5I. Electrophysiological characterization of human SGN-like neurons. FIG. 5A. IR- DIC images during patch-clamping (top) and corresponding confocal images (bottom) identifyingtype I and type II human SGN-like neurons recorded at D88 - 95 and > D120 in vitro. Scale bar = 25 pm. FIG. 5B. Representative traces of current-clamp recordings in MA and UA type I human SGN- like neurons at D88 - 95 and > D120. Red traces represent the current step corresponding to rheobase. FIG. 5C. Schematic illustrating AP parameters analyzed. FIG. 5D. Comparison of membrane time constant, rheobase, and latency to the first observed AP at rheobase between MA- and UA-type I human SGN-like neurons. Sample sizes: D88 - 95, MA (n = 11 ), UA (n = 16); > D120, MA (n = 10), and UA (n = 14). The circles and squares indicate SK8-A and UCSD cell lines, respectively, across all patch clamping datasets. FIG. 5E. AP properties of type I human SGN-like neurons at the maximum injected current step. Sample sizes: D88 - 95, MA (n = 11), UA (n = 16); > D120, MA (n = 10), UA (n = 14). FIG. 5F. Cell body diameter (top) and membrane capacitance (Cm; bottom) of type I human SGN-like neurons. Sample sizes: D88 - 95 (n = 27), > D120 (n = 24). FIG.5G. Representative traces of voltage-clamp recordings in type I human SGN-like neurons at D88 - 95 (top) and > D120 (bottom); red traces indicate maximum responses. FIG. 5H. Voltage-current plots for putative Na+- (top) and K+-mediated currents (bottom); red dashed squares indicate maximum-evoked amplitudes. FIG. 5I. Comparison of maximum-evoked amplitudes of putative Na+- (top) and K+-mediated currents (bottom) in type I human SGN-like neurons. Sample sizes: D88 - 95 (n= 16), > D120 (n = 22). *P< 0.05, *'P< 0.01 , ***P< 0.001 , *“P< 0.0001. Error bars refer to standard error of the mean (standard error of mean) in all panels. AP action potential, SGN spiral ganglion neuron, IR-DIC representative infrared differential interference contrast, MA multi-spike accommodating neurons, UA unitary-spike accommodating neurons, Vm membrane potential.

[0052] FIGS. 6A-6P. Human SGN-like neurons form functional synaptic connections with mouse hair cells. FIG. 6A. A schematic illustrating the co-culture setup of human SGN-like neurons with mouse cochlear hair cells. FIG. 6B. Bright-field images of denervated mouse hair cells co-cultured with human SGN-like neurons, placed at least 500 pm apart. Scale bar = 100 pm. FIG. 6C. In cocultures, growth of D81 human SGN-like neurons (human-specific NEFL+) toward MYO7A+hair cells (top) and side view of three-dimensional (3D) co-culture images (bottom). Scale bars = 20 pm. FIG.6D. Higher magnification view of the contact site (x-y plane) along with orthogonal (x-z, y-z) projections, confirming tight proximity between hair cells and human SGN-like neurons. Scale bar = 5 pm. FIG. 6E. Multiple connections between PRPH+type II D95 human SGN-like neurons and MYO7A+mouse hair cells. Scale bar = 10 pm. FIG. 6F. Expression of presynaptic CtBP2+and postsynaptic GLUR2+ / PSD95+proteins in surgical specimens of vestibular end organs from the human inner ear. Scale bar = 10 pm. FIG. 6G. Localization of CtBP2 and PSD95 synaptic puncta in co-cultures, along with MYO7A+hair cells and hNEF+human SGN-like neuron dendrites. Scale bar = 5 pm. FIG. 6H. Examples of paired synaptic (CtBP2 and PSD95) immunopuncta in co-culture ofmouse hair cells and human SGN-like neurons, marking putative synapses (yellow overlap). FIG. 6I.Fluorescent Cal520 Ca2+signals from D193 human SGN-like neurons co-cultured with P5 mouse hair cells. Left: maximum intensity projection over 4.5 min. Right: maximum intensity projection over 7.5 min in the presence of CNQX and AP5 (AMPA / NMDA receptor blockers). Scale bar = 100 pm.FIG. 6J. Representative Ca2+transients from three human SGN-like neurons (I), showing loss of Ca2+signal with CNQX / AP5 treatment. Ca2+signals were normalized to the mean baseline intensity.FIG. 6K. Representative experiment showing average relative change in Ca2+fluorescence intensity (red line) from 10 human SGN-like neurons (D193; gray lines) co-cultured with P5 mouse hair cells. Ca2+signal is abolished by CNQX and AP5. FIG. 6L. Quantification of the number of Ca2+transients in the co-culture before and after CNQX / AP5. Data from three preparations at D74, D80, D108, D193, and D264, for a total of n = 60 neurons. FIGS. 6M-6O. Parallel experiments in human SGN- like neurons solo culture at D193. CNQX and AP5 treatment does not abolish Ca2+activity. FIG. 6P.Quantification of Ca2+transients in human SGN-like neurons solo culture. Data from three preparations at D74, D80, D108, D193, and D264, n = 50 human SGN-like neurons. *"P< 0.0001 (Mann-Whitney test), ns not significant. SGN spiral ganglion neuron, IR-DIC representative infrared differential interference contrast, MA multi-spike accommodating neurons, UA unitary-spike accommodating neurons, D day, Ca2+calcium ion, NEFL neurofilament, PRPH peripheral neuronal marker peripherin, P postnatal day, AMPA alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, NMDA N-methyl-D-aspartate.

[0053] FIGS. 7A-7L. The human SGN-like neurons form functional synaptic connections with CN neurons as demonstrated by Ca2+imaging. FIG. 7A. A schematic illustrating the co-culture setup of human SGN-like neurons with mouse CN neurons derived from the brainstem. FIG. 7B. Left: lateral view of a sagittal mouse brain hemi-section (olfactory bulb removed) including the cochlea, auditory nerve, and CN. Right: coronal brain section including the auditory nerve, CN, and inferior colliculus, which is one of several possible auditory pathways located in midbrain. Inferior colliculus was used as an anatomical landmark for CN location in the coronal plane. Scale bar = 1 mm. FIG. 7C. Bright- field images of P5 mouse CN neurons (red dots) emerging from CN tissue. Yellow circles highlight a connection between multipolar CN neurons and D60 human SGN-like neurons at co-culture D6. Scale bar = 20 pm. FIG.7D. Co-culture of D78 human SGN-like neurons with P5 mouse CN neurons at D24, immunostained for presynaptic (VGLUT1) and postsynaptic (PSD95) puncta. A humanspecific NEFL antibody (hNEFL; purple) labels only human neurons, while a general NEFL antibody (NEFL; gray) labels both mouse and human neurons. Scale bar = 20 pm. FIG. 7E. Ca2+imaging of D108 human SGN-like neurons co-cultured with P5 CN neurons. Left: maximum intensity projection over 4.5 min. Right: maximum intensity projection over 7.5 min after CNQX / AP5 treatment. Scalebar = 50 pm. FIG. 7F. Example Ca2+transients from three CN neurons in (FIG. 7E), illustrating Ca2+signal loss after CNQX / AP5. Ca2+signals were normalized to the mean baseline intensity. FIG. 7G.Representative experiment showing the average relative change in Ca2+signal from 10 CN neurons (P5) co-cultured with D108 human SGN-like neurons, showing loss of signal upon synaptic inhibition.FIG. 7H. Quantification of the number of Ca2+transients in the co-culture before and after CNQX / AP5 treatment. Data from three different preparations (D61, D63, D108, D129, D131 , and D213) for a total of 55 CN neurons. FIGS. 7I-7L. As in (FIGS. 7E-7H), but for CN neurons-only culture (P5, control). Scale bar = 100 pm. No discernible Ca2+transients were observed under these conditions. n = 20 CN neurons (P5), with or without CNQX / AP5 treatment. ”*P < 0.0001 (the Mann-Whitney test). D day, Ca2+calcium ion, NEFL neurofilament light chain, CN cochlear nucleus, P postnatal day, SGN spiral ganglion neuron, CNQX 6-cyano-7-nitroquinoxaline-2, 3-dione, AP5 D(-)-2-amino-5- phosphonopentanoic acid, VGLUT1 solute carrier family 17 member 7, PSD95 postsynaptic density protein 95.

[0054] FIGS. 8A-8H. Higher seeding density on D11 promotes the formation of ONP-like cells and subsequent neuronal development. FIGS. 8A-8B. Low seeding density (25,000 cells / cm2) of pre- placodal ectoderm and neural crest precursors (on D11 ) after sorting negatively affected subsequent differentiation into human SGN-like cells. The low seeding density also affected the length of neurites. The longest sizes of neurons stained with NEFL (FIG. 8B) from axon to dendrite were < 200 pm on D63. SK8-A hiPSCs were used for this experiment. Scale bar = 100 pm. FIGS. 8C-8D.Higher seeding density (60,000 cells / cm2) on D11 after sorting was the appropriate cell density to form vesicles of ONP-like cells (yellow dotted circles) on D25. NEFL staining (FIG. 8D) on D63 revealed clusters of human SGN-like neurons resembling mouse SGN explant cultures in vitro [9]. SK8-A hiPSCs were used for this experiment. Scale bar = 100 pm. FIGS. 8E-8H. Representative phase-contrast image on D25 (FIG. 8E) showing ONP-like cells (yellow dotted circles) arising from NGFR+cells seeded at 60,000 cells / cm2on D11. These ONP-like cells expressed the neuroblast marker NEUROD1 (FIG. 8F), but no TLJBB3+neurons were observed (FIG. 8G). The D25 cultures also contained cells expressing otic lineage markers, including such as PAX2, PAX8 (FIG. 8H), and SOX2 (FIG. 8G). Scale bar = 100 pm. All experiments were performed with at least 3 biological replicates and 3 technical replicates. D day, ONP otic neural progenitor, hiPSCs human induced pluripotent stem cells, SGN spiral ganglion neurons, NEFL neurofilament, NGFR nerve growth factor receptor, TLJBB3 tubulin beta 3 class III, PAX2 paired box gene 2, SOX2 SRY-box transcription factor 2, DAPI 4',6-diamidino-2-phenylindole.

[0055] FIGS. 9A-9Q. Maturation and myelination of human SGN-like neurons. FIG. 9A-9C. Early- stage human SGN-like cells had two main sets of fibers extending from opposite sides of the cellbody, including small sub-branches. On D35, approximately (89.4 ± 9.5)% of human SGN-like neurons (FIG. 9A) expressed both PRPH and TUBB3. Scale bar = 50 pm (FIG. 9A) and 20 pm (FIGS. 9B-9C). FIG. 9D-9M. Expression of the glutaminergic neuronal marker VGLUT 1. Scale bar = 50 pm (FIG. 9D) and 20 pm (FIG. 9M). FIGS. 9E-9F. Maturing human SGN-like neurons displayed a bipolar morphology and separated into TUBB3+type I and PRPH+, GATA3+(FIG. 9E), or TH+(FIG.9F) type II subtypes. Scale bar = 50 pm (FIG. 9E) and 20 pm (FIG. 9F). FIG. 9G. MPZ expression in myelinating Schwann cell-like cells on D204 and its higher magnification view. Scale bar = 50 pm.FIG.9H. Representative bright-field images of D90 human SGN-like neurons derived from the UCSD hiPSC line. Scale bar = 50 pm. FIGS. 9I-9L. Expression of pan-neuronal marker MAP2 (i), type I SGN marker TLIBB3 (FIG. 9J), and type II markers PRPH (FIG.9K), and TH (FIG.9L) in D90 human SGN-like neurons. Scale bar = 100 pm (FIG. 9K), 50 pm (i), 20 pm (FIGS. 9 J, 9L). FIG. 9N.Expression of CALB1, a type la SGN marker confirmed in mouse SGNs at postnatal stage 17

[0010] , Scale bar = 20 pm. FIGS. 9O-9Q. Wrapping of type II human SGN-like neural fibers by nonmyelinating Schwann cell-like cells. Scale bar = 20 pm (FIG. 90), 10 pm (FIG. 9Q), 5 pm (FIG. 9P).All experiments were performed using at least 3 biological replicates and 3 technical replicates. D day, SGN spiral ganglion neurons, SD standard deviation, PRPH peripheral neuronal marker peripherin, TUBB3 tubulin beta 3 class III, hiPSC human-induced pluripotent stem cells, MAP2 microtubule-associated protein 2, CALB1 calbindinl, MPZ myelin protein zero, GAT A3 GATA binding protein 3, TH tyrosine hydroxylase, VGLUT1 vesicular glutamate transporter 1 , NGFR nerve growth factor receptor, SC Schwann cell, DAPI 4',6-diamidino-2-phenylindole.

[0056] FIGS. 10A-10J. Quality control and scRNA-seq data. FIG. 10A. Violin plots indicating the number of genes detected in each cell (nFeature_RNA). The cut-off thresholds for D25_1 (SK8-A), D25 2 (UCSD), D60 (SK8-A), D90 (SK8-A), D120 (SK8-A) were < 200 and > 6000, < 200 and > 8000, < 200 and > 7000, < 200 and > 6500, and < 200 and > 6500, respectively. The red dashed line indicates cut-off fractions. FIG. 10B. Violin plots showing the total number of molecules detected within a cell (nCount_RNA). FIG. 10C. Violin plots showing the percentage of mitochondrial genes. Above, 10% in D25_1, 15% in D25_2, 20% in D60, 15% in D90, and 15% in D120 were filtered out.FIG. 10D. Violin plots showing the percentage of ribosomal RNA. FIG. 10E. Violin plots showing the percentage of hemoglobin genes. FIG. 10F. Violin plots showing the percentage of platelet genes from each sample. FIG. 10G. UMAP plots showing doublets detected by DoubletFinder, and violin plots showing the number of genes detected in doublets. Doublets were removed and only the predicted singlet cells were used for further analysis. FIG. 10H. UMAP plots of UCSD D25 cells. FIG.101. Feature plots displaying key gene markers for classifying cell subtypes in UCSD D25. FIG. 10J.Feature plot visualizing the expression of undifferentiated CVG, SGN, and vestibular ganglionneuron (VGN) markers in the otic neural primordium cluster of UCSD D25 cells. scRNA-seq singlecell RNA sequencing, D day, CVG cochleovestibular ganglia, UMAP uniform manifold approximation and projection, SGN spiral ganglion neuron, VGN vestibular ganglion neuron, SOX2 SRY-box transcription factor 2, TUBB3 tubulin beta 3 class III, TOP2A DNA topoisomerase II alpha, MGP matrix gla protein, MIAT myocardial infarction associated transcript, MEIS2 meis homeobox 2, PROX1 prospero homeobox 1, TLX3 T cell leukemia homeobox 3, SALL3 spalt like transcription factor 3, PVALB parvalbumin.

[0057] FIG. 11A-11F. Differential expression of genes related to neurotransmission. FIG. 11 A.UMAP plot of D60 human SGN-like neurons and mouse SGNs collected at E14 - P1 [6]. FIG. 11 B.Violin plots illustrating differential expression of genes encoding synaptic vesicles, neurotransmitter transporters, and Ga2+binding proteins in human SGN-like neurons at D60. FIG. 11C. Violin plots representing expression of key genes related to glutamate, GABA, cholinergic, dopaminergic, and purinergic receptors in human SGN-like neurons at D60. FIG. 11D. Expression levels of selected genes related to synaptic vesicles, neurotransmitter transports, and Ca2+binding proteins between D25 and D120 human SGN-like neurons. FIGS. 11E-11F. Expression levels of genes encoding glutamate (FIG. 11 E) and GABA (FIG. 11 F) receptors between D25 and D120 human SGN-like neurons. UMAP uniform manifold approximation and projection, D day, SGN spiral ganglion neurons, Ca2+calcium ion, GABA gamma-aminobutyric acid, E embryonic day, P postnatal day, VAMP1 vesicle associated membrane protein 1, VAMP2 vesicle associated membrane protein 2, VAMP7 vesicle associated membrane protein 7, SYNGR3 synaptogyrin 3, SYN1 synapsin I, SYN2 synapsin II, SYP synaptophysin, SYT1 synaptotagmin 1, SYT2 synaptotagmin 2, SYT4 synaptotagmin 4, SYT5 synaptotagmin 5, SYT 11 synaptotagmin 11 , SYT 13 synaptotagmin 13, SYT 14 synaptotagmin 14, SYT16 synaptotagmin 16, SLC1A2 solute carrier family 1 member 2, SLC1A1 solute carrier family 1 member 1, SLC1A6 solute carrier family 1 member 6, SLC17A6 solute carrier family 17 member 6, SLC18A3 solute carrier family 18 member 3, CALM1 calmodulin 1 , CALM2 calmodulin 2, CALM3 calmodulin 3, CALB2 calbindin 2, CIB2 calcium and integrin binding protein 2, NCS1 neuronal calcium sensor 1 , NECAB1 neuronal calcium binding protein 1 , NECAB2 neuronal calcium binding protein 2, GRIK5 glutamate ionotropic receptor kainate type subunit 5, GRIA2 glutamate ionotropic receptor ampa type subunit 2, GRIA4 glutamate ionotropic receptor ampa type subunit 4, GRIN2B glutamate ionotropic receptor nmda type subunit 2b, GRID2 glutamate ionotropic receptor delta type subunit 2, CHRM2 cholinergic receptor muscarinic 2, CHRM3 cholinergic receptor muscarinic 3, GABBR1 gamma-aminobutyric acid type b receptor subunit 1 , GABBR2 gamma- aminobutyric acid type b receptor subunit 2, GABR3 gamma-aminobutyric acid type a receptor subunit gamma 3, GABRB3 gamma-aminobutyric acid type a receptor subunit beta 3, DRD2dopamine receptor d2, P2RX3 purinergic receptor p2x 3, NSF n-ethylmaleimide sensitive factor, STX7 syntaxin 7, STX12 syntaxin 12, SNAP25 synaptosome associated protein 25, NRXN1 neurexin 1, GRM1 glutamate metabotropic receptor 1, GRM2 glutamate metabotropic receptor 2, GRM3 glutamate metabotropic receptor 3, GRM4 glutamate metabotropic receptor 4, GRM5 glutamate metabotropic receptor 5, GRM6 glutamate metabotropic receptor 6, GRM7 glutamate metabotropic receptor 7, GRM8 glutamate metabotropic receptor 8, GRIK1 glutamate ionotropic receptor kainate type subunit 1 , GRIK2 glutamate ionotropic receptor kainate type subunit 2, GRIK3 glutamate ionotropic receptor kainate type subunit 3, GRIK4 glutamate ionotropic receptor kainate type subunit 4, GRIK5 glutamate ionotropic receptor kainate type subunit 5, GRIA1 glutamate ionotropic receptor AMPA type subunit 1 , GRIA2 glutamate ionotropic receptor AMPA type subunit 2, GRIA3 glutamate ionotropic receptor AMPA type subunit 3, GRIA4 glutamate ionotropic receptor AMPA type subunit 4, GRIN1 glutamate ionotropic receptor NMDA type subunit 1, GRIN2A glutamate ionotropic receptor NMDA type subunit 2a, GRIN2B glutamate ionotropic receptor NMDA type subunit 2b, GRIN2C glutamate ionotropic receptor NMDA type subunit 2c, GRIN2D glutamate ionotropic receptor NMDA type subunit 2d, GRIN3A glutamate ionotropic receptor NMDA type subunit 3a, GRIN3B glutamate ionotropic receptor NMDA type subunit 3b, GRID1 glutamate ionotropic receptor delta type subunit 1, GRID2 glutamate ionotropic receptor delta type subunit 2, GABBR1 gamma aminobutyric acid type B receptor subunit 1, GABBR2 gamma aminobutyric acid type B receptor subunit 2, GABRA1 gamma aminobutyric acid type A receptor subunit alpha 1, GABRA2 gamma aminobutyric acid type A receptor subunit alpha 2, GABRA3 gamma aminobutyric acid type A receptor subunit alpha 3, GABRA4 gamma aminobutyric acid type A receptor subunit alpha 4, GABRA5 gamma aminobutyric acid type A receptor subunit alpha 5, GABRA6 gamma aminobutyric acid type A receptor subunit alpha 6, GABRB1 gamma aminobutyric acid type A receptor subunit beta 1 , GABRB2 gamma aminobutyric acid type A receptor subunit beta 2, GABRB3 gamma aminobutyric acid type A receptor subunit beta 3, GABRD gamma aminobutyric acid type A receptor subunit delta, GABRE gamma aminobutyric acid type A receptor subunit epsilon, GABRG1 gamma aminobutyric acid type A receptor subunit gamma 1, GABRG2 gamma aminobutyric acid type A receptor subunit gamma 2, GABRG3 gamma aminobutyric acid type A receptor subunit gamma 3, GABRP gamma aminobutyric acid type A receptor subunit pi, GABRQ gamma aminobutyric acid type A receptor subunit theta, GABRR1 gamma aminobutyric acid type A receptor subunit rho 1 , GABRR2 gamma aminobutyric acid type A receptor subunit rho 2, GABRR3 gamma aminobutyric acid type A receptor subunit rho 3.

[0058] FIGS. 12A-12H. Firing properties, AP parameters at rheobase, and voltage responses to hyperpolarizing steps in type I human SGN-like neurons. FIGS. 12A-12B. Spike counts as a functionof injected current (FIG. 12A) and average ISI (FIG. 12B) for MA type I human SGN-like neurons. D88 - 95, n = 11 ; > D120, n = 10. FIGS. 12C-12D. AP properties analyzed at rheobase (FIG. 12C) and intrinsic properties (FIG. 12D) of type I human SGN-like neurons. D88 - 95: MA, n = 11 and UA, n = 16; > D120: MA, n = 10 and UA, n = 14. FIG. 12E. mRNA expression levels of HON channels.FIG. 12F. Representative traces of current-clamp recordings in type I human SGN-like neurons in response to hyperpolarizing current steps. FIGS. 12G-12H. Plot of the difference in membrane potential (AVm) between the peak of the hyperpolarizing response and the end of the current step as a function of hyperpolarizing current steps; red dashed square indicates average AVm at the -80 pA step, for D88 - 95 and > D120 (FIG. 12G). Comparison of the average AVm at the -80 pA step for D88 - 95 and >D120 (FIG. 12H). D88 - 95, n = 23; > D120, n = 23. 'P < 0.05, "P < 0.01 , ***P < 0.001,mP < 0.0001. Error bars refer to standard error of the mean (SEM) in all panels. AP action potential, SGN spiral ganglion neurons, ISI inter-spike interval, MA multi-spike accommodating neurons, D day, UA unitary-spike accommodating neurons, HCN hyperpolarization-activated cyclic nucleotide-gated.

[0059] FIGS. 13A-13G. Co-culture of hiPSC-derived human SGN-like neurons and mouse denervated hair cells. FIG. 13A. Denervated hair cells from Thy1-GFP BL6 mice were used for coculture with D44 human SGN-like neurons. No neurons expressing Thy1-GFP connected with hair cells. Scale bar = 20 pm. FIG. 13B. Absence of neurite (NEFL+) and PSD95+markers in denervated hair cells after 14 d of in vitro culture. The yellow inset shows higher magnification of mouse denervated hair cells expressing hair cell (MYO7A+) and presynaptic (CtBP2+) markers. Scale bar = 10 pm. FIG. 13C. Lack of hNEFL (a specific antibody for human neurofilament) expression in mouse cochlea explants. Scale bar = 100 pm. Fl.G. 13D Positive detection of hNEFL in human SGN-like neurons. Scale bar = 200 pm. FIG. 13E. Overview of co-culture showing physical contact between hair cells and both type I and type II human SGN-like neurons. Scale bar = 100 pm. FIG. 13F. Contact between type II human SGN-like neurons (PRPH+) and outer hair cells (PRESTIN+) in co-culture. All inner and outer hair cells expressed CALB2. Scale bar = 5 pm. FIG. 13G. Immunostained images showing c-Fos expression in co-cultures of P4 mouse hair cells and D251 human SGN-like neurons. Human cells were specifically identified using HNA. Scale bar = 50 pm. All experiments were performed using at least 3 biological replicates. CBA / CaJ mice were used for experiments in FIGS.13B, 13C, 13E, and 13F, while NOD / SCID mice were used for FIG. 13G. hiPSC human-induced pluripotent stem cells, SGN spiral ganglion neurons, D day, NEFL neurofilament, PSD95 post- synaptic protein, PRPH peripheral neuronal marker peripherin, HNA human nuclear antigen, CALB2 calbindin 2, MYO7A myosin VIIA, CtBP2 C-terminal binding protein 2, PSD95 postsynaptic density protein 95, PRESTIN solute carrier family 26 member 5, TUBB3 tubulin beta 3 class III.

[0060] FIGS. 14A-14D. Co-culture of hiPSC-derived human SGN-like neurons and mouse CN neurons. FIG. 14A. Bright-field images of P5 CN tissue with CN neurons (red dots) emerging from the CN explant. Scale bar = 20 pm. FIG. 14B. Co-culture of D73 human SGN-like neurons (hNEFL+, NEFL+) with different types of mouse CN neurons (hNEFL-, NEFL+) from 8-week-old mouse. Scale bar = 20 pm. FIG. 14C. Synaptic connections between mouse CN neurons (NEFL hNEFL ) from 8- week-old mice and D73 human SGN-like neurons (hNEFL+) after 28 d in co-culture. Scale bar = 50 pm. FIG. 14D. Representative immunostained images showing c-Fos expression in co-cultures of D81 human SGN-like neurons with P4 CN neurons. Scale bar = 50 pm. All experiments were performed using at least 3 biological replicates. CBA / CaJ mice were used for panels FIG. 14A-14C, and NOD / SCID mice were used for panel FIG. 14D. hiPSCs human-induced pluripotent stem cells, CN cochlear nucleus, D day, SGN spiral ganglion neurons, NEFL neurofilament, P postnatal day.DETAILED DESCRIPTION OF THE INVENTION

[0061] Compositions and methods are provided for generating spiral ganglion neuron-like cells from stem cells. Methods of using such spiral ganglion neuron-like cells in auditory research, disease modeling of sensorineural hearing loss, drug screening, and cell therapy are also provided.

[0062] Before the present compositions and methods are described, it is to be understood that this invention is not limited to particular methods or compositions described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0063] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0064] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materialsare now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. It is understood that the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0065] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0066] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a neuron" includes a plurality of such neurons and reference to "the induced pluripotent stem cell" includes reference to one or more induced pluripotent stem cells and equivalents thereof, known to those skilled in the art, and so forth.

[0067] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.Definitions

[0068] The term "about", particularly in reference to a given quantity, is meant to encompass deviations of plus or minus five percent.

[0069] The term “spiral ganglion neuron-associated disorder” or “SGN-associated disorder” refers to any disorder caused by SGN mutations, damage, degeneration, or loss of function, which may be associated with hearing loss or deafness. SGN-associated disorders include, but are not limited to, sensorineural hearing loss, auditory neuropathy, auditory neuropathy spectrum disorder, syndromic hearing loss, non-syndromic hearing loss, otosclerosis, and age-related hearing loss (presbycusis). SGN-associated disorders may be caused by noise-induced damage to SGNs, ototoxicity, aging, inflammation, or hereditary, developmental, or degenerative conditions.

[0070] The term "stem cell" refers to a cell that retains the ability to renew itself through mitotic cell division and that can differentiate into a diverse range of specialized cell types. Mammalian stem cells can be divided into three broad categories: embryonic stem cells, which are derived from blastocysts, adult stem cells, which are found in adult tissues, and cord blood stem cells, which arefound in the umbilical cord. In a developing embryo, stem cells can differentiate into all of the specialized embryonic tissues. In adult organisms, stem cells and progenitor cells act as a repair system for the body by replenishing specialized cells. Totipotent stem cells are produced from the fusion of an egg and sperm cell. Cells produced by the first few divisions of the fertilized egg are also totipotent. These cells can differentiate into embryonic and extraembryonic cell types. Pluripotent stem cells are the descendants of totipotent cells and can differentiate into cells derived from any of the three germ layers. Multipotent stem cells can produce only cells of a closely related family of cells (e.g., hematopoietic stem cells differentiate into red blood cells, white blood cells, platelets, etc.). Unipotent cells can produce only one cell type, but have the property of self-renewal, which distinguishes them from non-stem cells. Induced pluripotent stem cells are a type of pluripotent stem cell derived from adult cells that have been reprogrammed into an embryonic-like pluripotent state. Induced pluripotent stem cells can be derived, for example, from adult somatic cells such as peripheral blood mononuclear cells, fibroblasts, keratinocytes, epithelial cells, endothelial progenitor cells, mesenchymal stem cells, adipose derived stem cells, leukocytes, hematopoietic stem cells, bone marrow cells, or hepatocytes. In some embodiments, somatic cells are obtained from a biopsy of the inner ear of a patient.

[0071] As used herein, “reprogramming factors” refers to one or more, i.e., a cocktail, of biologically active factors that act on a cell to alter transcription, thereby reprogramming a cell to multipotency or to pluripotency. Reprogramming factors may be provided individually or as a single composition, that is, as a premixed composition, of reprogramming factors to the cells, e.g., somatic cells from an individual with a family history or genetic make-up of interest, such as a patient who has a disorder causing hearing loss or deafness. The factors may be provided at the same molar ratio or at different molar ratios. The factors may be provided once or multiple times in the course of culturing the cells of the subject invention. In some embodiments the reprogramming factor is a transcription factor, including without limitation, Oct3 / 4; Sox2; Klf4; c-Myc; Nanog; and Lin-28.

[0072] The somatic cells may include, without limitation, peripheral blood mononuclear cells, fibroblasts, keratinocytes, epithelial cells, endothelial progenitor cells, mesenchymal stem cells, adipose derived stem cells, leukocytes, hematopoietic stem cells, bone marrow cells, or hepatocytes, etc., which are contacted with reprogramming factors, as defined above, in a combination and quantity sufficient to reprogram the cell to pluripotency. Reprogramming factors may be provided to the somatic cells individually or as a single composition, that is, as a premixed composition, of reprogramming factors. In some embodiments the reprogramming factors are provided as a plurality of coding sequences on a vector.

[0073] Differentiation of iPSCs into SGN-like cells may be promoted by using various growth factors and other differentiation agents. For example, growth factors such as bone morphogenetic protein 4 (BMP4), basic fibroblast growth factor 2 (FGF2), insulin-like growth factor-1 (IGF-1 ), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and epidermal growth factor (EGF) in combination with other differentiation agents such as Wnt inhibitors, ROCK inhibitors, inhibitors of ALK2 and ALK3, sonic hedgehog (SHH), and retinoic acid (RA) can be used to promote differentiation of iPSCs into SGN-like cells (see Example 1).

[0074] The SGN-like cells are harvested at an appropriate stage of development, which may be determined based on the expression of markers and phenotypic characteristics of the desired mature differentiated cell type. Cultures may be empirically tested by staining for the presence of the markers of interest, by morphological determination, etc. For example, SGN-like cells can be identified by markers specific for cells of the SGN lineage, including, without limitation, tubulin beta 3 class III (TUBB3), neurofilament light polypeptide (NEFL), neurofilament heavy polypeptide (NEFH), POU class 4 homeobox 1 (POU4F1 ), neuronal differentiation 1 transcription factor (NEUROD1 ), and SRY- box transcription factor 2 (SOX2). The SGN-like cells may be further characterized by staining for the presence of markers that distinguish Type I and Type II SGN cells. Markers specific for Type I SGN cells, include, without limitation, prospero homeobox 1 (PROX1), sodium / potassium- transporting ATPase subunit alpha-3 (NKAa3), calbindin-2 (CALB2), and Ly6 / PLAUR domaincontaining protein 1 (LYPD1). Markers specific for Type II SGN cells, include, without limitation, peripherin (PRPH), tachykinin precursor 1 (TAC1 ), MAF bZIP transcription factor (MAFB), and GATA binding protein 3 (GAT A3). The mature SGN-like cells may be purified by positive selection for one or more markers expressed on mature SGN-like cells. The cells are optionally enriched before or after the positive selection step by drug selection, panning, density gradient centrifugation, etc. In addition, a negative selection can be performed, where the selection is based on expression of one or more of the markers found on the stem cells (e.g., hiSPCs) or somatic cells they are derived from (e.g., PBMCs, fibroblasts, epithelial cells, endothelial progenitor cells, leukocytes, hematopoietic stem cells, mesenchymal stem cells, bone marrow cells, hepatocytes), and the like. Selection may utilize panning methods, magnetic particle selection, particle sorter selection, and the like.

[0075] The somatic cells, the iPSCs derived therefrom, or the mature SGN-like cells may be genetically modified for a variety of purposes, e.g., to introduce a genetic mutation associated with a disorder causing hearing loss or deafness, modify, delete, or insert a gene, or provide marker genes, etc. Vectors may be introduced that express an exogenous gene, reprogramming factors, GRISPR systems, antisense nucleic acids, or ribozymes. Various techniques known in the art may be used to introduce nucleic acids into the target cells, e.g., electroporation, calcium precipitatedDNA, fusion, transfection, lipofection, infection and the like. The particular manner in which the DNA is introduced is not critical to the practice of the invention.

[0076] The phrase “mammalian cell” refers to any cell originating from mammalian tissue. The cell can be a primary cell obtained directly from a mammalian subject. The cell may also be a cell derived from the culture and expansion of a cell obtained from a subject. For example, the cell may be a stem cell, progenitor cell, or adult cell. Immortalized cells are also included within this definition. In some embodiments, the cell has been genetically engineered to express a recombinant protein and / or nucleic acid. The term “mammalian” includes, without limitation, human, equine, bovine, porcine, canine, feline, rodent (e.g., mice, rats, hamster), and primate.

[0077] By "container" is meant a glass, plastic, or metal vessel that can provide an aseptic environment for culturing cells.

[0078] The terms "peptide", “oligopeptide”, "polypeptide", and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms also apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Both full-length proteins and fragments thereof are encompassed by the definition. The terms also include post-expression modifications of the polypeptide, for example, phosphorylation, glycosylation, acetylation, hydroxylation, oxidation, and the like as well as chemically or biochemically modified or derivatized amino acids and polypeptides having modified peptide backbones. The terms also include fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues; immunologically tagged proteins; and the like. The terms include polypeptides including one or more of a fatty acid moiety, a lipid moiety, a sugar moiety, and a carbohydrate moiety.

[0079] “Isolated” refers to an entity of interest that is in an environment different from that in which it may naturally occur. “Isolated” is meant to include entities that are within samples that are substantially enriched for the entity of interest and / or in which the entity of interest is partially or substantially purified.

[0080] "Substantially purified" generally refers to isolation of a substance (compound, cell, protein, nucleic acid) such that the substance comprises the majority percent of the sample in which it resides. Typically in a sample, a substantially purified component comprises 50%, preferably 80%- 85%, more preferably 90-95% of the sample. Techniques for purifying substances of interest are well-known in the art and include, for example, ion-exchange chromatography, affinity chromatography and sedimentation according to density.

[0081] The terms "subject", "individual" or "patient" are used interchangeably herein and refer to a vertebrate, preferably a mammal. By "vertebrate” is meant any member of the subphylum Chordata, including, without limitation, humans and other primates, including nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like. The term does not denote a particular age. Thus, both adult and newborn individuals are intended to be covered.

[0082] A "therapeutically effective amount" or “therapeutic dose” is an amount sufficient to effect desired clinical results (i.e., achieve therapeutic efficacy). A therapeutically effective dose or amount can be administered in one or more administrations.

[0083] "Pharmaceutically acceptable excipient or carrier" refers to an excipient that may optionally be included in the compositions of the invention and that causes no significant adverse toxicological effects to the patient.

[0084] "Pharmaceutically acceptable salt" includes, but is not limited to, amino acid salts, salts prepared with inorganic acids, such as chloride, sulfate, phosphate, diphosphate, bromide, and nitrate salts, or salts prepared from the corresponding inorganic acid form of any of the preceding, e.g., hydrochloride, etc., or salts prepared with an organic acid, such as malate, maleate, fumarate, tartrate, succinate, ethylsuccinate, citrate, acetate, lactate, methanesulfonate, benzoate, ascorbate, para-toluenesulfonate, palmoate, salicylate and stearate, as well as estolate, gluceptate and lactobionate salts. Similarly salts containing pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium (including substituted ammonium).Stem Cell-Derived SGN-like Cells

[0085] The present disclosure provides spiral ganglion neuron (SGN)-like cells and methods of producing SGN-like cells from stem cells. The SGN-like cells may be used in cell replacement therapy or regenerative therapy for treating SGN-associated disorders involving diseases, conditions, and injuries of the ear resulting in hearing loss or deafness. The SGN-like cells may contain disease-relevant mutations to allow their use as disease models for screening therapeutics for treating SGN-associated disorders.

[0086] In certain embodiments, the SGN-like cells are produced from stem cells by a method comprising: (a) culturing the stem cells in a first medium comprising bone morphogenetic protein 4 (BMP4), a transforming growth factor-p (TGF-p) type I receptor inhibitor, and basic fibroblast growthfactor 2 (FGF2); (b) culturing cells, produced from the stem cells in the first medium, in a second medium comprising the FGF2, the TGF-p type I receptor inhibitor, a Wnt inhibitor, and an inhibitor of ALK2 and ALK3, wherein the cells differentiate into pre-placodal ectoderm and neural crest precursors; (c) isolating the pre-placodal ectoderm and neural crest precursors; (d) culturing the isolated pre-placodal ectoderm and neural crest precursors in a third medium comprising Wnt family member 3A (WNT3A), the FGF2, insulin-like growth factor-1 (IGF-1), and a non-selective Rho- associated protein kinase (ROCK) inhibitor; (e) culturing the cells, produced from the pre-placodal ectoderm and neural crest precursors in the third medium, in a fourth medium comprising sonic hedgehog (SHH), retinoic acid (RA), epidermal growth factor (EGF), the FGF2, and the IGF-1 , wherein the cells differentiate into otic neural progenitor cells; and (f) culturing the otic neural progenitor cells in a fifth medium comprising brain-derived neurotrophic factor (BDNF), neurotrophin- 3 (NT-3), the IGF-1, cyclic adenosine monophosphate (cAMP), and a selective ROCK inhibitor to induce neuronal maturation of the otic neural progenitor cells into the SGN-like cells.

[0087] Wnt inhibitors include, without limitation, CK18 inhibitors such as / V-(6-methyl-1 ,3- benzothiazol-2-yl)-2-[(4-oxo-3-phenyl-6,7-dihydrothieno[3,2-d]pyrimidin-2-yl)sulfanyl]acetamide (IWP-2) and 2-[[3-(2-methoxyphenyl)-4-oxo-6,7-dihydrothieno[3,2-d]pyrimidin-2-yl]sulfanyl]-N-(6- methyl-1 ,3-benzothiazol-2-yl)acetamide (IWP-4).

[0088] TGF-p type I receptor inhibitors may be used, for example, to block TGF- -induced differentiation, maintain pluripotency, and enhance cell expansion. Exemplary TGF-p type I receptor inhibitors include, without limitation, 4-[4-(1 ,3-benzodioxol-5-yl)-5-pyridin-2-yl-1 H-imidazol-2- yl]benzamide (SB-431542), 3-(6-methyl-2-pyridinyl)-N-phenyl-4-quinolin-4-ylpyrazole-1 - carbothioamide (A83-01), and 2-[5-(6-methyl-2-pyridinyl)-1 H-pyrazol-4-yl]-1 ,5-naphthyridine (RepSox).

[0089] Inhibitors of ALK2 and ALK3 include, without limitation, 4-[6-(4-piperazin-1- ylphenyl)pyrazolo[1 ,5-a]pyrimidin-3-yl]quinoline (LDN-193189), 5-[6-(4-piperazin-1- ylphenyl)pyrazolo[1 ,5-a]pyrimidin-3-yl]quinoline (LDN-212854), and dorsomorphin.

[0090] A ROCK inhibitor may be used to improve survival and maintain pluripotency of the iPSCs.ROCK inhibitors include, but are not limited to, small molecule inhibitors such as N-[(1S)-2-hydroxy- 1-phenylethyl]-N'-[4-(4-pyridinyl)phenyl]-urea (AS1892802), fasudil hydrochloride (also known as HA 1077), N-[3-[[2-(4-amino-1 ,2,5-oxadiazol-3-yl)-1 -ethyl- 1 H-imidazo[4,5-c]pyridin-6-yl]oxy]phenyl]-4- [2-(4-morpholinyl)ethoxy]benzamide (GSK269962), 4-[4-(Trifluoromethyl)phenyl]-N-(6-Fluoro-1 H- indazol-5-yl)-2-methyl-6-oxo-1 ,4,5,6-tetrahydro-3-pyridinecarboxamide (GSK 429286), (5)-(+)-2- Methyl-1 -[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1 H-1 ,4-diazepine dihydrochloride (H 1152 dihydrochloride), (S)-(+)-4-Glycyl-2-methyl-1 -[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1 H-1 ,4-diazepine dihydrochloride (glycyl-H 1152 dihydrochloride), N-[(3-Hydroxyphenyl)methyl]-N'-[4-(4- pyridinyl)-2-thiazolyl]urea dihydrochloride (RKI 1447 dihydrochloride), (3S)-1-[[2-(4-Amino-1 ,2,5- oxadiazol-3-yl)-1-ethyl-1 H-imidazo[4,5-c]pyridin-7-yl]carbonyl]-3-pyrrolidinamine dihydrochloride (SB772077B dihydrochloride), N-[2-[2-(Dimethylamino)ethoxy]-4-(1 H-pyrazol-4-yl)phenyl-2,3- dihydro-1 ,4-benzodioxin-2-carboxamide dihydrochloride (SR 3677 dihydrochloride), and trans-4- [(1 R)-1 -Aminoethyl]-N-4-pyridinylcyclohexanecarboxamide dihydrochloride (Y-27632 dihydrochloride), N-Benzyl[2-(pyrimidin-4-yl)amino]thiazole-4-carboxamide (Thiazovivin), a isoquinolinesulfonamide compound (Rho Kinase Inhibitor), N-(4-Pyridyl)-N'-(2,4,6- trichlorophenyl)urea (Rho Kinase Inhibitor II), 3-(4-Pyridyl)-1 H-indole (Rho Kinase Inhibitor III, Rockout), and 4-pyrazoleboronic acid pinacol ester; anti-ROCK antibodies such as Rock-1 (B1), Rock-1 (C-19), Rock-1 (H-11), Rock-1 (G-6), Rock-1 (H-85), Rock-1 (K-18), Rock-2 (C-20), Rock-2 (D-2), Rock-2 (D-11), Rock-2 (N-19), Rock-2 (H-85), and Rock-2 (30-J) (commercially available from Santa Cruz Biotechnology); ROCK CRISPR / Cas9 knockout plasmids such as Rock-1 CRISPR / Cas9 KO plasmid (h), Rock-2 CRISPR / Cas9 KO plasmid (h), Rock-1 CRISPR / Cas9 KO plasmid (m), and Rock-2 CRISPR / Cas9 KO plasmid (m); a ROCK siRNA, shRNA plasmid and / or shRNA lentiviral particle gene silencer such as Rock-1 siRNA (h): sc-29473, Rock-1 siRNA (m): sc-36432, Rock-1 siRNA (r): sc-72179, Rock-2 siRNA (h): sc-29474, Rock-2 siRNA (m): sc-36433, and Rock-2 siRNA (r): se-108088 (commercially available from Santa Cruz Biotechnology). Exemplary non-selective ROCK inhibitors include, without limitation, 4-[(1R)-1-aminoethyl]-N-pyridin-4-ylcyclohexane-1- carboxamide (Y-27632), thiazovivin, and fasudil. Exemplary selective ROCK inhibitors include, without limitation, 4-methyl-5-[[(2S)-2-methyl-1 ,4-diazepan-1-yl]sulfonyl]isoquinoline (H1152), ripasudil, Chroman 1 , N-[3-[2-(4-amino-1,2,5-oxadiazol-3-yl)-1-ethylimidazo[4,5-c]pyridin-6- yl]oxyphenyl]-4-(2-morpholin-4-ylethoxy)benzamide (GSK 269962A), and (1 R)-1 -(1 -isoquinolin-5- ylsulfonylpiperidin-4-yl)ethanamine (BA-1049 also known as NRL-1049).

[0091] In certain embodiments, the ROCK inhibitor decreases the level and / or activity of ROCK in cells or cell culture medium by at least or about 5%, at least or about 10%, at least or about 20%, at least or about 30%, at least or about 40%, at least or about 50%, at least or about 60%, at least or about 70%, at least or about 80%, at least or about 90%, or at least or about 95%. In some embodiments, a ROCK inhibitor completely inhibits the level and / or activity of ROCK in the cells or cell culture medium.

[0092] The stem cells can be embryonic stem cells, which are derived from blastocysts, adult stem cells, which are found in adult tissues, cord blood stem cells, which are found in the umbilical cord, or induced pluripotent stem cells, which can be derived from adult somatic cells. In some embodiments, the stem cells are iPSCs generated from somatic cells. The IPSCs can be generatedby reprogramming the somatic cells obtained from the subject into pluripotent stem cells followed by redifferentiation into SGN-like cells according to the methods described herein. Somatic cells can be induced into forming pluripotent stem cells, for example, by treating them with reprograming factors such as Yamanaka factors, including but not limited to, OCT3, OCT4, SOX2, KLF4, c-MYC, NANOG, and LIN28 (see, e.g., Takahashi et al. (2007) Cell. 131 (5):861 -872; herein incorporated by reference in its entirety). Somatic cells are contacted with reprogramming factors in a combination and quantity sufficient to reprogram the cells to pluripotency. Reprogramming factors may be provided to the somatic cells individually or as a single composition, that is, as a premixed composition, of reprogramming factors. In some embodiments the reprogramming factors are provided as a plurality of coding sequences on a vector.

[0093] Methods for "introducing a cell reprogramming factor into somatic cells” are not limited in particular, and known procedures can be selected and used as appropriate. For example, when a cell reprogramming factor as described above is introduced into somatic cells of the above- mentioned type in the form of proteins, such methods include ones using protein introducing reagents, fusion proteins with protein transfer domains (PTDs), electroporation, and microinjection. When a cell reprogramming factor as described above is introduced into somatic cells of the above- mentioned type in the form of nucleic acids encoding the cell reprogramming factor, a nucleic acid(s), such as cDNA(s), encoding the cell reprogramming factor can be inserted in an appropriate expression vector comprising a promoter that functions in somatic cells, which then can be introduced into somatic cells by procedures such as infection, lipofection, liposomes, electroporation, calcium phosphate coprecipitation, DEAE-dextran, microinjection, and electroporation. Examples of an "expression vector" include viral vectors, such as lentiviruses, retroviruses, adenoviruses, adeno- associated viruses, and herpes viruses; and expression plasmids for animal cells. For example, retroviral or Sendai virus (SeV) vectors are commonly used to introduce a nucleic acid(s) encoding a cell reprogramming factor as described above into somatic cells.

[0094] After in vitro expansion, stem cells can be differentiated into SGN-like cells by culturing the stem cells in the presence of various growth factors such as bone morphogenetic protein 4 (BMP4), basic fibroblast growth factor 2 (FGF2), insulin-like growth factor-1 (IGF-1), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and epidermal growth factor (EGF) in combination with other differentiation agents such as Wnt inhibitors, ROCK inhibitors, inhibitors of ALK2 and ALK3, sonic hedgehog (SHH), and retinoic acid (RA) in the culture, as described in Example 1.

[0095] The SGN-like cells are harvested at an appropriate stage of development, which may be determined based on the expression of markers and phenotypic characteristics of the desired SGNtype (Type I or Type II), e.g., at from about 1 to 2 months in culture. Cultures may be empirically tested by staining for the presence of one or more SGN markers of interest and / or by morphological determination. For example, SGN-like cells can be identified by markers specific for cells of the SGN lineage, including, without limitation, tubulin beta 3 class III (TUBB3), neurofilament light polypeptide (NEFL), neurofilament heavy polypeptide (NEFH), POU class 4 homeobox 1 (POU4F1), neuronal differentiation 1 transcription factor (NEUROD1), and SRY-box transcription factor 2 (SOX2). The SGN-like cells may be further characterized by staining for the presence of markers that distinguish Type I and Type II SGN cells. Markers specific for Type I SGN cells, include, without limitation, prospero homeobox 1 (PROX1), sodium / potassium-transporting ATPase subunit alpha-3 (NKAa3), calbindin-2 (GALB2), and Ly6 / PLAUR domain-containing protein 1 (LYPD1). Markers specific for Type II SGN cells, include, without limitation, peripherin (PRPH), tachykinin precursor 1 (TAC1 ), MAF bZIP transcription factor (MAFB), and GATA binding protein 3 (GATA3). The cells are optionally enriched before or after a positive selection step by drug selection, panning, density gradient centrifugation, etc. In another embodiment, a negative selection is performed, where the selection is based on expression of one or more markers found on the somatic cells, stem cells, or SGN precursors (e.g., otic neural progenitor cells). Selection may utilize panning methods, magnetic particle selection, particle sorter selection, and the like.

[0096] In some embodiments, the SGN-like cells are cultured for at least or about 1 week, at least or about 2 weeks, at least or about 3 weeks, at least or about 4 weeks, at least or about 5 weeks, at least or about 6 weeks, at least or about 7 weeks, at least or about 8 weeks, at least or about 9 weeks, at least or about 10 weeks, at least or about 12 weeks, at least or about 14 weeks, at least or about 16 weeks, at least or about 18 weeks, at least or about 20 weeks. In some embodiments, the SGN-like cells are cryopreserved for storage before use.

[0097] In some embodiments, the stem cells, used for generation of SGN-like cells, are iPSCs derived from somatic cells obtained from normal individuals. In other embodiments, the stem cells, used for generation of SGN-like cells, are iPSCs derived from somatic cells obtained from an individual comprising at least one allele encoding a mutation linked to an SGN-associated disorder, which can be used to generate a disease model.

[0098] A sample comprising somatic cells for generating iPSCs is obtained from a subject. The somatic cells may include, without limitation, peripheral blood mononuclear cells, fibroblasts, keratinocytes, epithelial cells, endothelial progenitor cells, mesenchymal stem cells, adipose derived stem cells, leukocytes, hematopoietic stem cells, bone marrow cells, and hepatocytes, and other cell types capable of generating patient-derived IPSCs that can be differentiated into SGN-like cells. The biological sample comprising somatic cells can be any sample from bodily fluids, tissue or cells thatcontain suitable somatic cells. A biological sample can be obtained from a subject by conventional techniques. For example, blood can be obtained by venipuncture, and solid tissue samples can be obtained by surgical techniques according to methods well known in the art. In some embodiments, somatic cells are obtained from a biopsy of the inner ear of a patient.

[0099] In some embodiments, the stem cells, somatic cells from which iPSCs are derived, or the SGN-like cells produced from stem cells may be genetically modified to introduce a mutation linked to an SGN-associated disorder. For example, a recombinant polynucleotide encoding a mutated protein may be introduced into a cell by viral mediated gene transfer using viral vectors such as, but not limited to, lentivirus, adenovirus, retroviruses, adeno-associated virus, or herpes virus vectors. Alternatively, cells can be genetically modified using engineered nucleases such as, but not limited to, GRISPR / CAS9, meganucleases, zinc finger nucleases, or transcription activator-like effector nucleases (TALENs) for gene editing. See, e.g., CRISPR Gene Editing: Methods and Protocols (edited by Luo, Humana, 2019), Genome Editing and Engineering: From TALENs, ZFNs and CRISPRs to Molecular Surgery (edited by Appasani and Church, Cambridge University Press, 2018); herein incorporated by reference in their entireties.

[0100] For cell therapy applications, in which a subject is treated with SGN-like cells for an SGN- associated disorder, adult stem cells or somatic cells (from which iPSCs are generated to produce the SGN-like cells) are preferably obtained from the subject that will be receiving the stem cell- derived SGN-like cells. Alternatively, the adult stem cells or somatic cells, from which iPSCs are generated, can be obtained directly from a donor, a culture of cells from a donor, or from established cell culture lines. Cells are preferably of the same immunological profile as the subject receiving the SGN-like cells for treatment of an SGN-associated disorder. Adult stem cells or somatic cells can be obtained, for example, by biopsy from a close relative or matched donor. The cells will generally be of the same species as the subject receiving the SGN-like cells.Genome Modification to Introduce Disease- Relevant Genetic Changes

[0101] Disease-relevant mutations can be introduced into the genome of the mature stem cell- derived SGN-like cells, or the stem cells or progenitor cells from which they are derived using any method known in the art to produce a disease model. In some embodiments, a CRISPR / Cas system is used to make genetic changes to a gene of interest in the mature SGN-like cells, or the stem cells or progenitor cells from which they are derived, for example, to introduce a mutation associated with an SGN-associated disorder causing hearing loss or deafness useful for disease modeling and drug screening. For example, a CRISPR / Cas system can be used to delete, inactivate, or mutate a gene, or eliminate or reduce gene expression or protein activity. Genome modification can be performed,for example, using homology directed repair (HDR) with a donor polynucleotide comprising a sequence comprising an intended genome edit flanked by a pair of homology arms responsible for targeting the donor polynucleotide to the target locus to be edited in a cell. The donor polynucleotide typically comprises a 5' homology arm that hybridizes to a 5' genomic target sequence and a 3' homology arm that hybridizes to a 3' genomic target sequence. The homology arms are referred to herein as 5' and 3' (i.e., upstream and downstream) homology arms, which relates to the relative position of the homology arms to the nucleotide sequence comprising the intended edit within the donor polynucleotide. The 5' and 3' homology arms hybridize to regions within the target locus in the genomic DNA to be modified, which are referred to herein as the "5' target sequence" and "3' target sequence," respectively.

[0102] The homology arm must be sufficiently complementary for hybridization to the target sequence to mediate homologous recombination between the donor polynucleotide and genomic DNA at the target locus. For example, a homology arm may comprise a nucleotide sequence having at least about 80-100% sequence identity to the corresponding genomic target sequence, including any percent identity within this range, such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto, wherein the nucleotide sequence comprising the intended edit is integrated into the genomic DNA by HDR at the genomic target locus recognized (i.e., sufficiently complementary for hybridization) by the 5' and 3' homology arms.

[0103] In certain embodiments, the corresponding homologous nucleotide sequences in the genomic target sequence (i.e., the "5' target sequence" and "3' target sequence") flank a specific site for cleavage and / or a specific site for introducing the intended edit. The distance between the specific cleavage site and the homologous nucleotide sequences (e.g., each homology arm) can be several hundred nucleotides. In some embodiments, the distance between a homology arm and the cleavage site is 200 nucleotides or less (e.g., 0, 10, 20, 30, 50, 75, 100, 125, 150, 175, and 200 nucleotides). In most cases, a smaller distance may give rise to a higher gene targeting rate. In a preferred embodiment, the donor polynucleotide is substantially identical to the target genomic sequence, across its entire length except for the sequence changes to be introduced to a portion of the genome that encompasses both the specific cleavage site and the portions of the genomic target sequence to be altered.

[0104] A homology arm can be of any length, e.g., 10 nucleotides or more, 50 nucleotides or more, 100 nucleotides or more, 250 nucleotides or more, 300 nucleotides or more, 350 nucleotides or more, 400 nucleotides or more, 450 nucleotides or more, 500 nucleotides or more, 1000 nucleotides (1 kb) or more, 5000 nucleotides (5 kb) or more, 10000 nucleotides (10 kb) or more, etc. In someinstances, the 5' and 3' homology arms are substantially equal in length to one another, e.g., one may be 30% shorter or less than the other homology arm, 20% shorter or less than the other homology arm, 10% shorter or less than the other homology arm, 5% shorter or less than the other homology arm, 2% shorter or less than the other homology arm, or only a few nucleotides less than the other homology arm. In other instances, the 5' and 3' homology arms are substantially different in length from one another, e.g., one may be 40% shorter or more, 50% shorter or more, sometimes 60% shorter or more, 70% shorter or more, 80% shorter or more, 90% shorter or more, or 95% shorter or more than the other homology arm.

[0105] The donor polynucleotide is used in combination with an RNA-guided nuclease, which is targeted to a particular genomic sequence (i.e. , genomic target sequence to be modified) by a guide RNA (gRNA). A target-specific guide RNA comprises a nucleotide sequence that is complementary to a genomic target sequence, and thereby mediates binding of the nuclease-gRNA complex by hybridization at the target site. For example, the gRNA can be designed with a sequence complementary to a target sequence in a gene of interest. In some embodiments, the gRNA is designed with a sequence complementary to a specific target site for introduction of a mutation to target the nuclease-gRNA complex to the site where the mutation will be introduced into the genome of a cell. The mutation may comprise an insertion, a deletion, or a substitution. For example, the mutation may include a single nucleotide variation, gene fusion, translocation, inversion, duplication, frameshift, missense, nonsense, or other mutation. The targeted allele may be a common genetic variant or a rare genetic variant. In certain embodiments, the gRNA is designed to selectively bind to a minor allele with single base-pair discrimination, for example, to allow binding of the nuclease- gRNA complex to a single nucleotide polymorphism (SNP). In particular, the gRNA may be designed to target disease-relevant mutations of interest for the purpose of genome editing to delete or deactivate the gene in an SGN-like cell.

[0106] In certain embodiments, the RNA-guided nuclease used for genome modification is a CRISPR system Gas nuclease. Any RNA-guided Gas nuclease capable of catalyzing site-directed cleavage of DNA to allow integration of donor polynucleotides by the HDR mechanism can be used in genome editing, including CRISPR system type I, type II, or type III Gas nucleases. Examples of Gas proteins include Cas1, Cas1 B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Casio, Cas10d, Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasG), Csc1, Csc2, Csa5, Csn2, Csm2, Gsm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Gmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14,Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1 , Csf2, Csf3, Csf4, and Cu1966, and homologs or modified versions thereof.

[0107] In certain embodiments, a type II CRISPR system Cas9 endonuclease is used. Cas9 nucleases from any species, or biologically active fragments, variants, analogs, or derivatives thereof that retain Cas9 endonuclease activity (i.e., catalyze site-directed cleavage of DNA to generate double-strand breaks) may be used to perform genome modification as described herein. The Cas9 need not be physically derived from an organism, but may be synthetically or recombinantly produced. Cas9 sequences from a number of bacterial species are well known in the art and listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries for Cas9 from: Streptococcus pyogenes (WP 002989955, WP 038434062, WP 011528583); Campylobacter jejuni (WP 022552435, YP 002344900), Campylobacter coll (WP_060786116); Campylobacter fetus (WP_059434633); Corynebacterium ulcerans (NC_015683, NC_017317); Corynebacterium diphtheria (NC_016782, NC_016786); Enterococcus faecalis (WP_033919308); Spiroplasma syrphidicola (NC_021284); Prevotella intermedia (NC_017861); Spiroplasma taiwanense (NC_021846); Streptococcus iniae (NC_021314); Belliella baltica (NC_018010); Psychroflexus torquisl (NC_018721); Streptococcus thermophilus (YP_820832), Streptococcus mutans (WP 061046374, WP 024786433); Listeria innocua (NP 472073); Listeria monocytogenes (WP_061665472); Legionella pneumophila (WP 062726656); Staphylococcus aureus (WP_001573634); Francisella tularensis (WP 032729892, WP_014548420), Enterococcus faecalis (WP_033919308); Lactobacillus rhamnosus (WP_048482595, WP_032965177); and Neisseria meningitidis (WP 061704949, YP 002342100); all of which sequences (as entered by the date of filing of this application) are herein incorporated by reference. Any of these sequences or a variant thereof comprising a sequence having at least about 70-100% sequence identity thereto, including any percent identity within this range, such as 70, 71 , 72, 73, 74, 75, 76, 77 , 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, can be used for genome editing, as described herein. See also Fonfara et al. (2014) Nucleic Acids Res. 42(4):2577-90; Kapitonov et al. (2015) J. Bacteriol. 198(5):797-807, Shmakov et al. (2015) Mol. Cell. 60(3):385-397, and Chylinski et al. (2014) Nucleic Acids Res. 42(10):6091-6105); for sequence comparisons and a discussion of genetic diversity and phylogenetic analysis of Cas9.

[0108] The CRISPR-Cas system naturally occurs in bacteria and archaea where it plays a role in RNA-mediated adaptive immunity against foreign DNA. The bacterial type II CRISPR system uses the endonuclease, Cas9, which forms a complex with a guide RNA (gRNA) that specifically hybridizes to a complementary genomic target sequence, where the Cas9 endonuclease catalyzescleavage to produce a double-stranded break. Targeting of Cas9 typically further relies on the presence of a 5' protospacer-adjacent motif (PAM) in the DNA at or near the gRNA-binding site.

[0109] The genomic target site will typically comprise a nucleotide sequence that is complementary to the gRNA, and may further comprise a protospacer adjacent motif (PAM). In certain embodiments, the target site comprises 20-30 base pairs in addition to a 3 base pair PAM. Typically, the first nucleotide of a PAM can be any nucleotide, while the two other nucleotides will depend on the specific Cas9 protein that is chosen. Exemplary PAM sequences are known to those of skill in the art and include, without limitation, NNG, NGN, NAG, and NGG, wherein N represents any nucleotide. In certain embodiments, the allele targeted by a gRNA comprises a mutation that creates a PAM within the allele, wherein the PAM promotes binding of the Cas9-gRNA complex to the allele.

[0110] In certain embodiments, the gRNA is 5-50 nucleotides, 10-30 nucleotides, 15-25 nucleotides, 18-22 nucleotides, or 19-21 nucleotides in length, or any length between the stated ranges, including, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, or 35 nucleotides in length. The guide RNA may be a single guide RNA comprising crRNA and tracrRNA sequences in a single RNA molecule, or the guide RNA may comprise two RNA molecules with crRNA and tracrRNA sequences residing in separate RNA molecules.

[0111] In another embodiment, the CRISPR nuclease from Prevotella and Francisella 1 (Cpf 1 , also known as Cas12a) is used. Cpf 1 is another class II CRISPR / Cas system RNA-guided nuclease with similarities to Cas9 and may be used analogously. Unlike Cas9, Cpf 1 does not require a tracrRNA and only depends on a crRNA in its guide RNA, which provides the advantage that shorter guide RNAs can be used with Cpf 1 for targeting than Cas9. Cpf 1 is capable of cleaving either DNA or RNA. The PAM sites recognized by Cpf 1 have the sequences 5'-YTN-3' (where "Y" is a pyrimidine and "N" is any nucleobase) or 5'-TTN-3', in contrast to the G-rich PAM site recognized by Cas9. Cpf1 cleavage of DNA produces double-stranded breaks with a sticky-ends having a 4 or 5 nucleotide overhang. For a discussion of Cpf1, see, e.g., Ledford et al. (2015) Nature. 526 (7571 ):17-17, Zetsche et al. (2015) Cell. 163 (3):759-771, Murovec et al. (2017) Plant Biotechnol. J. 15(8):917- 926, Zhang et al. (2017) Front. Plant Sci. 8:177, Fernandes et al. (2016) Postepy Biochem.62(3):315-326; herein incorporated by reference.

[0112] Cas12b (C2c1) is another class II CRISPR / Cas system RNA-guided nuclease that may be used. C2c1 , similarly to Cas9, depends on both a crRNA and tracrRNA for guidance to target sites. For a description of Cas12b, see, e.g., Shmakov et al. (2015) Mol Cell. 60(3):385-397, Zhang et al. (2017) Front Plant Sci. 8:177; herein incorporated by reference.

[0113] In yet another embodiment, an engineered RNA-guided Fokl nuclease may be used. RNA- guided Fokl nucleases comprise fusions of inactive Cas9 (dCas9) and the Fokl endonuclease (Fokl-dCas9), wherein the dCas9 portion confers guide RNA-dependent targeting on Fokl. For a description of engineered RNA-guided Fokl nucleases, see, e.g., Havlicek et al. (2017) Mol. Ther.25(2):342-355, Pan et al. (2016) Sci Rep. 6:35794, Tsai et al. (2014) Nat Biotechnol. 32(6):569-576; herein incorporated by reference.

[0114] An RNA-guided nuclease can be provided in the form of a protein, such as the nuclease complexed with a gRNA, or provided by a nucleic acid encoding the RNA-guided nuclease, such as an RNA (e.g., messenger RNA) or DNA (expression vector). In some embodiments, the RNA-guided nuclease and the gRNA are both provided by vectors. Both can be expressed by a single vector or separately on different vectors. The vector(s) encoding the RNA-guided nuclease an gRNA may be included in a CRISPR expression system to target a gene of interest in SGN-like cells.

[0115] Codon usage may be optimized to improve production of an RNA-guided nuclease in a particular cell or organism. For example, a nucleic acid encoding an RNA-guided nuclease or reverse transcriptase can be modified to substitute codons having a higher frequency of usage in a human cell, a non-human cell, a mammalian cell, a rodent cell, a mouse cell, a rat cell, or any other host cell of interest, as compared to the naturally occurring polynucleotide sequence. When a nucleic acid encoding the RNA-guided nuclease is introduced into cells (e.g., SGN-like cells), the protein can be transiently, conditionally, or constitutively expressed in the cell.

[0116] In another embodiment, CRISPR interference (CRISPRi) is used to repress gene expression.CRISPRi is performed with a complex of a catalytically inactive Cas9 (dCas9) with a guide RNA that targets the gene of interest. An engineered nuclease-deactivated Cas9 (dCas9) is used to allow sequence-specific targeting without cleavage. Nuclease-deactivated forms of Cas9 may be engineered by mutating catalytic residues at the active site of Cas9 to destroy nuclease activity. Any such nuclease deficient Cas9 protein from any species may be used as long as the engineered dCas9 retains gRNA-mediated sequence-specific targeting. In particular, the nuclease activity of Cas9 from Streptococcus pyogenes can be deactivated by introducing two mutations (D10A and H841A) in the RuvC1 and HNH nuclease domains. Other engineered dCas9 proteins may be produced by similarly mutating the corresponding residues in other bacterial Cas9 isoforms. For a description of engineered nuclease-deactivated forms of Cas9, see, e.g., Qi et al. (2013) Cell 152:1173-1183, Dominguez et al. (2016) Nat. Rev. Mol. Cell. Biol. 17(1 ):5-15; herein incorporated by reference in their entireties.

[0117] The dCas9 protein can be designed to target a gene of interest by altering its guide RNA sequence. A target-specific single guide RNA (sgRNA) comprises a nucleotide sequence that is complementary to a target site, and thereby mediates binding of the dCas9-sgRNA complex by hybridization at the target site. CRISPRi can be used to sterically repress transcription by blockingeither transcriptional initiation or elongation by designing a sgRNA with a sequence complementary to a promoter or exonic sequence. The sgRNA may be complementary to the non-template strand or the template strand, but preferably is complementary to the non-template strand to more strongly repress transcription.

[0118] The target site will typically comprise a nucleotide sequence that is complementary to the sgRNA, and may further comprise a protospacer adjacent motif (PAM). In certain embodiments, the target site comprises 20-30 base pairs in addition to a 3 base pair PAM. Typically, the first nucleotide of a PAM can be any nucleotide, while the two other nucleotides will depend on the specific Cas9 protein that is chosen. Exemplary PAM sequences are known to those of skill in the art and include, without limitation, NNG, NGN, NAG, and NGG, wherein N represents any nucleotide.

[0119] In certain embodiments, the sgRNA comprises 5-50 nucleotides, 10-30 nucleotides, 15-25 nucleotides, 18-22 nucleotides, 19-21 nucleotides, and any length between the stated ranges, including, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.

[0120] The sgRNAs are readily synthesized by standard techniques, e.g., solid phase synthesis via phosphoramidite chemistry, as disclosed in U.S. Patent Nos. 4,458,066 and 4,415,732, incorporated herein by reference; Beaucage et aL, Tetrahedron (1992) 48:2223-2311 ; and Applied Biosystems User Bulletin No. 13 (1 April 1987). Other chemical synthesis methods include, for example, the phosphotriester method described by Narang et al., Meth. Enzymol. (1979) 68:90 and the phosphodiester method disclosed by Brown et aL, Meth. Enzymol. (1979) 68:109.

[0011] In some embodiments, the dCas9 is fused to a transcriptional repressor domain capable of further repressing transcription of the gene of interest, e.g., by inducing heterochromatinization. For example, a Kruppel associated box (KRAB) can be fused to dCas9 to repress transcription of a target gene in human cells (see, e.g., Gilbert et al. (2013) Cell. 154 (2): 442-45, O'Geen et al. (2017) Nucleic Acids Res. 45(17):9901 -9916; herein incorporated by reference).

[0122] Alternatively, dCas9 can be used to introduce epigenetic changes that reduce expression of a gene of interest by fusion of dCas9 to an epigenetic modifier such as a chromatin-modifying epigenetic enzyme. The promoter for the gene of interest can be silenced, for example, by methylation or acetylation (e.g., histone H3 lysine 9 [H3K9] methylation, histone H3 lysine 27 [H3K27] methylation, and / or DNA methylation). For example, fusion of dCas9 to a DNA methyltransferase such as DNA methyltransferase 3 alpha (DNMT3A) or a chimeric Dnmt3a / Dnmt3L methyltransferase (DNMT3A3L) allows targeted DNA methylation. Fusion of dCas9 to histone demethylase LSD1 allows targeted histone demethylation (see, e.g., Liu et al. (2016) Cell 167(1 ):233-247, Lo et al.(2017) FIOOORes. 6. pii: F1000 Faculty Rev-747, and Stepper et al. (2017) Nucleic Acids Res.45(4):1703-1713; herein incorporated by reference).

[0123] In yet other embodiments, an RNA-targeting CRISPR-Cas13 system is used to perform RNA interference to reduce expression of a gene of interest. Members of the Cas13 family are RNA- guided RNases containing two HEPN domains having RNase activity. In particular, Cas13a (C2c2), Cas13b (C2c6), and Cas13d can be used for RNA knockdown. Cas13 proteins can be made to target and cleave transcribed RNA using a gRNA with complementarity to the target transcript sequence. The gRNA is typically about 64 nucleotides in length with a short hairpin crRNA and a 28-30 nucleotide spacer that is complementary to the target site on the RNA transcript. Cas13 recognition and cleavage of a target transcript results in degradation of the transcript as well as nonspecific degradation of any nearby transcripts. See, e.g., Abudayyeh et al. (2017) Nature 550:280-284, Hameed et al. (2019) Microb. Pathog. 133:103551, Wang et al. (2019) Biotechnol Adv. 37(5):708- 729, Aman et al. (2018) Viruses 10(12). pii: E732, and Zhang et al. (2018) Cell 175(1 ):212-223; herein incorporated by reference.

[0014] In certain embodiments, a CRISPR system is used to introduce one or more mutations linked to an SGN-associated disorder into the mature SGN-like cells (or the iPSCs or progenitor cells they are derived from) to produce cells that can be used as a disease model. Mutations linked to SGN- associated disorders include autosomal dominant, autosomal recessive, sex-linked, and mitochondrial mutations. Such mutations may reduce the numbers of SGN cells, cause metabolic stress, cell degeneration, or cell death, or disrupt formation of SGN synapses with hair cells or central nervous system neurons (e.g., cochlear nucleus neurons). In particular, mutations in the TMPRSS3, NLRP3, FGF13, TBC1 D24, GATA2, GAT A3, DFNB59, GRM7, and ATP6V1B2 genes have been linked to SGN-associated disorders causing hearing loss or deafness. Representative mutations linked to SGN-associated disorders and hearing loss or deafness are listed on The Hereditary Hearing Loss Homepage and associated database (hereditaryhearingloss.org / ), currently maintained by the Molecular Otolaryngology and Renal Research Laboratories of the University of Iowa; all of which mutations (as entered by the date of filing of this application) are herein incorporated by reference.Screening Assays

[0125] SGN-like cells can be subjected to a plurality of candidate agents or other therapeutic intervention. Candidate agents include, without limitation, small molecules, i.e., drugs, genetic constructs that increase or decrease expression of an RNA of interest, CRISPR systems, optogenetic perturbation, electrical changes, and the like. Methods are also provided for determiningthe activity of a candidate agent on a disease-relevant cell, the method comprising contacting SGN- like cells comprising at least one allele encoding a mutation associated with an SGN-associated disorder with the candidate agent; and determining the effect of the agent on morphologic, genetic or functional parameters. Screening assays may involve determining the effect of contacting the SGN-like cells with the candidate agent on migration, synapse formation (e.g., with hair cells or cochlear nucleus neurons), interactions of SGN-like cells with glial cells, etc. in culture, which may be tested with one or a panel of cellular environments, where the cellular environment includes one or more of: electrical stimulation, alterations in ionicity, stimulation with a candidate agent of interest, contact with other cells including without limitation, hair cells, other neurons, neural progenitors, or glial cells, contact with infectious agents, e.g., bacterial, viral, fungal, or parasitic infectious agents, and the like, and where cells may vary in genotype, in prior exposure to an environment of interest, in the dose of agent that is provided, etc. Usually at least one control is included, for example, a negative control and a positive control. Culture of cells is typically performed in a sterile environment, for example, at 37°C. in an incubator containing a humidified 92-95% air / 5-8% CO2 atmosphere. Cell culture may be carried out in nutrient mixtures containing undefined biological fluids such as fetal calf serum, or media which is fully defined and serum free. The effect of the altering of the environment is assessed by monitoring multiple output parameters, including morphological, electrophysiological, functional, and genetic changes.

[0126] Examples of analytic methods comprise, for example, assessing synaptic integration of SGN- like cells by using array tomography to detect pre- and post-synaptic proteins. To further examine synaptic puncta 'synaptograms' consisting of a series of high-resolution sections through a single synapse may be obtained. Electrophysiology measurements including voltage clamp recordings of synaptic responses can be performed on SGN-like cells.

[0017] Live imaging of cells, including during cell migration, may be performed and cells modified to express a detectable marker. Calcium sensitive dyes can be used, e.g., Fura-2 calcium imaging; Fluo-4 calcium imaging, GCaMP6 calcium imaging, voltage imaging using voltage indicators such as voltage-sensitive dyes (e.g., di-4-ANEPPS, di-8-ANEPPS, and RH237) and / or genetically- encoded voltage indicators (e.g., ASAP1 , Archer) can be used on the SGN-like cells or cells associated with or derived therefrom.

[0128] Methods of analysis at the single cell level are also of interest, e.g., as described above: live imaging (including confocal or light-sheet microscopy), single cell gene expression or single cell RNA sequencing, calcium imaging, immunocytochemistry, patch-clamping, flow cytometry and the like. Various parameters can be measured to determine the effect of a drug or treatment on the SGN-like cells or cells associated with or derived therefrom.

[0129] Parameters include quantifiable components of cells, particularly components that can be accurately measured, desirably in a high throughput system. A parameter can also be any cell component or cell product including cell surface determinant, receptor, protein or conformational or posttranslational modification thereof, lipid, carbohydrate, organic or inorganic molecule, nucleic acid, e.g., mRNA, DNA, etc. or a portion derived from such a cell component or combinations thereof. Although most parameters will provide a quantitative readout, in some instances a semi-quantitative or qualitative result will be acceptable. Readouts may include a single determined value, or may include mean, median value or the variance, etc. Variability is expected and a range of values for each of the set of test parameters will be obtained using standard statistical methods with a common statistical method used to provide single values.

[0130] Parameters of interest include detection of cytoplasmic, cell surface or secreted biomolecules, frequently biopolymers, e.g., polypeptides, polysaccharides, polynucleotides, lipids, etc. Cell surface and secreted molecules are a preferred parameter type as these mediate cell communication and cell effector responses and can be more readily assayed. In one embodiment, parameters include specific epitopes. Epitopes are frequently identified using specific monoclonal antibodies or receptor probes. In some cases, the molecular entities comprising the epitope are from two or more substances and comprise a defined structure; examples include combinatorically determined epitopes associated with heterodimeric integrins. A parameter may be detection of a specifically modified protein or oligosaccharide. A parameter may be defined by a specific monoclonal antibody or a ligand or receptor binding determinant.

[0131] Candidate agents of interest are biologically active agents that encompass numerous chemical classes, primarily organic molecules, which may include organometallic molecules, inorganic molecules, genetic sequences, etc. In particular, candidate drugs, select therapeutic antibodies and protein-based therapeutics with preferred biological response functions can be evaluated. Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, frequently at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules, including peptides, polynucleotides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.

[0132] Included are pharmacologically active drugs, genetically active molecules, etc. Compounds of interest include chemotherapeutic agents, anti-inflammatory agents, neurotransmitters, hormones or hormone antagonists, ion channel modifiers, and neuroactive agents. Exemplary pharmaceuticalagents include those described in, "The Pharmacological Basis of Therapeutics," Goodman and Gilman, McGraw-Hill, New York, N.Y., (1996), Ninth edition, under the sections: Drugs Acting at Synaptic and Neuroeffector Junctional Sites; Cardiovascular Drugs; Vitamins, Dermatology; and Toxicology, all incorporated herein by reference.

[0133] Test compounds may include all of the classes of molecules described above, and may further comprise samples of unknown content. Of interest are complex mixtures of naturally occurring compounds derived from natural sources such as plants. While many samples will comprise compounds in solution, solid samples that can be dissolved in a suitable solvent may also be assayed. Samples of interest include environmental samples, e.g., ground water, sea water, mining waste, etc.; biological samples, e.g., lysates prepared from crops, tissue samples, etc.; manufacturing samples, e.g., time course during preparation of pharmaceuticals; as well as libraries of compounds prepared for analysis; and the like. Samples of interest include compounds being assessed for potential therapeutic value, i.e. , drug candidates.

[0134] The term samples also includes the fluids described above to which additional components have been added, for example components that affect the ionic strength, pH, total protein concentration, etc. In addition, the samples may be treated to achieve at least partial fractionation or concentration. Biological samples may be stored if care is taken to reduce degradation of the compound, e.g., under nitrogen, frozen, or a combination thereof. The volume of sample used is sufficient to allow for measurable detection, usually from about 0.1 to 1 ml of a biological sample is sufficient.

[0135] Compounds, including candidate agents, are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds, including biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.

[0136] As used herein, the term "genetic agent" refers to polynucleotides and analogs thereof, which agents are tested in the screening assays by addition of the genetic agent to a cell. The introduction of the genetic agent results in an alteration of the total genetic composition of the cell. Genetic agents such as DNA can result in an experimentally introduced change in the genome of a cell, generallythrough the integration of the sequence into a chromosome, for example using CRISPR mediated genomic engineering (see for example Shmakov et al. (2017) Nature Reviews Microbiology 15:169). Genetic changes can also be transient, where the exogenous sequence is not integrated but is maintained as an episomal agents. Genetic agents, such as antisense oligonucleotides, can also affect the expression of proteins without changing the cell's genotype, by interfering with the transcription or translation of mRNA. The effect of a genetic agent is to increase or decrease expression of one or more gene products in the cell.

[0137] Introduction of an expression vector encoding a polypeptide can be used to express the encoded product in cells lacking the sequence, or to over-express the product. Various promoters can be used that are constitutive or subject to external regulation, where in the latter situation, one can turn on or off the transcription of a gene. These coding sequences may include full-length cDNA or genomic clones, fragments derived therefrom, or chimeras that combine a naturally occurring sequence with functional or structural domains of other coding sequences. Alternatively, the introduced sequence may encode an anti-sense sequence; be an anti-sense oligonucleotide; RNAi, encode a dominant negative mutation, or dominant or constitutively active mutations of native sequences; altered regulatory sequences, etc.

[0138] Antisense and RNAi oligonucleotides can be chemically synthesized by methods known in the art. Preferred oligonucleotides are chemically modified from the native phosphodiester structure, in order to increase their intracellular stability and binding affinity. A number of such modifications have been described in the literature, which alter the chemistry of the backbone, sugars or heterocyclic bases. Among useful changes in the backbone chemistry are phosphorothioates; phosphorodithioates, where both of the non-bridging oxygens are substituted with sulfur; phosphoroamidites; alkyl phosphotriesters and boranophosphates. Achiral phosphate derivatives include 3'-O'-5'-S-phosphorothioate, 3'-S-5'-O-phosphorothioate, 3'-CH2-5'-0-phosphonate and 3'- NH-5'-O-phosphoroamidate. Peptide nucleic acids replace the entire ribose phosphodiester backbone with a peptide linkage. Sugar modifications are also used to enhance stability and affinity, e.g., morpholino oligonucleotide analogs.

[0139] Agents are screened for biological activity by adding the agent to at least one and usually a plurality of SGN-like cells, in one or in a plurality of environmental conditions, e.g., following stimulation with an agonist, following electric or mechanical stimulation, etc. The change in parameter readout in response to the agent is measured, desirably normalized, and the resulting screening results may then be evaluated by comparison to reference screening results, e.g., with cells having other mutations of interest, normal SGNs, type I SGNs, type II SGNs, and the like. The reference screening results may include readouts in the presence and absence of differentenvironmental changes, screening results obtained with other agents, which may or may not include known drugs, etc.

[0140] The agents are conveniently added in solution, or readily soluble form, to the medium of cells in culture. The agents may be added in a flow-through system, as a stream, intermittent or continuous, or alternatively, adding a bolus of the compound, singly or incrementally, to an otherwise static solution. In a flow-through system, two fluids are used, where one is a physiologically neutral solution, and the other is the same solution with the test compound added. The first fluid is passed over the cells, followed by the second. In a single solution method, a bolus of the test compound is added to the volume of medium surrounding the cells. The overall concentrations of the components of the culture medium should not change significantly with the addition of the bolus, or between the two solutions in a flow through method.

[0141] Preferred agent formulations do not include additional components, such as preservatives, that may have a significant effect on the overall formulation. Thus, preferred formulations consist essentially of a biologically active compound and a physiologically acceptable carrier, e.g., water, ethanol, DMSO, etc. However, if a compound is liquid without a solvent, the formulation may consist essentially of the compound itself.

[0142] A plurality of assays may be run in parallel with different agent concentrations to obtain a differential response to the various concentrations. As known in the art, determining the effective concentration of an agent typically uses a range of concentrations resulting from 1:10, or other log scale, dilutions. The concentrations may be further refined with a second series of dilutions, if necessary. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration or below the level of detection of the agent or at or below the concentration of agent that does not give a detectable change in the phenotype.

[0143] Various methods can be utilized for quantifying the presence of selected parameters, in addition to the functional parameters described above. For measuring the amount of a molecule that is present, a convenient method is to label a molecule with a detectable moiety, which may be fluorescent, luminescent, radioactive, enzymatically active, etc., particularly a molecule specific for binding to the parameter with high affinity fluorescent moieties are readily available for labeling virtually any biomolecule, structure, or cell type. Immunofluorescent moieties can be directed to bind not only to specific proteins but also specific conformations, cleavage products, or site modifications like phosphorylation. Individual peptides and proteins can be engineered to fluoresce, e.g., by expressing them as green fluorescent protein chimeras inside cells (for a review see Jones et al. (1999) Trends Biotechnol. 17(12):477-81). Thus, antibodies can be genetically modified to provide a fluorescent dye as part of their structure

[0144] Depending upon the label chosen, parameters may be measured using other than fluorescent labels, using such immunoassay techniques as radioimmunoassay (RIA) or enzyme linked immunosorbance assay (ELISA), homogeneous enzyme immunoassays, and related non-enzymatic techniques. These techniques utilize specific antibodies as reporter molecules, which are particularly useful due to their high degree of specificity for attaching to a single molecular target. U.S. Pat. No.4,568,649 describes ligand detection systems, which employ scintillation counting. These techniques are particularly useful for protein or modified protein parameters or epitopes, or carbohydrate determinants. Cell readouts for proteins and other cell determinants can be obtained using fluorescent or otherwise tagged reporter molecules. Cell based ELISA or related non-enzymatic or fluorescence-based methods enable measurement of cell surface parameters and secreted parameters. Capture ELISA and related non-enzymatic methods usually employ two specific antibodies or reporter molecules and are useful for measuring parameters in solution. Flow cytometry methods are useful for measuring cell surface and intracellular parameters, as well as shape change and granularity and for analyses of beads used as antibody- or probe-linked reagents. Readouts from such assays may be the mean fluorescence associated with individual fluorescent antibody- detected cell surface molecules or cytokines, or the average fluorescence intensity, the median fluorescence intensity, the variance in fluorescence intensity, or some relationship among these.

[0145] Both single cell multiparameter and multicell multi parameter multiplex assays, where input cell types are identified and parameters are read by quantitative imaging and fluorescence and confocal microscopy are used in the art, see Confocal Microscopy Methods and Protocols (Methods in Molecular Biology Vol. 122.) Paddock, Ed., Humana Press, 1998. These methods are described in U.S. Pat. No. 5,989,833 issued Nov. 23, 1999.

[0146] Of particular interest for the disclosed SGN screening system are parameters related to the electrical properties of the cells and therefore directly informative about neuronal function and activity. Methods to measure neuronal activity may sense the occurrence of action potentials (spikes). The characteristics of the occurrence of a single spike or multiple spikes either in timely clustered groups (bursts) or distributed over longer time (spike train) of a single neuron or a group of neurons indicate neuronal activation patterns and thus reflect functional neuronal properties, which can be described my multiple parameters. Such parameters can be used to quantify and describe changes in neuronal activity.

[0147] Neuronal activity parameters include, without limitation, total number of spikes (per recording period); mean firing rate (of spikes); inter-spike interval (distance between sequential spikes); total number of bursts (per recording period); burst frequency; number of spikes per burst; burst duration (in milliseconds); inter-burst interval (distance between sequential bursts); burst percentage (theportion of spikes occurring within a burst); total number of network bursts (spontaneous synchronized network activity); network burst frequency; number of spikes per network burst; network burst duration; inter-network-burst interval; inter-spike interval within network bursts; network burst percentage (the portion of bursts occurring within a network burst); salutatory migration, etc.

[0148] Quantitative readouts of neuronal activity parameters may include baseline measurements in the absence of agents or a pre-defined genetic control condition and test measurements in the presence of a single or multiple agents or a genetic test condition. Furthermore, quantitative readouts of neuronal activity parameters may include long-term recordings and may therefore be used as a function of time (change of parameter value). Readouts may be acquired either spontaneously or in response to or presence of stimulation or perturbation of the complete neuronal network or selected components of the network. The quantitative readouts of neuronal activity parameters may further include a single determined value, the mean or median values of parallel, subsequent or replicate measurements, the variance of the measurements, various normalizations, the cross-correlation between parallel measurements, etc. and every statistic used to a calculate a meaningful and informative factor.

[0149] Comprehensive measurements of neuronal activity using electrical or optical recordings of the parameters described herein may include spontaneous activity and activity in response to targeted electrical or optical stimulation of all neuronal cells or a subpopulation of neuronal cells (e.g., all SGN-like cells, type I SGN-like cells, or type II SGN-like cells). Furthermore, spontaneous or induced neuronal activity can be measured in the self-assembled functional environment and circuitry of the neural culture or under conditions of selective perturbation or excitation of specific subpopulations of neuronal cells as discussed above. In some embodiments, the neural culture may include SGN-like cells in combination with hair cells and / or glial cells.

[0150] In the provided assays, comprehensive measurements of neuronal activity can be conducted at different time points along neuronal maturation and usually include a baseline measurement directly before contacting the neural culture with the agents of interest and a subsequent measurement under agent exposure. Moreover, long-term effects of agents on neural maturation and development can be assessed by contacting the immature neural culture at an early time point with agents of interest and acquiring measurements of the same cultures after further maturation at a later time point compared to control cultures without prior agent exposure.

[0151] In some embodiments, standard recordings of neuronal activity of mature neural cultures of SGN-like cells are conducted after about 2 weeks, after about 3 weeks, after about 4 weeks, after about 6 weeks, after about 8 weeks following differentiation of the stem cells into SGN-like cells. Recordings of neuronal activity may encompass the measurement of additive, synergistic oropposing effects of agents that are successively applied to the cultures, therefore the duration recording periods can be adjusted according to the specific requirements of the assay. In some embodiments the measurement of neuronal activity is performed for a predetermined concentration of an agent of interest, whereas in other embodiments measurements of neuronal activity can be applied for a range of concentrations of an agent of interest.

[0152] In some embodiments, the assays described herein are used to evaluate changes in SGN- like cell function in response to optogenetic perturbation of neural activity. In certain embodiments, optogenetics is used to induce cell-specific perturbations between central nervous system neurons and the SGN-like cells. For example, optogenetics can be used to excite or inhibit one or more selected neurons of interest using light. For a description of optogenetics techniques, see, e.g., Abe et aL, 2012; Desai et aL, 2011; Duffy et aL, 2015; Gerits et aL, 2012; Kahn et al., 2013; Lee et aL, 2010; Liu et al., 2015; Ohayon et al., 2013; Weitz et al., 2015; Weitz and Lee, 2013; herein incorporated by reference.

[0153] In some embodiments the provided assays are used to assess maturation of the neural culture of SGN-like cells. Maturation of SGN-like cells can be measured based on morphology by optically assessing parameters such as dendritic arborization, axon elongation, total area of neuronal cell bodies, number of primary processes per neuron, total length of processes per neuron, number of branching points per primary process as well as density and size of synaptic puncta stained by synaptic markers such as synapsin-1, synaptophysin, bassoon, PSD95, and Homer. Moreover, general neuronal maturation and differentiation can be assessed by measuring expression of marker proteins alone or in combination using FACS analysis, immunoblotting, or fluorescence microscopy imaging, patch clamping. Maturation and differentiation of SGN subtypes can further be tested by measuring expression of specific proteins, including, without limitation, tubulin beta 3 class III (TUBB3), neurofilament light polypeptide (NEFL), neurofilament heavy polypeptide (NEFH), POU class 4 homeobox 1 (POU4F1 ), neuronal differentiation 1 transcription factor (NEUROD1 ), and SRY- box transcription factor 2 (SOX2). For type I SGN cells, this includes staining, e.g., for one or more markers selected from prospero homeobox 1 (PROX1), sodium / potassium-transporting ATPase subunit alpha-3 (NKAa3), calbindin-2 (CALB2), and Ly6 / PLAUR domain-containing protein 1 (LYPD1), etc. For type II SGN cells, this includes staining, e.g., for one or more markers selected from peripherin (PRPH), tachykinin precursor 1 (TAC1), MAF bZIP transcription factor (MAFB), and GATA binding protein 3 (GATA3).

[0154] The results of an assay can be entered into a data processor to provide a dataset. Algorithms may be used for the comparison and analysis of data obtained under different conditions. The effect of factors and agents is read out by determining changes in multiple parameters. The data will includethe results from assay combinations with the agent(s), and may also include one or more of comparison to a control state, a simulated state, and the results from other assay combinations using other agents or performed under other conditions. For rapid and easy comparisons, the results may be presented visually in a graph or chart, and can include numbers, color representations, etc.

[0155] The dataset may be prepared from values obtained by measuring parameters of SGN-like cells in the presence and absence of different cells, e.g., hair cells, glial cells, other neurons, genetically modified cells, cells cultured in the presence of specific factors or agents that affect neuronal function, as well as comparing the presence of the agent of interest and at least one other state, usually the control state, which may include the state without the agent or with a different agent. The parameters include functional states such as synapse formation and calcium ions in response to stimulation, whose levels vary in the presence of the factors. The results may be normalized against a standard, usually a "control value or state," to provide a normalized data set. Values obtained from test conditions can be normalized by subtracting the unstimulated control values from the test values, and dividing the corrected test value by the corrected stimulated control value. Other methods of normalization can also be used; and the logarithm or other derivative of measured values or ratio of test to stimulated or other control values may be used. Data is normalized to control data on the same cell type under control conditions, but a dataset may comprise normalized data from one, two or multiple cell types and assay conditions. The dataset can comprise values of the levels of sets of parameters obtained under different assay combinations. Compilations are developed that provide the values for a sufficient number of alternative assay combinations to allow comparison of values.

[0156] A database can be compiled from sets of experiments, for example, a database can contain data obtained from a panel of assay combinations, with multiple different environmental changes, where each change can be a series of related compounds, or compounds representing different classes of molecules.

[0157] Mathematical systems can be used to compare datasets, and to provide quantitative measures of similarities and differences between them. For example, the datasets can be analyzed by pattern recognition algorithms or clustering methods (e.g., hierarchical or k-means clustering, etc.) that use statistical analysis (correlation coefficients, etc.) to quantify relatedness. These methods can be modified (by weighting, employing classification strategies, etc.) to optimize the ability of a dataset to discriminate different functional effects. For example, individual parameters can be given more or less weight when analyzing the dataset, in order to enhance the discriminatory ability of the analysis. The effect of altering the weights assigned each parameter is assessed, and an iterative process is used to optimize pathway or cellular function discrimination.

[0158] The comparison of a dataset obtained from a test compound, and a reference dataset(s) is accomplished by the use of suitable deduction protocols, Al systems, statistical comparisons, etc. Preferably, the dataset is compared with a database of reference data. Similarity to reference data involving known pathway stimuli or inhibitors can provide an initial indication of the cellular pathways targeted or altered by the test stimulus or agent.

[0159] A reference database can be compiled. These databases may include reference data from panels that include known agents or combinations of agents that target specific pathways, as well as references from the analysis of cells treated under environmental conditions in which single or multiple environmental conditions or parameters are removed or specifically altered. Reference data may also be generated from panels containing cells with genetic constructs that selectively target or modulate specific cellular pathways. In this way, a database is developed that can reveal the contributions of individual pathways to a complex response.

[0160] The effectiveness of pattern search algorithms in classification can involve the optimization of the number of parameters and assay combinations. The disclosed techniques for selection of parameters provide for computational requirements resulting in physiologically relevant outputs. Moreover, these techniques for pre-filtering data sets (or potential data sets) using cell activity and disease-relevant biological information improve the likelihood that the outputs returned from database searches will be relevant to predicting agent mechanisms and in vivo agent effects.

[0161] For the development of an expert system for selection and classification of biologically active drug compounds or other interventions, the following procedures are employed. For every reference and test pattern, typically a data matrix is generated, where each point of the data matrix corresponds to a readout from a parameter, where data for each parameter may come from replicate determinations, e.g., multiple individual cells of the same type. As previously described, a data point may be quantitative, semi-quantitative, or qualitative, depending on the nature of the parameter.

[0162] The readout may be a mean, average, median or the variance or other statistically or mathematically derived value associated with the measurement. The parameter readout information may be further refined by direct comparison with the corresponding reference readout. The absolute values obtained for each parameter under identical conditions will display a variability that is inherent in live biological systems and also reflects individual cellular variability as well as the variability inherent between individuals.

[0163] Classification rules are constructed from sets of training data (i.e., data matrices) obtained from multiple repeated experiments. Classification rules are selected as correctly identifying repeated reference patterns and successfully distinguishing distinct reference patterns.Classification rule-learning algorithms may include decision tree methods, statistical methods, naive Bayesian algorithms, and the like.

[0164] A knowledge database will be of sufficient complexity to permit novel test data to be effectively identified and classified. Several approaches for generating a sufficiently encompassing set of classification patterns, and sufficiently powerful mathematical / statistical methods for discriminating between them can accomplish this.

[0165] The data from SGN-like cells treated with specific drugs known to interact with particular targets or pathways provide a more detailed set of classification readouts. Data generated from cells that are genetically modified using over-expression techniques and anti-sense techniques, permit testing the influence of individual genes on the phenotype.

[0166] A preferred knowledge database contains reference data for the SGN-like cells, environments and parameters. For complex environments, data reflecting small variations in the environment may also be included in the knowledge database, e.g., environments where one or more factors or cell types of interest are excluded or included or quantitatively altered in, for example, concentration or time of exposure, etc.Pharmaceutical Compositions

[0167] Pharmaceutical compositions comprising SGN-like cells, produced by the methods described herein, are also provided. The SGN-like cells can be combined, as appropriate, with pharmaceutically acceptable excipients, carriers, or media, in particular, sterile water and physiological saline, hydrogels, vegetable oils, resolvents, buffers, acids, bases, carbohydrates, inorganic salts, antimicrobial agents, antioxidants, emulsifiers, suspending agents, surfactants, stabilizers, vehicles, binders, diluents, tonicity agents, soothing agents, bulking agents, disintegrants, coating agents, lubricants, coloring agents, solution adjuvants, or other additives, and, optionally, other medicinal agents, and combinations thereof. Exemplary physiologically acceptable carriers include sterile, pyrogen-free water and sterile, pyrogen-free, phosphate buffered saline or other buffer such as HEPES or TRIS to maintain pH at appropriate physiological levels.

[0168] Cell aggregates or cell clusters may be dissociated by an enzymatic treatment with at least one protease. For example, cell aggregates or cell clusters may be dissociated with trypsin, dispase, accutase, and / or collagenase (e.g., collagenase I, II, III, and IV, etc.).

[0169] In some embodiments, the pharmaceutical composition comprising the SGN-like cells is a sustained-release formulation, or a formulation that is administered using a sustained-release device. Such devices are well known in the art, and include, for example, transdermal patches, and miniature implantable pumps that can provide for delivery of the SGN-like cells over time in acontinuous, steady-state fashion at a variety of doses to achieve a sustained-release effect with a non-sustained-release pharmaceutical composition.Cellular Therapy with SGN-like Cells

[0170] Stem cell-derived SGN-like cells, produced as described herein, are administered to a subject in a therapeutically effective amount. The phrase “therapeutically effective amount” refers to the administration of the SGN-like cells to a subject, either alone or in combination with hair cells and / or other neurons, or as a part of a pharmaceutical composition and either in a single dose or as part of a series of doses, in an amount that is capable of having a detectable, positive effect on any symptom, aspect, or characteristics of an SGN-associated disease, disorder or condition when administered to a patient. The therapeutically effective amount can be ascertained by measuring relevant physiological effects. For example, in the case of an SGN-associated disorder causing hearing loss or deafness, a therapeutically effective amount of the SGN-like cells improves hearing or slows or halts further loss of hearing. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular type of SGN-associated disorder being treated, the mode of administration, and the like. An appropriate "effective" amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation, based upon the information provided herein.

[0171] The present disclosure contemplates the administration of the SGN-like cells, and compositions thereof, in any appropriate manner. The SGN-like cells may be engrafted, transplanted, or implanted into an ear of a subject. Suitable routes of administration include local injection or surgical implantation directly into an ear, such as in the cochlea of the inner ear (e.g., in the Rosenthal's canal, the modiolus, or the organ of Corti). The method may comprise engrafting an effective amount (e.g., a therapeutically effective amount) of the SGN-like cells or a pharmaceutical composition containing them into the cochlea of the inner ear of the subject. The engrafted cells may replace damaged or diseased SGN cells in the cochlea. In certain embodiments, multiple therapeutically effective doses of compositions comprising SGN-like cells are administered to a subject.

[0172] In certain embodiments, the SGN-like cells that are administered to a subject are autologous, allogeneic, or xenogeneic. Usually, but not always, the subject who receives the SGN-like cells (i.e. , the recipient) is also the subject from whom somatic cells for generating iPSCs or adult stem cells are harvested or obtained, which provides the advantage that the SGN-like cells produced from the iPSCs or adult stem cells are autologous. However, stem cells or somatic cells can be obtained fromanother subject (i.e. , donor), a culture of cells from a donor, or from established cell culture lines and differentiated into SGN-like cells, as described herein. Cells may be obtained from the same or a different species than the subject to be treated, but preferably are of the same species, and more preferably of the same immunological profile as the subject. Such cells can be obtained, for example, from a biological sample comprising somatic cells from a close relative or matched donor, induced into pluripotency to produce iPSCs, differentiated into SGN-like cells, and administered to a subject in need of treatment for an SGN-associated disorder. The patients or subjects who donate or receive the somatic cells, from which the iPSCs are generated, are typically mammalian, and usually human. However, this need not always be the case, as veterinary applications are also contemplated.Kits

[0173] Kit are provided to perform the subject methods for producing the SGN-like cells from stem cells, screening the SGN-like cells, or using the SGN-like cells in cellular therapy. In some embodiments, the kit comprises SGN-like cells. In some embodiments, the kit comprises agents for producing SGN-like cells from stem cells, including bone morphogenetic protein 4 (BMP4), a transforming growth factor-p (TGF- ) type I receptor inhibitor (e.g., SB-431542), basic fibroblast growth factor 2 (FGF2), a Wnt inhibitor (e.g., IWP-2), an inhibitor of ALK2 and ALK3 (e.g., LDN- 193189), Wnt family member 3A (WNT3A), sonic hedgehog (SHH), retinoic acid (RA), epidermal growth factor (EGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and ROCK inhibitors (e.g., Y-27632, H1152).

[0174] A kit may comprise media suitable for inducing differentiation of stem cells into SGN-like cells and culturing the SGN-like cells. In certain embodiments, the kit comprises a first medium comprising 10 ng / ml BMP4, 1 pmol / L SB-431542, and 10 ng / ml FGF2; a second medium comprising 100 nmol / L LDN-193189, 1 pmol / L SB-431542, 2 mmol / L IWP-2, and 10 ng / ml FGF2; a third medium comprising 100 ng / ml WNT3A, 10 ng / ml FGF2, 50 ng / ml IGF-1, and 10 pmol / L Y-27632; a fourth medium comprising 500 ng / ml SHH, 0.5 pmol / L RA, 20 ng / ml EGF, 10 ng / ml FGF2, and 50 ng / ml IGF-1 ; and a fifth medium comprisinglO ng / ml BDNF, 10 ng / ml NT-3, 10 ng / ml IGF-1, and 10 ng / ml H1152. Additionally, the kit may include buffers, tissue culture plates, flasks, test tubes, vials, and the like, and optionally one or more other factors, antibiotics, or other media supplements, and the like.

[0175] Such kits generally will comprise, in suitable means, distinct containers for each individual reagent or solution. Suitable containers for the compositions include, for example, bottles, vials, syringes, and test tubes. Containers can be formed from a variety of materials, including glass or plastic. A container may have a sterile access port (for example, the container may be a vial having a stopper pierceable by a hypodermic injection needle).

[0176] The kit may also provide a delivery device for administration of SGN-like cells to a patient. For example, kits may comprise a container having a sterile access port (e.g., the container may be a vial having a stopper pierceable by a hypodermic injection needle). The kit can further comprise a container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It can also contain other materials useful to the end-user, including other pharmaceutically acceptable formulating solutions such as buffers, diluents, filters, needles, and syringes or other delivery device.

[0177] In addition to the above components, the subject kits may further include (in certain embodiments) instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, and the like. Yet another form of these instructions is a computer-readable medium, e.g., diskette, compact disk (CD), DVD, flash drive, and the like, on which the information has been recorded. Yet another form of these instructions that may be present is a website address which may be used via the internet to access the information at a removed site.Examples of Non-Limiting Aspects of the Disclosure

[0178] Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure numbered 1-50 are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below:1. A method of producing spiral ganglion neuron (SGN)-like cells from stem cells, the method comprising:(a) culturing the stem cells in a first medium comprising bone morphogenetic protein 4 (BMP4), a transforming growth factor-|3 (TGF-f>) type I receptor inhibitor, and basic fibroblast growth factor 2 (FGF2);(b) culturing cells, produced from the stem cells in the first medium, in a second medium comprising the FGF2, the TGF- type I receptor inhibitor, a Wnt inhibitor, and an inhibitor of ALK2 and ALK3, wherein the cells differentiate into pre-placodal ectoderm and neural crest precursors;(c) isolating the pre-placodal ectoderm and neural crest precursors;(d) culturing the isolated pre-placodal ectoderm and neural crest precursors in a third medium comprising Wnt family member 3A (WNT3A), the FGF2, insulin-like growth factor-1 (IGF-1 ), and a non-selective Rho-associated protein kinase (ROCK) inhibitor;(e) culturing the cells, produced from the pre-placodal ectoderm and neural crest precursors in the third medium, in a fourth medium comprising sonic hedgehog (SHH), retinoic acid (RA), epidermal growth factor (EGF), the FGF2, and the IGF-1, wherein the cells differentiate into otic neural progenitor cells; and(f) culturing the otic neural progenitor cells in a fifth medium comprising brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), the IGF-1, cyclic adenosine monophosphate (cAMP), and a selective ROCK inhibitor to induce neuronal maturation of the otic neural progenitor cells into the SGN-like cells.2. The method of aspect 1 , wherein the stem cells are embryonic stem cells, adult stem cells, or induced pluripotent stem cells (iPSCs).3. The method of aspect 2, wherein the iPSCs are human induced pluripotent stem cells (hiPSCs).4. The method of aspect 3, wherein the hiPSCs are SK8-A hiPSCs.5. The method of any one of aspects 1-4, wherein the Wnt inhibitor is A / -(6-methyl-1 ,3-benzothiazol-2-yl)-2-[(4-oxo-3-phenyl-6,7-dihydrothieno[3,2-d]pyrimidin-2-yl)sulfanyl]acetamide (IWP-2).6. The method of any one of aspects 1-5, wherein the TGF-J3 type I receptor inhibitor is 4-[4-(1 ,3-benzodioxol-5-yl)-5-pyridin-2-yl-1 H-imidazol-2-yl]benzamide (SB-431542).7. The method of any one of aspects 1-6, wherein the inhibitor of ALK2 and ALK3 is 4-[6-(4-piperazin-1 -ylphenyl)pyrazolo[1 ,5-a]py rimidi n-3-y l]qu i no I i ne (LDN-193189).8. The method of any one of aspects 1-7, wherein the non-selective ROCK inhibitor is 4-[(1 R)-1 -aminoethyl]-N-pyridin-4-ylcyclohexane-1 -carboxamide (Y-27632).9. The method of any one of aspects 1-8, wherein the selective ROCK inhibitor is 4-methyl-5-[[(2S)-2-methyl-1 ,4-diazepan-1-yl]sulfonyl]isoquinoline (H1152).10. The method of any one of aspects 1 -9, wherein the pre-placodal ectoderm and neural crest precursors comprise TRA-1-60 pluripotent stem cell marker negative-nerve growth factor receptor positive (TRA-1-60-NGFR+) cells.11 . The method of aspect 10, wherein said isolating the pre-placodal ectoderm and neural crest precursors comprises using fluorescence-activated cell sorting or magnetic-activated cell sorting.12. The method of aspect 11 , wherein the magnetic-activated cell sorting is performed using magnetic microbeads conjugated to anti-TRA-1-60 antibodies and anti-NGFR antibodies.13. The method of any one of aspects 10-12, wherein step (d) comprising initially seeding the third medium with about 60,000 TRA-1-60 NGFR+cells / cm2for said culturing.14. The method of any one of aspects 1-13, wherein step (a) comprises said culturing the stem cells in the first medium for about 3 days.15. The method of any one of aspects 1-14, wherein step (b) comprises said culturing the stem cells in the second medium for about 5 days.16. The method of any one of aspects 1 -15, wherein step (d) comprises said culturing the cells in the third medium for about 7 days.17. The method of any one of aspects 1 -16, wherein step (e) comprises said culturing the TRA-1-60 NGFR+cells in the fourth medium for about 7 days.18. The method of any one of aspects 1-17, wherein step (f) comprises said culturing the otic neural progenitor cells in the fifth medium for about 6 days.19. The method of any one of aspects 1-18, wherein the first medium comprises 10 ng / ml BMP4, 1 pmol / L SB-431542, and 10 ng / ml FGF2.20. The method of any one of aspects 1-19, wherein the second medium comprises 100 nmol / L LDN-193189, 1 pmol / L SB-431542, 2 mmol / L IWP-2, and 10 ng / ml FGF2.21. The method of any one of aspects 1-20, wherein the third medium comprises 100 ng / ml WNT3A, 10 ng / ml FGF2, 50 ng / ml IGF-1, and 10 pmol / L Y-27632.22. The method of any one of aspects 1-21 , wherein the fourth medium comprises 500 ng / ml SHH, 0.5 pmol / L RA, 20 ng / ml EGF, 10 ng / ml FGF2, and 50 ng / ml IGF-1.23. The method of any one of aspects 1 -22, wherein the fifth medium comprises 10 ng / ml BDNF, 10 ng / ml NT-3, 10 ng / ml IGF-1, and 10 ng / ml H1152.24. The method of any one of aspects 1-23, further comprising isolating the SGN-like cells.25. The method of any one of aspects 1-24, further comprising characterizing the SGN-like cells by performing single-cell RNA sequencing, immunocytochemistry, whole-cell patchclamping, gene expression profiling, calcium ion (Ca2+) imaging, or any combination thereof.26. The method of any one of aspects 1-25, further comprising co-culturing the SGN-like cells with hair cells, glial cells, cochlear nucleus neurons, or any combination thereof.27. The method of any one of aspects 1-26, wherein the SGN-like cells comprise type I SGN-like cells and type II SGN-like cells.28. The method of aspect 27, further comprising isolating the type I SGN-like cells or the type II SGN-like cells.29. The method of any one of aspects 1-28, further comprising detecting one or more cellular markers to identify the SGN-like cells, wherein the one or more cellular markers are selected from tubulin beta 3 class III (TUBB3), neurofilament light polypeptide (NEFL), neurofilament heavy polypeptide (NEFH), POU class 4 homeobox 1 (POU4F1), neuronal differentiation 1 transcription factor (NEUROD1), SRY-box transcription factor 2 (SOX2), prospero homeobox 1 (PROX1),sodium / potassium-transporting ATPase subunit alpha-3 (NKAa3), calbindin-2 (CALB2), and Ly6 / PLAUR domain-containing protein 1 (LYPD1). peripherin (PRPH), tachykinin precursor 1 (TAG1), MAF bZIP transcription factor (MAFB), and GATA binding protein 3 (GAT A3).30. The method of any one of aspects 1-29, wherein the stem cells comprise a genetic mutation associated with hearing loss or deafness.31. The method of any one of aspects 1-30, wherein the SGN-like cells comprise a genetic mutation associated with hearing loss or deafness.32. The method of any one of aspects 4-31 , wherein the hiPSCs are derived from a somatic cell from a patient who has a genetic mutation associated with hearing loss or deafness.33. The method of aspect 32, wherein the somatic cell is obtained from a biopsy of the inner ear of the patient.34. The method of any one of aspects 1 -33, wherein the genetic mutation associated with hearing loss or deafness is introduced into the stem cells or the SGN-like cells by gene editing.35. The method of any one of aspects 30-34, wherein the genetic mutation associated with hearing loss or deafness is in a gene selected from TMPRSS3, NLRP3, FGF13, TBC1D24, GATA2, GATA3, DFNB59, GRM7, and ATP6V1 B2.36. A composition comprising SGN-like cells produced by the method of any one of aspects 1 -34.37. The composition of aspect 36, further comprising glial cells, hair cells, cochlear nucleus neurons, or any combination thereof.38. The composition of aspect 36 or 37, further comprising a pharmaceutically acceptable excipient.39. A method of screening a candidate agent to determine its effects on SGN-like cells, the method comprising:contacting the SGN-like cells produced by the method of any one of aspects 1-35 with the candidate agent; anddetermining the effects of the candidate agent on morphological, electrophysiological, genetic, or functional parameters of the SGN-like cells.40. The method of aspect 39, wherein the SGN-like cells comprise a genetic mutation associated with hearing loss or deafness.41 . The method of aspect 39 or 40, wherein said determining the effects of the candidate agent comprises performing single-cell RNA sequencing, immunocytochemistry, whole-cell patchclamping, gene expression profiling, calcium ion (Ca2+) imaging, confocal microscopy, atomic force microscopy, super-resolution microcopy, light-sheet microscopy, two-photon microscopy, fluorescence microscopy, migration assays, axonal growth and pathfinding assays, or any combination thereof.42. The method of any one of aspects 39-41 , further comprising contacting the SGN-like cells with glial cells, hair cells, cochlear nucleus neurons, or any combination thereof.43. The method of aspect 42, further comprising detecting the effects of the candidate agent on synapses between the SGN-like cells and the cochlear nucleus neurons or the hair cells.44. The method of aspect 43, wherein the synapses are glutamatergic synapses.45. The method of any one of aspects 42-44, further comprising detecting the effects of the candidate agent on growth of neurites of the SGN-like cells toward the hair cells.46. The method of any one of aspects 42-45, further comprising detecting the effects of the candidate agent on interactions of the glial cells and the SGN-like cells.47. The method of any one of aspects 39-46, wherein the SGN-like cells are cultured in a two-dimensional (2D) culture system.48. A method of treating a spiral ganglion neuron-associated disorder in a subject, the method comprising administering a therapeutically effective amount of the composition of aspect 38 locally to the ear of the subject.49. A composition comprising the SGN-like cells produced by the method of any one of aspects 1-35 for use in treating a spiral ganglion neuron-associated disorder.50. Use of the SGN-like cells produced by the method of any one of aspects 1-35 in the manufacture of a medicament for treating a spiral ganglion neuron-associated disorder in a subject.EXPERIMENTAL

[0179] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

[0180] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

[0181] The present invention has been described in terms of particular embodiments found or proposed by the present inventor to comprise preferred modes for the practice of the invention. It will be appreciated by those of skill in the art that, in light of the present disclosure, numerous modifications and changes can be made in the particular embodiments exemplified without departing from the intended scope of the invention. All such modifications are intended to be included within the scope of the appended claims.Example 1Engineering of functional auditory neurons from human induced pluripotent stem cellsIntroduction

[0182] The sensory hair cells that transduce sound within the mammalian inner ear are innervated by dendrites of spiral ganglion neurons (SGNs) (1, 2). Axon bundles of SGNs project from the cochlea to the central nervous system as cranial nerve VIII to transmit auditory signals for higher processing (3-5). Loss or damage of the SGNs due to diverse etiologies, including aging (6, 7), noise exposure (8), ototoxic drugs (9), infection (10), and hereditary defects (11 , 12), is a leading cause of permanent hearing impairment in humans. SGN degeneration is often thought to occur secondarily due to the loss of hair cells that normally provide essential trophic support (13, 14). However, several studies in humans have demonstrated that primary degeneration of SGNs can occur in the presence of morphologically intact or minimally damaged hair cells, particularly in cases of aging (15), exposure to certain ototoxic drugs (9, 16), viral infections (10, 17), certain rheumatologic conditions (10), and genetic mutations affecting neuronal survival (10, 18, 19) and synaptic transmission (12). Notably, in age-related hearing loss, primary SGN degeneration accounts for an estimated 15 - 30% of cases in humans (7, 15).

[0183] A fundamental obstacle in the study of human SGNs, and the development of therapeutic strategies for their restoration, is the lack of established and well-characterized in vitro models. It is not possible to non-destructively obtain human inner ear tissue biopsies due to the small size of the human cochlea, its complex three-dimensional (3D) anatomy, and encasement in dense bone. To overcome this challenge, there have been multiple attempts to create mouse primary SGN cell lines (20) or, more recently, to generate otic neurons from human stem cells (21-29). To date, these methods have yielded cells of uncertain identity and function. In particular, the absence of SGN- specific markers, critical for defining human SGNs from other possible neuron types, has led to ambiguity regarding the identity of cells generated from pluripotent stem cells in prior protocols (21- 26). Thus, there is an urgent need for further improvement in the method of SGN generation as well as a multi-factorial, quantitative understanding of the cells’ identity to establish their utility in otologic research.

[0184] Here, by mimicking the cell-to-cell signaling occurring during human inner ear development in vivo, we engineered an optimized protocol for the generation of functional human SGN-like neurons via otic neurosensory progenitor (ONP)-like cells derived from human induced pluripotentstem cells (hiPSCs). This study aimed to characterize their morphological, molecular, electrophysiological, and functional features to demonstrate their cellular identity and support their utility as effective in vitro models of human SGNs.Methods2.1 hiPSC culture

[0185] hiPSC lines SK8-A, generated in our laboratory (30), and UCSD112i-2-11 (UCSD), purchased from WiCell (Madison, Wl, USA), were used before passage number 50. Cells were maintained on Matrigel human embryonic stem cell (hESC) qualified matrix (Cat# 354277, Corning, USA) in mTeSRI (Cat# 85850, StemCell Technologies, Canada) or mTeSR plus medium (Cat# 100- 0276, StemCell Technologies, Canada) supplemented with 1x penicillin-streptomycin (Cat# 15140122, Gibco, USA). Detailed culture conditions, reagents, and catalog numbers are provided in2.2 Differentiation of hiPSCs

[0186] For differentiation, we modified a previously published protocol (22) and optimized it for better differentiation into SGNs using chemically defined media (CDM). In our study, undifferentiated hiPSC lines SK8-A and UCSD were dissociated with TrypLE Select (Cat# 12563011 , Gibco, USA) and seeded at 10,000 cells / cm2(SK8-A) or 8000 - 10,000 cells / cm2(UCSD) onto growth factor-reduced Matrigel (Cat# 356230, Corning, USA) in mTeSR. The concentration of Y-27632 (Cat# 1254, TOCRIS, UK), an inhibitor of Rho-associated protein kinase used to enhance stem cell survival in culture, was maintained at 10 pmol / L throughout DO - 2. On D3, the medium was replaced with a CDM containing 10 ng / ml bone morphogenetic protein 4 (BMP4; Cat#314-BP, R&D Systems, USA), 1 pmol / L SB-431542 (Cat# 1614, TOCRIS, UK), and 10 ng / ml basic fibroblast growth factor 2 (FGF2, Cat# 233-FB, R&D Systems, USA). The CDM contained a 50:50 mixture of DMEM / F12 (Cat# 11330032, Gibco, USA) and Neurobasal medium (Cat# 21103049, Gibco, USA), additionally supplemented with N2 [1% (v / v) final concentration, Cat# 17502048, Gibco, USA], B27 [2% (v / v) final concentration, Cat# 17504044, Gibco, USA], 2 mmol / L L-glutamine (Cat# 25030032, Gibco, USA), 0.1 mmol / L 2-mercaptoethanol (Cat# 21985023, Gibco, USA), and 50 ng / ml Normocin (Cat# ant-nr, InvivoGen, USA). On D6, the medium was changed to CDM supplemented with 100 nmol / L LDN-193189 (Cat# 04-0074-02, Stemgent, USA), 1 pmol / L SB-431542, 2 mmol / L IWP-2 (Cat# 3533, TOCIRS, UK), and 10 ng / ml FGF2.

[0187] On D11 , TRA-1 -60 (pluripotent stem cell marker)7nerve growth factor receptor (NGFR)+cells were collected by using magnetic sorting with anti-TRA-1-60 (Cat# 130-100-832, Miltenyi Biotec,Germany) and anti-NGFR (Cat# 130-097-127, Miltenyi Biotec, Germany) microbeads. A full version of the sorting protocol is available on Miltenyi Biotec’s website (https: / / www.miltenyibiotec.com / US- en / ). After purification, the cells were seeded at 60,000 cells / cm2on growth factor-reduced Matrigel- coated plates in CDM with 100 ng / ml Wnt3a (Cat# 5036-WN, R&D Systems, USA), 10 ng / ml FGF2, 50 ng / ml insulin-like growth factor-1 (IGF-1, Cat# 291 -G1, R&D Systems, USA), and 10 pmol / L Y- 27632. On D18, the medium was replaced with CDM containing 500 ng / ml sonic hedgehog (SHH, Cat# 1845-SH, R&D Systems, USA), 0.5 pmol / L retinoic acid (RA, Cat# 0695, TOCRIS, UK), 20 ng / ml epidermal growth factor (EGF, Cat# 236-EG, R&D Systems, USA), 10 ng / ml FGF2, and 50 ng / ml IGF-1. On D22, coverslips or culture plates were coated with 0.1 mg / ml poly-ornithine (Cat# P3655, Sigma, USA) diluted in 1x borate buffer (Cat# 28341, Thermo Scientific, USA) for > 3 h at room temperature followed by 20 pg / ml laminin (Cat# 3400-010-02, R&D Systems, USA) in Hank’s Balanced Salt Solution (HBSS; Cat# 14025076, Gibco, USA) for 3 d at 4°C. During D3 - 25, the medium was changed every day. On D25, cells were treated with TrypLE Select and gently detached by cell scrapers. The cells were seeded at 500,000 cells / cm2on poly-ornithine and laminin-coated coverslips or plates in Neurobasal medium supplemented with 1% N2, 2% B27, 1 mmol / L GlutaMAX (Cat# 35050061, Gibco, USA), 0.5 mmol / L dibutyryl-cyclic adenosine monophosphate (cAMP, Cat# SC-201567, Santa Cruz Biotechnology, USA), 10 ng / ml brain-derived neurotrophic factor (BDNF, Cat# 248-BDB, R&D Systems, USA), 10 ng / ml NT-3 (Cat# 267-N3, R&D Systems, USA), 10 ng / ml IGF-1, 50 ng / ml Normocin, and 10 ng / ml H1152 (Cat# 2414, TOCRIS, UK). The medium without H1152 was replaced every 2 d from D26 to D30. Half of the medium without H1152 was replaced every 3 d afterward.2.3 Human subjects

[0188] Human vestibular inner ear tissue was obtained during surgical labyrinthectomies and translabyrinthine resections of vestibular schwannomas (n = 2). Tissue was used for immunohistological comparison with hiPSC-derived SGN-like neurons.2.4 Single-cell RNA sequencing (scRNA-seq)

[0189] SGN-like cells differentiated from SK8-A and UCSD hiPSCs were dissociated at multiple developmental time points (D25, D60, D90, and D120) and processed for scRNA-seq using the Chromium platform (10* Genomics). Libraries were prepared according to manufacturer protocols and sequenced to sufficient depth to ensure robust transcriptomic coverage.2.5 scRNA-seq data analysis

[0190] Raw sequencing data were processed using 10x Genomics Cell Ranger 2.1.0 pipeline (support.10xgenomics.com / ) and analyzed in Seurat. Low-quality cells and doublets were removed based on standard filtering criteria. Dimensionality reduction, clustering, and marker identification were performed using established workflows. Cross-species integration with published mouse datasets was conducted using Harmony to assess developmental correspondence. Detailed filtering thresholds, software versions, and analysis parameters are provided in Methods.2.6 Immunocytochemistry and imaging

[0191] Cells and co-culture samples were fixed, immunolabeled, and imaged using confocal microscopy. Standard blocking, antibody incubation, and imaging procedures were used. Z-stack imaging and 3D reconstruction were applied where appropriate to assess synaptic contacts.2.7 Electrophysiological recordings

[0192] Whole-cell patch-clamp recordings were performed on hiPSC-derived SGN-like neurons at D88 - 95 or D120 - 234. Neuronal intrinsic properties, action potential (AP) firing patterns, and voltage-gated currents were assessed under current- and voltage-clamp configurations.2.8 Animal models and co-culture systems

[0193] A total of 36 mice of either sex were used in this study, including 31 CBA / CaJ wild-type mice and 5 NOD / SCID wild-type mice. Of these, 35 mice were postnatal (P3 - 6) and 1 mouse was adult (8 weeks). Postnatal and adult mice were used for cochlear explant and cochlear nucleus (CN) coculture experiments. Explants were positioned near hiPSC-derived SGN-like neurons to assess neurite outgrowth and synaptic connectivity. Cultures were maintained under controlled conditions and analyzed by immunofluorescence.2.9 Ca2+imaging

[0194] Ca2+imaging was used to assess synaptic activity in human SGN-like neurons co-cultured with mouse hair cells or CN neurons. Fluorescent Ca2+indicators were applied, and activity was recorded before and after pharmacological blockade of glutamatergic transmission.2.10 Statistical analysis

[0195] Unless the purpose was solely for structural observation, immunostaining was quantified using Imaged and reported as the proportion of cells positive for the stain. All immunostaining imagesare representative of at least 3 replicates, except for those involving human inner ear tissue, which used two technical replicates due to scarcity of the tissue.

[0196] Electrophysiology recordings were obtained by combining data of 4 and 3 independent experiments with cell lines D88 - 95 and > D120 SK8-A, respectively, and 1 and 4 independent experiments with cell lines D88 - 95 and > D120 UCSD, respectively.

[0197] For Ca2+imaging experiments of human SGN-like neurons, statistical comparisons were performed using unpaired Mann-Whitney tests. Human SGN-like neurons generated from three different passage numbers of hiPSCs were analyzed. Ca2+signals were normalized to the mean baseline intensity.

[0198] All statistical analyses were conducted in GraphPad Prism 9.2.0 software. A P < 0.05 was used to determine significance. The number of technical and biological replicates in each experiment are also listed in the figure captions.Results3.1 Generation of human ONP-like cells from hiPSCs

[0199] Mammalian inner ear cells and their neighboring cells arise from two embryonic origins: the otic placode from the pre-placodal region (31) and cranial neural crest (ONG) cells from the neural tube (32, 33). The otic placode gives rise to an otic vesicle, which is the origin of most cell types in the inner ear. The cells in the otic vesicle undergo proliferation and then differentiation, with higher expression of SHH in the ventral versus dorsal cochlear duct specifying the future cochlea and vestibule, respectively (34, 35). CNC cells are a source of glial cells and periotic mesenchyme in the inner ear in vivo (36, 37). With these developmental cues in mind, we generated pre-otic fate cells and their neighboring cells, including pre-placodal ectoderm and neural crest cells, from hiPSCs (SK8-A) (FIG. 1 A). On D11 of differentiation, we performed magnetic-activated cell sorting to isolate NGFR+cells, which are known to be enriched for pre-placodal ectoderm and neural crest (38, 39) during human development (40). As the survival and appropriate motility of these cells are highly sensitive to cell-cell interactions (41) and low density promotes non-neural ectoderm (42), we improved upon a previous protocol (22) by optimizing the cell density at 60,000 cells / cm2on D11.

[0200] After cell sorting, we observed that low seeding density (25,000 cells / cm2) of precursors of pre-placodal ectoderm and neural crest on D11 negatively impacted development into SGN-like cells (FIG. 8A). The cell numbers gradually decreased beginning around D25, with only < 5 cells / cm2surviving by D63 (FIG. 1 B). Low seeding density also negatively affected neurite length. The longest length of neurons stained with tubulin beta 3 class III (TUBB3) and neurofilament (NEFL), from axonto dendrite, was < 200 pm (FIG. 1 B; FIG. 8B). We subsequently determined that seeding 60,000 cells / cm2of precursors of pre-placodal ectoderm and neural crest on D11 after sorting was the appropriate cell density (FIGS. 8C, 8D); 98.3% [with standard deviation (SD) 1.8%] of sorted cells were NGFR+on D12 (FIG. 1C). To induce ONP-like cells and mimic essential in vivo signaling cues ultimately directing to an SGN fate, the pre-placodal ectoderm and neural crest cells were grown in medium containing SHH between D18 - 25 (22). This protocol reproducibly generated a donutshaped cluster of ONP-like cells [neurogenic differentiation 1 (NEUROD1)+] and otic progenitors [paired box 2 (PAX2)+, PAX8+, and SRY-box transcription factor 2 (SOX2)+] (FIGS. 1D-1G), which was replicated in another hiPSC cell line (UCSD) (FIGS. 8E-8H).3.2 Generation of neurons with human SGN-like properties from ONP-like cells

[0201] Under a cocktail of BDNF, neurotrophin-3 (NT3), and IGF-1 , which are endogenous neurotrophins essential for the development and maintenance of SGNs (22, 43), neurites began to grow out from the neuronal body around D35. Between D35 and D90, the neurites transitioned from having complex branching patterns to a classic bipolar configuration as small branches disappeared (FIG. 9), resembling the loss of branches in vivo as SGNs extend their neurites into the cochlea and central nervous system (44). At D90, the expression of the glutamatergic neuronal marker vesicular glutamate transporter 1 (VGLUT1), which packages glutamate into synaptic vesicles, further suggested that these hiPSC-derived neurons have shared properties with SGNs (FIGS. 9D, 9M).

[0202] In vivo, mature SGNs consist of two main populations that are functionally distinct and can be identified by the absence (type I) or presence (type II) of the peripheral neuronal marker peripherin (PRPH) (45). In the organ of Corti, the sensory epithelium of the cochlea, type I myelinated neurons innervate inner hair cells, whereas type II non-myelinating neurons innervate outer hair cells (1, 2, 46). In our in vitro system, at D35, (89.4 ± 9.5)% of early-stage human SGN-like neurons expressed both the pan-neuronal marker TUBB3 and PRPH, suggesting that specific type I and type II neurons are not differentiated at this stage in culture. However, PRPH expression was gradually lost between D35 and D90; at D90, (94.6 ± 3.9)% of cells expressed only TUBB3 and not PRPH, fitting a molecular phenotype of type I SGNs (FIGS. 2A, 2B). The small percentage of neurons that remained PRPH+expressed additional markers of type II SGNs, such as GATA binding protein 3 (GATA3) or tyrosine hydroxylase, further supporting their molecular type II identity (FIGS. 9E, 9F). This differentiation process in vitro corresponds to both mouse (47, 48) and human (49) auditory neuron development in the cochlea. The protein expression pattern at approximately D35 was comparable to that seen at roughly around 10 weeks of gestation (W10), and approximately D90 was comparable to W18 - 20,which is when PRPH expression distinguishes type I (PRPH ) from type II (PRPH+) SGNs in vivo (49).

[0203] In the human cochlea, peripheral glial cells envelop or myelinate the SGNs and promote their survival, aid in synapse formation and pruning, and provide nutrient and metabolic support (50, 51). We performed immunostaining to investigate whether the glial cells were engaged in a specific organized structure with the human SGN-like neurons. From around D90, the glial cell marker glial fibrillary acidic protein (GFAP) was observed in clusters of SGN-like cell somas and in neurites (FIG.2C). At higher magnification, the cell bodies of human SGN-like neurons were observed to be partially surrounded by satellite glia-like cells (FIG. 2D). In addition, 3D cross-section images showed that myelinating [myelin protein zero (MPZ)+] and non-myelinating (NGFR+) Schwann cells wrapped the axons of types I and II human SGN-like neurons, respectively (FIG. 2E; FIGS. 9G, 9P, 9Q). The formation of myelinating fibers along human SGN-like neurons was comparable to Schwann cells’ ensheathment observed in human adult inner ear tissue (52) (FIGS.2F, 2G). Together, this evidence demonstrates that our culture system simulates the gradual morphological development of SGNs in the cochlea and their interaction with peripheral glial cells, as previously observed in humans (49). Importantly, this differentiation process was phenocopied using the UCSD cell line (FIGS. 8, 9).3.3 Lineage origins of the human SGN-like neurons

[0204] To understand the molecular diversity of cell lineages arising in developing auditory neurons (FIG. 3A), we performed scRNA-seq at D25. Downstream analyses were conducted after initial quality control and doublet removal (FIGS. 10A-10G). D25 clustering (3896 cells), visualized via uniform manifold approximation and projection (UMAP), suggested two transcriptionally distinct groups of cells originating from the proteolipid protein 1 (PLP1)+CNC (43.7%) and SOX2+otic vesicle (55.8%) (FIG. 3B).

[0205] The composition of the cell types translated to a differentiation efficiency rate of 99.5% inner ear-related cells and only 0.5% keratinizing epithelium. The CNC-originated group comprised three clusters: CNC cells [PLP1+Forkhead box D3 (FOXD3)+, 6.0%], mesenchyme [Twist-related protein 1 (TWIST1)+Lumican (LUM)+TGFB Decorin (DCN)+, 20.3%), and mesenchyme-cycling (TWISTFLUIWTGFB DCN* DNA topoisomerase II alpha (TOP2Ay. 17.4%) (FIG. 3C), similar to prior reports in mouse (53). Periotic mesenchymal cells are the most numerous cell types within the cochlea, influencing SGN peripheral axon formation, organization, and innervation in vivo; gene mutations in these cells cause hearing loss (11). Accordingly, more than one-third (37.7%) of the mesenchymal population in this culture is expected to support the generation of SGN-like cells (FIG.3B). No cell clusters expressed early mesodermal or endodermal lineage markers such as T-boxtranscription factor T (TBXT) or S0X17, suggesting that the mesenchyme cells in the culture arose from a CNC rather than a mesodermal lineage.

[0206] The otic vesicle-originating group diverged into otic vesicle-like cells corresponding to ventro- anterior [Wnt family member 4 (WNT4)+Hes family bHLH transcription factor 5 (HES5)+, 27.7%] and ventro-medial [Fibroblast growth factor 8 (FGF8)+PAX2 18.9%) fates in vivo (FIGS. 3B, 3C) (54). The ventro-anterior cells were closely aligned with cochleovestibular ganglia (CVG), resembling in vivo neuroblasts (the origin of SGNs and vestibular ganglion neurons), delaminating from the otic vesicle through epithelial-mesenchymal transition (EMT) and differentiating into neurons, as previously described (55). No cell clusters expressed markers involved in suppressing CVG formation, such as TBX1 , in the dorsal domain of the otic vesicle (56). As in vivo, the cluster of otic neural primordium consisted of EMT cells [TWIST1+Keratin 18 (KRT18)+Vimentin (VIM)+], delaminating neuroblasts (NEUROD1+HES ), and cells undergoing neurogenesis (TLIBB3* DoublecortinlDCX)+POU Class 4 Homeobox 1 (POU4F1)+] (FIGS. 3D-3F). Similar to observations from mouse E10.5 neuroblasts (57), the broad expression of neuronal marker TUBB3 was evident in otic neural primordium, particularly in the neurogenesis cluster; doublecourtin (DCX), a late neuroblast- and young neuron-marker (58), was also most abundantly expressed in the neurogenesis cluster (FIG. 3F). Additionally, cells involved in EMT and neuroblast phases exhibited high expression of proliferative markers such as TOP2A (FIG. 3F). However, as the neurogenesis process progressed, these cells lost their proliferative capacity, differentiating into neurons. Within the otic neural primordium cluster, a larger proportion of these neurons expressed genes suggesting SGN-like identity, such as meis homeobox 2 (MEIS2, 48.7%) and prospero homeobox protein 1 (PROX1, 21 .0%), compared to those associated with early vestibular ganglion neurons such as T Cell leukemia homeobox 3 (TLX3, 9.0%), spalt like transcription factor 3 (SALL3, 0%), and parvalbumin (PVALB, 0%) (59) (FIG. 3G). Additionally, 41.7% of the cells expressed INSM1 (Additional file 5), which is reported to be expressed in both otic neural progenitors and early SGNs (60). The low expression of genes specific to hindbrain neurons, geniculate ganglion of the facial nerve adjacent to the inner ear, and epibranchial placode (FIG. 3H) reinforces the specificity of the auditory neuron differentiation. To ensure experimental robustness, we repeated the scRNA-seq experiments with another cell line (UCSD) on D25, yielding consistent results (FIGS. 10H-10J).3.4 Diversity of human SGN-like neurons

[0207] To map a transcriptional trajectory of the development of the human SGN-like neurons, scRNA-seq was conducted at three additional differentiation time points: D60, D90, and D120. The neuron clusters were isolated based on the expression of a range of pan-neuronal markers such asTUBB3 and sodium / potassium-transporting ATPase subunit beta-1 ATP1B1) (FIG. 4A). Although the UMAP visualization of the entire dataset suggests maturity progression from D25 to D60, D90, and D120, with the absence of a type II cluster at D25, we did not observe distinct, segregating clusters during D60 - 120 (FIG. 4B). This implies a similar gene expression pattern among D60 - 120 stages, albeit with some changes insufficient to delineate new distinct developmental clusters.

[0208] At D60, the efficiency rate of inner ear-related cell differentiation remained high (98.4%) in culture. Examining human SGN-like neuron heterogeneity, we visualized isolated TUBB clusters (26.1%) via UMAP, revealing 6 distinct neuronal clusters, including early and intermediate human SGN-like neurons (FIG. 4C). Presumed types I and II human SGN-like neurons fell into distinct groups enriched for genes such as calcium / calmodulin-dependent protein kinase II inhibitor 1 (CAMK2N1) and PRPH^iG.4D). Overall, types I and II human SGN-like neurons composed 94.7% and 5.3% of the cell population, respectively, similar to histological estimates of their proportions in vivo (61).

[0209] To determine whether the human SGN-like neurons are undergoing differentiation or if specific subtypes are emerging, we conducted a correlation analysis between our D60 human SGN- like neurons and previously published data from E14 - P1 mouse SGNs (48). Early and intermediate human SGN-like neurons closely aligned with E14 - 16, while type I neurons overlapped with E18- P1 , a stage at which all SGN subtypes are transcriptionally identifiable (FIG. 4E; FIG. 11 A). Indeed, the potential type I neurons could be further separated based on unique and combinatorial molecular profiles into clusters a [glycine receptor alpha 3 (GLRA3)+, roundabout guidance receptor 2 (R0B02)+, and rabphilin 3A (RPH3A+)], 0 [ROBO , LIM Homeobox 9 (LHX9)+, and LY6 / PLAUR domain containing 1 (LYPD1)+], and y [LYPD1+, semaphorin 5A (SEMA5A)+, and POU4F1+] (FIG.4F), although it is premature to conclude whether the clusters represent various differentiation stages or are comparable to mature SGN subtypes. In addition, each sub-cluster had differential expression of genes related to neuronal transmission, including synaptic vesicles, neurotransmitter transporters, Ca2+binding proteins, and neurotransmitter receptors; and the difference was usually more prominent between types I and II (FIGS. 11 B, 11C).

[0210] Regarding afferent cochlear neurotransmission, the primary neurotransmitter released by hair cells is glutamate (62, 63). Our findings revealed the expression of various glutamatergic receptor subunits known to be expressed in SGNs (FIG. 4G; FIG. 11E). Glutamate ionotropic receptor AMPA type subunit 2 (GRIA2) and subunit 4 (GRIA4) are the two main a-amino-3-hydroxy- 5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits found in SGNs (64). GRIA2 makes AMPA receptors Ca2+-impermeable and is postulated to be protective from glutamatergic excitotoxicity and hearing loss later in life (65). Expression of GRIA3, although lower than that ofGRIA2 and GRIA4 (FIG. 11 E), is important for regulating the AMPA receptor subunit stoichiometry in ribbon synapses, modulating ribbon morphology and minimizing GluA2-lacking AMPA receptors that flux Ca2+(65). Glutamate ionotropic receptor NMDA type subunit 2B (GRIN2B) is an early developmental N-methyl-D-aspartate (NMDA) receptor subunit responsible for neuronal survival and glutamatergic transmission (66); its expression in SGNs has been demonstrated previously in rats (64). In addition to glutamatergic receptors, we report high expression of gamma-aminobutyric acid (GABA)-B receptors in both the type I and type II human SGN-like neurons (FIG. 4G; FIG. 11 F), as reported previously in rodents (67). GABA-B receptors are found in SGN afferents under inner and outer hair cells, and these receptors retrogradely modulate outer hair cells amplifier function in type II SGNs (67). The expression of synaptic receptors remained relatively constant during D60 - 120 (FIGS. 11D-11F).

[0211] Expression of synaptic vesicle-associated protein release machinery (e.g., SYT11, SYN1, and SYNGR1), neurotransmitter transporters (e.g., SLC17A6), and Ca2+binding proteins (e.g., CALM1 and CALM3), were also found in the human SGN-like neurons (FIG. 4G). Together, the transcriptomic data indicate that the human SGN-like neurons acquire a “kit” of synaptic receptors and vesicle release molecules that enable human SGN-like neurons to receive glutamatergic excitatory transmission, modulate their synaptic partners via GABA-B metabotropic receptors, and release glutamate themselves.

[0212] The maturity of the human SGN-like neurons is also reflected in the set of ion channel subunits they express (68, 69). Ion channel composition determines neuronal intrinsic excitability and spiking properties, and modulates integration of synaptic inputs (68). The human SGN-like neurons strongly up-regulated various potassium (K+) and sodium (Na+) channel subunits (FIG. 4H) around D120, much later than the observed expression of synaptic receptors and proteins, suggesting that neuronal intrinsic excitability matures in vitro after D120.

[0213] In addition to the human SGN-like neurons, there was a glia cluster (1.2% of cells) expressing pan-Schwann cell markers [S100 calcium binding protein B (S100B) and Erb-B2 receptor tyrosine kinase 3 (ERBB3)], a key Schwann cell lineage transcription factor (SGX10), myelin proteins (PLP1 and MPZ), and other regulators of myelination [crystallin alpha B (CRYAB)] (FIG. 4I). A subset of these cells (31.9%) also expressed NGFR (FIG. 4I), a marker of type II SGNs, non-myelinating Schwann cells, and satellite glial cells (70).

[0214] Taken together, these findings suggest that the human SGN-like neurons generated via our method are comparable to type I and type II in vivo based on their transcriptomic signatures of synaptic and intrinsic excitability-mediating genes, alongside a distinct population of surrounding glial cells.3.5 Intrinsic excitability of human SGN-like neurons: electrophysiological characterization

[0215] Whole-cell patch-clamp was used to determine whether the human SGN-like neurons can elicit APs and evaluate other electrophysiological properties important for overall excitability. Recordings were performed on human SGN-like neurons generated from SK8-A and UCSD hiPSC lines at two time points, D88 -95 and D120-235 (> D120), to compare parameters before and after up-regulation of K+- and Na+-channel subunits (FIG. 4H). During recordings, human SGN-like neurons were filled with biocytin for post-hoc classification of either type I or type II by staining for PRPH (FIG. 5A). Type II human SGN-like neurons constituted approximately 10% of the recorded cells (4 / 31 at D88 - 95 and 2 / 26 at >D120), as reported in vivo (69); therefore, we focused on characterizing the more prevalent type I neurons (approximately 90% of recorded cells).

[0216] First of all, we tested for spiking response patterns in current-clamp mode. Two distinct neuronal groups were revealed based on their responses to step current injections: multi-spike accommodating neurons [MA; n = 21 (41%)] and unitary-spike accommodating neurons [UA; n = 30 (59%)] (FIG. 5B). UA neurons did not fire more than two APs in response to current steps across a range of stimuli intensities, while MA neurons fired multiple APs and increased their firing frequency with incremental current amplitudes (FIG. 12A). These results indicate that, like developing SGNs from other mammalian species, type I human SGN-like neurons exhibit two main subclasses based on their firing properties, likely coinciding with expression pattern of different K+- and H-type currents (68, 71-73). MA and UA type I human SGN-like neurons differed in their membrane time constant, rheobase (the minimum current intensity necessary to evoke an AP), and latency to the first evoked AP (time constant statistically different only at > D120) (FIGS. 5C, 5D). These findings are consistent with prior reports in rodent SGNs (71). Notably, the rheobase values increased by almost two-fold between D88 - 95 and > D120 (FIG. 5D), along with hyperpolarized resting membrane potential (FIG. 12D), consistent with the upregulation of K+channels (i.e., KCNQs) responsible for maintaining negative resting membrane potential (74) (FIG. 4H).

[0217] Given the increase in the expression of Na+and K+channel subunits responsible for fast rise [e.g., sodium voltage gated channel alpha subunit 1 (SCN1A) coding for Nav1.1] and rapid repolarization [e.g., potassium voltage gated channel subfamily A member (KCNA)1 - 5 for Kv1-5 and potassium voltage gated channel modifier subfamily G member 4 (KCNG4) for Kv6.4.] of APs, and enabling high frequency firing [e.g., potassium voltage gated channel subfamily C member 2 (KCNC2) and 4; Kv3.2 and 3.4 (75)] in the human SGN-like neurons between D90 and D120 (FIG.4H), we compared changes in several AP parameters between these timepoints to see if the direction of changes is as predicted by the transcriptome (FIGS. 5C, 5E; FIGS. 12B, 12C). Indeed, as Na+channel subunit expression [e.g., sodium voltage gated channel alpha subunit 1 (SCNA1A)] increased from D90 to D120, firing threshold lowered and AP amplitude almost doubled for both MA and UA, resulting in an overshooting AP characteristic of neurons in vivo. AP half-width decreased along with an increase in K+channel abundance (Kv1 and Kv3 channels; KGNAs and KCNCs) and their lower activation threshold (KCNG4 subunit) (76, 77) (FIG. 5E). In addition to the changing active properties due to ion channel composition, passive properties also changed; between D88 - 95 and > D120, there was an increase in somatic diameter, and consequently, membrane capacitance (Cm) was increased (FIG. 5F). Increase in Cm likely contributed to the longer latency to first AP (FIG. 5E).

[0218] To directly evaluate changes in ion channel currents, we performed whole-cell recordings of type I human SGN-like neurons in voltage-clamp configuration. We observed fast inward currents that peaked at holding voltages of -30 mV and long-lasting steady outward currents (FIGS. 5G, 5H), which is consistent with the reported current-voltage (I - V) profiles for inward Na+- and Ca2+-current and outward K+-mediated currents in mouse and rat SGNs (68, 78, 79). The amplitudes of putative Na+- and K+-mediated currents were larger in > D120 as compared to D88 - 95 human SGN-like neurons (FIGS. 5G-5I), in accordance with the increase in mRNA levels for these channels at these two differentiation time points (FIG. 4H). Type I human SGN-like neurons also exhibited a voltage sag in response to large hyperpolarizing current steps in current-clamp configuration, indicating the presence of the mixed conductance Na+ / K+-mediated lhcurrents as previously reported (80). Consistent with the presence of the voltage sag, increasing expression of hyperpolarization activated cyclic nucleotide gated potassium channel (HCN) 1 - 3 subunits that build HCN channels was detected in increasing numbers of human SGN-like neurons from D60 - 120 (FIG. 12E), similar to developing mouse ear (81). Despite the increase in HCN expression, voltage sag significantly decreased between D88-95 and > D120 (P< 0.0001), likely due to the increase in leak K+channels, like potassium two pore domain channel subfamily K member 9 (KCNK9) (FIG. 4H), contributing to shunting of currents at membrane potentials around rest (82) (FIGS. 12F-12H).

[0219] Together, the results indicate that the active electrophysiological properties of the human SGN-like neurons coincide with the developmental increase in mRNA expression of ion channel subunits responsible for the reliable generation and propagation of APs (Navi .1 and Kv1 s), enabling and modulating high-frequency firing properties of critical importance for encoding auditory inputs [Kv1s, Kv3s, and human Ether-a-go-go-related gene (hERGs)] (77, 83), and fine-tuning of the neurons’ excitability (HCNs and leak channels). These changes, along with growth that affects passive properties of neurons, point to a maturation sequence for the human SGN-like neurons that results in functional profiles similar to those described for type I SGNs ex vivo (71-73).3.6 Human SGN-like neurons form functional synapses with hair cells

[0220] In mammals, SGNs are the bridge between the detection of physical sound by hair cells and the perception of that sound by the brain (1 , 2). Therefore, to validate the functionality of our human SGN-like neurons, we first examined their potential to form synaptic connections with hair cells, their only presynaptic partner in vivo. To achieve this, we used a mouse cochlear hair cell explant coculture system (FIG. 6A) (23, 24, 26). Mouse hair cell cochlear explants could potentially contain mouse SGNs. Therefore, before proceeding with the functional experiment, we confirmed the absence of mouse SGNs in our co-cultures by test-running explants from Thy1 -GFP BL6 transgenic mice with GFP+SGNs. Explants cultured for 14 d displayed no GFP-positive neurons, indicating that endogenous mouse SGNs did not survive in these conditions (FIG. 13A). Therefore, any neuronal activity observed in the co-cultures was exclusively due to the human SGN-like neurons.

[0221] For the co-culture experiment, CBA / CaJ mouse cochlear explants were dissected from the auditory neurons’ peripheral processes, thereby denervating the explant and leaving primarily hair cells. After 14 d of solo culture of the denervated explant, mouse hair cells retained presynaptic ribbons, while the post-synaptic side was largely gone (FIG. 13B), as previously reported (84). Next, cells derived from our protocol (D81 - 134; which included SGN-like neurons as well as some mesenchyme and glial cells) were introduced to the denervated mouse hair cells culture. After 14 d of co-culture, the human SGN-like neurons extended their neurites radially toward the explant and made direct contact with the hair cells, as confirmed by the hair cell marker myosin 7a (MYO7A) and human-specific NEFL antibody (FIGS. 6B-6D; FIGS. 13C, 13D). In 4 experiments, an average of 8.83% and 3.88% of hair cells were innervated by TUBB3+type I or PRPH+type II human SGN-like neurons, respectively.

[0222] While the TUBB3+neurons exclusively innervated a single hair cell, PRPH+neurons formed connections with multiple solute carrier family 26 member 5 (PRESTIN)+mouse outer hair cells (FIG.6E; FIGS. 13E, 13F). This observation is consistent with in vivo cochlear innervation patterns, where type I SGNs innervate a single inner hair cell while type II SGN form synapses with several outer hair cells (85). Similar to the pattern of synaptic connections observed in adult human inner ear tissue (FIG. 6F), the co-cultures had (38.8 ± 23.5)% paired pre- and post-synaptic [C-terminal binding protein 2 (GtBP2)+postsynaptic density protein 95 (PSD95)+] puncta (FIGS. 6G, 6H). Due to the culture system (i.e., requiring cell adherence to the dish), the newly formed synapses were not always located at the base of the hair cells, as would be expected in vivo, and instead formed on various regions of the cell body. This is consistent with the results of prior studies of cultured inner ear tissue, which have observed the apical repositioning of ribbon synapses (86, 87).

[0223] To test if paired CtBP2+PSD95+puncta between mouse hair cells and human SGN-like neurons in the co-culture correspond to functional glutamatergic synapses, we performed Ca2+imaging of human SGN-like neurons. This approach leveraged the fact that hair cells from P5 mouse cochlea spontaneously fire Ca2+spikes, which trigger glutamate release onto SGNs, leading to bursts of APs via synaptic activation (88, 89). Indeed, on the co-culture of human SGN-like neurons with P5 mouse hair cells, we observed Ca2+transients in the soma of human SGN-like neurons located in the vicinity of hair cells (FIG. 61). To confirm that these Ca2+transients are due to glutamatergic synaptic transmission from hair cells, we applied 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX) and (2R)-2-Amino-5-phosphonopentanoic acid (AP5), antagonists of AMPA / kainate and NMDA receptors, respectively. The CNQX / AP5 cocktail abolished Ca2+transients (FIGS. 6J-6L), demonstrating that observed Ca2+transients were mediated by glutamate released from spontaneously active hair cells.

[0224] Ca2+imaging of human SGN-like neurons cultured alone also showed spontaneous Ca2+transients. However, approximately 70% of these transients were unaffected by glutamatergic receptor blockers (FIGS. 6M-6P). This observation is consistent with intrinsic spontaneous activity seen in vivo, known to play an important role in the refinement of auditory circuits before the onset of hearing (90-93). Such intrinsic activity may be mediated by hyperpolarization-activated cationic channels or voltage-gated K+channels (94), both of which are expressed in human SGN-like neurons.

[0225] To extend these functional assays, we examined c-Fos expression in the co-culture system.Synaptic input triggers Ca2+influx into postsynaptic neurons, activating pathways such as the MAPK / ERK, which drive c-fos gene transcription and nuclear c-Fos protein expression (95, 96). Based on this mechanism, we hypothesized that synaptic activity between hair cells and human SGN-like neurons would induce c-Fos. Indeed, in co-culture, (30.81 ± 13.63)% of human SGN-like neurons expressed c-Fos, whereas this proportion was reduced to (6.60 ± 6.07)% following CNQX / AP5 treatment (FIG. 13G). The human SGN-like neurons cultured alone showed less, but detectable, c-Fos expression (4.92 ± 0.36)%, congruent with less frequent intrinsic spontaneous activity observed in Ca2+imaging.

[0226] Together, these findings suggest that human SGN-like neurons are capable of forming functional glutamate-mediated synaptic connections with hair cells in co-culture. This capability is supported by the early expression of AMPA and NMDA receptor subunits (GRIA2 - 4 and GRIN2B) (FIG. 11E), as well as by previously published in vivo functional studies of mouse auditory development (88).3.7 Human SGN-like neurons form functional synapses onto CN neurons

[0227] In mammals, SGNs directly project to the CN of the brainstem, the first brain structure of the central auditory pathway (97). We tested whether the human SGN-like neurons could project to and / or attract neurons from dissected mouse CN (FIG. 7A). As early as D6 in co-culture, neurons were observed to emerge from the CN explant to form connections with the human SGN-like neurons (FIGS. 7B, 7C; FIG. 14A). The neuron types can be distinguished by their unique morphology, as CN neurons are multipolar while the human SGN-like neurons are bipolar, and by staining with human-specific and human- and mouse-reactive NEFL antibodies. The CN neurons exhibited several different shapes, including bushy and stellate cell-like cells, likely corresponding to anteroventral and posteroventral CN, and fusiform cell-like cells likely corresponding to the dorsal CN (98) (FIG. 14B). In this system, we observed presynaptic markers [synaptophysin (SY)+and VGLUT1+] associated with human SGN-like neurites and a post-synaptic marker (PSD95+) associated with CN neurites in close proximity to each other, suggesting that human SGN-like neurons form synapses with CN neurons (FIGS. 7D; FIG. 14C).

[0228] As with mouse hair cell to human SGN-like neuron co-culture, we performed Ca2+imaging to determine if human SGN-like neurons make functional synapses onto CN neurons. Prior to coculturing, human SGN-like neurons were labeled with Dil to distinguish them from CN neurons. In the co-culture, Ca2+transients observed in CN neurons were abolished in the presence of CNQX and AP5 (FIGS. 7E-7H), suggesting their synaptic origin. In contrast to co-culture, Ca2+imaging of CN neurons cultured alone revealed no Ca2+transients (FIGS. 7I-7L) , suggesting that glutamatergic contacts between CN neurons, if they exist, do not result in significant depolarization to trigger Ca2+transients. Consistent with these findings, (10.78 ± 5.94)% of CN neurons in co-culture expressed c- Fos, whereas expression was reduced to (1.91 ± 0.47)% following the CNQX / AP5 treatment (FIG.14D). CN neurons cultured alone (control) exhibited almost no detectable c-Fos expression (0.96 ± 0.70)%.

[0229] Together, these findings indicate that the CNQX / AP5-sensitive Ca2+activity in mouse CN neurons when co-cultured with human SGN-like neurons is likely due to glutamatergic input from human SGN-like neurons into CN neurons.Discussion

[0230] Although hearing loss disables over 5% of the world’s population (99), there are currently no effective pharmaceutical or cellular therapies approved for sensorineural hearing loss. Discovery and testing of such therapies can be propelled via reliable and physiologically relevant in vitro human inner ear models. Here, we present a robust differentiation protocol for generating human SGN-likeneurons from hiPSCs, designed to mimic the signaling cues from surrounding tissue present during human auditory neuron development in vivo. We carefully interrogated the lineage, molecular signatures, gene and protein expression, and functional properties of the resulting cells to increase confidence that they possess SGN characteristics. This approach enabled a level of validation and in-depth analysis of human SGN-like neurons previously possible only in rodent models (71 , 100).

[0231] Our differentiation protocol resulted in neurons exhibiting electrophysiological properties previously reported in ex vivo SGNs (71, 72), and evidenced by the presence of Na+- and K+- mediated currents, hyperpolarized resting potentials, overshooting APs, and firing patterns consistent with mature SGN subtypes (MA and UA). These changes are consistent with the increase in expression of mRNA encoding for various Na+and K+channels. Moreover, the passive and active properties of human SGN-like neurons described here, parallel neuronal developmental mechanisms previously described for central neurons in various brain regions (101, 102). Notably, the human SGN-like neurons generated in this study elicit overshooting APs and firing patterns that closely resembling ex vivo recordings of mature SGNs (71 , 72).

[0232] In addition to maturing intrinsic excitability properties, human SGN-like neurons have the necessary molecular machinery to form functional synapses. The expression of mRNA encoding various AMPA and NMDA receptor subunits supports their capacity to establish glutamatergic synapses with both hair cells and CN neurons. These synapses contain functional CNQX- and AP5- sensitive AMPA and NMDA receptors, similar to what is shown in ex vivo work in animal models (88, 103, 104). This demonstrated ability to functionally integrate with both pre- or post-synaptic partners represents a critical milestone toward establishing physiologically accurate models of human hearing.

[0233] Our methodology represents a fundamental improvement over prior protocols for the generation of human SGN-like neurons from hiPSCs, particularly because of the high purity of inner ear-related cells achieved, the in-depth description of cell type identities to inform future studies, and simplification of the culture system to maximize its utility. Our protocol allowed the differentiation of hiPSCs into early inner ear linage (i.e., otic vesicle- and CNC-related) cells at > 99% efficiency on D25, and into human SGN-like neurons and surrounding (i.e., glia and mesenchymal) cells at > 98% efficiency on D60. Although additional studies are needed, our findings suggest that these human SGN-like neurons are developing along the trajectory that enables their relaying of tonotopic information. This is supported by two observations. First, we identified at least two subpopulations of type II SGN-like neurons (TH+PRPH+and TH PRPH+). In vivo, TH+type II SGNs are known to be distributed along a tonotopic gradient, with a higher proportion at the cochlear apex compared to the base (105). Second, human SGN-like neurons formed synaptic connections with multiple types ofGN neurons, including bushy, stellate, and fusiform cells, which likely originate from anteroventral, posteroventral, and dorsal regions of CN, respectively. These findings suggest that human SGN-like neurons may have a capacity to innervate CN in a manner consistent with tonotopic organization. The unprecedented maturity of the human SGN-like cells, roughly equivalent to SGNs at the second trimester of gestation, is likely due to their co-generation with Schwann cells, satellite glia, and periotic mesenchymal cells whose roles may have been underestimated in prior protocols. In vivo, these cell types reciprocally interact with SGNs during inner ear development and are essential for their maturation (11, 106-108). Thus, the strategy of co-generating human SGN-like neurons with “neighboring” cells likely enabled this protocol’s high efficiency and reproducibility, and the unprecedented maturity of target human SGN-like neurons.

[0234] This system could offer powerful cellular models to decipher the precise mechanisms underlying hearing disorders and enable future targeted therapies. For example, our high-efficiency, monolayer differentiation protocol can be a reliable source of human SGN-like neurons suitable for drug screening. Although 3D organoids have been spotlighted for this purpose due to their multicellular structure, they are less compatible with high-throughput screening. In contrast, our two- dimensional (2D) culture system provides benefits for easier scaling, real-time monitoring, functional tests, and quality control in a realistic way, as has already been demonstrated using dissociated mouse SGNs (109). These features are particularly valuable during drug screening when testing thousands of compounds in parallel.

[0235] Given the repeated failure of clinical trials for hearing loss therapies that initially demonstrated promising results in animal models, a major strength of our platform is its use of human-derived cells, thereby improving the potential for clinical translation. A particularly compelling near-term application is the concept of an engineered biopsy of the inner ear using a patient’s own hiPSCs. This strategy could be especially valuable considering the large number of genes associated with hearing loss, the current inability to access living inner ear tissue for routine biopsy, and the fact that the histopathology of the human cochlea has been described for only about one-tenth of known deafness-causing mutations (110). In addition, our human SGN-like neurons may serve as a foundation for future cell-based therapies. Although further studies will be required to determine whether these neurons can integrate into existing neural circuits and restore function, the present work demonstrates that they can form functional synaptic connections with both peripheral and central targets, which would be an essential prerequisite for the restoration of hearing following neuronal damage. Clinical translation of such an approach would require the development of minimally invasive round window injection techniques when aimed at reestablishing biological hearing. Importantly, therapeutic applications may be realized sooner in the context of cochlearimplants. As the hearing ability and speech recognition in cochlear implant users are thought to correlate with the number of surviving SGNs (111, 112), patient-specific SGNs might be used co- therapeutically with cochlear implants to achieve better auditory outcomes. The current protocol provides an ideal platform fortesting this translational application in the future, as well as addressing other important questions in otologic research.

[0236] Although this study establishes a robust protocol for generating SGN-like neurons from hiPSCs, arguably the closest to their in vivo counterpart to date, several limitations remain before the platform can be advanced toward translational relevance. First, a more thorough exploration of communication between human SGN-like neurons and their partner cells is necessary. In this study, we assessed cell-cell communication using synaptic marker staining and Ca2+imaging in co-culture systems. However, the newly formed synapses were not always positioned at the base of the hair cells, as is observed in vivo; and long-term synaptic activity remains to be demonstrated. In addition, because Ca2+imaging relied primarily on spontaneous activity, only a limited proportion of cells were activated. Future approaches such as picospritzer, multi-chamber microfluidic system (to stimulate distinct populations with KOI), or optogenetic tools (e.g., channel rhodopsin expressing cells) may enable more controlled stimulation. Importantly, systematic testing of bidirectional signaling in cocultures involving mouse hair cells, human SGN-like neurons, and mouse GN neurons will be critical before moving to in vivo studies. Second, functional characterization of human SGN and glia interactions is needed. While our immunostaining demonstrated satellite glia-like cells surrounding human SGN-like neurons and Schwann cell-like cells wrapping of axons, functional assays were not performed. Perturbation or co-culture assays could elucidate the roles of glia in synapse formation, axon guidance, and neuronal maintenance. For example, assessing SGN neurite growth toward mouse hair cell targets while selectively ablating Schwann cells (e.g., with fluoroacetate) could provide valuable insights, with neurite length as a primary readout. Third, while our current 2D culture system offers a practical platform, particularly for drug screening, transitioning to 3D organoids or scaffold-based systems will be important to more closely recapitulate the native inner ear environment. Such approaches could better mimic the mechanical, biochemical, and structural cues that shape auditory neuron development and function in vivo. Finally, in vivo survival and transplantation studies will be required to determine the clinical potential of hiPSC-derived SGN-like neurons. Initial experiments in healthy animal models should assess long-term survival, integration into cochlear circuits, and synapse formation with both hair cells and CN neurons. Subsequent transplantation into auditory neuron injury models, followed by functional assessments such as auditory brainstem responses and distortion product otoacoustic emissions, will provide essentialevidence of therapeutic potential. Together, these directions highlight both the promise of this platform and the critical next steps needed for its translation into future therapies.Conclusions

[0237] This study presents a reliable and scalable 2D differentiation protocol for generating human SGN-like cells from hiPSCs, recapitulating key developmental stages of the human inner ear. The resulting neurons exhibit structural, molecular, and functional characteristics similar to primary SGNs found in vivo, including the ability to form functional glutamatergic synapses with both peripheral and central auditory targets. Importantly, this platform offers several advantages for translational applications: it enables access to otherwise unattainable human auditory neurons without compromising hearing, supports functional analysis of patient-specific genetic mutations, and is compatible with high-throughput drug testing. Given that hearing loss is a major cause of disability among both civilian and military populations, our system provides a practical foundation for accelerating therapeutic discovery and advancing personalized regenerative strategies.References

[0238] 1. Spoendlin H. The innervation of the organ of Corti. J Laryngol Otol. 1967;81(7):717- 38.

[0239] 2. Berglund AM, Ryugo DK. Hair cell innervation by spiral ganglion neurons in the mouse. J Comp Neurol. 1987;255(4):560-70.

[0240] 3. Kiang NY, Watanabe T, Thomas EC, Clark LF. Stimulus coding in the cat's auditory nerve. Preliminary report. Ann Otol Rhinol Laryngol. 1962;71 :1009-26.

[0241] 4. Fekete DM, Rouiller EM, Liberman MC, Ryugo DK. The central projections of intracellularly labeled auditory nerve fibers in cats. J Comp Neurol. 1984;229(3):432-50.

[0242] 5. Brown MC, Berglund AM, Kiang NY, Ryugo DK. Central trajectories of type II spiral ganglion neurons. J Comp Neurol. 1988;278(4):581 -90.

[0243] 6. Schuknecht HF. FURTHER OBSERVATIONS ON THE PATHOLOGY OF PRESBYCUSIS. Arch Otolaryngol. 1964;80:369-82.

[0244] 7. Schuknecht HF, Gacek MR. Cochlear pathology in presbycusis. Ann Otol Rhinol Laryngol. 1993;102(1 Pt 2):1-16.

[0245] 8. Kujawa SG, Liberman MC. Acceleration of age-related hearing loss by early noise exposure: evidence of a misspent youth. J Neurosci. 2006;26(7):2115-23.

[0246] 9. Hinojosa R, Lerner SA. Cochlear neural degeneration without hair cell loss in two patients with aminoglycoside ototoxicity. J Infect Dis. 1987; 156(3) :449-55.

[0247] 10. Sagers JE, Landegger LD, Worthington S, Nadol JB, Stankovic KM. Human Cochlear Histopathology Reflects Clinical Signatures of Primary Neural Degeneration. Sci Rep.2017;7(1):4884.

[0248] 11. Brooks PM, Rose KP, MacRae ML, Rangoussis KM, Gurjar M, Hertzano R, et al.Pou3f4-expressing otic mesenchyme cells promote spiral ganglion neuron survival in the postnatal mouse cochlea. J Comp Neurol. 2020;528(12):1967-85.

[0249] 12. Ruel J, Emery S, Nouvian R, BersotT, Amilhon B, Van Rybroek JM, et al. Impairment of SLC17A8 encoding vesicular glutamate transporter-3, VGLUT3, underlies nonsyndromic deafness DFNA25 and inner hair cell dysfunction in null mice. Am J Hum Genet. 2008;83(2):278-92.

[0250] 13. Johnsson LG. Sequence of degeneration of Corti's organ and its first-order neurons.Ann Otol Rhinol Laryngol. 1974;83(3):294-303.

[0251] 14. Takeno S, Wake M, Mount RJ, Harrison RV. Degeneration of spiral ganglion cells in the chinchilla after inner hair cell loss induced by carboplatin. Audiol Neurootol. 1998;3(5) :281 -90.

[0252] 15. Pauler M, Schuknecht HF, Thornton AR. Correlative studies of cochlear neuronal loss with speech discrimination and pure-tone thresholds. Arch OtorhinolaryngoL 1986;243(3):200-6.

[0253] 16. Sone M, Schachern PA, Paparella MM. Loss of spiral ganglion cells as primary manifestation of aminoglycoside ototoxicity. Hear Res. 1998;115(1-2):217-23.

[0254] 17. Nadol JB, Jr. Patterns of neural degeneration in the human cochlea and auditory nerve: implications for cochlear implantation. Otolaryngol Head Neck Surg. 1997;117(3 Pt 1):220-8.

[0255] 18. Bahmad F, Jr., Merchant SN, Nadol JB, Jr., Tranebjaerg L. Otopathology in Mohr- Tranebjaerg syndrome. Laryngoscope. 2007;117(7):1202-8.

[0256] 19. Wu CC, Lin YH, Liu TC, Lin KN, Yang WS, Hsu CJ, et al. Identifying Children With Poor Cochlear Implantation Outcomes Using Massively Parallel Sequencing. Medicine (Baltimore).2015;94(27):e1073.

[0257] 20. Meas SJ, Nishimura K, Scheibinger M, Dabdoub A. In vitro Methods to Cultivate Spiral Ganglion Cells, and Purification of Cellular Subtypes for Induced Neuronal Reprogramming. Front Neurosci. 2018;12:822.

[0258] 21 . Kurihara S, Fujioka M, Hirabayashi M, Yoshida T, Hosoya M, Nagase M, et al. Otic organoids containing spiral ganglion neuron-like cells derived from human-induced pluripotent stem cells as a model of drug-induced neuropathy. Stem Cells Transl Med. 2022;11 (3):282-96.

[0259] 22. Matsuoka AJ, Morrissey ZD, Zhang C, Homma K, Belmadani A, Miller CA, et al.Directed differentiation of human embryonic stem cells toward placode-derived spiral ganglion-like sensory neurons. Stem Cells Trans Med. 2017;6(3):923-36.

[0260] 23. Gunewardene N, Bergen NV, Crombie D, Needham K, Dottori M, Nayagam BA. Directing human induced pluripotent stem cells into a neurosensory lineage for auditory neuron replacement. Biores Open Access. 2014;3(4):162-75.

[0261] 24. Nayagam BA, Edge AS, Needham K, Hyakumura T, Leung J, Nayagam DA, et al. An in vitro model of developmental synaptogenesis using cocultures of human neural progenitors and cochlear explants. Stem Cells Dev. 2013;22(6):901 -12.

[0262] 25. Needham K, Hyakumura T, Gunewardene N, Dottori M, Nayagam BA.Electrophysiological properties of neurosensory progenitors derived from human embryonic stem cells. Stem Cell Res. 2014;12(1 ):241 -9.

[0263] 26. Shi F, Corrales CE, Liberman MC, Edge AS. BMP4 induction of sensory neurons from human embryonic stem cells and reinnervation of sensory epithelium. Eur J Neurosci.2007;26(11):3016-23.

[0264] 27. Heuer RA, Nella KT, Chang HT, Coots KS, Oleksijew AM, Roque CB, et al. Three- dimensional otic neuronal progenitor spheroids derived from human embryonic stem cells. Tissue Eng Part A. 2021 ;27(3-4):256-69.

[0265] 28. Boddy SL, Romero-Guevara R, Ji AR, Unger C, Corns L, Marcotti W, et al. Generation of otic lineages from integration-free human-induced pluripotent stem cells reprogrammed by mRNAs. Stem Cells Int. 2020;2020:3692937.

[0266] 29. Chen W, Jongkamonwiwat N, Abbas L, Eshtan SJ, Johnson SL, Kuhn S, et al.Restoration of auditory evoked responses by human ES-cell-derived otic progenitors. Nature.2012;490(7419):278-82.

[0267] 30. Jeong M, Ocwieja KE, Han D, Wackym PA, Zhang Y, Brown A, et al. Direct SARS- CoV-2 infection of the human inner ear may underlie COVID-19-associated audiovestibular dysfunction. Commun Med. 2021 ;1 (1):44.

[0268] 31 . Jacobson AG. The Determination and Positioning of the Nose, Lens and Ear. li. The Role of the Endoderm. J Exp Zool. 1963;154:285-91.

[0269] 32. His W. Die Haute und Hbhlen des Kbrpers: Academisches Programm:Schweighauser; 1865.

[0270] 33. Sandell LL, Butler Tjaden NE, Barlow AJ, Trainor PA. Cochleovestibular nerve development is integrated with migratory neural crest cells. Dev Biol. 2014;385(2):200-10.

[0271] 34. Liu Z, Owen T, Zhang L, Zuo J. Dynamic expression pattern of sonic hedgehog in developing cochlear spiral ganglion neurons. Dev Dyn. 2010;239(6):1674-.

[0272] 35. Riccomagno MM, Martinu L, Mulheisen M, Wu DK, Epstein DJ. Specification of the mammalian cochlea is dependent on Sonic hedgehog. Genes Dev. 2002;16(18):2365-78.

[0273] 36. Trainor PA, Tam PP. Cranial paraxial mesoderm and neural crest cells of the mouse embryo: co-distribution in the craniofacial mesenchyme but distinct segregation in branchial arches. Development. 1995 ; 121 (8):2569-82.

[0274] 37. Freyer L, Aggarwal V, Morrow BE. Dual embryonic origin of the mammalian otic vesicle forming the inner ear. Development. 2011 ;138(24):5403-14.

[0275] 38. Heuer JG, Fatemie-Nainie S, Wheeler EF, Bothwell M. Structure and developmental expression of the chicken NGF receptor. Dev Biol. 1990;137(2):287-304.

[0276] 39. Abe H, Wataya H, Amano O, Kondo H. Localization of nerve growth factor receptor in developing inner ear of rats. Acta Otolaryngol. 1991 ;111 (4):691 -8.

[0277] 40. Roccio M, Perny M, Ealy M, Widmer HR, Heller S, Senn P. Molecular characterization and prospective isolation of human fetal cochlear hair cell progenitors. Nat Commun.2018 ;9( 1 ) :4027.

[0278] 41. Szabo A, Mayor R. Mechanisms of neural crest migration. Annu Rev Genet.2018;52:43-63.

[0279] 42. Leung AW, Kent Merest D, Li JY. Differential BMP signaling controls formation and differentiation of multipotent preplacodal ectoderm progenitors from human embryonic stem cells. Dev Biol. 2013;379(2):208-20.

[0280] 43. Mou K, Hunsberger CL, Cleary JM, Davis RL. Synergistic effects of BDNF and NT-3 on postnatal spiral ganglion neurons indexing terms: Inner ear; cochlea; primary auditory neuron. J Comp Neurol. 1997;386:529-39.

[0281] 44. Koundakjian EJ, Appier J L, Goodrich LV. Auditory neurons make stereotyped wiring decisions before maturation of their targets. J Neurosci. 2007;27(51):14078-88.

[0282] 45. Hafidi A. Peripherin-like immunoreactivity in type II spiral ganglion cell body and projections. Brain Res. 1998;805(1 -2):181 -90.

[0283] 46. Spoendlin H. Degeneration behaviour of the cochlear nerve. Arch Klin Exp Ohren Nasen Kehlkopfheilkd. 1971 ;200(4):275-91 .

[0284] 47. Petitpre C, Faure L, Uhl P, Fontanet P, Filova I, Pavlinkova G, et al. Single-cell RNA- sequencing analysis of the developing mouse inner ear identifies molecular logic of auditory neuron diversification. Nat Commun. 2022;13(1):3878.

[0285] 48. Sanders TR, Kelley MW. Specification of neuronal subtypes in the spiral ganglion begins prior to birth in the mouse. Proc Natl Acad Sci USA. 2022;119(48):e2203935119.

[0286] 49. Locher H, Frijns JHM, van Iperen L, de Groot JCMJ, Huisman MA, Chuva de Sousa Lopes SM. Neurosensory development and cell fate determination in the human cochlea. Neural Dev. 2013;8:20.

[0287] 50. Zuchero JB, Barres BA. Glia in mammalian development and disease. Development.2015;142(22):3805-9.

[0288] 51 . Locher H, de Groot JO, van Iperen L, Huisman MA, Frijns JH, Chuva de Sousa Lopes SM. Distribution and development of peripheral glial cells in the human fetal cochlea. PLoS One.2014;9(1):e88066.

[0289] 52. Rattay F, Potrusil T, Wenger C, Wise AK, Glueckert R, Schrott-Fischer A. Impact of morphometry, myelinization and synaptic current strength on spike conduction in human and cat spiral ganglion neurons. PloS One. 2013 ;8( 11 ):e79256.

[0290] 53. Rose KP, Manilla G, Milon B, Zalzman O, Song Y, Coate TM, et al. Spatially distinct otic mesenchyme cells show molecular and functional heterogeneity patterns before hearing onset. iScience. 2023;26(10):107769.

[0291] 54. Durruthy-Durruthy R, Gottlieb A, Hartman BH, Waidhaus J, Laske RD, Altman R, et al. Reconstruction of the mouse otocyst and early neuroblast lineage at single-cell resolution. Cell.2014;157(4):964-78.

[0292] 55. Lu CC, Appier JM, Andres Houseman E, Goodrich LV. Developmental profiling of spiral ganglion neurons reveals insights into auditory circuit assembly. J Neurosci.2011 ;31 (30):10903-18.

[0293] 56. Raft S, Nowotschin S, Liao J, Morrow BE. Suppression of neural fate and control of inner ear morphogenesis by Tbx1. Development. 2004 ; 131 (8):1801 -12.

[0294] 57. Matern MS, Durruthy-Durruthy R, Bird O, Darmanis S, Scheibinger M, Groves AK, et al. Transcriptional dynamics of delaminating neuroblasts in the mouse otic vesicle. Cell Rep.2023;42(6):112545.

[0295] 58. Gleeson JG, Lin PT, Flanagan LA, Walsh CA. Doublecortin is a microtubule- associated protein and is expressed widely by migrating neurons. Neuron. 1999;23(2):257-71.

[0296] 59. Sun Y, Wang L, Zhu T, Wu B, Wang G, Luo Z, et al. Single-cell transcriptomic landscapes of the otic neuronal lineage at multiple early embryonic ages. Cell Rep.2022 ;38(12):110542.

[0297] 60. Lorenzen SM, Duggan A, Osipovich AB, Magnuson MA, Garcia-Anoveros J. Insml promotes neurogenic proliferation in delaminated otic progenitors. Meeh Dev. 2015;138 Pt 3:233-45.

[0298] 61 . Nayagam BA, Muniak MA, Ryugo DK. The spiral ganglion: Connecting the peripheral and central auditory systems. Hear Res. 2011 ;278(1-2):2-20.

[0299] 62. Puel JL. Chemical synaptic transmission in the cochlea. Prog Neurobiol. 1995;47(6).

[0300] 63. Ryan AF, Schwartz IR. Preferential glutamine uptake by cochlear hair cells:implications for the afferent cochlear transmitter. Brain Res. 1984;290(2):376-9.

[0301] 64. Niedzielski AS, Wenthold RJ. Expression of AMPA, kainate, and NMDA receptor subunits in cochlear and vestibular ganglia. J Neurosci. 1995 ; 15(3 Pt 2):2338-53.

[0302] 65. Rutherford MA, Bhattacharyya A, Xiao M, Cai HM, Pal I, Rubio ME. GluA3 subunits are required for appropriate assembly of AMPAR GluA2 and GluA4 subunits on cochlear afferent synapses and for presynaptic ribbon modiolar-pillar morphology. Elife. 2023;12.

[0303] 66. Paoletti P, Bellone C, Zhou Q. NMDA receptor subunit diversity: impact on receptor properties, synaptic plasticity and disease. Nat Rev Neurosci. 2013;14(6):383-400.

[0304] 67. Maison SF, Casanova E, Holstein GR, Bettier B, Liberman MC. Loss of GABAB receptors in cochlear neurons: threshold elevation suggests modulation of outer hair cell function by type II afferent fibers. J Assoc Res Otolaryngol. 2009;10(1):50-63.

[0305] 68. Conrad LJ, Grandi FC, Carlton AJ, Jeng JY, de Tomasi L, Zarecki P, et al. The upregulation of K(+) and HCN channels in developing spiral ganglion neurons is mediated by cochlear inner hair cells. J Physiol. 2024;602(20):5329-51.

[0306] 69. Shrestha BR, Chia C, Wu L, Kujawa SG, Liberman MC, Goodrich LV. Sensory Neuron Diversity in the Inner Ear Is Shaped by Activity. Cell. 2018;174(5):1229-46 e17.

[0307] 70. Liu W, Glueckert R, Kinnefors A, Schrott-Fischer A, Bitsche M, Rask-Andersen H.Distribution of P75 neurotrophin receptor in adult human cochlea-an immunohistochemical study. Cell Tissue Res. 2012;348(3):407-15.

[0308] 71. Petitpre C, Wu H, Sharma A, Tokarska A, Fontanet P, Wang Y, et al. Neuronal heterogeneity and stereotyped connectivity in the auditory afferent system. Nature Commun.2018;9(1 ):1 -13.

[0309] 72. Reid MA, Flores-Otero J, Davis RL. Firing patterns of type II spiral ganglion neurons in vitro. J Neurosci. 2004;24(3):733-42.

[0310] 73. Crozier RA, Davis RL. Unmasking of spiral ganglion neuron firing dynamics by membrane potential and neurotrophin-3. J Neurosci. 2014;34(29):9688-702.

[0311] 74. Huang H, Trussell LO. KCNQ5 channels control resting properties and release probability of a synapse. Nat Neurosci. 2011 ;14(7):840-7.

[0312] 75. Rudy B, McBain CJ. Kv3 channels: voltage-gated K+ channels designed for high- frequency repetitive firing. Trends Neurosci. 2001 ;24(9):517-26.

[0313] 76. Ottschytsch N, Raes A, Van Hoorick D, Snyders DJ. Obligatory heterotetramerization of three previously uncharacterized Kv channel alpha-subunits identified in the human genome. Proc Natl Acad Sci U S A. 2002;99(12):7986-91.

[0314] 77. Petitpre C, Wu H, Sharma A, Tokarska A, Fontanet P, Wang Y, et al. Neuronal heterogeneity and stereotyped connectivity in the auditory afferent system. Nat Common.2018 ;9( 1 ) :3691.

[0315] 78. Browne L, Smith KE, Jagger DJ. Identification of persistent and resurgent sodium currents in spiral ganglion neurons cultured from the mouse cochlea. eNeuro. 2017;4(6).

[0316] 79. Markowitz AL, Kalluri R. Gradients in the biophysical properties of neonatal auditory neurons align with synaptic contact position and the intensity coding map of inner hair cells. eLife.2020;9(e55378):1-33.

[0317] 80. Mo ZL, Davis RL. Heterogeneous voltage dependence of inward rectifier currents in spiral ganglion neurons. J Neurophysiol. 1997;78(6):3019-27.

[0318] 81. Kim YH, Holt JR. Functional contributions of HCN channels in the primary auditory neurons of the mouse inner ear. J Gen Physiol. 2013;142(3):207-23.

[0319] 82. Bando Y, Hirano T, Tagawa Y. Dysfunction of KCNK potassium channels impairs neuronal migration in the developing mouse cerebral cortex. Cereb Cortex. 2014;24(4):1017-29.

[0320] 83. Kaczmarek LK. Modulation of potassium conductances optimizes fidelity of auditory information. Proc Natl Acad Sci U S A. 2023;120(12):e2216440120.

[0321] 84. Tong M, Brugeaud A, Edge AS. Regenerated synapses between postnatal hair cells and auditory neurons. J Assoc Res Otolaryngol. 2013;14(3):321 -9.

[0322] 85. Spoendlin H. The innervation of the cochlear receptor. In: Moller A, editor. Basic Mechanisms in Hearing. New York, NY: Academic Press; 1973. p. 185-234.

[0323] 86. Hickman TT, Hashimoto K, Liberman LD, Liberman MC. Cochlear synaptic degeneration and regeneration after noise: Effects of age and neuronal subgroup. Front Cell Neurosci. 2021 ;15:684706.

[0324] 87. Katsumi S, Sahin Ml, Lewis RM, Iyer JS, Landegger LD, Stankovic KM. Intracochlear perfusion of tumor necrosis factor-alpha induces sensorineural hearing loss and synaptic degeneration in guinea pigs. Front Neurol. 2019;10:1353.

[0325] 88. Zhang-Hooks Y, Agarwal A, Mishina M, Bergles DE. NMDA Receptors Enhance Spontaneous Activity and Promote Neuronal Survival in the Developing Cochlea. Neuron.2016;89(2):337-50.

[0326] 89. De Faveri F, Ceriani F, Marcotti W. In vivo spontaneous Ca(2+) activity in the prehearing mammalian cochlea. Nat Common. 2025;16(1 ):29.

[0327] 90. Lippe WR. Rhythmic spontaneous activity in the developing avian auditory system. J Neurosci. 1994;14(3 Pt 2):1486-95.

[0328] 91 . T ritsch NX, Yi E, Gale JE, Glowatzki E, Bergles DE. The origin of spontaneous activity in the developing auditory system. Nature. 2007;450(7166):50-5.

[0329] 92. Sonntag M, Englitz B, Kopp-Scheinpflug C, Rubsamen R. Early postnatal development of spontaneous and acoustically evoked discharge activity of principal cells of the medial nucleus of the trapezoid body: an in vivo study in mice. J Neurosci. 2009;29(30):9510-20.

[0330] 93. Babola TA, Li S, Gribizis A, Lee BJ, Issa JB, Wang HC, et al. Homeostatic Control of Spontaneous Activity in the Developing Auditory System. Neuron. 2018;99(3):511-24 e5.

[0331] 94. Liu Q, Lee E, Davis RL. Heterogeneous intrinsic excitability of murine spiral ganglion neurons is determined by Kv1 and HCN channels. Neuroscience. 2014;257:96-110.

[0332] 95. Xia Z, Dudek H, Miranti CK, Greenberg ME. Calcium influx via the NMDA receptor induces immediate early gene transcription by a MAP kinase / ERK-dependent mechanism. J Neurosci. 1996;16(17):5425-36.

[0333] 96. Vanhoutte P, Barnier JV, Guibert B, Pages C, Besson MJ, Hipskind RA, et al.Glutamate induces phosphorylation of Elk-1 and CREB, along with c-fos activation, via an extracellular signal-regulated kinase-dependent pathway in brain slices. Mol Cell Biol.1999;19(1 ):136-46.

[0334] 97. Leake PA, Snyder RL. T opographic organization of the central projections of the spiral ganglion in cats. J Comp Neurol. 1989;281 (4):612-29.

[0335] 98. Pickles JO. Auditory pathways: anatomy and physiology. Handb Clin Neurol.2015;129:3-25.

[0336] 99. World Health Organization. Deafness and hearing loss 2023 [Available from:https: / / www.who.int / news-room / fact-sheets / detail / deafness-and-hearing-loss.

[0337] 100. Shrestha BR, Chia C, Wu L, Kujawa SG, Liberman MC, Goodrich LV. Sensory neuron diversity in the inner ear is shaped by activity. Cell. 2018;174(5):1229-46. e17.

[0338] 101. Dougherty KA. Differential developmental refinement of the intrinsic electrophysiological properties of CA1 pyramidal neurons from the rat dorsal and ventral hippocampus. Hippocampus. 2020;30(3):233-49.

[0339] 102. Perez-Garcia P, Pardillo-Diaz R, Geribaldi-Doldan N, Gomez-Oliva R, Dominguez- Garcia S, Castro C, et al. Refinement of active and passive membrane properties of layer v pyramidal neurons in rat primary motor cortex during postnatal development. Front Molec Neurosci. 2021 ;14.

[0340] 103. Ruel J, Chen C, Pujol R, Bobbin RP, Puel JL. AMPA-preferring glutamate receptors in cochlear physiology of adult guinea-pig. J Physiol. 1999;518 ( Pt 3)(Pt 3):667-80.

[0341] 104. Reijntjes DOJ, Pyott SJ. The afferent signaling complex: Regulation of type I spiral ganglion neuron responses in the auditory periphery. Hear Res. 2016;336:1-16.

[0342] 105. Wu JS, Vyas P, Glowatzki E, Fuchs PA. Opposing expression gradients of calcitonin- related polypeptide alpha (Calca / Cgrpa) and tyrosine hydroxylase (Th) in type II afferent neurons of the mouse cochlea. J Comp Neurol. 2018;526(3):425-38.

[0343] 106. Hanani M, Spray DC. Emerging importance of satellite glia in nervous system function and dysfunction. Nat Rev Neurosci. 2020;21 (9):485-98.

[0344] 107. Hosoya M, Fujioka M, Murayama AY, Okano H, Ogawa K. The common marmoset as suitable nonhuman alternative for the analysis of primate cochlear development. Febs j.2021 ;288(1):325-53.

[0345] 108. Coate TM, Raft S, Zhao X, Ryan AK, Crenshaw EB, 3rd, Kelley MW. Otic mesenchyme cells regulate spiral ganglion axon fasciculation through a Pou3f4 / EphA4 signaling pathway. Neuron. 2012;73(1):49-63.

[0346] 109. Whition DS, Grover M, Dunne SF, Richter S, Luan C-H, Richter C-P. Novel high content screen detects compounds that promote neurite regeneration from cochlear spiral ganglion neurons. Sci Rep. 2015;5(1 ):15960.

[0347] 110. Bommakanti K, Iyer JS, Stankovic KM. Cochlear histopathology in human genetic hearing loss: State of the science and future prospects. Hear Res. 2019;382:107785.

[0348] 111. Seyyedi M, Viana LM, Nadol JB, Jr. Within-subject comparison of word recognition and spiral ganglion cell count in bilateral cochlear implant recipients. Otol Neurotol. 2014;35(8):1446- 50.

[0349] 112. Nadol JB, Jr., Young YS, Glynn RJ. Survival of spiral ganglion cells in profound sensorineural hearing loss: implications for cochlear implantation. Ann Otol Rhinol Laryngol.1989;98(6):411-6.

[0350] 113. Orvis J, Gottfried B, Kancherla J, Adkins RS, Song Y, Dror AA, et al. gEAR: Gene Expression Analysis Resource portal for community-driven, multi-omic data exploration. Nat Methods. 2021 ;18(8):843-4.Supplemental MethodsHuman induced pluripotent stem cell (hiPSC) culture

[0351] hiPSC lines SK8-A, generated in our laboratory [1], and UCSD112i-2-11 (UCSD), purchased from WiCell (Madison, Wl, USA), were used before passage number 50. Cells were maintained on Matrigel human embryonic stem cell (hESC) qualified matrix (Cat# 354277, Corning, USA) in mTeSRI (Cat# 85850, StemCell Technologies, Canada) or mTeSR plus medium (Cat# 100-0276, StemCell Technologies, Canada) supplemented with 1x penicillin-streptomycin (Cat# 15140122,Gibco, USA). Cells were passaged at around 80% confluency and hiPSC colonies were treated with ReLeSR (Cat# 100-0484, StemCell Technologies, Canada) for detaching by tapping the side of plates. Detached cell clumps were plated on new Matrigel-coated 6-well plates. The medium was replenished every day (mTeSRI) or every other day (mTeSR plus). Detailed information about the origin and characterization of cell line UCSD is available at: wicell.org / home / stem-cells / catalog-of- stem-cell-lines / ucsd112i-2-11 ,cmsx?closable=true.

[0352] The culture method described in this study was replicated by two independent investigators in separate locations [M.J. (Massachusetts and California) and Petra Stojkovic (Massachusetts)] using the cell lines SK8-A and UCSD. Thus, we confirmed that the success of this protocol is not limited by a person, cell lines, or place.Human vestibular tissue

[0353] To examine the morphology and key protein expression of mature human SGNs, fresh inner ear vestibular tissue was collected during surgical labyrinthectomies and translabyrinthine resections of vestibular schwannomas (n = 2) occurring as part of routine clinical management. The notation regarding this tissue, as reported by the surgeon at the time of excision, was “vestibular end organs” which together include saccular and utricular maculae and cristae of the semicircular canals; further details as to the specific organ were not available.Dissociation of human SGN-like cells for scRNA-seq

[0354] To dissociate the samples generated from hiPSCs SK8-A and UCSD into single cells, randomly selected wells containing the cells on differentiated D25 (n = 3 for both lines), D60 (n = 6), D90 (n = 6), and D120 (n = 6) were pooled. In brief, collected cells were incubated with TrypLE Select for about 5 min in a 37 °C incubator until detachment of the edge of cells was observed. After removing TrypLE Select from the plate, 10% fetal bovine serum in the Neurobasal medium was added and gentle pipetting using p1000 tips until no visible cell aggregation. The suspension was filtered through a 100 pm cell strainer to eliminate any chunk of cell aggregates. Additionally, Debris Removal Solution (Cat# 130-109-398, Miltenyi Biotec, Germany), a density gradient reagent, was used to remove debris from viable cells. Then, cells were resuspended in 1 x Dulbecco’s PBS (Cat# 14190144, Gibco, USA) containing 0.04% bovine serum albumin (Cat# A8412, Sigma, USA). Cell viability and live-cell counting were determined by manual counting using a hemocytometer and cell BioRad TC20. The final cell concentration was 1000 cells / pl with cell viability above 80%.scRNA-seq cDNA library preparation and sequencing

[0355] Single-cell 3’ RNA-seq experiments were conducted using the Chromium single-cell system (10x Genomics, USA) and the NexSeq 2000 (Illumina, USA). The dissociated cells were added to a single-cell master mix (targeting 10,000 cells), following the Chromium Single Cell G000183 Chromium Single Cell3’ version 3 user guide, revision C (1 Ox Genomics, USA). Along with the singlecell gel beads and oil partitioned in separate wells of a Single Cell B Chip, the single-cell reaction mixture was loaded to the Chromium Controller for Gel Bead-in-Emulsion generation and barcoding, followed by cDNA synthesis and library preparation. At each step, the quality of the cDNA and library was examined by Tapestation 4200 (Agilent Technologies, USA). The resulting library was sequenced in a custom program for 28-bp plus 91 -bp paired-end sequencing on an Illumina NextSeq 2000 to a reading depth of more than 30,000 reads per cell.scRNA-seq data analysis

[0356] The 10x Genomics Cell Ranger 2.1.0 pipeline (http: / / support.10xgenomics.com / ) was used to process raw sequence data. In brief, Cell Ranger with bcl2fastq (https: / / support.illumina.com / ) was used to demultiplex raw base sequence calls generated from the sequencer into sample-specific FASTQ files. The FASTQ files were then aligned to the reference genome with RNA-seq aligner Spliced Transcripts Alignment to a Reference (STAR) software. The aligned reads were traced back to the individual cells, and the gene expression level of individual genes is quantified based on the number of unique molecular indices detected in each cell. Filtered gene-cell barcode matrices were generated by Cell Ranger for further analysis. Cells with fewer than 200 unique genes identified and cells with more than 6000 - 8000 total RNA molecules detected were all removed from the analysis. In addition, cells with higher than 10 - 20% mitochondrial reads were filtered and removed from the analysis. To detect doublets, DoubletFinder [3] with standard parameters was used, and doublet rate was predicted based on number of cells captured (3.2 - 7.2%).

[0357] After removing low-quality cells, the gene expression levels for each cell were normalized by the total number of unique molecular indices in the cell and multiplied by a scaling factor of 10,000. After log-transformation, we used Seurat v.4.0.4-5.0.0 for cell clustering using principal component analysis on highly variable genes. Cell cluster visualization was done in uniform manifold approximation and projection (UMAP) space, which offers preservation of the data’s global structure. The gene markers for each cluster were identified through differential expression analysis by comparing cells in the cluster to all other cells. Cell cluster identities were manually defined with the cluster-specific marker genes. To analyze the subgroups of clusters, we applied functions “subset” from Seurat. The expression of a range of pan-neuronal markers such as tubulin beta 3 class III(TUBB3) and sodium / potassium-transporting ATPase subunit beta-1 (ATP1B1) was used to subset neuron clusters marked as red dot circles. The R package ggplot2 was used to plot the average gene expression. Violin plots and feature plots were used to visualize specific gene expressions across clusters and different sample conditions. Integration via the harmony algorithm [4], which has been shown to adequately remove batch effects while conserving biological variation, was used to compare D25 and D60 SGN-like cells to embryonic day (E) 13.5 [5] and E14 - postnatal day (P) 1 [6] mouse datasets, respectively.Immunocytochemistry and imaging

[0358] Cells grown on coverslips were fixed with 4% paraformaldehyde (Cat# AAJ19943K2, ThermoFisher Scientific, USA) for 10 min at room temperature. For permeabilization, cells were washed 3 times with PBS (Cat# 14080-055, Gibco, USA) and incubated in PBST, which is 0.1% Triton X-100 (Cat# T8787, Sigma, USA) in 1x PBS solution, for 10 min at room temperature. Unspecific binding was blocked with 5% normal horse serum (Cat# ab7484, Abeam, USA) or 5% goat serum (Cat# PCN5000, Gibco, USA) in PBST for 1 h. Samples were then incubated overnight at 4 °C with specific primary antibodies diluted in 1% bovine serum albumin (Cat# A9647, Sigma, USA) in PBST, washed 3 times with PBS, and incubated with secondary antibodies in PBST. Vectashield (Cat# H1000, Vector Laboratories, USA) with DAPI (Cat# 4083, Cell Signaling, USA) was used to mount the samples and visualize cellular nuclei. Negative control experiments without the primary antibodies were processed in parallel. Microscopy was performed using a Leica SP8 confocal microscope (Leica Microsystems, Germany) or ZEISS LSM 880 (Carl Zeiss AG, Germany).

[0359] For co-culture staining, a similar protocol was used. Briefly, coverslips on hiPSC-derived SGNs and mouse explants were rinsed in PBS, fixed with 4% paraformaldehyde in PBS for 20 min, washed with PBS, and blocked in a blocking buffer consisting of 5% normal horse serum with 0.5% Triton X-100 at room temperature on a horizontal shaker for 30 min. Samples were then incubated overnight at 4 °C with specific primary antibodies diluted in 1% normal horse serum in 0.5% Triton X-100, washed 3 times with PBS, and incubated with secondary antibodies in PBST for 90 min. Specimens were imaged with a Leica SP8 confocal microscope (Leica Microsystems, Germany). After taking an overview of the specimen at 20x magnification, images were reconstituted in a 3D mode to count hair cells and find connections between mouse hair cells and SGN neurites derived from hiPSCs. Then, the area of synaptic connection was visualized by 63x with 2.4x digital zoom at a Z-step-size of 0.3 pm. To avoid imaging with overlapping or close wavelengths simultaneously, samples were sequentially scanned.Electrophysiological recordings

[0360] Whole-cell electrophysiological recordings were made from human SGN-like neurons generated from two different hiPSCs. Recorded neurons were maintained for D88 - 95 or D120 - 234 in vitro. During recordings, neurons were continuously superfused in artificial cerebrospinal fluid containing (in mmol / L): 125 NaCI, 2.5 KCI, 1.25 NaH2PC>4, 25 NaHCOs, 25 D(+)-glucose, 1 MgCh and 2 CaCI2, buffered with 5% carbogen. Neurons were visualized under infrared differential interference contrast (IR-DIC) with a microscope (Scientifica, UK; Olympus Americas, USA) using an Orca Flash 4.0 CMOS digital camera C13440 (Hamamatsu Photonics, Japan). Patch pipettes with resistances of 3 - 4 MOhm were pulled using a Flaming / Brown Micropipette Puller P-97 (Sutter Instruments, USA) and filled with intracellular solution containing (in mmol / L): 115 K-gluconate, 7 KCI, 10 N-(2-Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 0.05 ethyleneglycol- bis(P-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA), 2 Na2-ATP, 2 Mg-ATP, and 0.5 Na2-GTP (pH adjusted to 7.3 with KOH; osmolarity 285 mOsm). For post-hoc identification, 0.1 - 0.3% biocytin was loaded into cells via patch-pipettes. Biocytin was visualized after conjugation with streptavidin- Alexa fluor 488.

[0361] Recordings were made at room temperature (22 °C) and liquid junction potentials were not corrected. Series resistance (Rs), membrane resistance, capacitance, and time constant were calculated from a 10 mV hyperpolarizing step with a duration of 100 ms applied in voltage-clamp configuration. Whole-cell Analyzed data only included recordings with Rs < 30 MOhm. Data were acquired and low-pass filtered at 3 kHz with a Multiclamp 700B amplifier (Molecular Devices, USA), in combination with custom software written in Matlab (Mathworks, USA) based on scripts developed in the Sabatini (https: / / github.com / bernardosabatinilab) and Sanes laboratories. A USB NI-6343 digital-to-analog converter (National Instruments, USA) was employed to digitize data at 10 kHz. The analysis was performed offline with custom-written Matlab routines.

[0362] The intrinsic neuronal properties and action potential (AP) features were determined by applying a series of hyperpolarizing and depolarizing steps while in current-clamp configuration. For voltage-clamp recordings, neurons were kept at held at -70 mV and voltage stepped from -100 mV to 40 mV in 10 mV increments. Putative potassium currents were calculated as the difference in current amplitude between the last 10 ms of the stimulation protocol and the baseline amplitude after leak current was subtracted. Putative sodium currents were calculated as the difference in current amplitude between the inflection point after the first capacitive transient and the negative current. For current-clamp recordings, current injections from -80 pA to 400 pA in steps of 20 pA were applied. Rheobase was defined as the minimal depolarizing current to elicit an AP. If rheobase was not reached before 400 pA, current injections from -80 pA to 800 pA were applied. AP properties wereanalyzed both at rheobase and at the maximum current step to which steady APs were elicited. Neurons were classified as multi-spike accommodating neurons (MA) when 3 or more APs were evoked, and as unitary-spike accommodating neurons (UA) when less than 3 APs in a given current injection step were observed across the whole stimulation protocol. AP threshold for the first AP within a step was defined as the membrane potential value at the point before the peak of each AP in which dV / dt was 7.5% of the maximum dV / dt. The latency to the first AP during a current injection step was calculated as the time between the start of the stimulus and the time of the AP threshold, which was considered the onset of the AP. The averaged inter-spike interval (IS I) was measured as the mean difference in onset times between APs within a step, in MA neurons only. For each current injection step, the average half-width was determined as the mean of the width of all APs in response to that current step at 50% of the membrane potential value between the threshold and the peak of each AP. Average AP height was determined as the mean of the difference between the membrane potential value at the peak of each AP and the value at the subsequent trough.Animals

[0363] A total of 36 mice of either sex were used in this study, comprising 31 CBA / CaJ wild-type mice and 5 NOD / SCID wild-type mice. Of these, 35 were aged P3 - 6 and 1 was aged 8 weeks. Mice were procured from Jackson Laboratory (Bar Harbor, ME, USA). All experimental procedures with mice were approved by the Institutional Animal Care and Use Committees of Massachusetts Eye and Ear Infirmary in Boston, MA, USA and Stanford University in Stanford, CA, USA (approved protocol ID: 33998). All procedures with animals were conducted according to the National Academies of Sciences Guide for the Care and Use of Laboratory Animals (8thedition, NAS Press, USA).Co-culture with mouse hair cells

[0364] Cochlear explants were obtained from P3 - 6 CBA / CaJ or NOD / SCID wild-type mice (Jackson Laboratory; n = 30). At this neonatal stage, sex cannot be reliably determined; therefore, both sexes were included. Dissection of cochlear explants was performed largely as previously described [7], Briefly, pups were decapitated, and the external auditory canals were incised with a scalpel blade. The skin was folded anteriorly to expose the cranium. The cranium was opened along the sagittal suture using a #15 scalpel blade. A vertical cut was made posterior to the orbits to remove and discard the snout. The forebrain, cerebellum, and brainstem were removed through blunt dissection using #4 forceps. The cranium was placed into a plastic Petri dish filled with cold HBSS, and the cochlea was completely exposed, located adjacent to the stapedial artery (a tortuous arterywithin the temporal bone). The cochlea was then bluntly separated from the temporal bone using #4 forceps. The cochlea was carefully dissected from the vestibular system, and the cochlear otic capsule (typically cartilaginous at this age) was dissected with #4 forceps.

[0365] The spiral ligament, adherent to the organ of Corti, was separated from the rest of the cochlea and the modiolus, using a micro knife or two forceps, starting from the base and moving toward the apex. The organ of Corti was carefully dissected into a more apical and more basal part, containing sensory hair cells and dendrites of SGNs. The tectorial and Reissner’s membrane and cell bodies of SGNs were removed. In an additional set of experiments, cochlear explants from P4 CBA / CaJ and Thy1-GFP BL6 (Jackson Laboratory) were denervated to dissect the sensory epithelium (organ of Corti, containing hair cells) from the spiral limbus (containing neurites of SGN), using microdissection techniques previously described [8].

[0366] After isolating the cochlear explants or denervated hair cells, they were transferred onto a 12 mm diameter coverslip (Cat# GG12-1.5-oz, Neuvitro, USA) on which hiPSC-derived cells were cultured. The explants were placed approximately 500 pm away from a cluster of human SGN-like cell somas, allowing space for the neurites of human SGN-like neurons to extend toward the mouse hair cells. To prevent the explants from detaching, only a minimal amount (200 - 250 pl) of SGN differentiation medium (D25) was added, just enough to cover the explants. The medium was replenished approximately 24 h later, after the explants had attached to the coverslip. The medium was changed every 2 - 3 d based on its color (indicating pH changes) and evaporation rate. The cultures were monitored daily under a bright-field inverted microscope to assess the cells’ condition and the potential connection between mouse hair cells and human SGN-like neurons. The cocultures were maintained in a 37 °C, 5% CO2 incubator for up to 14 d and were fixed with 4% paraformaldehyde for a downstream immunostaining analysis.

[0367] To quantify synaptic puncta, we counted all C-terminal binding protein 2 (CtBP2)+ / postsynaptic density protein 95 (PSD95)-, CtBP27PSD95+, and CtBP2+ / PSD95+. All these combined numbers are denominators for calculating percentage of paired synaptic puncta. Cocultures were maintained for up to 14 d. As a control, cochlea explants or denervated hair cells were cultured in isolation (n = 6, 3 mice for each) in the same way and duration.Co-culture with mouse cochlear nucleus (CN)

[0368] CN tissue was collected from P3 - 6 pups (n= 17) and 8-week-old CBA / CaJ mice (n = 1) of either sex. Pups were anesthetized by inducing hypothermia and promptly decapitated when unconscious. The head was placed in a 60 mm Petri dish containing 70% ethanol and the skull was exposed with a mediosagittal incision on scalp with a #15 blade. After cutting external auditory canalsbilaterally the cranium was opened along the sagittal suture from anterior to posterior. The cranial incision was then deepened ventrally toward the base of the skull. The snout was removed with a coronal cut posterior to the orbits and two halves of the cranium were placed in a 60 mm Petri dish containing ice-cold HBSS (Gat# 14025092, Gibco, USA). The forebrain, cerebellum, and brainstem were gently removed from the skull halves by blunt dissection with #5 forceps and transferred to a dish containing fresh ice-cold HBSS. The brainstem was then separated from the cerebral and cerebellar hemispheres. The cochlear nuclei are identified by their relation to several anatomical landmarks on the lateral portion of the brainstem: a) the auditory nerve stump is ventral, b) the inferior cerebellar peduncle is dorsal, and c) the sulcus of the anterior-inferior cerebellar artery is posterior to the structure of interest. A 150 - 250 pm thick parasagittal slice of the cochlear nuclei is made with a 45° stab microsurgical knife (PE3045, Oasis, USA) following the structure of the nuclei. The sample was subsequently cut into smaller 150 pm x 150 pm 150 pm fragments. Some of the samples were pipetted using 200 pl tips for further dissociation.

[0369] Cochlear nuclei explant pieces were placed onto 12 mm diameter coverslips containing human SGN-like cells. Typically, each CN explant was divided into 3 portions and placed on 3 separate coverslips containing human SGN-like cells. The co-cultured human SGN-like cells and CN were kept in SGN differentiation medium (D25) in an incubator at 37 °C and 5% CO2. Two days after co-culture, unattached cells or debris from the CN explants were carefully removed and replenished the culture medium. The cultures were monitored daily under a bright-field inverted microscope to assess the cells’ condition and the potential connection between mouse CN neurons and human SGN-like neurons. Half of the medium volume (approximately 250 pl) was replaced every 3 d, and the co-cultures were maintained for up to 28 d before being processed for immunofluorescence imaging.Calcium ion (Ca2+) imaging

[0370] Ca2+imaging with fluorescent Ca2+dyes was performed to evaluate synaptic dependence of spiking activity of human SGN-like neurons or CBA / CaJ mouse CN neurons (P5 or P6) co-cultured with CBA / CaJ mouse hair cells (P5). Cells were labeled with 10 pmol / L Gal-520 AM (Cat# ab171868, Abeam, USA) in artificial perilymph supplemented with 25 pmol / L Sulfinpyrazone (Cat# S9509, Sigma, USA) and 0.1% Pluronic F-127 (Cat# P6866, Thermo Scientific, USA). The labeling was performed at 37 °C for 90 min. The artificial perilymph solution was freshly prepared, containing (in mmol / L): 144 NaCI (Cat# S7653, Sigma, USA), 5.8 KCI (Cat# P3911, Sigma, USA), 0.7 NaH2PO4(Cat# S8282, Sigma, USA), 10 HEPES (Cat# H3375, Sigma, USA), 0.9 MgCI2-6H2O (Cat# M0250,Sigma, USA), 1.3 CaCl2-2H2O (Cat# C5080, Sigma, USA), and 5.6 D-glucose (Cat# G7021 , Sigma, USA). The pH of the solution was adjusted to 7.4.

[0371] Images of fluorescent neurons were captured every 30 s on Zeiss Axiovert A1 inverted fluorescence microscope. This temporal resolution was sufficient to discern spiking-dependent Ca2+transients and whether they were downstream of synaptic transmission. Baseline Ca2+fluorescence was recorded for 4.5 min before the addition of 10 pmol / L 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX) (Cat# 1045, Tocris, UK) and 50 pmol / L D-(-)-2-Amino-5-phosphonopentanoic acid (D-AP5; Cat# 0106, Tocris, UK); imaging in CNQX / D-AP5 was for 7.5 min. Ca2+fluorescence intensity of each human SGN-like neuron or mouse CN neuron was measured with Imaged software as the average pixel brightness over the soma of each neuron. The fold change, presented on the y-axis of the graphs was calculated as the ratio of Ca2+fluorescence intensity of each cell to the average baseline intensity.References

[0372] 1. Jeong M, Ocwieja KE, Han D, Wackym PA, Zhang Y, Brown A, et al. Direct SARS- CoV-2 infection of the human inner ear may underlie COVID-19-associated audiovestibular dysfunction. Commun Med. 2021 ;1 (1):44.

[0373] 2. Matsuoka AJ, Morrissey ZD, Zhang C, Homma K, Belmadani A, Miller CA, et al.Directed differentiation of human embryonic stem cells toward placode-derived spiral ganglion-like sensory neurons. Stem Cells Trans Med. 2017;6(3):923-36.

[0374] 3. McGinnis CS, Murrow LM, Gartner ZJ. DoubletFinder: doublet detection in single-cell RNA sequencing data using artificial nearest neighbors. Cell Syst. 2019;8(4):329-37.e4.

[0375] 4. Luecken MD, Buttner M, Chaichoompu K, Danese A, Interlandi M, Mueller MF, et al.Benchmarking atlas-level data integration in single-cell genomics. Nat Methods. 2022;19(1 ):41 -50.

[0376] 5. Sun Y, Wang L, Zhu T, Wu B, Wang G, Luo Z, et al. Single-cell transcriptomic landscapes of the otic neuronal lineage at multiple early embryonic ages. Cell Rep.2022 ;38(12):110542.

[0377] 6. Sanders TR, Kelley MW. Specification of neuronal subtypes in the spiral ganglion begins prior to birth in the mouse. Proc Natl Acad Sci U S A. 2022;119(48):e2203935119.

[0378] 7. Landegger LD, Dilwali S, Stankovic KM. Neonatal murine cochlear explant technique as an in vitro screening tool in hearing research. J Vis Exp. 2017;124:55704.

[0379] 8. Parker M, Brugeaud A, Edge ASB. Primary culture and plasmid electroporation of the murine organ of corti. J Vis Exp. 2010(36):1685.

[0380] 9. Meas SJ, Nishimura K, Scheibinger M, Dabdoub A. In vitro methods to cultivate spiral ganglion cells, and purification of cellular subtypes for induced neuronal reprogramming. Front Neurosci. 2018;12:822.

[0381] 10. Petitpre C, Wu H, Sharma A, Tokarska A, Fontanet P, Wang Y, et al. Neuronal heterogeneity and stereotyped connectivity in the auditory afferent system. Nat Commun.2018;9(1):3691.Table 1. Medium compositionDay 0: mTeSRCatalog Stock Final Total (100 Product name Supplier No. concentration concentration ml) mTeSR Stem Cell 100-0276 98.9 ml TechnologiesY-27632 TOCRIS 1254 10 mmol / L lOpmol / L 100 pl Penicillin- Gibco 15140122 100* lx 1 ml streptomycinTable 2. List of primary antibodiesAntigen Manufacturer Cat# Host Clonality Reactivity Dilution Markers for and isotypeNGFR Millipore AB1554 Rabbit polyclonal h, m, r 0.388889 Precursors of Sigma preplacodal ectoderm (PPE) and neural crest cell, type II spiral ganglion neuron (SGN), non-myelin Schwann cell, and satellite glial cell NESTIN Millipore MAB5326 Mouse monoclonal h 0.180556 Precursors of Sigma IgGl PPE and neural crest cell NEURO Novus H00004760 Mouse monoclonal h, m 1:50 Neuroblast DI lgG2aPAX2 R&D systems AF3364 Goat polyclonal h 1:50 Early otic IgG lineage cell PAX8 Abeam AB97477 Rabbit polyclonal h, m, r 0.180556 Early otic lineage cell SOX2 BD 561469 Mouse monoclonal h, m 1:50 Otic epithelium,Biosciences IgGl neuralprogenitorSOX2 Cell Signaling CST2748s Rabbit polyclonal h, m 0.319444 Otic epithelium, Technology neural progenitor SOXIO Cell Signaling #89356 Rabbit monoclonal h 0.388889 Neural crest Technology IgGGATA3 Cell Signaling 5852 Rabbit monoclonal h, m 0.597222 Precursors of Technology IgG PPE and neural crest cell, type II SGN TUBB3 Millipore AB9354 Chicke polyclonal h, m, r 0.736111 SGN Sigma nTUBB3 BioLegend 801203 Mouse monoclonal h, m, r 0.388889 SGNlgG2aPRPH Novus NBP1- Chicke polyclonal h, m, r, p, 1.430556 Type II SGN Biologicals 05423 n bMAP2 Santa Cruz sc-74421 Mouse monoclonal h, m, r 0.180556 Neuron IgGlCX43 Millipore MAB3067 Mouse monoclonal h, m, r, p, 0.215278 Gap junction Sigma IgGl dMPZ Bioss bs-0337R Rabbit polyclonal h, m, r, d 0.111111 Myelinating Schwann cell MBP Novus NBP2- Mouse monoclonal h, m 0.319444 Myelinating Biologicals 22121 IgGl Schwann cell TH Novus NB330-109 Rabbit polyclonal h, m, r, 0.736111 Type II SGN Biologicals dr, maS100B Novus NBP2- Mouse monoclonal h, m, r, b 0.097222 Schwann cell Biologicals 53187 lgG2aMyosin Proteus 25-6790 Rabbit polyclonal h, r, m, p, 0.388889 Hair cell 7a Biosciences av, amNeurofil Millipore AB5539 Chicke polyclonal b, f, m, h, niiniZ SGN ament H Sigma n p, rCtBP2 BD 612044 Mouse monoclonal h, m, r, d 0.736111 Presynapse Biosciences IgGlPSD95 Neuromab 75-028 Mouse monoclonal h, r, m 0.736111 Postsynapse lgG2ahNF-H Abnova MAB5186 Mouse monoclonal h 1.083333 SGNIgGlhGluR2 Novus NBP2- Mouse monoclonal h 0.388889 Postsynapse Biologicals 61775 lgG2bVGLUT1 Synaptic 135-303 Rabbit polyclonal h, r, m, b 0.736111 Glutamatergic Systems neuron VGLUT1 Abeam ab227805 Rabbit monoclonal h, r, m 1 ?g / ml Glutamatergic neuron GFAP Cell Signaling 80788 Rabbit monoclonal h, m, r, 0.215278 Glial cells Technology mkh human, r rat, m mouse, p pig, av avian, am amphibian, b bovine, f feline, d dog, dr Drosophilia, i insect, ma mammal, mk monkey, NGFR nerve growth factor receptor, NEURODI a donut-shaped cluster of ONP-like cells, GATA3 GATA binding protein 3, TUBB3 Tubulin beta 3 class III, PRPH peripheral neuronal markerperi pherin , MAP2 microtubule-associated protein 2, CX43 connexin 43, MPZ myelin protein zero, TH tyrosine hydroxylase, VGLUT1 theglutamatergic neuronal marker vesicular glutamate transporter 1, GFAP glial fibrillary acidic proteinTable 3. Gene expression on D25.Gene P-value Avgjo pct.l pct.2 P-value_adj Clusterg2 FCCOL3A1 4.03E-295 2.186 0.943 0.332 1.47E-290 CNC- Mesenchyme S100A11 9.28E-260 1.695 0.984 0.57 3.40E-255 CNC- Mesenchyme TUBA1B 6.54E-248 -1.364 0.934 0.982 2.39E-243 CNC- MesenchymeLUM 3.51E-247 1.768 0.914 0.338 1.28E-242 CNC- Mesenchyme H2AFZ 8.52E-244 -1.386 0.904 0.978 3.12E-239 CNC- Mesenchyme HMGB2 6.37E-228 -2.372 0.395 0.865 2.33E-223 CNC- Mesenchyme TWIST1 6.97E-224 1.501 0.871 0.298 2.55E-219 CNC- Mesenchyme HMGB1 3.20E-213 -1.136 0.944 0.978 1.17E-208 CNC- MesenchymeTPT1 3.15E-204 0.646 1 0.994 1.15E-199 CNC- Mesenchyme CENPF 9.71E-199 -2.426 0.215 0.774 3.55E-194 CNC- MesenchymeBGN 2.40E-194 1.503 0.764 0.243 8.79E-190 CNC- MesenchymeFRZB 1.03E-192 1.407 0.787 0.271 3.76E-188 CNC- Mesenchyme TGFBI 2.97E-191 1.414 0.802 0.289 1.09E-186 CNC- Mesenchyme APOE 2.11E-186 1.689 0.898 0.489 7.73E-182 CNC- Mesenchyme STMN1 6.76E-184 -0.998 0.949 0.985 2.47E-179 CNC- Mesenchyme TMSB4X 9.03E-183 0.939 1 0.985 3.31E-178 CNC- Mesenchyme HMGN2 2.17E-181 -1.441 0.777 0.934 7.94E-177 CNC- MesenchymeMGP 5.18E-180 2.228 0.692 0.202 1.90E-175 CNC- Mesenchyme CKS1B 8.76E-177 -1.764 0.412 0.827 3.21E-172 CNC- Mesenchyme HAS2 4.12E-174 1.475 0.759 0.27 1.51E-169 CNC- MesenchymeT0P2A 1.66E-171 -2.172 0.172 0.704 6.06E-167 CNC- Mesenchyme UBE2C 1.86E-171 -2.589 0.177 0.706 6.80E-167 CNC- Mesenchyme PTMA 3.37E-170 -0.695 0.992 0.994 1.23E-165 CNC- Mesenchyme HSP90AA1 4.29E-169 -1.011 0.932 0.976 1.57E-164 CNC- Mesenchyme FTL 1.87E-159 0.745 1 0.991 6.85E-155 CNC- Mesenchyme PEG10 7.40E-157 1.341 0.91 0.599 2.71E-152 CNC- Mesenchyme NUSAP1 1.84E-156 -1.827 0.116 0.646 6.75E-152 CNC- Mesenchyme NR2F1 2.29E-156 1.183 0.938 0.593 8.37E-152 CNC- Mesenchyme CALD1 4.02E-156 1.152 0.926 0.612 1.47E-151 CNC- Mesenchyme CKB 3.23E-155 -2.196 0.216 0.704 1.18E-150 CNC- Mesenchyme SAT1 7.74E-152 1.507 0.926 0.622 2.83E-147 CNC- Mesenchyme UBE2S 1.65E-151 -1.805 0.486 0.827 6.05E-147 CNC- Mesenchyme DCN 3.71E-149 1.253 0.697 0.245 1.36E-144 CNC- Mesenchyme CDC42EP5 2.91E-143 1.15 0.755 0.331 1.07E-138 CNC- Mesenchyme MKI67 6.34E-141 -1.718 0.091 0.598 2.32E-136 CNC- Mesenchyme SLC1A3 1.41E-14O 1.318 0.712 0.283 5.15E-136 CNC- Mesenchyme PTTG1 1.83E-140 -1.871 0.348 0.756 6.71E-136 CNC- Mesenchyme ACTG1 4.98E-138 0.576 1 0.99 1.82E-133 CNC- Mesenchyme OLFML3 6.87E-136 1.153 0.562 0.165 2.51E-131 CNC- Mesenchyme FTH1 9.13E-136 0.628 1 0.988 3.34E-131 CNC- Mesenchyme IGFBP2 2.06E-135 0.878 0.992 0.899 7.54E-131 CNC- Mesenchyme IFITM3 1.18E-132 1.155 0.83 0.471 4.32E-128 CNC- Mesenchyme CCNB1 2.11E-132 -1.969 0.112 0.597 7.71E-128 CNC- Mesenchyme TUBB4B 3.89E-128 -1.564 0.548 0.826 1.42E-123 CNC- MesenchymeHMGA1 1.09E-126 -1.321 0.458 0.801 4.01E-122 CNC- Mesenchyme RPL10 2.69E-126 0.411 0.999 0.997 9.83E-122 CNC- Mesenchyme RANBP1 3.19E-124 -1.062 0.718 0.901 1.17E-119 CNC- Mesenchyme TPX2 4.00E-124 -1.604 0.11 0.573 1.46E-119 CNC- Mesenchyme CALM2 8.68E-123 -1.01 0.847 0.957 3.18E-118 CNC- Mesenchyme HOXB-AS3 1.49E-122 -2.524 0.126 0.578 5.47E-118 CNC- Mesenchyme CKS2 1.91E-121 -1.488 0.404 0.758 6.99E-117 CNC- Mesenchyme ITM2A 7.47E-120 1.168 0.626 0.229 2.74E-115 CNC- Mesenchyme DLGAP5 1.14E-119 -1.563 0.057 0.524 4.17E-115 CNC- Mesenchyme SNCA 1.57E-119 1.098 0.684 0.293 5.74E-115 CNC- Mesenchyme TUBB 2.67E-119 -0.634 0.97 0.987 9.78E-115 CNC- Mesenchyme ADD3 1.79E-118 1.107 0.76 0.393 6.56E-114 CNC- Mesenchyme CDH11 1.47E-116 1.036 0.643 0.258 5.38E-112 CNC- Mesenchyme ANXA1 1.37E-114 1.227 0.499 0.143 5.00E-110 CNC- Mesenchyme ASPM 9.90E-110 -1.552 0.071 0.509 3.62E-105 CNC- Mesenchyme BIRC5 1.62E-109 -1.378 0.145 0.575 5.92E-105 CNC- Mesenchyme FOXCI 2.24E-108 0.939 0.66 0.271 8.20E-104 CNC- Mesenchyme RBP1 5.05E-108 -1.384 0.659 0.872 1.85E-103 CNC- Mesenchyme H1FX 3.18E-106 -1.102 0.628 0.86 1.16E-101 CNC- Mesenchyme MAD2L1 1.45E-105 -1.283 0.179 0.598 5.30E-101 CNC- Mesenchyme HNRNPA2B 5.27E-105 -0.8 0.888 0.958 1.93E-100 CNC-1 Mesenchyme COL6A3 1.65E-104 1.008 0.388 0.09 6.05E-100 CNC- Mesenchyme C9orfl6 3.55E-104 1.095 0.899 0.661 1.30E-99 CNC- Mesenchyme CCNB2 3.21E-103 -1.423 0.11 0.532 1.18E-98 CNC- MesenchymeKPNA2 3.31E-103 -1.53 0.322 0.677 1.21E-98 CNC- Mesenchyme NASP 4.65E-103 -1.003 0.64 0.865 1.70E-98 CNC- Mesenchyme CEBPD 1.11E-102 0.971 0.456 0.131 4.08E-98 CNC- Mesenchyme HIST1H4C 1.13E-102 -2.535 0.693 0.823 4.13E-98 CNC- Mesenchyme TYMS 6.62E-102 -1.185 0.428 0.751 2.42E-97 CNC- Mesenchyme RPS12 2.13E-99 0.362 1 0.997 7.81E-95 CNC- Mesenchyme RAN 5.46E-99 -0.726 0.909 0.955 2.00E-94 CNC- Mesenchyme PRRX1 1.22E-98 0.917 0.573 0.221 4.47E-94 CNC- Mesenchyme FLRT2 1.38E-97 0.994 0.468 0.15 5.05E-93 CNC- Mesenchyme AKAP12 3.50E-97 1.19 0.832 0.538 1.28E-92 CNC- Mesenchyme ARL6IP1 1.12E-96 -1.612 0.436 0.726 4.10E-92 CNC- Mesenchyme ODC1 1.09E-95 -1.195 0.34 0.685 3.97E-91 CNC- Mesenchyme CDKN3 9.68E-95 -1.381 0.107 0.505 3.54E-90 CNC- Mesenchyme S0X2 3.73E-94 -1.438 0.14 0.53 1.37E-89 CNC- Mesenchyme CDK1 4.95E-94 -1.276 0.117 0.516 1.81E-89 CNC- Mesenchyme H2AFX 6.84E-94 -1.251 0.158 0.551 2.50E-89 CNC- Mesenchyme EVA1B 1.35E-91 0.919 0.61 0.271 4.93E-87 CNC- Mesenchyme MDK 1.72E-91 -0.607 0.981 0.988 6.30E-87 CNC- Mesenchyme DEK 4.40E-91 -1.015 0.485 0.784 1.61E-86 CNC- Mesenchyme TMSB15A 8.79E-91 -0.993 0.442 0.77 3.22E-86 CNC- Mesenchyme PSIP1 1.45E-90 -1.065 0.475 0.767 5.32E-86 CNC- Mesenchyme HOXB9 1.44E-89 -1.527 0.051 0.429 5.29E-85 CNC- Mesenchyme ITGA1 3.67E-89 0.911 0.354 0.087 1.34E-84 CNC- Mesenchyme CENPV 4.96E-89 -1.129 0.452 0.746 1.82E-84 CNC- MesenchymeARHGAP29 1.28E-88 0.889 0.486 0.168 4.68E-84 CNC- Mesenchyme JPT1 1.29E-88 -1.017 0.64 0.84 4.74E-84 CNC- Mesenchyme CENPW 2.51E-88 -1.064 0.328 0.677 9.17E-84 CNC- Mesenchyme VCAN 6.13E-88 0.818 0.855 0.56 2.24E-83 CNC- Mesenchyme CD63 1.43E-87 0.673 0.982 0.911 5.25E-83 CNC- Mesenchyme EDNRA 2.83E-87 0.9 0.437 0.14 1.04E-82 CNC- Mesenchyme IER3 1.18E-85 0.9 0.622 0.282 4.33E-81 CNC- Mesenchyme PCOLCE 2.12E-85 0.857 0.581 0.244 7.75E-81 CNC- Mesenchyme HMGB3 8.41E-85 -1.109 0.293 0.646 3.08E-80 CNC- Mesenchyme LAMA4 1.12E-84 0.887 0.468 0.168 4.10E-80 CNC- Mesenchyme UBE2T 1.92E-84 -1.092 0.294 0.644 7.02E-80 CNC- Mesenchyme RPL28 9.65E-84 0.343 0.999 0.994 3.53E-79 CNC- Mesenchyme TUBB2B 2.73E-83 -1.211 0.561 0.803 9.99E-79 CNC- Mesenchyme PRC1 3.06E-83 -1.132 0.063 0.425 1.12E-78 CNC- Mesenchyme HMMR 4.65E-81 -1.199 0.048 0.405 1.70E-76 CNC- Mesenchyme C1QTNF4 9.47E-81 0.913 0.641 0.324 3.47E-76 CNC- Mesenchyme MYL12A 1.78E-80 0.875 0.861 0.647 6.51E-76 CNC- Mesenchyme PCLAF 3.16E-80 -1.115 0.234 0.591 1.16E-75 CNC- Mesenchyme SNRPE 4.16E-80 -0.754 0.766 0.9 1.52E-75 CNC- Mesenchyme IGFBP5 8.85E-80 1.048 0.593 0.275 3.24E-75 CNC- Mesenchyme ALCAM 1.14E-79 0.837 0.458 0.164 4.18E-75 CNC- Mesenchyme NUF2 1.47E-79 -1.123 0.049 0.4 5.37E-75 CNC- Mesenchyme SMC4 1.41E-78 -1.068 0.203 0.552 5.16E-74 CNC- Mesenchyme TFPI 1.11E-77 0.743 0.428 0.144 4.06E-73 CNC- MesenchymeHIST1H1D 1.18E-77 -1.535 0.178 0.523 4.32E-73 CNC- Mesenchyme TMSB10 2.10E-77 0.538 0.992 0.962 7.70E-73 CNC- Mesenchyme CDC20 5.87E-77 -1.224 0.064 0.41 2.15E-72 CNC- Mesenchyme ARL4C 6.18E-77 -1.145 0.064 0.412 2.26E-72 CNC- Mesenchyme FAP 9.32E-77 0.681 0.42 0.139 3.41E-72 CNC- Mesenchyme HIST1H1B 1.27E-76 -1.386 0.097 0.444 4.66E-72 CNC- Mesenchyme CCNA2 1.60E-76 -1.06 0.054 0.397 5.85E-72 CNC- Mesenchyme NUCKS1 1.80E-76 -0.863 0.73 0.872 6.59E-72 CNC- Mesenchyme PHLDA2 3.54E-76 0.823 0.333 0.088 1.29E-71 CNC- Mesenchyme COLEC12 6.34E-75 0.77 0.412 0.137 2.32E-70 CNC- Mesenchyme TPM1 2.73E-74 0.924 0.817 0.583 9.99E-70 CNC- Mesenchyme SERF2 4.11E-74 0.476 0.994 0.966 1.50E-69 CNC- Mesenchyme KIF2OB 4.40E-74 -1.061 0.114 0.457 1.61E-69 CNC- Mesenchyme ITGA4 3.09E-73 0.773 0.376 0.117 1.13E-68 CNC- Mesenchyme GTS El 3.87E-73 -1.016 0.058 0.389 1.42E-68 CNC- Mesenchyme FRMD4A 1.03E-72 0.814 0.597 0.301 3.79E-68 CNC- Mesenchyme SPC25 3.67E-72 -0.95 0.043 0.369 1.34E-67 CNC- Mesenchyme AURKB 6.21E-72 -0.987 0.059 0.389 2.27E-67 CNC- Mesenchyme SNRPD1 2.44E-71 -0.731 0.771 0.881 8.92E-67 CNC- Mesenchyme MIR1OOHG 2.58E-71 0.737 0.463 0.183 9.43E-67 CNC- Mesenchyme TIMP1 3.13E-71 0.968 0.721 0.446 1.15E-66 CNC- Mesenchyme CFI 4.74E-71 0.718 0.324 0.09 1.74E-66 CNC- Mesenchyme CKAP2 1.12E-70 -1.054 0.155 0.491 4.10E-66 CNC- Mesenchyme SELENOM 1.49E-7O 0.824 0.54 0.255 5.45E-66 CNC- MesenchymeCENPH 2.43E-70 -0.953 0.197 0.532 8.89E-66 CNC- Mesenchyme 0ST4 7.44E-70 0.586 0.958 0.863 2.72E-65 CNC- Mesenchyme SNRPF 1.16E-69 -0.658 0.808 0.909 4.25E-65 CNC- Mesenchyme PTGES3 1.81E-69 -0.728 0.739 0.885 6.62E-65 CNC- Mesenchyme CPED1 2.71E-69 0.702 0.393 0.133 9.93E-65 CNC- Mesenchyme LAMB1 4.60E-69 0.8 0.456 0.181 1.68E-64 CNC- Mesenchyme PHLDA1 1.05E-68 0.755 0.499 0.207 3.83E-64 CNC- Mesenchyme NELL2 2.34E-68 0.761 0.486 0.203 8.58E-64 CNC- Mesenchyme EEF1A1 6.34E-68 0.259 1 1 2.32E-63 CNC- Mesenchyme FGFBP3 1.07E-67 -1.158 0.288 0.592 3.93E-63 CNC- Mesenchyme LSM4 1.99E-67 -0.765 0.638 0.823 7.30E-63 CNC- Mesenchyme ERH 2.65E-67 -0.665 0.819 0.91 9.68E-63 CNC- Mesenchyme SM0C2 3.89E-67 0.604 0.265 0.062 1.42E-62 CNC- Mesenchyme SG02 2.08E-66 -1.027 0.054 0.364 7.60E-62 CNC- Mesenchyme YBX1 2.16E-66 -0.453 0.977 0.981 7.89E-62 CNC- Mesenchyme CDCA8 2.74E-66 -0.971 0.03 0.332 1.00E-61 CNC- Mesenchyme HIST1H1C 2.84E-66 -1.236 0.134 0.449 1.04E-61 CNC- Mesenchyme RPL13 4.17E-66 0.261 0.999 0.998 1.53E-61 CNC- Mesenchyme COL5A2 5.18E-66 0.926 0.524 0.245 1.90E-61 CNC- Mesenchyme TPM4 1.06E-65 0.756 0.837 0.61 3.90E-61 CNC- Mesenchyme NDC80 6.71E-65 -0.899 0.045 0.351 2.45E-60 CNC- Mesenchyme SERPINF1 2.45E-64 0.728 0.515 0.233 8.98E-60 CNC- Mesenchyme SNRPB 2.91E-64 -0.774 0.631 0.819 1.06E-59 CNC- Mesenchyme SRSF3 6.63E-64 -0.663 0.79 0.904 2.43E-59 CNC- MesenchymeMDFI 1.25E-63 0.766 0.581 0.299 4.57E-59 CNC- Mesenchyme CDCA3 2.16E-63 -0.924 0.032 0.327 7.90E-59 CNC- Mesenchyme FN1 1.51E-62 1.108 0.482 0.217 5.54E-58 CNC- Mesenchyme ZFP36L2 3.03E-62 0.759 0.561 0.281 1.11E-57 CNC- Mesenchyme PCDH7 5.77E-62 0.709 0.475 0.208 2.11E-57 CNC- Mesenchyme SEMA3E 7.76E-62 0.575 0.297 0.082 2.84E-57 CNC- Mesenchyme SET 1.09E-61 -0.613 0.855 0.937 3.98E-57 CNC- Mesenchyme CENPE 1.13E-61 -0.95 0.052 0.347 4.13E-57 CNC- Mesenchyme TUBA1A 1.45E-61 -0.829 0.944 0.978 5.30E-57 CNC- Mesenchyme LSM5 2.36E-61 -0.778 0.551 0.765 8.65E-57 CNC- Mesenchyme CSRP2 2.82E-61 -0.835 0.487 0.736 1.03E-56 CNC- Mesenchyme RPA3 2.97E-61 -0.86 0.403 0.668 1.09E-56 CNC- Mesenchyme MALAT1 4.23E-61 0.37 1 1 1.55E-56 CNC- Mesenchyme PFDN5 7.58E-61 0.472 0.972 0.92 2.78E-56 CNC- Mesenchyme PIMREG 1.21E-60 -0.916 0.043 0.332 4.41E-56 CNC- Mesenchyme PCDH18 2.06E-60 0.658 0.449 0.188 7.54E-56 CNC- Mesenchyme AURKA 2.30E-60 -0.91 0.032 0.319 8.42E-56 CNC- Mesenchyme JUND 2.62E-60 0.594 0.936 0.827 9.59E-56 CNC- Mesenchyme CALR 3.53E-60 0.681 0.909 0.751 1.29E-55 CNC- Mesenchyme CENPA 6.36E-60 -0.89 0.042 0.327 2.33E-55 CNC- Mesenchyme MXRA5 7.40E-60 0.681 0.388 0.146 2.71E-55 CNC- Mesenchyme ECT2 9.00E-60 -0.917 0.058 0.346 3.30E-55 CNC- Mesenchyme VIM 1.13E-59 0.484 0.996 0.976 4.13E-55 CNC- Mesenchyme SNAI2 8.00E-59 0.584 0.282 0.08 2.93E-54 CNC- MesenchymeDAB2 1.52E-58 0.721 0.408 0.163 5.58E-54 CNC- Mesenchyme NFIA 2.06E-58 0.741 0.516 0.242 7.54E-54 CNC- Mesenchyme A2M 2.66E-58 0.676 0.322 0.104 9.75E-54 CNC- Mesenchyme COL6A2 3.51E-58 0.634 0.427 0.175 1.29E-53 CNC- Mesenchyme CD24 3.63E-58 -1.106 0.112 0.408 1.33E-53 CNC- Mesenchyme ITM2C 5.31E-58 0.701 0.64 0.377 1.94E-53 CNC- Mesenchyme KIF11 6.43E-58 -0.856 0.059 0.343 2.35E-53 CNC- Mesenchyme H2AFV 8.97E-58 -0.745 0.583 0.786 3.28E-53 CNC- Mesenchyme FCGRT 5.25E-57 0.577 0.298 0.091 1.92E-52 CNC- Mesenchyme SFRP2 1.17E-56 -1.195 0.136 0.432 4.26E-52 CNC- Mesenchyme IGFBP7 1.82E-56 0.923 0.235 0.056 6.66E-52 CNC- Mesenchyme DTYMK 2.57E-56 -0.884 0.269 0.551 9.41E-52 CNC- Mesenchyme RPL34 2.50E-55 0.287 0.999 0.994 9.15E-51 CNC- Mesenchyme PDGFRB 3.39E-55 0.59 0.306 0.098 1.24E-50 CNC- Mesenchyme LAPTM4A 3.71E-55 0.7 0.764 0.545 1.36E-50 CNC- Mesenchyme MIS18BP1 4.11E-55 -0.871 0.153 0.444 1.50E-50 CNC- Mesenchyme MT-C01 9.05E-55 0.267 0.997 0.995 3.31E-50 CNC- Mesenchyme SFRP1 1.23E-54 0.719 0.606 0.344 4.51E-50 CNC- Mesenchyme HOXB2 1.52E-54 -0.973 0.051 0.318 5.56E-50 CNC- Mesenchyme LRRC17 3.42E-54 0.705 0.458 0.209 1.25E-49 CNC- Mesenchyme RRM2 3.57E-54 -0.782 0.056 0.326 1.30E-49 CNC- Mesenchyme CDH2 5.78E-54 -0.879 0.086 0.362 2.12E-49 CNC- Mesenchyme RAMP2 6.80E-54 0.621 0.402 0.165 2.49E-49 CNC- Mesenchyme CENPU 1.70E-53 -0.78 0.125 0.407 6.21E-49 CNC- MesenchymeSNRPG 5.03E-53 -0.609 0.763 0.874 1.84E-48 CNC- Mesenchyme PMP22 6.32E-53 0.685 0.367 0.142 2.31E-48 CNC- Mesenchyme OAF 8.90E-53 0.676 0.606 0.347 3.26E-48 CNC- Mesenchyme LHFPL6 1.45E-52 0.632 0.394 0.16 5.29E-48 CNC- Mesenchyme KIF2C 1.61E-52 -0.765 0.033 0.291 5.88E-48 CNC- Mesenchyme NR3C1 2.63E-52 0.68 0.413 0.177 9.64E-48 CNC- Mesenchyme CFH 9.82E-52 0.484 0.192 0.04 3.59E-47 CNC- Mesenchyme SMC3 1.98E-51 -0.714 0.481 0.708 7.23E-47 CNC- Mesenchyme PSRC1 1.55E-50 -0.76 0.03 0.279 5.66E-46 CNC- Mesenchyme DBF4 4.13E-50 -0.817 0.154 0.428 1.51E-45 CNC- Mesenchyme ZWINT 4.56E-50 -0.742 0.107 0.379 1.67E-45 CNC- Mesenchyme H3F3A 6.10E-50 -0.281 1 0.997 2.23E-45 CNC- Mesenchyme PBK 1.23E-49 -0.77 0.071 0.327 4.52E-45 CNC- Mesenchyme ITGA8 1.64E-49 0.614 0.317 0.113 6.01E-45 CNC- Mesenchyme PLK1 4.00E-49 -0.795 0.033 0.276 1.47E-44 CNC- Mesenchyme CTSC 9.59E-49 0.667 0.332 0.126 3.51E-44 CNC- Mesenchyme DDR2 1.06E-48 0.612 0.343 0.134 3.88E-44 CNC- Mesenchyme ECM1 1.21E-48 0.441 0.187 0.04 4.45E-44 CNC- Mesenchyme TIMP3 2.17E-48 0.622 0.38 0.157 7.94E-44 CNC- Mesenchyme ITM2B 3.32E-48 0.618 0.842 0.675 1.21E-43 CNC- Mesenchyme B2M 3.75E-48 0.665 0.848 0.685 1.37E-43 CNC- Mesenchyme RPS4X 4.72E-48 0.273 0.999 0.995 1.73E-43 CNC- Mesenchyme MGST1 5.84E-48 -0.783 0.451 0.678 2.14E-43 CNC- Mesenchyme HIC1 9.45E-48 0.455 0.232 0.064 3.46E-43 CNC- MesenchymeITGB3BP 1.99E-47 -0.777 0.213 0.478 7.28E-43 CNC- Mesenchyme WWTR1 3.01E-47 0.611 0.426 0.198 1.10E-42 CNC- Mesenchyme CENPN 3.31E-47 -0.718 0.129 0.395 1.21E-42 CNC- Mesenchyme NCAPG 3.45E-47 -0.716 0.061 0.31 1.26E-42 CNC- Mesenchyme MAML2 4.38E-47 0.606 0.409 0.181 1.60E-42 CNC- Mesenchyme RPL37 5.40E-47 0.272 0.997 0.991 1.98E-42 CNC- Mesenchyme ANGPTL1 6.43E-47 0.525 0.254 0.078 2.35E-42 CNC- Mesenchyme ANP32B 8.27E-47 -0.614 0.667 0.824 3.03E-42 CNC- Mesenchyme GDF15 9.07E-47 1.128 0.331 0.128 3.32E-42 CNC- Mesenchyme PRKDC 1.13E-46 -0.638 0.638 0.807 4.15E-42 CNC- Mesenchyme S0X4 1.15E-46 0.453 0.931 0.805 4.20E-42 CNC- Mesenchyme PPIB 1.16E-46 0.535 0.902 0.79 4.24E-42 CNC- Mesenchyme KNL1 1.66E-46 -0.759 0.061 0.303 6.08E-42 CNC- Mesenchyme PCSK9 2.03E-46 0.605 0.28 0.095 7.42E-42 CNC- Mesenchyme NEK2 2.49E-46 -0.735 0.013 0.236 9.11E-42 CNC- Mesenchyme BASP1 3.22E-46 0.46 0.806 0.586 1.18E-41 CNC- Mesenchyme SERPINH1 6.61E-46 0.651 0.568 0.337 2.42E-41 CNC- Mesenchyme ANP32E 6.93E-46 -0.771 0.442 0.659 2.54E-41 CNC- Mesenchyme MND1 7.31E-46 -0.745 0.105 0.353 2.68E-41 CNC- Mesenchyme SLC25A3 9.72E-46 0.377 0.991 0.959 3.56E-41 CNC- Mesenchyme SNHG29 1.26E-45 0.359 0.975 0.947 4.63E-41 CNC- Mesenchyme NMU 2.08E-45 -0.795 0.058 0.298 7.60E-41 CNC- Mesenchyme SYNE2 2.09E-45 -0.777 0.235 0.495 7.63E-41 CNC- Mesenchyme SG01 4.92E-45 -0.694 0.044 0.277 1.80E-40 CNC- MesenchymeKIF22 8.49E-45 -0.715 0.104 0.354 3.11E-40 CNC- Mesenchyme RHOC 1.52E-44 0.599 0.676 0.44 5.55E-40 CNC- Mesenchyme SEC61B 2.58E-44 0.483 0.914 0.83 9.46E-40 CNC- Mesenchyme HIST1H1E 3.91E-44 -0.821 0.093 0.338 1.43E-39 CNC- Mesenchyme ORC6 5.94E-44 -0.702 0.164 0.421 2.17E-39 CNC- Mesenchyme KIFC1 1.35E-43 -0.679 0.042 0.267 4.95E-39 CNC- Mesenchyme TACC3 1.98E-43 -0.674 0.048 0.276 7.24E-39 CNC- Mesenchyme HNRNPD 2.59E-43 -0.639 0.616 0.778 9.47E-39 CNC- Mesenchyme PA2G4 7.36E-43 -0.692 0.524 0.711 2.69E-38 CNC- Mesenchyme BUB3 7.92E-43 -0.795 0.251 0.495 2.90E-38 CNC- Mesenchyme SPATS2L 8.38E-43 0.628 0.399 0.185 3.07E-38 CNC- Mesenchyme SRP9 1.62E-42 -0.587 0.703 0.845 5.91E-38 CNC- Mesenchyme SSR2 1.63E-42 0.554 0.828 0.682 5.95E-38 CNC- Mesenchyme HTRA1 1.98E-42 0.631 0.348 0.148 7.24E-38 CNC- Mesenchyme PHGDH 2.28E-42 -0.751 0.417 0.643 8.36E-38 CNC- Mesenchyme EMP3 2.32E-42 0.597 0.669 0.437 8.50E-38 CNC- Mesenchyme FAM114A1 5.11E-42 0.562 0.336 0.139 1.87E-37 CNC- Mesenchyme NUDT1 1.70E-41 -0.714 0.264 0.506 6.22E-37 CNC- Mesenchyme MCM7 1.87E-41 -0.713 0.191 0.444 6.84E-37 CNC- Mesenchyme HSPA5 2.64E-41 0.574 0.809 0.631 9.67E-37 CNC- Mesenchyme GRK5 3.32E-41 0.483 0.265 0.093 1.21E-36 CNC- Mesenchyme PCDH9 4.02E-41 0.535 0.297 0.113 1.47E-36 CNC- Mesenchyme H2AFY 4.57E-41 -0.645 0.547 0.743 1.67E-36 CNC- Mesenchyme PIF1 1.35E-40 -0.65 0.014 0.214 4.95E-36 CNC- MesenchymeMYLIP 2.18E-40 0.488 0.271 0.098 8.00E-36 CNC- Mesenchyme C0L1A1 2.58E-40 0.761 0.294 0.115 9.44E-36 CNC- Mesenchyme LAMA1 2.69E-40 0.567 0.419 0.207 9.84E-36 CNC- Mesenchyme HNRNPAB 3.13E-40 -0.62 0.596 0.766 1.15E-35 CNC- Mesenchyme COL4A2 5.87E-40 0.675 0.552 0.329 2.15E-35 CNC- Mesenchyme KNSTRN 6.91E-40 -0.677 0.058 0.276 2.53E-35 CNC- Mesenchyme SEC61G 9.30E-40 0.448 0.915 0.841 3.40E-35 CNC- Mesenchyme RARRES2 1.11E-39 -0.753 0.126 0.362 4.08E-35 CNC- Mesenchyme CBX5 1.53E-39 -0.585 0.607 0.774 5.59E-35 CNC- Mesenchyme IGFBPL1 2.08E-39 -0.861 0.024 0.226 7.62E-35 CNC- Mesenchyme KIF4A 2.15E-39 -0.609 0.034 0.24 7.88E-35 CNC- Mesenchyme HIST1H1A 2.32E-39 -0.85 0.062 0.275 8.51E-35 CNC- Mesenchyme RRM1 3.01E-39 -0.648 0.186 0.427 1.10E-34 CNC- Mesenchyme RAD51AP1 8.40E-39 -0.63 0.081 0.303 3.07E-34 CNC- Mesenchyme VRK1 1.92E-38 -0.593 0.097 0.325 7.03E-34 CNC- Mesenchyme COL4A1 2.00E-38 0.698 0.385 0.189 7.34E-34 CNC- Mesenchyme CENPK 2.06E-38 -0.67 0.15 0.382 7.55E-34 CNC- Mesenchyme KIF14 2.14E-38 -0.57 0.019 0.213 7.85E-34 CNC- Mesenchyme SKA2 2.23E-38 -0.682 0.309 0.54 8.15E-34 CNC- Mesenchyme SMC2 4.32E-38 -0.659 0.17 0.403 1.58E-33 CNC- Mesenchyme AP1S2 4.55E-38 -0.806 0.404 0.609 1.66E-33 CNC- Mesenchyme DEPDC1 6.93E-38 -0.648 0.043 0.246 2.54E-33 CNC- Mesenchyme HOXB3 1.04E-37 -0.662 0.033 0.232 3.81E-33 CNC- Mesenchyme DNAJC1 1.18E-37 0.587 0.6 0.387 4.33E-33 CNC- MesenchymeKIF23 1.39E-37 -0.59 0.035 0.235 5.09E-33 CNC- Mesenchyme IFITM2 1.42E-37 0.597 0.593 0.375 5.18E-33 CNC- Mesenchyme TMPO 1.52E-37 -0.684 0.424 0.633 5.58E-33 CNC- Mesenchyme SEPTIN6 1.69E-37 0.571 0.569 0.349 6.17E-33 CNC- Mesenchyme COL11A1 2.11E-37 0.584 0.423 0.22 7.72E-33 CNC- Mesenchyme IER2 2.21E-37 0.665 0.634 0.425 8.11E-33 CNC- Mesenchyme CENPM 2.35E-37 -0.57 0.048 0.254 8.58E-33 CNC- Mesenchyme ZNF503 2.53E-37 -0.722 0.167 0.398 9.27E-33 CNC- Mesenchyme BMP2 2.84E-37 0.498 0.232 0.079 1.04E-32 CNC- Mesenchyme HNRNPA3 2.85E-37 -0.506 0.823 0.893 1.04E-32 CNC- Mesenchyme HES6 2.93E-37 -1.283 0.152 0.373 1.07E-32 CNC- Mesenchyme SRSF7 3.65E-37 -0.599 0.624 0.788 1.34E-32 CNC- Mesenchyme MAGED2 3.84E-37 0.525 0.823 0.668 1.41E-32 CNC- Mesenchyme EZH2 4.18E-37 -0.663 0.144 0.371 1.53E-32 CNC- Mesenchyme COL1A2 6.62E-37 0.653 0.221 0.072 2.42E-32 CNC- Mesenchyme TTK 8.89E-37 -0.577 0.019 0.207 3.25E-32 CNC- Mesenchyme OSTC 9.53E-37 0.466 0.884 0.786 3.49E-32 CNC- Mesenchyme HOXC6 9.55E-37 -0.593 0.02 0.21 3.50E-32 CNC- Mesenchyme HOXC9 1.00E-36 -0.633 0.023 0.212 3.66E-32 CNC- Mesenchyme RAD21 1.36E-36 -0.693 0.405 0.607 4.97E-32 CNC- Mesenchyme HOXB8 1.50E-36 -0.598 0.02 0.209 5.47E-32 CNC- Mesenchyme COL6A1 2.10E-36 0.521 0.438 0.232 7.69E-32 CNC- Mesenchyme HNRNPR 2.19E-36 -0.585 0.621 0.782 8.02E-32 CNC- Mesenchyme NPC2 3.07E-36 0.529 0.755 0.56 1.12E-31 CNC- MesenchymeSTC1 3.17E-36 0.476 0.163 0.041 1.16E-31 CNC- Mesenchyme SPRY4 3.27E-36 0.502 0.278 0.109 1.20E-31 CNC- Mesenchyme CALU 4.49E-36 0.596 0.696 0.509 1.64E-31 CNC- Mesenchyme TNFRSF21 7.12E-36 0.399 0.177 0.049 2.61E-31 CNC- Mesenchyme PNRC1 8.70E-36 0.536 0.472 0.264 3.19E-31 CNC- Mesenchyme CKAP2L 1.05E-35 -0.546 0.025 0.211 3.84E-31 CNC- Mesenchyme DHRS3 1.19E-35 0.554 0.409 0.212 4.36E-31 CNC- Mesenchyme DHFR 1.55E-35 -0.671 0.153 0.373 5.68E-31 CNC- Mesenchyme RHOJ 1.69E-35 0.45 0.278 0.11 6.18E-31 CNC- Mesenchyme C0L8A1 2.93E-35 0.351 0.157 0.039 1.07E-30 CNC- Mesenchyme SOX9 2.95E-35 0.531 0.403 0.206 1.08E-30 CNC- Mesenchyme PRDX5 3.01E-35 0.431 0.913 0.827 1.10E-30 CNC- Mesenchyme FHL3 3.37E-35 0.486 0.285 0.118 1.24E-30 CNC- Mesenchyme EXOSC8 4.81E-35 -0.652 0.338 0.547 1.76E-30 CNC- Mesenchyme SEMA3A 7.40E-35 0.489 0.333 0.15 2.71E-30 CNC- Mesenchyme IFITM1 1.29E-34 0.487 0.273 0.109 4.71E-30 CNC- Mesenchyme ANXA5 1.52E-34 0.538 0.707 0.524 5.55E-30 CNC- Mesenchyme CCT5 2.45E-34 -0.595 0.468 0.672 8.97E-30 CNC- Mesenchyme H0XC8 2.52E-34 -0.593 0.018 0.194 9.23E-30 CNC- Mesenchyme PLAT 2.55E-34 0.438 0.247 0.091 9.34E-30 CNC- Mesenchyme MXD3 2.55E-34 -0.555 0.037 0.224 9.34E-30 CNC- Mesenchyme HSPE1 3.20E-34 -0.483 0.777 0.86 1.17E-29 CNC- Mesenchyme LGALS3 3.57E-34 0.403 0.177 0.051 1.31E-29 CNC- Mesenchyme Clorfl22 4.03E-34 0.536 0.698 0.521 1.48E-29 CNC- MesenchymeBUB1B 6.24E-34 -0.541 0.043 0.23 2.28E-29 CNC- Mesenchyme GMNN 6.32E-34 -0.601 0.25 0.471 2.31E-29 CNC- Mesenchyme AL359555.4 7.85E-34 0.334 0.149 0.037 2.87E-29 CNC- Mesenchyme RPL35 9.49E-34 -0.265 0.997 0.99 3.47E-29 CNC- Mesenchyme SHISAL2B 1.12E-33 -0.605 0.015 0.188 4.10E-29 CNC- Mesenchyme FRMD6 1.60E-33 0.432 0.297 0.126 5.86E-29 CNC- Mesenchyme DRAXIN 1.78E-33 -0.561 0.029 0.209 6.50E-29 CNC- Mesenchyme CDCA2 1.94E-33 -0.502 0.019 0.192 7.10E-29 CNC- Mesenchyme SRGAP3 2.12E-33 -0.704 0.1 0.304 7.76E-29 CNC- Mesenchyme SNHG18 2.61E-33 0.399 0.273 0.109 9.56E-29 CNC- Mesenchyme PARP1 2.78E-33 -0.635 0.395 0.587 1.02E-28 CNC- Mesenchyme FZD3 3.48E-33 -0.672 0.116 0.323 1.27E-28 CNC- Mesenchyme H0TAIRM1 4.01E-33 -0.584 0.034 0.215 1.47E-28 CNC- Mesenchyme CEP55 4.73E-33 -0.533 0.018 0.189 1.73E-28 CNC- Mesenchyme PPIC 5.05E-33 0.368 0.168 0.048 1.85E-28 CNC- Mesenchyme HJURP 5.25E-33 -0.509 0.019 0.191 1.92E-28 CNC- Mesenchyme RTKN2 7.18E-33 -0.541 0.045 0.231 2.63E-28 CNC- Mesenchyme USP1 7.90E-33 -0.622 0.174 0.389 2.89E-28 CNC- Mesenchyme HSPD1 1.11E-32 -0.46 0.806 0.876 4.07E-28 CNC- Mesenchyme TROAP 1.25E-32 -0.547 0.039 0.222 4.57E-28 CNC- Mesenchyme BUB1 1.26E-32 -0.588 0.039 0.22 4.60E-28 CNC- Mesenchyme MZT1 1.58E-32 -0.65 0.197 0.414 5.80E-28 CNC- Mesenchyme GNG11 2.16E-32 0.552 0.523 0.326 7.90E-28 CNC- Mesenchyme TCF4 2.72E-32 0.539 0.657 0.461 9.95E-28 CNC- MesenchymeRRBP1 2.75E-32 0.564 0.501 0.311 1.01E-27 CNC- Mesenchyme EGFR 2.84E-32 0.379 0.229 0 .084 1.04E-27 CNC- Mesenchyme TUBA1C 5.07E-32 -0.854 0.21 0 .416 1.86E-27 CNC- Mesenchyme KLHL4 5.88E-32 0.402 0.182 0 .056 2.15E-27 CNC- Mesenchyme NREP 6.23E-32 0.459 0.696 0 .496 2.28E-27 CNC- Mesenchyme SUM02 8.09E-32 -0.306 0.973 0 .978 2.96E-27 CNC- Mesenchyme PAMR1 8.51E-32 0.324 0.152 0 .04 3.12E-27 CNC- Mesenchyme DSTN 1.02E-31 0.487 0.751 0 .587 3.74E-27 CNC- Mesenchyme C0L14A1 1.30E-31 -0.602 0.024 0 .194 4.77E-27 CNC- Mesenchyme XRCC5 1.56E-31 -0.482 0.736 0 .84 5.72E-27 CNC- Mesenchyme CPD 1.61E-31 0.55 0.355 0 .178 5.90E-27 CNC- Mesenchyme SPRY1 1.65E-31 0.443 0.66 0 .449 6.03E-27 CNC- Mesenchyme LMNB1 1.70E-31 -0.596 0.136 0 .345 6.21E-27 CNC- Mesenchyme NTS 3.43E-31 0.487 0.212 0 .074 1.25E-26 CNC- Mesenchyme ITGB1 5.90E-31 0.487 0.754 0 .608 2.16E-26 CNC- Mesenchyme C21orf58 1.27E-30 -0.548 0.083 0.271 4.66E-26 CNC- Mesenchyme CDC25C 1.52E-30 -0.484 0.032 0.199 5.56E-26 CNC- Mesenchyme NBL1 2.64E-30 0.377 0.23 0.088 9.68E-26 CNC- Mesenchyme C7 2.90E-30 0.461 0.182 0.06 1.06E-25 CNC- Mesenchyme AHNAK 2.96E-30 0.455 0.268 0.112 1.08E-25 CNC- Mesenchyme NID2 3.40E-30 0.356 0.211 0.076 1.25E-25 CNC- Mesenchyme PRRX2 4.14E-30 0.421 0.309 0.141 1.52E-25 CNC- Mesenchyme HIST2H2AC 5.19E-30 -0.516 0.03 0.196 1.90E-25 CNC- Mesenchyme PWWP3B 6.68E-30 0.317 0.131 0.032 2.44E-25 CNC- MesenchymePPIA 1.16E-29 -0.289 0.989 0.98 4.23E-25 CNC- Mesenchyme HAPLN1 1.90E-29 0.45 0.211 0.077 6.94E-25 CNC- Mesenchyme KCTD12 1.91E-29 0.438 0.29 0.13 6.99E-25 CNC- Mesenchyme VCL 2.08E-29 0.537 0.49 0.304 7.62E-25 CNC- Mesenchyme KIF15 2.28E-29 -0.507 0.042 0.21 8.36E-25 CNC- Mesenchyme PRDX2 2.49E-29 -0.375 0.926 0.943 9.13E-25 CNC- Mesenchyme METRN 2.78E-29 -0.469 0.76 0.849 1.02E-24 CNC- Mesenchyme HELLS 4.60E-29 -0.632 0.269 0.466 1.68E-24 CNC- Mesenchyme CD 164 4.66E-29 0.48 0.458 0.271 1.71E-24 CNC- Mesenchyme AM0TL1 5.03E-29 0.485 0.352 0.184 1.84E-24 CNC- Mesenchyme PRR11 6.04E-29 -0.48 0.029 0.188 2.21E-24 CNC- Mesenchyme UCP2 6.63E-29 -0.53 0.077 0.258 2.43E-24 CNC- Mesenchyme ANP32A 1.03E-28 -0.507 0.568 0.716 3.76E-24 CNC- Mesenchyme C0MMD6 1.08E-28 0.359 0.934 0.856 3.96E-24 CNC- Mesenchyme MYEF2 1.39E-28 -0.579 0.15 0.348 5.10E-24 CNC- Mesenchyme LIMA1 1.53E-28 0.428 0.354 0.182 5.59E-24 CNC- Mesenchyme CD99 1.92E-28 0.469 0.741 0.578 7.03E-24 CNC- Mesenchyme CNTNAP2 2.31E-28 -0.588 0.049 0.216 8.45E-24 CNC- Mesenchyme SERBP1 2.74E-28 -0.416 0.773 0.856 1.00E-23 CNC- Mesenchyme ADD3-AS1 3.45E-28 0.306 0.135 0.036 1.26E-23 CNC- Mesenchyme KIF18A 3.85E-28 -0.479 0.028 0.183 1.41E-23 CNC- Mesenchyme EPS8 4.04E-28 0.47 0.396 0.222 1.48E-23 CNC- Mesenchyme ADGRF5 4.43E-28 0.278 0.119 0.028 1.62E-23 CNC- Mesenchyme RRAS 4.83E-28 0.469 0.417 0.236 1.77E-23 CNC- MesenchymeNCAPD2 4.83E-28 -0.511 0.077 0.253 1.77E-23 CNC- Mesenchyme ENY2 5.67E-28 -0.473 0.702 0.803 2.07E-23 CNC- Mesenchyme LAMC3 6.16E-28 0.377 0.179 0.061 2.25E-23 CNC- Mesenchyme ANGPT1 8.03E-28 0.354 0.154 0.046 2.94E-23 CNC- Mesenchyme PCNA 9.55E-28 -0.628 0.274 0.47 3.50E-23 CNC- Mesenchyme PKM 1.08E-27 -0.556 0.598 0.732 3.94E-23 CNC- Mesenchyme RACGAP1 1.19E-27 -0.467 0.025 0.177 4.35E-23 CNC- Mesenchyme CPE 1.20E-27 0.48 0.548 0.363 4.38E-23 CNC- Mesenchyme SFPQ 1.30E-27 -0.524 0.62 0.754 4.75E-23 CNC- Mesenchyme EIF1AX 1.45E-27 -0.463 0.715 0.815 5.30E-23 CNC- Mesenchyme LRP10 1.47E-27 0.385 0.274 0.123 5.36E-23 CNC- Mesenchyme SQSTM1 1.53E-27 0.465 0.476 0.3 5.58E-23 CNC- Mesenchyme AC004540. 1.83E-27 -0.622 0.117 0.299 6.70E-23 CNC- 2 Mesenchyme FBXO5 1.83E-27 -0.531 0.105 0.284 6.71E-23 CNC- Mesenchyme P4HA1 1.87E-27 0.507 0.381 0.213 6.86E-23 CNC- Mesenchyme TRIM59 3.04E-27 -0.548 0.083 0.259 1.11E-22 CNC- Mesenchyme NRP2 3.26E-27 0.53 0.402 0.231 1.19E-22 CNC- Mesenchyme BANF1 3.60E-27 -0.438 0.759 0.832 1.32E-22 CNC- Mesenchyme CYTOR 3.74E-27 0.442 0.256 0.112 1.37E-22 CNC- Mesenchyme CCDC34 4.21E-27 -0.576 0.294 0.485 1.54E-22 CNC- Mesenchyme ENO1 5.45E-27 -0.447 0.764 0.864 1.99E-22 CNC- Mesenchyme FBN1 5.89E-27 0.392 0.265 0.119 2.16E-22 CNC- Mesenchyme ARID5B 5.92E-27 0.349 0.244 0.103 2.17E-22 CNC- Mesenchyme NPM1 5.97E-27 -0.263 0.982 0.986 2.19E-22 CNC- MesenchymeANXA2 6.71E-27 0.674 0.547 0.38 2.46E-22 CNC- Mesenchyme ITGA10 6.79E-27 0.295 0.136 0.038 2.49E-22 CNC- Mesenchyme ARHGDIB 7.46E-27 0.389 0.203 0.078 2.73E-22 CNC- Mesenchyme NME1 7.60E-27 -0.539 0.614 0.731 2.78E-22 CNC- Mesenchyme RGL1 8.00E-27 0.339 0.201 0.075 2.93E-22 CNC- Mesenchyme CD81 8.21E-27 0.397 0.85 0.722 3.00E-22 CNC- Mesenchyme MARCKSL1 8.91E-27 -0.314 0.961 0.974 3.26E-22 CNC- Mesenchyme SINHCAF 9.10E-27 -0.552 0.295 0.493 3.33E-22 CNC- Mesenchyme GLT8D2 1.06E-26 0.362 0.206 0.08 3.88E-22 CNC- Mesenchyme RFX4 1.07E-26 -0.497 0.027 0.175 3.91E-22 CNC- Mesenchyme IGDCC3 1.26E-26 -0.607 0.395 0.567 4.63E-22 CNC- Mesenchyme FABP7 1.41E-26 -0.803 0.319 0.503 5.17E-22 CNC- Mesenchyme ALX1 1.73E-26 0.465 0.311 0.154 6.32E-22 CNC- Mesenchyme STATS 1.83E-26 0.463 0.432 0.257 6.71E-22 CNC- Mesenchyme NAE1 1.84E-26 -0.534 0.372 0.552 6.73E-22 CNC- Mesenchyme MYDGF 2.07E-26 0.452 0.622 0.452 7.59E-22 CNC- Mesenchyme RARB 2.21E-26 0.459 0.274 0.129 8.10E-22 CNC- Mesenchyme TMEFF1 2.38E-26 -0.526 0.074 0.243 8.70E-22 CNC- Mesenchyme CARHSP1 2.42E-26 -0.54 0.36 0.547 8.85E-22 CNC- Mesenchyme CKAP5 2.55E-26 -0.535 0.162 0.352 9.32E-22 CNC- Mesenchyme PPFIBP1 2.59E-26 0.493 0.352 0.189 9.49E-22 CNC- Mesenchyme CTSK 2.72E-26 0.344 0.189 0.07 9.97E-22 CNC- Mesenchyme CCND1 3.34E-26 -0.706 0.437 0.59 1.22E-21 CNC- Mesenchyme ARHGAP11 3.59E-26 -0.428 0.034 0.185 1.32E-21 CNC- A MesenchymeLINC01151 4.38E-26 0.433 0.152 0.047 1.60E-21 CNC- Mesenchyme ACYP1 4.53E-26 -0.485 0.144 0.333 1.66E-21 CNC- Mesenchyme LGALS1 5.51E-26 0.683 0.443 0.273 2.02E-21 CNC- Mesenchyme PAX3 5.76E-26 -0.532 0.086 0.255 2.11E-21 CNC- Mesenchyme LSM3 5.76E-26 -0.467 0.538 0.696 2.11E-21 CNC- Mesenchyme PLAGL1 7.48E-26 -0.624 0.201 0.381 2.74E-21 CNC- Mesenchyme FAN Cl 8.19E-26 -0.466 0.069 0.234 3.00E-21 CNC- Mesenchyme SRRM1 8.39E-26 -0.475 0.615 0.745 3.07E-21 CNC- Mesenchyme HES4 8.69E-26 -0.688 0.237 0.418 3.18E-21 CNC- Mesenchyme IGFBP4 1.04E-25 0.466 0.352 0.194 3.81E-21 CNC- Mesenchyme NCAPH 1.35E-25 -0.403 0.027 0.169 4.94E-21 CNC- Mesenchyme ACTG2 1.47E-25 0.307 0.11 0.026 5.36E-21 CNC- Mesenchyme YWHAQ 1.60E-25 -0.488 0.596 0.731 5.84E-21 CNC- Mesenchyme ASRGL1 1.89E-25 -0.462 0.048 0.201 6.93E-21 CNC- Mesenchyme F0XM1 2.34E-25 -0.382 0.029 0.174 8.58E-21 CNC- Mesenchyme P4HB 2.46E-25 0.483 0.572 0.405 9.00E-21 CNC- Mesenchyme GYPC 2.59E-25 0.438 0.62 0.448 9.48E-21 CNC- Mesenchyme THY1 2.99E-25 0.321 0.22 0.09 1.10E-20 CNC- Mesenchyme CDCA4 3.70E-25 -0.442 0.091 0.26 1.35E-20 CNC- Mesenchyme MMP2 3.76E-25 0.473 0.393 0.232 1.37E-20 CNC- Mesenchyme TM4SF1 4.06E-25 0.388 0.124 0.034 1.48E-20 CNC- Mesenchyme TPD52L1 4.23E-25 0.418 0.293 0.143 1.55E-20 CNC- Mesenchyme TCIM 4.30E-25 0.256 0.119 0.031 1.57E-20 CNC- Mesenchyme PPP1R1A 4.45E-25 -0.494 0.03 0.173 1.63E-20 CNC- MesenchymeSPP1 4.54E-25 0.587 0.172 0.061 1.66E-20 CNC- Mesenchyme PLAUR 5.47E-25 0.378 0.177 0.064 2.00E-20 CNC- Mesenchyme FSTL1 6.10E-25 0.459 0.462 0.291 2.23E-20 CNC- Mesenchyme FBLIM1 9.35E-25 0.362 0.24 0.106 3.42E-20 CNC- Mesenchyme STIM2 1.14E-24 0.461 0.285 0.141 4.18E-20 CNC- Mesenchyme GINS2 1.39E-24 -0.535 0.223 0.408 5.09E-20 CNC- Mesenchyme HHIP 1.64E-24 -0.487 0.029 0.17 6.02E-20 CNC- Mesenchyme SAP30 2.10E-24 -0.514 0.183 0.361 7.70E-20 CNC- Mesenchyme KDELR2 3.11E-24 0.475 0.6 0.444 1.14E-19 CNC- Mesenchyme SSR4 3.36E-24 0.363 0.807 0.691 1.23E-19 CNC- Mesenchyme CRYM 3.52E-24 0.271 0.14 0.044 1.29E-19 CNC- Mesenchyme EFNA5 3.66E-24 0.392 0.302 0.152 1.34E-19 CNC- Mesenchyme LIN28A 3.67E-24 -0.387 0.02 0.153 1.34E-19 CNC- Mesenchyme L0XL2 4.08E-24 0.339 0.174 0.064 1.49E-19 CNC- Mesenchyme FABP3 4.39E-24 0.434 0.246 0.111 1.61E-19 CNC- Mesenchyme DEPDC1B 5.60E-24 -0.4 0.028 0.165 2.05E-19 CNC- Mesenchyme SPTBN1 5.94E-24 0.482 0.487 0.324 2.17E-19 CNC- Mesenchyme DNAJC9 8.48E-24 -0.472 0.159 0.34 3.10E-19 CNC- Mesenchyme LINC02381 8.66E-24 -0.488 0.039 0.181 3.17E-19 CNC- Mesenchyme TPI1 8.74E-24 -0.397 0.813 0.884 3.20E-19 CNC- Mesenchyme MIS18A 9.11E-24 -0.497 0.203 0.39 3.33E-19 CNC- Mesenchyme HMGN1 9.22E-24 -0.315 0.894 0.937 3.38E-19 CNC- Mesenchyme R0B01 1.10E-23 0.408 0.342 0.187 4.03E-19 CNC- Mesenchyme CXCL14 1.14E-23 0.373 0.148 0.048 4.18E-19 CNC- MesenchymeCLSPN 1.43E-23 -0.495 0.128 0.297 5.24E-19 CNC- Mesenchyme SESN3 1.59E-23 0.522 0.487 0.325 5.82E-19 CNC- Mesenchyme PDE5A 2.00E-23 0.324 0.172 0.063 7.31E-19 CNC- Mesenchyme GSTP1 2.01E-23 -0.533 0.981 0.98 7.36E-19 CNC- Mesenchyme DSE 2.10E-23 0.401 0.345 0.192 7.69E-19 CNC- Mesenchyme WIPI1 2.63E-23 0.429 0.307 0.161 9.62E-19 CNC- Mesenchyme KIF20A 3.80E-23 -0.396 0.024 0.155 1.39E-18 CNC- Mesenchyme VWF 4.31E-23 0.38 0.149 0.05 1.58E-18 CNC- Mesenchyme WFIKKN1 4.39E-23 -0.414 0.013 0.137 1.61E-18 CNC- Mesenchyme JAK1 4.43E-23 0.419 0.429 0.266 1.62E-18 CNC- Mesenchyme TLE1 4.54E-23 0.466 0.419 0.256 1.66E-18 CNC- Mesenchyme ARL2 4.60E-23 -0.5 0.403 0.564 1.68E-18 CNC- Mesenchyme ATAD2 5.88E-23 -0.526 0.179 0.354 2.15E-18 CNC- Mesenchyme DPYSL5 6.85E-23 -0.412 0.032 0.167 2.51E-18 CNC- Mesenchyme WNT4 7.33E-23 -0.448 0.014 0.137 2.68E-18 CNC- Mesenchyme IER5L 7.82E-23 0.512 0.586 0.426 2.86E-18 CNC- Mesenchyme NTM 7.85E-23 0.368 0.221 0.097 2.87E-18 CNC- Mesenchyme CLEC1A 8.02E-23 0.293 0.129 0.039 2.94E-18 CNC- Mesenchyme FGF18 8.10E-23 -0.536 0.018 0.143 2.96E-18 CNC- Mesenchyme MELK 8.61E-23 -0.373 0.033 0.168 3.15E-18 CNC- Mesenchyme NSD2 1.00E-22 -0.446 0.153 0.327 3.67E-18 CNC- Mesenchyme LAMC1 1.03E-22 0.469 0.364 0.211 3.76E-18 CNC- Mesenchyme ABCA9 1.06E-22 0.276 0.144 0.048 3.86E-18 CNC- Mesenchyme IFI16 1.23E-22 0.305 0.189 0.076 4.49E-18 CNC- MesenchymePLS3 1.26E-22 0.426 0.524 0.358 4.61E-18 CNC- Mesenchyme C0X17 1.61E-22 -0.536 0.297 0.469 5.89E-18 CNC- Mesenchyme ASF1B 1.67E-22 -0.391 0.037 0.171 6.13E-18 CNC- Mesenchyme HSP90B1 1.71E-22 0.346 0.941 0.879 6.27E-18 CNC- Mesenchyme SAT2 1.73E-22 0.39 0.746 0.6 6.32E-18 CNC- Mesenchyme AC103702. 2.17E-22 -0.359 0.013 0.132 7.94E-18 CNC-1 Mesenchyme ATAD5 2.25E-22 -0.447 0.085 0.238 8.22E-18 CNC- Mesenchyme DUSP6 2.39E-22 0.485 0.646 0.486 8.76E-18 CNC- Mesenchyme PRMT1 2.91E-22 -0.46 0.655 0.768 1.06E-17 CNC- Mesenchyme ADGRL4 2.91E-22 0.255 0.112 0.031 1.06E-17 CNC- Mesenchyme TTYH1 2.94E-22 -0.598 0.189 0.353 1.08E-17 CNC- Mesenchyme SRSF2 3.03E-22 -0.474 0.413 0.586 1.11E-17 CNC- Mesenchyme TSPAN18 4.41E-22 -0.418 0.056 0.198 1.61E-17 CNC- Mesenchyme SPDL1 4.44E-22 -0.425 0.073 0.222 1.62E-17 CNC- Mesenchyme PRDX1 7.14E-22 -0.362 0.914 0.939 2.61E-17 CNC- Mesenchyme RPS17 7.59E-22 -0.334 0.871 0.909 2.78E-17 CNC- Mesenchyme SMC1A 8.39E-22 -0.474 0.235 0.411 3.07E-17 CNC- Mesenchyme PKDCC 8.62E-22 0.376 0.283 0.146 3.15E-17 CNC- Mesenchyme ANLN 1.01E-21 -0.361 0.03 0.158 3.70E-17 CNC- Mesenchyme ALDH7A1 1.03E-21 -0.472 0.249 0.42 3.77E-17 CNC- Mesenchyme DBI 1.28E-21 -0.369 0.818 0.872 4.68E-17 CNC- Mesenchyme YEATS4 1.28E-21 -0.427 0.096 0.251 4.69E-17 CNC- Mesenchyme PSMA4 1.50E-21 -0.438 0.668 0.765 5.47E-17 CNC- Mesenchyme H0XB4 1.74E-21 -0.349 0.019 0.139 6.36E-17 CNC- MesenchymeGPSM2 1.75E-21 -0.412 0.043 0.175 6.42E-17 CNC- Mesenchyme TMEM263 2.17E-21 0.366 0.298 0.159 7.92E-17 CNC- Mesenchyme PRICKLEI 2.26E-21 0.402 0.258 0.128 8.27E-17 CNC- Mesenchyme ICAM1 2.49E-21 0.268 0.133 0.043 9.13E-17 CNC- Mesenchyme VDAC3 2.54E-21 -0.459 0.466 0.623 9.31E-17 CNC- Mesenchyme GLRX5 2.70E-21 -0.458 0.527 0.669 9.90E-17 CNC- Mesenchyme FEN1 2.95E-21 -0.423 0.088 0.24 1.08E-16 CNC- Mesenchyme AEBP1 2.99E-21 0.36 0.266 0.135 1.10E-16 CNC- Mesenchyme CACYBP 3.10E-21 -0.441 0.369 0.543 1.14E-16 CNC- Mesenchyme ARPC2 4.37E-21 0.407 0.824 0.711 1.60E-16 CNC- Mesenchyme SEC31A 4.78E-21 0.429 0.487 0.337 1.75E-16 CNC- Mesenchyme CST3 4.91E-21 0.589 0.769 0.668 1.80E-16 CNC- Mesenchyme ARMCX6 5.71E-21 0.353 0.331 0.185 2.09E-16 CNC- Mesenchyme DACT3 6.45E-21 0.399 0.298 0.162 2.36E-16 CNC- Mesenchyme EPHA3 6.61E-21 0.329 0.16 0.061 2.42E-16 CNC- Mesenchyme DIAPH3 6.95E-21 -0.364 0.048 0.179 2.54E-16 CNC- Mesenchyme INCENP 7.26E-21 -0.325 0.014 0.127 2.66E-16 CNC- Mesenchyme EMILIN1 7.53E-21 0.264 0.154 0.057 2.76E-16 CNC- Mesenchyme SRSF9 7.55E-21 -0.36 0.812 0.866 2.76E-16 CNC- Mesenchyme NCL 7.64E-21 -0.358 0.898 0.915 2.80E-16 CNC- Mesenchyme COL26A1 7.93E-21 0.37 0.259 0.129 2.90E-16 CNC- Mesenchyme AKR1B1 7.99E-21 -0.488 0.225 0.392 2.92E-16 CNC- Mesenchyme SLC25A6 8.01E-21 0.34 0.856 0.789 2.93E-16 CNC- Mesenchyme FAM83D 9.73E-21 -0.305 0.011 0.123 3.56E-16 CNC- MesenchymeEIF4A3 1.05E-20 -0.503 0.277 0.443 3.85E-16 CNC- Mesenchyme LSM7 1.07E-20 -0.352 0.812 0.877 3.90E-16 CNC- Mesenchyme TFAP2B 1.11E-20 0.338 0.27 0.136 4.08E-16 CNC- Mesenchyme CIT 1.18E-20 -0.332 0.023 0.141 4.32E-16 CNC- Mesenchyme TSC22D3 1.45E-20 0.405 0.389 0.239 5.30E-16 CNC- Mesenchyme CDCA5 1.54E-20 -0.352 0.025 0.144 5.65E-16 CNC- Mesenchyme SERPINA3 1.61E-20 0.33 0.104 0.028 5.89E-16 CNC- Mesenchyme TAGLN3 1.66E-20 -1.183 0.071 0.207 6.08E-16 CNC- Mesenchyme HES5 1.72E-20 -0.59 0.032 0.154 6.29E-16 CNC- Mesenchyme MSM01 1.74E-20 0.392 0.765 0.637 6.38E-16 CNC- Mesenchyme INKAI 1.81E-20 0.317 0.162 0.063 6.64E-16 CNC- Mesenchyme RBPMS 2.07E-20 0.363 0.343 0.2 7.56E-16 CNC- Mesenchyme CBX1 2.17E-20 -0.425 0.609 0.732 7.96E-16 CNC- Mesenchyme ESCO2 2.84E-20 -0.334 0.029 0.15 1.04E-15 CNC- Mesenchyme RHEB 2.89E-20 -0.431 0.571 0.704 1.06E-15 CNC- Mesenchyme OIP5 3.26E-20 -0.345 0.023 0.14 1.19E-15 CNC- Mesenchyme A1BG 3.66E-20 0.321 0.225 0.106 1.34E-15 CNC- Mesenchyme YES1 3.72E-20 0.396 0.394 0.244 1.36E-15 CNC- Mesenchyme PLEKHF1 4.69E-20 0.319 0.141 0.051 1.72E-15 CNC- Mesenchyme TCEAL9 4.70E-20 0.429 0.604 0.455 1.72E-15 CNC- Mesenchyme POU3F2 5.51E-20 -0.402 0.039 0.166 2.02E-15 CNC- Mesenchyme PLD3 5.84E-20 0.381 0.501 0.35 2.14E-15 CNC- Mesenchyme SEC24D 6.32E-20 0.311 0.199 0.088 2.31E-15 CNC- Mesenchyme HNRNPM 7.05E-20 -0.461 0.53 0.665 2.58E-15 CNC- MesenchymeS1PR3 7.47E-20 0.335 0.197 0.088 2.73E-15 CNC- Mesenchyme AT0X1 7.70E-20 0.398 0.764 0.662 2.82E-15 CNC- Mesenchyme SSBP4 1.06E-19 0.464 0.58 0.435 3.87E-15 CNC- Mesenchyme MYBL2 1.22E-19 -0.383 0.069 0.204 4.46E-15 CNC- Mesenchyme TMEM160 1.26E-19 -0.432 0.388 0.547 4.60E-15 CNC- Mesenchyme NR2F1-AS1 1.49E-19 0.329 0.302 0.165 5.46E-15 CNC- Mesenchyme PELI1 1.62E-19 0.35 0.229 0.112 5.92E-15 CNC- Mesenchyme SNRNP40 1.71E-19 -0.475 0.285 0.437 6.27E-15 CNC- Mesenchyme CYP1B1 1.92E-19 0.296 0.134 0.047 7.02E-15 CNC- Mesenchyme SKA3 2.12E-19 -0.32 0.024 0.137 7.77E-15 CNC- Mesenchyme CSTB 2.14E-19 0.391 0.698 0.585 7.82E-15 CNC- Mesenchyme POC1A 2.27E-19 -0.338 0.03 0.147 8.32E-15 CNC- Mesenchyme FABP5 2.56E-19 0.491 0.802 0.718 9.37E-15 CNC- Mesenchyme MRPL51 2.67E-19 -0.376 0.763 0.807 9.77E-15 CNC- Mesenchyme HDLBP 3.65E-19 0.388 0.489 0.333 1.34E-14 CNC- Mesenchyme EZR 3.93E-19 -0.424 0.098 0.241 1.44E-14 CNC- Mesenchyme HNRNPU 5.40E-19 -0.399 0.698 0.786 1.97E-14 CNC- Mesenchyme LDHB 5.59E-19 -0.269 0.939 0.955 2.04E-14 CNC- Mesenchyme SRPK1 6.22E-19 -0.428 0.264 0.427 2.28E-14 CNC- Mesenchyme NUDCD2 6.93E-19 -0.466 0.316 0.477 2.54E-14 CNC- Mesenchyme XRCC6 8.12E-19 -0.408 0.491 0.631 2.97E-14 CNC- Mesenchyme CNBP 8.42E-19 -0.446 0.553 0.678 3.08E-14 CNC- Mesenchyme DACT2 8.79E-19 0.271 0.136 0.05 3.22E-14 CNC- Mesenchyme DCHS1 9.16E-19 0.309 0.251 0.13 3.35E-14 CNC- MesenchymeNDUFS6 9.59E-19 -0.358 0.745 0.824 3.51E-14 CNC- Mesenchyme FAM89A 9.89E-19 0.423 0.302 0.172 3.62E-14 CNC- Mesenchyme TK1 1.01E-18 -0.36 0.054 0.178 3.68E-14 CNC- Mesenchyme GCSH 1.07E-18 -0.411 0.542 0.664 3.91E-14 CNC- Mesenchyme NIDI 1.32E-18 0.309 0.287 0.158 4.84E-14 CNC- Mesenchyme HNRNPH3 1.35E-18 -0.42 0.652 0.753 4.95E-14 CNC- Mesenchyme MIR4435- 1.39E-18 0.252 0.138 0.051 5.07E-14 CNC-2HG Mesenchyme FNDC5 1.40E-18 0.326 0.271 0.142 5.14E-14 CNC- Mesenchyme PTMS 1.52E-18 -0.307 0.963 0.951 5.57E-14 CNC- Mesenchyme SH3BP5 1.58E-18 0.305 0.208 0.098 5.79E-14 CNC- Mesenchyme RBMS3 1.60E-18 0.291 0.256 0.133 5.87E-14 CNC- Mesenchyme SMARCA2 1.88E-18 0.257 0.153 0.06 6.88E-14 CNC- Mesenchyme DADI 1.88E-18 0.327 0.838 0.753 6.90E-14 CNC- Mesenchyme WDR54 2.19E-18 -0.451 0.251 0.411 8.00E-14 CNC- Mesenchyme CCDC140 2.21E-18 -0.292 0.005 0.1 8.08E-14 CNC- Mesenchyme C4orf3 2.46E-18 0.394 0.737 0.61 8.99E-14 CNC- Mesenchyme GSN 2.73E-18 0.342 0.284 0.156 1.00E-13 CNC- Mesenchyme SLC20A2 2.95E-18 0.343 0.203 0.095 1.08E-13 CNC- Mesenchyme HMGCR 3.42E-18 0.408 0.655 0.511 1.25E-13 CNC- Mesenchyme PSMB3 3.59E-18 -0.392 0.626 0.746 1.31E-13 CNC- Mesenchyme TGFB1I1 3.65E-18 0.279 0.163 0.068 1.33E-13 CNC- Mesenchyme SALL1 3.66E-18 -0.309 0.02 0.125 1.34E-13 CNC- Mesenchyme ANGPT2 3.70E-18 0.362 0.187 0.085 1.36E-13 CNC- Mesenchyme DLC1 4.83E-18 0.302 0.26 0.138 1.77E-13 CNC- MesenchymeSH3BGRL 5.10E-18 0.387 0.554 0.414 1.87E-13 CNC- Mesenchyme SSR3 5.15E-18 0.386 0.707 0.603 1.88E-13 CNC- Mesenchyme HMCN1 5.21E-18 0.333 0.275 0.151 1.91E-13 CNC- Mesenchyme H0XC4 5.84E-18 -0.288 0.01 0.108 2.14E-13 CNC- Mesenchyme BCL7A 5.96E-18 -0.42 0.163 0.316 2.18E-13 CNC- Mesenchyme ARHGEF39 6.12E-18 -0.283 0.009 0.104 2.24E-13 CNC- Mesenchyme BCL2L12 6.15E-18 -0.415 0.189 0.341 2.25E-13 CNC- Mesenchyme HEY1 6.68E-18 0.268 0.167 0.07 2.44E-13 CNC- Mesenchyme TM0D3 6.95E-18 0.377 0.434 0.289 2.54E-13 CNC- Mesenchyme PDGFRA 7.92E-18 0.304 0.179 0.08 2.90E-13 CNC- Mesenchyme SUPT16H 9.71E-18 -0.449 0.524 0.648 3.55E-13 CNC- Mesenchyme SNCG 1.09E-17 -0.665 0.034 0.144 3.99E-13 CNC- Mesenchyme CCT2 1.15E-17 -0.405 0.626 0.735 4.20E-13 CNC- Mesenchyme TECR 1.17E-17 -0.445 0.437 0.586 4.28E-13 CNC- Mesenchyme TPBG 1.19E-17 -0.744 0.288 0.425 4.37E-13 CNC- Mesenchyme TPM2 1.24E-17 0.446 0.384 0.242 4.52E-13 CNC- Mesenchyme CAPZB 1.27E-17 0.347 0.698 0.575 4.66E-13 CNC- Mesenchyme IDI1 1.29E-17 0.371 0.814 0.707 4.71E-13 CNC- Mesenchyme TMEM106C 1.38E-17 -0.383 0.191 0.342 5.06E-13 CNC- Mesenchyme RPL22L1 1.38E-17 0.356 0.636 0.492 5.07E-13 CNC- Mesenchyme RFC3 1.39E-17 -0.391 0.087 0.216 5.07E-13 CNC- Mesenchyme MLLT3 1.48E-17 -0.415 0.088 0.221 5.42E-13 CNC- Mesenchyme ENHO 1.51E-17 -0.336 0.053 0.172 5.52E-13 CNC- Mesenchyme CDC42EP3 1.53E-17 0.275 0.193 0.089 5.59E-13 CNC- MesenchymeRCAN1 1.60E-17 0.301 0.164 0.07 5.87E-13 CNC- Mesenchyme ECU 1.74E-17 -0.441 0.381 0.524 6.36E-13 CNC- Mesenchyme BRCA1 1.81E-17 -0.348 0.066 0.19 6.62E-13 CNC- Mesenchyme VEGFD 1.91E-17 0.345 0.189 0.088 6.99E-13 CNC- Mesenchyme SPAG5 2.34E-17 -0.294 0.019 0.119 8.56E-13 CNC- Mesenchyme DNM1 2.37E-17 0.346 0.301 0.176 8.66E-13 CNC- Mesenchyme SULT1C4 2.46E-17 0.296 0.215 0.106 9.00E-13 CNC- Mesenchyme MIR99AHG 2.69E-17 0.423 0.524 0.394 9.86E-13 CNC- Mesenchyme CCPG1 2.71E-17 0.386 0.254 0.137 9.92E-13 CNC- Mesenchyme ETV1 3.06E-17 0.376 0.375 0.238 1.12E-12 CNC- Mesenchyme H2AFY2 3.18E-17 -0.381 0.352 0.505 1.16E-12 CNC- Mesenchyme CXXC4 3.29E-17 -0.319 0.016 0.114 1.21E-12 CNC- Mesenchyme P4HA2 3.39E-17 0.32 0.213 0.107 1.24E-12 CNC- Mesenchyme IGFBP6 3.44E-17 0.278 0.157 0.066 1.26E-12 CNC- Mesenchyme GSTK1 3.71E-17 0.348 0.259 0.142 1.36E-12 CNC- Mesenchyme PFN2 3.87E-17 -0.451 0.437 0.565 1.42E-12 CNC- Mesenchyme HIST1H2BH 4.43E-17 -0.316 0.042 0.153 1.62E-12 CNC- Mesenchyme VBP1 4.47E-17 -0.428 0.333 0.485 1.64E-12 CNC- Mesenchyme LRR1 4.68E-17 -0.354 0.087 0.214 1.71E-12 CNC- Mesenchyme JMJD1C 4.84E-17 0.363 0.477 0.345 1.77E-12 CNC- Mesenchyme IFI27L2 4.90E-17 0.375 0.48 0.336 1.79E-12 CNC- Mesenchyme ALYREF 4.96E-17 -0.445 0.231 0.378 1.81E-12 CNC- Mesenchyme SBDS 5.32E-17 0.389 0.429 0.293 1.95E-12 CNC- Mesenchyme BEX3 5.45E-17 0.257 0.96 0.902 1.99E-12 CNC- MesenchymeC0L2A1 5.81E-17 0.463 0.641 0.524 2.13E-12 CNC- Mesenchyme RGS10 6.42E-17 0.272 0.245 0.128 2.35E-12 CNC- Mesenchyme G2E3 6.61E-17 -0.378 0.095 0.226 2.42E-12 CNC- Mesenchyme HNRNPAO 7.18E-17 -0.373 0.515 0.648 2.63E-12 CNC- Mesenchyme TPRKB 7.36E-17 -0.429 0.303 0.449 2.69E-12 CNC- Mesenchyme AL359091.1 7.53E-17 -0.374 0.032 0.137 2.76E-12 CNC- Mesenchyme CIP2A 7.80E-17 -0.332 0.052 0.166 2.86E-12 CNC- Mesenchyme CCT6A 9.55E-17 -0.387 0.572 0.687 3.49E-12 CNC- Mesenchyme EIF5 9.99E-17 -0.394 0.674 0.745 3.66E-12 CNC- Mesenchyme UBE2I 1.04E-16 -0.373 0.609 0.727 3.82E-12 CNC- Mesenchyme MAP1LC3A 1.08E-16 0.286 0.168 0.074 3.95E-12 CNC- Mesenchyme TLN1 1.14E-16 0.363 0.46 0.322 4.17E-12 CNC- Mesenchyme KDM5B 1.19E-16 0.314 0.665 0.527 4.36E-12 CNC- Mesenchyme SSRP1 1.19E-16 -0.409 0.292 0.439 4.37E-12 CNC- Mesenchyme GAS1 1.23E-16 -0.397 0.11 0.24 4.49E-12 CNC- Mesenchyme SLIT3 1.25E-16 0.301 0.213 0.107 4.59E-12 CNC- Mesenchyme ACBD3 1.30E-16 0.332 0.394 0.256 4.74E-12 CNC- Mesenchyme COMMD4 1.39E-16 -0.371 0.184 0.332 5.09E-12 CNC- Mesenchyme XPNPEP1 1.41E-16 0.327 0.278 0.158 5.15E-12 CNC- Mesenchyme HIST1H3G 1.44E-16 -0.289 0.013 0.104 5.28E-12 CNC- Mesenchyme NBDY 1.45E-16 0.364 0.701 0.583 5.30E-12 CNC- Mesenchyme ITPR2 1.54E-16 0.261 0.157 0.067 5.65E-12 CNC- Mesenchyme RABAC1 1.63E-16 0.339 0.742 0.624 5.98E-12 CNC- Mesenchyme RNF5 1.78E-16 -0.413 0.249 0.394 6.53E-12 CNC- MesenchymeNRIP1 1.88E-16 0.387 0.48 0.344 6.89E-12 CNC- Mesenchyme IGFBP3 1.94E-16 0.293 0.134 0.053 7.09E-12 CNC- Mesenchyme LINC01224 2.04E-16 -0.293 0.02 0.116 7.47E-12 CNC- Mesenchyme FKBP3 2.33E-16 -0.408 0.54 0.662 8.53E-12 CNC- Mesenchyme C0X5A 2.36E-16 -0.3 0.813 0.852 8.66E-12 CNC- Mesenchyme NUP37 2.38E-16 -0.374 0.131 0.266 8.71E-12 CNC- Mesenchyme T0X3 2.44E-16 -0.303 0.018 0.112 8.95E-12 CNC- Mesenchyme SRSF10 2.60E-16 -0.416 0.408 0.542 9.52E-12 CNC- Mesenchyme NQO1 2.66E-16 0.389 0.381 0.249 9.75E-12 CNC- Mesenchyme SNRNP25 2.70E-16 -0.401 0.29 0.439 9.90E-12 CNC- Mesenchyme CNN3 2.92E-16 0.322 0.866 0.78 1.07E-11 CNC- Mesenchyme HNRNPK 2.98E-16 -0.352 0.76 0.82 1.09E-11 CNC- Mesenchyme NSMCE4A 3.12E-16 -0.385 0.158 0.299 1.14E-11 CNC- Mesenchyme OSTF1 3.14E-16 0.253 0.164 0.073 1.15E-11 CNC- Mesenchyme YWHAE 3.27E-16 -0.323 0.832 0.87 1.20E-11 CNC- Mesenchyme GRAMD1A 3.66E-16 0.316 0.25 0.138 1.34E-11 CNC- Mesenchyme RFC4 4.12E-16 -0.338 0.076 0.197 1.51E-11 CNC- Mesenchyme FNDC3B 4.29E-16 0.33 0.212 0.109 1.57E-11 CNC- Mesenchyme DYRK4 4.36E-16 0.362 0.364 0.236 1.60E-11 CNC- Mesenchy...

Claims

What is claimed is:

1. A method of producing spiral ganglion neuron (SGN)-like cells from stem cells, the method comprising:(a) culturing the stem cells in a first medium comprising bone morphogenetic protein 4 (BMP4), a transforming growth factor-p (TGF-[3) type I receptor inhibitor, and basic fibroblast growth factor 2 (FGF2);(b) culturing cells, produced from the stem cells in the first medium, in a second medium comprising the FGF2, the TGF- type I receptor inhibitor, a Wnt inhibitor, and an inhibitor of ALK2 and ALK3, wherein the cells differentiate into pre-placodal ectoderm and neural crest precursors;(c) isolating the pre-placodal ectoderm and neural crest precursors;(d) culturing the isolated pre-placodal ectoderm and neural crest precursors in a third medium comprising Wnt family member 3A (WNT3A), the FGF2, insulin-like growth factor-1 (IGF-1), and a non-selective Rho-associated protein kinase (ROCK) inhibitor;(e) culturing the cells, produced from the pre-placodal ectoderm and neural crest precursors in the third medium, in a fourth medium comprising sonic hedgehog (SHH), retinoic acid (RA), epidermal growth factor (EGF), the FGF2, and the IGF-1, wherein the cells differentiate into otic neural progenitor cells; and(f) culturing the otic neural progenitor cells in a fifth medium comprising brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), the IGF-1, cyclic adenosine monophosphate (cAMP), and a selective ROCK inhibitor to induce neuronal maturation of the otic neural progenitor cells into the SGN-like cells.

2. The method of claim 1 , wherein the stem cells are embryonic stem cells, adult stem cells, or induced pluripotent stem cells (iPSCs).

3. The method of claim 2, wherein the iPSCs are human induced pluripotent stem cells (hiPSCs).

4. The method of claim 3, wherein the hiPSCs are SK8-A hiPSCs.

5. The method of any one of claims 1-4, wherein the Wnt inhibitor is / V-(6-methyl-1 ,3-benzothiazol-2-yl)-2-[(4-oxo-3-phenyl-6,7-dihydrothieno[3,2-d]pyrimidin-2-yl)sulfanyl]acetamide (IWP-2).

6. The method of any one of claims 1-5, wherein the TGF- type I receptor inhibitor is 4-[4-(1 ,3-benzodioxol-5-yl)-5-pyridin-2-yl-1 H-imidazol-2-yl]benzamide (SB-431542).

7. The method of any one of claims 1 -6, wherein the inhibitor of ALK2 and ALK3 is 4-[6-(4-piperazin-1 -ylphenyl)pyrazolo[1 ,5-a]pyrim idin-3-yl]quinoline (LDN-193189).

8. The method of any one of claims 1-7, wherein the non-selective ROCK inhibitor is 4-[(1 R)-1 -aminoethyl]-N-pyridin-4-ylcyclohexane-1 -carboxamide (Y-27632).

9. The method of any one of claims 1-8, wherein the selective ROCK inhibitor is 4-methyl-5-[[(2S)-2-methyl-1 ,4-diazepan-1-yl]sulfonyl]isoquinoline (H1152).

10. The method of any one of claims 1-9, wherein the pre-placodal ectoderm and neural crest precursors comprise TRA-1-60 pluripotent stem cell marker negative-nerve growth factor receptor positive (TRA-1-60 NGFR+) cells.11 . The method of claim 10, wherein said isolating the pre-placodal ectoderm and neural crest precursors comprises using fluorescence-activated cell sorting or magnetic-activated cell sorting.

12. The method of claim 11, wherein the magnetic-activated cell sorting is performed using magnetic microbeads conjugated to anti-TRA-1-60 antibodies and anti-NGFR antibodies.

13. The method of any one of claims 10-12, wherein step (d) comprising initially seeding the third medium with about 60,000 TRA-1 -60" NGFR+cells / cm2for said culturing.

14. The method of any one of claims 1-13, wherein step (a) comprises said culturing the stem cells in the first medium for about 3 days.

15. The method of any one of claims 1-14, wherein step (b) comprises said culturing the stem cells in the second medium for about 5 days.

16. The method of any one of claims 1-15, wherein step (d) comprises said culturing the cells in the third medium for about 7 days.

17. The method of any one of claims 1 -16, wherein step (e) comprises said culturing the TRA-1 -60" NGFR+cells in the fourth medium for about 7 days.

18. The method of any one of claims 1-17, wherein step (f) comprises said culturing the otic neural progenitor cells in the fifth medium for about 6 days.

19. The method of any one of claims 1-18, wherein the first medium comprises 10 ng / ml BMP4, 1 pmol / L SB-431542, and 10 ng / ml FGF2.

20. The method of any one of claims 1-19, wherein the second medium comprises 100 nmol / L LDN-193189, 1 pmol / L SB-431542, 2 mmol / L IWP-2, and 10 ng / ml FGF2.21 . The method of any one of claims 1 -20, wherein the third medium comprises 100 ng / ml WNT3A, 10 ng / ml FGF2, 50 ng / ml IGF-1, and 10 pmol / L Y-27632.

22. The method of any one of claims 1-21, wherein the fourth medium comprises 500 ng / ml SHH, 0.5 pmol / L RA, 20 ng / ml EGF, 10 ng / ml FGF2, and 50 ng / ml IGF-1.

23. The method of any one of claims 1-22, wherein the fifth medium comprises 10 ng / ml BDNF, 10 ng / ml NT-3, 10 ng / ml IGF-1, and 10 ng / ml H1152.

24. The method of any one of claims 1 -23, further comprising isolating the SGN-like cells.

25. The method of any one of claims 1 -24, further comprising characterizing the SGN-like cells by performing single-cell RNA sequencing, immunocytochemistry, whole-cell patch-clamping, gene expression profiling, calcium ion (Ca2+) imaging, or any combination thereof.

26. The method of any one of claims 1-25, further comprising co-culturing the SGN-like cells with hair cells, glial cells, cochlear nucleus neurons, or any combination thereof.

27. The method of any one of claims 1-26, wherein the SGN-like cells comprise type I SGN-like cells and type II SGN-like cells.

28. The method of claim 27, further comprising isolating the type I SGN-like cells or the type II SGN-like cells.

29. The method of any one of claims 1-28, further comprising detecting one or more cellular markers to identify the SGN-like cells, wherein the one or more cellular markers are selected from tubulin beta 3 class III (TUBB3), neurofilament light polypeptide (NEFL), neurofilament heavy polypeptide (NEFH), POU class 4 homeobox 1 (POU4F1), neuronal differentiation 1 transcription factor (NEUROD1), SRY-box transcription factor 2 (SOX2), prospero homeobox 1 (PROX1), sodium / potassium-transporting ATPase subunit alpha-3 (NKAa3), calbindin-2 (CALB2), and Ly6 / PLAUR domain-containing protein 1 (LYPD1). peripherin (PRPH), tachykinin precursor 1 (TAC1), MAF bZIP transcription factor (MAFB), and GATA binding protein 3 (GAT A3).

30. The method of any one of claims 1-29, wherein the stem cells comprise a genetic mutation associated with hearing loss or deafness.

31. The method of any one of claims 1-30, wherein the SGN-like cells comprise a genetic mutation associated with hearing loss or deafness.

32. The method of any one of claims 4-31 , wherein the hiPSCs are derived from a somatic cell from a patient who has a genetic mutation associated with hearing loss or deafness.

33. The method of claim 32, wherein the somatic cell is obtained from a biopsy of the inner ear of the patient.

34. The method of any one of claims 1-33, wherein the genetic mutation associated with hearing loss or deafness is introduced into the stem cells or the SGN-like cells by gene editing.

35. The method of any one of claims 30-34, wherein the genetic mutation associated with hearing loss or deafness is in a gene selected from TMPRSS3, NLRP3, FGF13, TBC1 D24, GATA2, GATA3, DFNB59, GRM7, and ATP6V1 B2.

36. A composition comprising SGN-like cells produced by the method of any one of claims 1-34.

37. The composition of claim 36, further comprising glial cells, hair cells, cochlear nucleus neurons, or any combination thereof.

38. The composition of claim 36 or 37, further comprising a pharmaceutically acceptable excipient.

39. A method of screening a candidate agent to determine its effects on SGN-like cells, the method comprising:contacting the SGN-like cells produced by the method of any one of claims 1-35 with the candidate agent; anddetermining the effects of the candidate agent on morphological, electrophysiological, genetic, or functional parameters of the SGN-like cells.

40. The method of claim 39, wherein the SGN-like cells comprise a genetic mutation associated with hearing loss or deafness.41 . The method of claim 39 or 40, wherein said determining the effects of the candidate agent comprises performing single-cell RNA sequencing, immunocytochemistry, whole-cell patchclamping, gene expression profiling, calcium ion (Ca2+) imaging, confocal microscopy, atomic force microscopy, super-resolution microcopy, light-sheet microscopy, two-photon microscopy, fluorescence microscopy, migration assays, axonal growth and pathfinding assays, or any combination thereof.

42. The method of any one of claims 39-41 , further comprising contacting the SGN-like cells with glial cells, hair cells, cochlear nucleus neurons, or any combination thereof.

43. The method of claim 42, further comprising detecting the effects of the candidate agent on synapses between the SGN-like cells and the cochlear nucleus neurons or the hair cells.

44. The method of claim 43, wherein the synapses are glutamatergic synapses.

45. The method of any one of claims 42-44, further comprising detecting the effects of the candidate agent on growth of neurites of the SGN-like cells toward the hair cells.

46. The method of any one of claims 42-45, further comprising detecting the effects of the candidate agent on interactions of the glial cells and the SGN-like cells.

47. The method of any one of claims 39-46, wherein the SGN-like cells are cultured in a two-dimensional (2D) culture system.

48. A method of treating a spiral ganglion neuron-associated disorder in a subject, the method comprising administering a therapeutically effective amount of the composition of claim 38 locally to the ear of the subject.

49. A composition comprising the SGN-like cells produced by the method of any one of claims 1-35 for use in treating a spiral ganglion neuron-associated disorder.

50. Use of the SGN-like cells produced by the method of any one of claims 1-35 in the manufacture of a medicament for treating a spiral ganglion neuron-associated disorder in a subject.

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