Unraveling the Mechanisms of Vestibular Neuron Formation from Human Induced Pluripotent Stem Cells.

The development of in vitro models that accurately recapitulate the complex cellular and molecular interactions of the inner ear is crucial for understanding inner ear development, function, and disease. In this study, we utilized a customized microfluidic platform to generate human induced pluripotent stem cell (hiPSC)-derived three-dimensional otic sensory neurons (OSNs). hiPSC-derived otic neuronal progenitors (ONPs) were cultured in hydrogel-embedded microfluidic channels over a 40-day period. Careful modulation of Wnt and Shh signaling pathways was used to influence dorsoventral patterning and direct differentiation toward a vestibular neuron lineage. After validating the microfluidic platform, OSN spheroid transcription factor and protein expression were assessed using real-time quantitative polymerase chain reaction (RT-qPCR), immunocytochemistry, and flow cytometry. The results demonstrated the successful differentiation of hiPSCs into ONPs and subsequent divergent differentiation into vestibular neuronal lineages, as evidenced by the expression of characteristic markers. Overall, our microfluidic platform provides a physiologically relevant environment for the culture and differentiation of hiPSCs, offering a valuable tool for studying inner ear development, disease and drug screening, and regenerative medicine applications.

The Hidden Enemy: Mal de Débarquement Syndrome and Its Impact on Military Operations.

Mal de Débarquement Syndrome (MdDS) is a poorly understood vestibular disorder that frequently affects military personnel exposed to motion during transportation and deployment. It is characterized by a persistent sensation of motion often experienced after disembarking from a ship or other mode of transportation. It can significantly affect a service member's balance, coordination, attention, and focus, which can then substantially impact their quality of life, ability to perform their military duties, and overall mission readiness. Despite its potential impact, comprehensive studies on MdDS are scarce, especially within the military. The unique conditions of military service, including frequent travel, long flights, maritime deployments, and high-stress environments, make the military well suited to study MdDS. Increased awareness and understanding of MdDS is crucial for everyone in the military-from medical personnel responsible for the diagnosis and treatment of MdDS to commanders who must consider the operational impact of impaired personnel.

In-situ hearing threshold estimation using Gaussian process classification.

One in six Americans suffers from hearing loss. While treatment with amplification is possible for many, the acceptance rate of hearing aids is low. Poor device fitting is one of the reasons. The hearing aid fitting starts with a detailed hearing assessment by a trained audiologist in a sound-controlled environment, using standard equipment. The hearing aid is adjusted step-by-step, following well-described procedures based on the audiogram. However, for many patients in rural settings, considerable travel time to a hearing center discourages them from receiving a hearing test and treatment. We hypothesize that hearing assessment with the patient's hearing aid can reliably substitute the hearing test in the clinic. Over-the-counter hearing aids could be programmed from a distance and fine-tuned by the hearing aid wearer. This study shows that a patient-controlled hearing assessment via a hearing aid in a non-clinical setting is not statistically different from an audiologist-controlled hearing assessment in a clinical setting. The differences in hearing obtained with our device and the Gaussian Process are within 3 dB of the standard audiogram. At 250 Hz, the sound delivery with the hearing aid used in this study added an additional reduction of sound level, which was not compensated.

Enhanced survival of hypoimmunogenic otic progenitors following intracochlear xenotransplantation: repercussions for stem cell therapy in hearing loss models.

Stem cell replacement holds the potential for sensorineural hearing loss (SNHL) treatment. However, its translation into clinical practice requires strategies for improving stem cell survival following intracochlear transplantation. Considering recent findings showing that the inner ear contains a resident population of immune cells, we hypothesized that immune evasion would improve the survival and residence time of transplanted stem cells in the cochlea, potentially leading to better outcomes. To test this, we leveraged genetic engineering techniques to develop hypoimmunogenic human-induced pluripotent stem cells (hi-iPSC), which lack human leukocyte antigen expression. We found that gene editing does not affect the biological properties of hi-iPSCs, including their capacity to differentiate into otic neural progenitors (ONPs). Compared to wild-type ONPs, more hypoimmunogenic ONPs (derived from hi-iPSCs) were found in the inner ear of immunocompetent mice ten days following cochlear xenotransplantation. This approach may open a new avenue for experimental and clinical SNHL treatments.

Application of supervised machine learning algorithms for the evaluation of utricular function on patients with Meniere's disease: utilizing subjective visual vertical and ocular-vestibular-evoked myogenic potentials.

BACKGROUND: Research on the otolith organs remains inconclusive. OBJECTIVES: This study seeks to further elucidate utricular function in patients with Meniere's disease (MD) in three ways: (1) We aimed to disambiguate the role of the Subjective Visual Vertical (SVV) and Ocular Vestibular Evoked Myogenic Potential (o-VEMP) tests regarding which utricular subsystem each is measuring. (2) We sought to characterize the acute and chronic state of MD by identifying differences in the relationship of SVV and o-VEMP results across patients with acute and chronic MD. (3) We attempted to find a machine-learning algorithm that could predict acute versus chronic MD using SVV and o-VEMP. METHODS: A prospective study with ninety subjects. RESULTS: (1) SVV and o-VEMP tests were found to have a moderate linear relationship in patients with acute MD, suggesting each test measures a different utricular subsystem. (2) Regression analyses statistically differed across the two patient populations, suggesting that SVV results were normalized in chronic MD patients. (3) Logistic regression and Naïve Bayes algorithms were found to predict acute and chronic MD accurately. SIGNIFICANCE: A better understanding of what diagnostic tests measure will lead to a better classification system for MD and more targeted treatment options in the future.

Bridging the electrode-neuron gap: finite element modeling of in vitro neurotrophin gradients to optimize neuroelectronic interfaces in the inner ear.

Although cochlear implant (CI) technology has allowed for the partial restoration of hearing over the last few decades, persistent challenges (e.g., poor performance in noisy environments and limited ability to decode intonation and music) remain. The "electrode-neuron gap" is inherent to these challenges and poses the most significant obstacle to advancing past the current plateau in CI performance. We propose the development of a "neuro-regenerative nexus"-a biological interface that doubly preserves native spiral ganglion neurons (SGNs) while precisely directing the growth of neurites arising from transplanted human pluripotent stem cell (hPSC)-derived otic neuronal progenitors (ONPs) toward the native SGN population. We hypothesized that the Polyhedrin Delivery System (PODS®-recombinant human brain-derived neurotrophic factor [rhBDNF]) could stably provide the adequate BDNF concentration gradient to hPSC-derived late-stage ONPs to facilitate otic neuronal differentiation and directional neurite outgrowth. To test this hypothesis, a finite element model (FEM) was constructed to simulate BDNF concentration profiles generated by PODS®-rhBDNF based on initial concentration and culture device geometry. For biological validation of the FEM, cell culture experiments assessing survival, differentiation, neurite growth direction, and synaptic connections were conducted using a multi-chamber microfluidic device. We were able to successfully generate the optimal BDNF concentration gradient to enable survival, neuronal differentiation toward SGNs, directed neurite extension of hPSC-derived SGNs, and synaptogenesis between two hPSC-derived SGN populations. This proof-of-concept study provides a step toward the next generation of CI technology. STATEMENT OF SIGNIFICANCE: Our study demonstrates that the generation of in vitro neurotrophin concentration gradients facilitates survival, neuronal differentiation toward auditory neurons, and directed neurite extension of human pluripotent stem cell-derived auditory neurons. These findings are indispensable to designing a bioactive cochlear implant, in which stem cell-derived neurons are integrated into a cochlear implant electrode strip, as the strategy will confer directional neurite growth from the transplanted cells in the inner ear. This study is the first to present the concept of a "neuro-regenerative nexus" congruent with a bioactive cochlear implant to eliminate the electrode-neuron gap-the most significant barrier to next-generation cochlear implant technology.

Kikuchi-Fujimoto disease presenting in a patient with SARS-CoV-2: a case report.

BACKGROUND: We present a yet to be described association of SARS-CoV-2 infection with Kikuchi-Fujimoto disease. CASE PRESENTATION: A 32-year-old physician with history of SARS-CoV-2 infection presented to the emergency department with 2 weeks of fever, chills, and right sided cervical lymphadenopathy. He was treated empirically for presumed folliculitis with worsening of symptoms leading to repeat presentation to the emergency department. Extensive workup was unrevealing of an infectious cause and needle biopsy of the lesion was unrevealing. An excisional lymph node biopsy revealed follicular hyperplasia with necrotic foci showing abundance of histiocytes at the edge of necrosis with CD8 predominance of T-cells. Final diagnosis was deemed to be Kikuchi-Fujimoto disease. Antibiotic therapy was discontinued, and the patient's symptoms resolved with steroid therapy and expectant management. CONCLUSIONS: This is the first report of a patient developing Kikuchi-Fujimoto disease following SARS-CoV-2 infection. Clinicians should be aware of Kikuchi-Fujimoto disease as a possibility when approaching patients with hyper-inflammatory states who present with cervical lymphadenopathy.

Recurrent macroglossia requiring tracheostomy after haemorrhagic basal ganglia stroke.

A 50-year-old African American woman with hypertension, congestive heart failure, chronic kidney disease and prior cerebral vascular accident was transferred from an outside hospital after being found unresponsive and subsequently intubated for severe orolingual swelling. Imaging showed left thalamic haemorrhagic stroke, and the lingual swelling was clinically concerning for angio-oedema, with which a lingual biopsy was consistent. Work-up was negative for hereditary or acquired angio-oedema, and imaging was negative for structural causes. Of note, the patient had an episode of severe orolingual swelling 3 months prior to this presentation after suffering left thalamic haemorrhage which self-resolved after approximately 2 months. In both episodes lingual swelling predated receipt of tissue plasminogen activator and she had discontinued ACE inhibitor therapy since her first episode of tongue swelling. Despite medical and supportive management, tongue swelling progressed during admission and the decision was made to allow the patient's tongue swelling to self-resolve.

Three-Dimensional Otic Neuronal Progenitor Spheroids Derived from Human Embryonic Stem Cells.

Stem cell-replacement therapies have been proposed as a potential tool to treat sensorineural hearing loss by aiding the regeneration of spiral ganglion neurons (SGNs) in the inner ear. However, transplantation procedures have yet to be explored thoroughly to ensure proper cell differentiation and optimal transplant procedures. We hypothesized that the aggregation of human embryonic stem cell (hESC)-derived otic neuronal progenitor (ONP) cells into a multicellular form would improve their function and their survival in vivo post-transplantation. We generated hESC-derived ONP spheroids-an aggregate form conducive to differentiation, transplantation, and prolonged cell survival-to optimize conditions for their transplantation. Our findings indicate that these cell spheroids maintain the molecular and functional characteristics similar to those of ONP cells, which are upstream in the SGN lineage. Moreover, our phenotypical, electrophysiological, and mechanical data suggest an optimal spheroid transplantation point after 7 days of in vitro three-dimensional (3D) culture. We have also developed a feasible transplantation protocol for these spheroids using a micropipette aided by a digital microinjection system. In summary, the present work demonstrates that the transplantation of ONP cells in spheroid form into the inner ear through micropipette 7 days after seeding for 3D spheroid culture is an expedient and viable method for stem cell replacement therapies in the inner ear.

Probable neurosarcoidosis presenting as acute on chronic otorrhoea: a difficult diagnosis.

An elderly African American woman presented to our clinic following 9 months of right-sided unilateral headache, otorrhoea and progressive hearing loss. Despite treatment with topical and oral antibiotics, her clinical condition worsened, and imaging showed mastoid coalescence with an associated subgaleal abscess. She underwent right mastoidectomy and was discharged 3 days later on broad-spectrum intravenous antibiotics despite negative operative cultures. Six weeks later, she was hospitalised with diplopia secondary to a right lateral rectus palsy. Imaging showed abscess resolution but progressive bony remodelling and enhancement of the lateral extending into anterior skull base. Chest CT demonstrated upper lobe predominant pulmonary micronodules, and mastoid biopsy on revision surgery was notable for non-caseating granulomas. Further extensive work-up could not identify an alternative cause, and a presumptive diagnosis of neurosarcoidosis was made. The patient was initiated on intravenous steroids, experienced symptomatic improvement and was thereafter transitioned to oral steroid taper on discharge.

Diagnostic value of refixation saccades in the Video Head Impulse Test (vHIT) in unilateral definite Meniere's disease.

Introduction: We currently interpret the video Head Impulse Test (vHIT) results mainly based on the gain value.Aim: The purpose of this study is to evaluate vHIT results for both gain and re-fixation saccades on unilateral definite Meniere's disease (MD) subjects in comparison with normal healthy subjects.Materials and Methods: Forty unilateral definite MD subjects and age-matched healthy subjects were recruited. Pure tone audiometry, the caloric test, and the vHIT test were performed on MD subjects. The vHIT test was performed on healthy subjects.Results: The velocity regression gain (VRG) of the affected ear in patients with MD is significantly lower than of those in healthy subjects. The total percentage of refixation saccades is significantly higher in patients with MD when compared to healthy subjects. VRG values were not well-correlated with the percentage of refixation saccades. VRG asymmetry values are also not well-correlated with the percentage of unilateral canal weakness. A moderately stronger correlation between the percentage of refixation saccades and percentage of unilateral canal weakness, with an r2 of 0.474.Conclusions: The present study suggests that while VRGs are still a diagnostic parameter of detecting MD, the presence of refixation saccades can also have diagnostic value, especially with normal VRGs, in detecting MD.

An engineered three-dimensional stem cell niche in the inner ear by applying a nanofibrillar cellulose hydrogel with a sustained-release neurotrophic factor delivery system.

Although the application of human embryonic stem cells (hESCs) in stem cell-replacement therapy remains promising, its potential is hindered by a low cell survival rate in post-transplantation within the inner ear. Here, we aim to enhance the in vitro and in vivo survival rate and neuronal differentiation of otic neuronal progenitors (ONPs) by generating an artificial stem cell niche consisting of three-dimensional (3D) hESC-derived ONP spheroids with a nanofibrillar cellulose hydrogel and a sustained-release brain-derivative neurotrophic factor delivery system. Our results demonstrated that the transplanted hESC-derived ONP spheroids survived and neuronally differentiated into otic neuronal lineages in vitro and in vivo and also extended neurites toward the bony wall of the cochlea 90 days after the transplantation without the use of immunosuppressant medication. Our data in vitro and in vivo presented here provide sufficient evidence that we have established a robust, reproducible protocol for in vivo transplantation of hESC-derived ONPs to the inner ear. Using our protocol to create an artificial stem cell niche in the inner ear, it is now possible to work on integrating transplanted hESC-derived ONPs further and also to work toward achieving functional auditory neurons generated from hESCs. Our findings suggest that the provision of an artificial stem cell niche can be a future approach to stem cell-replacement therapy for inner-ear regeneration. STATEMENT OF SIGNIFICANCE: Inner ear regeneration utilizing human embryonic stem cell-derived otic neuronal progenitors (hESC-derived ONPs) has remarkable potential for treating sensorineural hearing loss. However, the local environment of the inner ear requires a suitable stem cell niche to allow hESC-derived ONP engraftment as well as neuronal differentiation. To overcome this obstacle, we utilized three-dimensional spheroid formation (direct contact), nanofibrillar cellulose hydrogel (extracellular matrix), and a neurotrophic factor delivery system to artificially create a stem cell niche in vitro and in vivo. Our in vitro and in vivo data presented here provide sufficient evidence that we have established a robust, reproducible protocol for in vivo transplantation of hESC-derived ONPs to the inner ear.

Evaluation of the utricular function with the virtual-subject visual vertical system: comparison with ocular vestibular-evoked myogenic potentials.

Introduction: The subjective visual vertical (SVV) is the most frequently assessed modality of verticality perception and has been measured in a variety of clinical situations, including peripheral vestibular lesions.Aim: The main objectives are (1) to collect normative data of Virtual SVV™ from healthy subjects, and (2) to study the correlation between Virtual SVV™ and ocular vestibular-evoked myogenic potentials (o-VEMP) on healthy subjects.Materials and methods: Forty-three healthy subjects were recruited. Air conduction (AC)-elicited oVEMPs and bone conduction (BC)-elicited oVEMPs were measured. BC stimuli were produced with a RadioEar B-81 High Output Bone Transducer. Virtual SVV™ were also measured.Results: Virtual SVV™ data from our healthy subjects were consistent with previously published normative SVV data. Normal Virtual SVV™ data did not correlate with normal AC-elicited and BC-elicited oVEMPs.Conclusions: Virtual SVV™ data from our healthy subjects were consistent with previously published normative SVV data. Virtual SVV™ data from our 43 health subjects only had weak correlation with c-VEMP, AC-elicited and BC-elicited oVEMPs. These data serve as a baseline for a future study of patients with unilateral utricular dysfunction.Significance: The Virtual SVV™ can be an attractive substitute for traditional SVV in clinical settings.

Full Factorial Microfluidic Designs and Devices for Parallelizing Human Pluripotent Stem Cell Differentiation.

Human pluripotent stem cells (hPSCs) are promising therapeutic tools for regenerative therapies and disease modeling. Differentiation of cultured hPSCs is influenced by both exogenous factors added to the cultures and endogenously secreted molecules. Optimization of protocols for the differentiation of hPSCs into different cell types is difficult because of the many variables that can influence cell fate. We present microfluidic devices designed to perform three- and four-factor, two-level full factorial experiments in parallel for investigating and directly optimizing hPSC differentiation. These devices feature diffusion-isolated, independent culture wells that allow for control of both exogenous and endogenous cellular signals and that allow for immunocytochemistry (ICC) and confocal microscopy in situ. These devices are fabricated by soft lithography in conjunction with 3D-printed molds and are operable with a single syringe pump, eliminating the need for specialized equipment or cleanroom facilities. Their utility was demonstrated by on-chip differentiation of hPSCs into the auditory neuron lineage. More broadly, these devices enable multiplexing for experimentation with any adherent cell type or even multiple cell types, allowing efficient investigation of the effects of medium conditions, pharmaceuticals, or other soluble reagents.

Cervicofacial necrotising fasciitis by clindamycin-resistant and methicillin-resistant Staphylococcus aureus (MRSA) in a young healthy man.

An otherwise healthy 24-year-old man presented with 1 week of fever, facial pain and swelling. He initially sought care at an outside hospital, where he was diagnosed with folliculitis and sent home with oral antibiotics. On arrival at our institution, CT neck was ordered, which demonstrated diffuse submental phlegmon, prompting incision and drainage. After initial improvement, the patient experienced high fevers and increased swelling just 12 hours later. The decision was made to take the patient for operative exploration, and wide debridement was performed due to suspicion for necrotising fasciitis intraoperatively that was ultimately confirmed on final pathology. Final speciation of intraoperative culture demonstrated a clindamycin-resistant and methicillin-resistant strain of Staphylococcus aureus The patient was managed with intravenous antibiotics, additional debridement and careful wound care. Delayed partial closure of wound was eventually performed once patient showed marked and persistent clinical improvement. The patient was discharged on hospital day 12 with close follow-up.

Developmental profiling of microRNAs in the human embryonic inner ear.

Due to the extreme inaccessibility of fetal human inner ear tissue, defining of the microRNAs (miRNAs) that regulate development of the inner ear has relied on animal tissue. In the present study, we performed the first miRNA sequencing of otic precursors in human specimens. Using HTG miRNA Whole Transcriptome assays, we examined miRNA expression in the cochleovestibular ganglion (CVG), neural crest (NC), and otic vesicle (OV) from paraffin embedded (FFPE) human specimens in the Carnegie developmental stages 13-15. We found that in human embryonic tissues, there are different patterns of miRNA expression in the CVG, NC and OV. In particular, members of the miR-183 family (miR-96, miR-182, and miR-183) are differentially expressed in the CVG compared to NC and OV at Carnegie developmental stage 13. We further identified transcription factors that are differentially targeted in the CVG compared to the other tissues from stages 13-15, and we performed gene set enrichment analyses to determine differentially regulated pathways that are relevant to CVG development in humans. These findings not only provide insight into the mechanisms governing the development of the human inner ear, but also identify potential signaling pathways for promoting regeneration of the spiral ganglion and other components of the inner ear.

Creating a stem cell niche in the inner ear using self-assembling peptide amphiphiles.

The use of human embryonic stem cells (hESCs) for regeneration of the spiral ganglion will require techniques for promoting otic neuronal progenitor (ONP) differentiation, anchoring of cells to anatomically appropriate and specific niches, and long-term cell survival after transplantation. In this study, we used self-assembling peptide amphiphile (PA) molecules that display an IKVAV epitope (IKVAV-PA) to create a niche for hESC-derived ONPs that supported neuronal differentiation and survival both in vitro and in vivo after transplantation into rodent inner ears. A feature of the IKVAV-PA gel is its ability to form organized nanofibers that promote directed neurite growth. Culture of hESC-derived ONPs in IKVAV-PA gels did not alter cell proliferation or viability. However, the presence of IKVAV-PA gels increased the number of cells expressing the neuronal marker beta-III tubulin and improved neurite extension. The self-assembly properties of the IKVAV-PA gel allowed it to be injected as a liquid into the inner ear to create a biophysical niche for transplanted cells after gelation in vivo. Injection of ONPs combined with IKVAV-PA into the modiolus of X-SCID rats increased survival and localization of the cells around the injection site compared to controls. Human cadaveric temporal bone studies demonstrated the technical feasibility of a transmastoid surgical approach for clinical intracochlear injection of the IKVAV-PA/ONP combination. Combining stem cell transplantation with injection of self-assembling PA gels to create a supportive niche may improve clinical approaches to spiral ganglion regeneration.

The Protean Neuropsychiatric and Vestibuloauditory Manifestations of Neurosarcoidosis.

BACKGROUND: A rare subset of sarcoidosis, neurosarcoidosis, is reported to occur in 5-7% of sarcoid patients and can manifest in a variety of ways. The most common are facial paralysis and optic neuritis, less commonly causing cochleovestibulopathy, blindness, anosmia, and other cranial nerve (CN) palsies. The sensory deficit may be severe and psychiatric symptoms may result from the effects of the disease or steroid treatment. Although MRI-compatible cochlear implants are now available, concerns about the feasibility of recoverable hearing with cochlear implantation in these patients as well as the practical difficulty of disease monitoring due to implant artifact must be considered. RESULTS: We present 3 recent cases from different institutions. The first is a 39-year-old man with a history of progressively worsening hearing loss, followed by visual loss, delusions, agitation, ataxia, and musical auditory hallucinations, diffuse leptomeningeal enhancement on MRI with a normal serum angiotensin-converting enzyme (ACE) level but elevated cerebrospinal fluid (CSF) ACE levels, suggesting neurosarcoidosis, was treated with corticosteroids, and underwent successful cochlear implantation. The second is a 36-year-old woman with rapid-onset horizontal diplopia, left mixed severe sensorineural hearing loss (SNHL) and tinnitus, diffuse leptomeningeal enhancement on MRI, and progressive palsy of the left CNs IV, VI, VII, IX, X and XI, with altered mental status requiring admission following high-dose intravenous corticosteroids. The third is a 15-year-old boy who presented with sudden, bilateral, profound SNHL, recurrent headaches, and left facial weakness refractory to antivirals, ultimately diagnosed with neurosarcoidosis following an aborted cochlear implantation where diffuse inflammation was found, and histopathology revealed Schaumann bodies; he was treated with methotrexate and later underwent successful cochlear implantation. CONCLUSIONS: Neurosarcoidosis is an elusive diagnosis and can cause hearing loss and psychiatric symptoms. Cochlear implantation for patients with severe hearing loss should be considered once the diagnosis is confirmed, as it is possible to achieve a successful level of hearing. Psychiatric symptoms can manifest with the onset of neurosarcoidosis, result from CN deficits, or develop as a side effect from long-term, high-dose corticosteroids, and should be monitored carefully in patients with neurosarcoidosis.

Cervical oesophageal perforation secondary to food consumption in a well-appearing patient.

A 71-year-old woman presented to the emergency department 8 days after ingesting fish with mild neck pain but otherwise demonstrated no signs of infection. X-rays were negative but CT imaging demonstrated a curvilinear radiodense object extending from the posterior cervical oesophagus through the right thyroid lobe terminating in the neck just a few millimetres from the external carotid artery. Rigid oesophagoscopy and direct laryngoscopy were negative and the neck was explored for the foreign body, which ultimately was encountered after a painstaking dissection of the right neck that included skeletonisation of the recurrent laryngeal nerve. Her postoperative recovery was uneventful and after a 3-day course of intravenous antibiotics she was discharged on oral antibiotics, in good condition and tolerating a soft diet.

Directed Differentiation of Human Embryonic Stem Cells Toward Placode-Derived Spiral Ganglion-Like Sensory Neurons.

The ability to generate spiral ganglion neurons (SGNs) from stem cells is a necessary prerequisite for development of cell-replacement therapies for sensorineural hearing loss. We present a protocol that directs human embryonic stem cells (hESCs) toward a purified population of otic neuronal progenitors (ONPs) and SGN-like cells. Between 82% and 95% of these cells express SGN molecular markers, they preferentially extend neurites to the cochlear nucleus rather than nonauditory nuclei, and they generate action potentials. The protocol follows an in vitro stepwise recapitulation of developmental events inherent to normal differentiation of hESCs into SGNs, resulting in efficient sequential generation of nonneuronal ectoderm, preplacodal ectoderm, early prosensory ONPs, late ONPs, and cells with cellular and molecular characteristics of human SGNs. We thus describe the sequential signaling pathways that generate the early and later lineage species in the human SGN lineage, thereby better describing key developmental processes. The results indicate that our protocol generates cells that closely replicate the phenotypic characteristics of human SGNs, advancing the process of guiding hESCs to states serving inner-ear cell-replacement therapies and possible next-generation hybrid auditory prostheses. © Stem Cells Translational Medicine 2017;6:923-936.