in silico transcriptome dissection of neocortical excitatory neurogenesis via joint matrix decomposition and transfer learning.

The rising quality and amount of multi-omic data across biomedical science demands that we build innovative solutions to harness their collective discovery potential. From publicly available repositories, we have assembled and curated a compendium of gene-level transcriptomic data focused on mammalian excitatory neurogenesis in the neocortex. This collection is open for exploration by both computational and cell biologists at nemoanalytics.org, and this report forms a demonstration of its utility. Applying our novel structured joint decomposition approach to mouse, macaque and human data from the collection, we define transcriptome dynamics that are conserved across mammalian excitatory neurogenesis and which map onto the genetics of human brain structure and disease. Leveraging additional data within NeMO Analytics via projection methods, we chart the dynamics of these fundamental molecular elements of neurogenesis across developmental time and space and into postnatal life. Reversing the direction of our investigation, we use transcriptomic data from laminar-specific dissection of adult human neocortex to define molecular signatures specific to excitatory neuronal cell types resident in individual layers of the mature neocortex, and trace their emergence across development. We show that while many lineage defining transcription factors are most highly expressed at early fetal ages, the laminar neuronal identities which they drive take years to decades to reach full maturity. Finally, we interrogated data from stem-cell derived cerebral organoid systems demonstrating that many fundamental elements of in vivo development are recapitulated with high-fidelity in vitro, while specific transcriptomic programs in neuronal maturation are absent. We propose these analyses as specific applications of the general approach of combining joint decomposition with large curated collections of analysis-ready multi-omics data matrices focused on particular cell and disease contexts. Importantly, these open environments are accessible to, and must be fueled with emerging data by, cell biologists with and without coding expertise.

The cells of the sensory epithelium, and not the stria vascularis, are the main cochlear cells related to the genetic pathogenesis of age-related hearing loss.

Age-related hearing loss (ARHL) is a major health concern among the elderly population. It is hoped that increasing our understanding of its underlying pathophysiological processes will lead to the development of novel therapies. Recent genome-wide association studies (GWASs) discovered a few dozen genetic variants in association with elevated risk for ARHL. Integrated analysis of GWAS results and transcriptomics data is a powerful approach for elucidating specific cell types that are involved in disease pathogenesis. Intriguingly, recent studies that applied such bioinformatics approaches to ARHL resulted in disagreeing findings as for the key cell types that are most strongly linked to the genetic pathogenesis of ARHL. These conflicting studies pointed either to cochlear sensory epithelial or to stria vascularis cells as the cell types most prominently involved in the genetic basis of ARHL. Seeking to resolve this discrepancy, we integrated the analysis of four ARHL GWAS datasets with four independent inner-ear single-cell RNA-sequencing datasets. Our analysis clearly points to the cochlear sensory epithelial cells as the key cells for the genetic predisposition to ARHL. We also explain the limitation of the bioinformatics analysis performed by previous studies that led to missing the enrichment for ARHL GWAS signal in sensory epithelial cells. Collectively, we show that cochlear epithelial cells, not stria vascularis cells, are the main inner-ear cells related to the genetic pathogenesis of ARHL.

DeepFreeze 3D-biofabrication for Bioengineering and Storage of Stem Cells in Thick and Large-Scale Human Tissue Analogs.

3D bioprinting holds great promise for meeting the increasing need for transplantable tissues and organs. However, slow printing, interlayer mixing, and the extended exposure of cells to non-physiological conditions in thick structures still hinder clinical applications. Here the DeepFreeze-3D (DF-3D) procedure and bioink for creating multilayered human-scale tissue mimetics is presented for the first time. The bioink is tailored to support stem cell viability, throughout the rapid freeform DF-3D biofabrication process. While the printer nozzle is warmed to room temperature, each layer solidifies at contact with the stage (-80 °C), or the subsequent layers, ensuring precise separation. After thawing, the encapsulated stem cells remain viable without interlayer mixing or delamination. The composed cell-laden constructs can be cryogenically stored and thawed when needed. Moreover, it is shown that under inductive conditions the stem cells differentiate into bone-like cells and grow for months after thawing, to form large tissue-mimetics in the scale of centimeters. This is important, as this approach allows the generation and storage of tissue mimetics in the size and thickness of human tissues. Therefore, DF-3D biofabrication opens new avenues for generating off-the-shelf human tissue analogs. It further holds the potential for regenerative treatments and for studying tissue pathologies caused by disease, tumor, or trauma.

Cochlear organoids reveal transcriptional programs of postnatal hair cell differentiation from supporting cells.

We explore the changes in chromatin accessibility and transcriptional programs for cochlear hair cell differentiation from postmitotic supporting cells using organoids from postnatal cochlea. The organoids contain cells with transcriptional signatures of differentiating vestibular and cochlear hair cells. Construction of trajectories identifies Lgr5+ cells as progenitors for hair cells, and the genomic data reveal gene regulatory networks leading to hair cells. We validate these networks, demonstrating dynamic changes both in expression and predicted binding sites of transcription factors (TFs) during organoid differentiation. We identify known regulators of hair cell development, Atoh1, Pou4f3, and Gfi1, and the analysis predicts the regulatory factors Tcf4, an E-protein and heterodimerization partner of Atoh1, and Ddit3, a CCAAT/enhancer-binding protein (C/EBP) that represses Hes1 and activates transcription of Wnt-signaling-related genes. Deciphering the signals for hair cell regeneration from mammalian cochlear supporting cells reveals candidates for hair cell (HC) regeneration, which is limited in the adult.

Spatially distinct otic mesenchyme cells show molecular and functional heterogeneity patterns before hearing onset.

The cochlea consists of diverse cellular populations working in harmony to convert mechanical stimuli into electrical signals for the perception of sound. Otic mesenchyme cells (OMCs), often considered a homogeneous cell type, are essential for normal cochlear development and hearing. Despite being the most numerous cell type in the developing cochlea, OMCs are poorly understood. OMCs are known to differentiate into spatially and functionally distinct cell types, including fibrocytes of the lateral wall and spiral limbus, modiolar osteoblasts, and specialized tympanic border cells of the basilar membrane. Here, we show that OMCs are transcriptionally and functionally heterogeneous and can be divided into four distinct populations that spatially correspond to OMC-derived cochlear structures. We also show that this heterogeneity and complexity of OMCs commences during early phases of cochlear development. Finally, we describe the cell-cell communication network of the developing cochlea, inferring a major role for OMC in outgoing signaling.

Metformin Protects Against Noise-Induced Hearing Loss in Male Mice.

HYPOTHESIS: Metformin treatment will protect mice from noise-induced hearing loss (NIHL). BACKGROUND: We recently identified metformin as the top-ranking, Food and Drug Administration-approved drug to counter inner ear molecular changes induced by permanent threshold shift-inducing noise. This study is designed to functionally test metformin as a potential otoprotective drug against NIHL. METHODS: Male and female B6CBAF1/J mice were obtained at 7 to 8 weeks of age. A cohort of the females underwent ovariectomy to simulate menopause and eliminate the effect of ovarian-derived estrogens. At 10 weeks of age, mice underwent a permanent threshold shift-inducing noise exposure (102.5 or 105 dB SPL, 8-16 kHz, 2 h). Auditory brainstem response (ABR) thresholds were obtained at baseline, 24 h after noise exposure, and 1 week after noise exposure. Mice were administered metformin (200 mg/kg/d) or a saline control in their drinking water after the baseline ABR and for the remainder of the study. After the 1-week ABR, mice were euthanized and cochlear tissue was analyzed. RESULTS: Metformin treatment reduced the 1-week ABR threshold shift at 16 kHz ( p < 0.01; d = 1.20) and 24 kHz ( p < 0.01; d = 1.15) as well as outer hair cell loss in the 32-45.5 kHz range ( p < 0.0001; d = 2.37) in male mice. In contrast, metformin treatment did not prevent hearing loss or outer hair cell loss in the intact or ovariectomized female mice. CONCLUSIONS: Metformin exhibits sex-dependent efficacy as a therapeutic for NIHL. These data compel continued investigation into metformin's protective effects and demonstrate the importance of evaluating the therapeutic efficacy of drugs in subjects of both sexes.

A guide to the BRAIN Initiative Cell Census Network data ecosystem.

Characterizing cellular diversity at different levels of biological organization and across data modalities is a prerequisite to understanding the function of cell types in the brain. Classification of neurons is also essential to manipulate cell types in controlled ways and to understand their variation and vulnerability in brain disorders. The BRAIN Initiative Cell Census Network (BICCN) is an integrated network of data-generating centers, data archives, and data standards developers, with the goal of systematic multimodal brain cell type profiling and characterization. Emphasis of the BICCN is on the whole mouse brain with demonstration of prototype feasibility for human and nonhuman primate (NHP) brains. Here, we provide a guide to the cellular and spatial approaches employed by the BICCN, and to accessing and using these data and extensive resources, including the BRAIN Cell Data Center (BCDC), which serves to manage and integrate data across the ecosystem. We illustrate the power of the BICCN data ecosystem through vignettes highlighting several BICCN analysis and visualization tools. Finally, we present emerging standards that have been developed or adopted toward Findable, Accessible, Interoperable, and Reusable (FAIR) neuroscience. The combined BICCN ecosystem provides a comprehensive resource for the exploration and analysis of cell types in the brain.

The Neuroscience Multi-Omic Archive: a BRAIN Initiative resource for single-cell transcriptomic and epigenomic data from the mammalian brain.

Scalable technologies to sequence the transcriptomes and epigenomes of single cells are transforming our understanding of cell types and cell states. The Brain Research through Advancing Innovative Neurotechnologies (BRAIN) Initiative Cell Census Network (BICCN) is applying these technologies at unprecedented scale to map the cell types in the mammalian brain. In an effort to increase data FAIRness (Findable, Accessible, Interoperable, Reusable), the NIH has established repositories to make data generated by the BICCN and related BRAIN Initiative projects accessible to the broader research community. Here, we describe the Neuroscience Multi-Omic Archive (NeMO Archive; nemoarchive.org), which serves as the primary repository for genomics data from the BRAIN Initiative. Working closely with other BRAIN Initiative researchers, we have organized these data into a continually expanding, curated repository, which contains transcriptomic and epigenomic data from over 50 million brain cells, including single-cell genomic data from all of the major regions of the adult and prenatal human and mouse brains, as well as substantial single-cell genomic data from non-human primates. We make available several tools for accessing these data, including a searchable web portal, a cloud-computing interface for large-scale data processing (implemented on Terra, terra.bio), and a visualization and analysis platform, NeMO Analytics (nemoanalytics.org).

Sex-Linked Biology and Gender-Related Research Is Essential to Advancing Hearing Health.

There is robust evidence that sex (biological) and gender (behavioral/social) differences influence hearing loss risk and outcomes. These differences are noted for animals and humans-in the occurrence of hearing loss, hearing loss progression, and response to interventions. Nevertheless, many studies have not reported or disaggregated data by sex or gender. This article describes the influence of sex-linked biology (specifically sex-linked hormones) and gender on hearing and hearing interventions, including the role of sex-linked biology and gender in modifying the association between risk factors and hearing loss, and the effects of hearing loss on quality of life and functioning. Most prevalence studies indicate that hearing loss begins earlier and is more common and severe among men than women. Intrinsic sex-linked biological differences in the auditory system may account, in part, for the predominance of hearing loss in males. Sex- and gender-related differences in the effects of noise exposure or cardiovascular disease on the auditory system may help explain some of these differences in the prevalence of hearing loss. Further still, differences in hearing aid use and uptake, and the effects of hearing loss on health may also vary by sex and gender. Recognizing that sex-linked biology and gender are key determinants of hearing health, the present review concludes by emphasizing the importance of a well-developed research platform that proactively measures and assesses sex- and gender-related differences in hearing, including in understudied populations. Such research focus is necessary to advance the field of hearing science and benefit all members of society.

The Genomics of Auditory Function and Disease.

Current estimates suggest that nearly half a billion people worldwide are affected by hearing loss. Because of the major psychological, social, economic, and health ramifications, considerable efforts have been invested in identifying the genes and molecular pathways involved in hearing loss, whether genetic or environmental, to promote prevention, improve rehabilitation, and develop therapeutics. Genomic sequencing technologies have led to the discovery of genes associated with hearing loss. Studies of the transcriptome and epigenome of the inner ear have characterized key regulators and pathways involved in the development of the inner ear and have paved the way for their use in regenerative medicine. In parallel, the immense preclinical success of using viral vectors for gene delivery in animal models of hearing loss has motivated the industry to work on translating such approaches into the clinic. Here, we review the recent advances in the genomics of auditory function and dysfunction, from patient diagnostics to epigenetics and gene therapy.

Senior Care Pharmacists, Audiologists, and Otologists: Improving Hearing Health for Older Patients Through Interprofessional Collaboration.

The purpose of this manuscript is to provide pharmacists with education on hearing loss that colleagues in audiology believe is most critical for pharmacists. As well as highlighting insightful interventions pharmacists can make in collaboration with hearing professionals, such as audiologists, otolaryngologists, and otologists, to improve patient care. This project was initiated by professional students at the University of Maryland in both Baltimore and College Park campuses, after completing the interprofessional elective course IPE Care in Geriatrics. Upon completion of the course, the authors performed an extensive literature search and reviewed publications pertaining to pharmacy, audiology, and their integration.Hearing loss can have a significant impact on a patient's quality of life. Older people are at an increased risk for experiencing hearing impairment, but often do not seek help from health care providers. Collaboration between audiologists, otolaryngologists, and pharmacists has the potential to improve patients' access to hearing health and break barriers for patients. Important interventions that pharmacists can make to better serve their patients with hearing loss include screening, enhancing communication, and hearing aid assistance. This article also provides guidance on identifying patients who would be candidates for over-the-counter hearing aids and patients who should be referred to a hearing professional. This skill will become increasingly relevant with the emergence of over-the-counter hearing aids.

Genome-wide association meta-analysis identifies 48 risk variants and highlights the role of the stria vascularis in hearing loss.

Hearing loss is one of the top contributors to years lived with disability and is a risk factor for dementia. Molecular evidence on the cellular origins of hearing loss in humans is growing. Here, we performed a genome-wide association meta-analysis of clinically diagnosed and self-reported hearing impairment on 723,266 individuals and identified 48 significant loci, 10 of which are novel. A large proportion of associations comprised missense variants, half of which lie within known familial hearing loss loci. We used single-cell RNA-sequencing data from mouse cochlea and brain and mapped common-variant genomic results to spindle, root, and basal cells from the stria vascularis, a structure in the cochlea necessary for normal hearing. Our findings indicate the importance of the stria vascularis in the mechanism of hearing impairment, providing future paths for developing targets for therapeutic intervention in hearing loss.

Advancing discovery in hearing research via biologist-friendly access to multi-omic data.

High-throughput cell type-specific multi-omic analyses have advanced our understanding of inner ear biology in an unprecedented way. The full benefit of these data, however, is reached from their re-use. Successful re-use of data requires identifying the natural users and ensuring proper data democratization and federation for their seamless and meaningful access. Here we discuss universal challenges in access and re-use of multi-omic data, possible solutions, and introduce the gEAR (the gene Expression Analysis Resource, umgear.org)-a tool for multi-omic data visualization, sharing and access for the ear field.

Estradiol Protects against Noise-Induced Hearing Loss and Modulates Auditory Physiology in Female Mice.

Recent studies have identified sex-differences in auditory physiology and in the susceptibility to noise-induced hearing loss (NIHL). We hypothesize that 17ß-estradiol (E2), a known modulator of auditory physiology, may underpin sex-differences in the response to noise trauma. Here, we gonadectomized B6CBAF1/J mice and used a combination of electrophysiological and histological techniques to study the effects of estrogen replacement on peripheral auditory physiology in the absence of noise exposure and on protection from NIHL. Functional analysis of auditory physiology in gonadectomized female mice revealed that E2-treatment modulated the peripheral response to sound in the absence of changes to the endocochlear potential compared to vehicle-treatment. E2-replacement in gonadectomized female mice protected against hearing loss following permanent threshold shift (PTS)- and temporary threshold shift (TTS)-inducing noise exposures. Histological analysis of the cochlear tissue revealed that E2-replacement mitigated outer hair cell loss and cochlear synaptopathy following noise exposure compared to vehicle-treatment. Lastly, using fluorescent in situ hybridization, we demonstrate co-localization of estrogen receptor-2 with type-1C, high threshold spiral ganglion neurons, suggesting that the observed protection from cochlear synaptopathy may occur through E2-mediated preservation of these neurons. Taken together, these data indicate the estrogen signaling pathways may be harnessed for the prevention and treatment of NIHL.

Lineage-tracing and translatomic analysis of damage-inducible mitotic cochlear progenitors identifies candidate genes regulating regeneration.

Cochlear supporting cells (SCs) are glia-like cells critical for hearing function. In the neonatal cochlea, the greater epithelial ridge (GER) is a mitotically quiescent and transient organ, which has been shown to nonmitotically regenerate SCs. Here, we ablated Lgr5+ SCs using Lgr5-DTR mice and found mitotic regeneration of SCs by GER cells in vivo. With lineage tracing, we show that the GER houses progenitor cells that robustly divide and migrate into the organ of Corti to replenish ablated SCs. Regenerated SCs display coordinated calcium transients, markers of the SC subtype inner phalangeal cells, and survive in the mature cochlea. Via RiboTag, RNA-sequencing, and gene clustering algorithms, we reveal 11 distinct gene clusters comprising markers of the quiescent and damaged GER, and damage-responsive genes driving cell migration and mitotic regeneration. Together, our study characterizes GER cells as mitotic progenitors with regenerative potential and unveils their quiescent and damaged translatomes.

A transcriptomic and epigenomic cell atlas of the mouse primary motor cortex.

Single-cell transcriptomics can provide quantitative molecular signatures for large, unbiased samples of the diverse cell types in the brain1-3. With the proliferation of multi-omics datasets, a major challenge is to validate and integrate results into a biological understanding of cell-type organization. Here we generated transcriptomes and epigenomes from more than 500,000 individual cells in the mouse primary motor cortex, a structure that has an evolutionarily conserved role in locomotion. We developed computational and statistical methods to integrate multimodal data and quantitatively validate cell-type reproducibility. The resulting reference atlas-containing over 56 neuronal cell types that are highly replicable across analysis methods, sequencing technologies and modalities-is a comprehensive molecular and genomic account of the diverse neuronal and non-neuronal cell types in the mouse primary motor cortex. The atlas includes a population of excitatory neurons that resemble pyramidal cells in layer 4 in other cortical regions4. We further discovered thousands of concordant marker genes and gene regulatory elements for these cell types. Our results highlight the complex molecular regulation of cell types in the brain and will directly enable the design of reagents to target specific cell types in the mouse primary motor cortex for functional analysis.

A cell-type-specific atlas of the inner ear transcriptional response to acoustic trauma.

Noise-induced hearing loss (NIHL) results from a complex interplay of damage to the sensory cells of the inner ear, dysfunction of its lateral wall, axonal retraction of type 1C spiral ganglion neurons, and activation of the immune response. We use RiboTag and single-cell RNA sequencing to survey the cell-type-specific molecular landscape of the mouse inner ear before and after noise trauma. We identify induction of the transcription factors STAT3 and IRF7 and immune-related genes across all cell-types. Yet, cell-type-specific transcriptomic changes dominate the response. The ATF3/ATF4 stress-response pathway is robustly induced in the type 1A noise-resilient neurons, potassium transport genes are downregulated in the lateral wall, mRNA metabolism genes are downregulated in outer hair cells, and deafness-associated genes are downregulated in most cell types. This transcriptomic resource is available via the Gene Expression Analysis Resource (gEAR; https://umgear.org/NIHL) and provides a blueprint for the rational development of drugs to prevent and treat NIHL.

Music level preference and perceived exercise intensity in group spin classes.

CONTEXT: Sound levels in fitness classes often exceed safe levels despite studies that show many participants find high sound levels stressful. AIMS: The objective is to determine if lower sound levels in spinning classes significantly impact exercise intensity and to determine if class participants prefer the music played at lower levels. SETTINGS AND DESIGN: Observational study of 1-hour group spin classes. METHODS AND MATERIALS: Sound levels were measured in 18 spin classes over two weeks. No adjustments were made in week-1 and sound levels were decreased by 3 dB in week-2. Participant preferences and data on post-class hearing changes were collected via post-class questionnaires (n = 213) and divided into three terciles based on the total sound exposure of corresponding classes. STATISTICAL ANALYSIS USED: Unweighted survey generalized linear models are used to sort the causal relationships between different variables simultaneously and participant responses. The Chi-square test is used to reveal statistically significant relationships between two or more categorical variables. RESULTS: When mean sound levels exceeded 98.4 dBC, respondents were 23 times more likely to report the music as too loud than too quiet (P < 0.05), and four times more likely to prefer a decrease, rather than an increase, in sound level (P < 0.05). There was no significant difference in respondents reporting high exercise intensity between the middle (95.7-98.1 dBC) and upper (98.4-101.0 dBC) terciles, 67.1% and 71.8%, respectively (P = 0.53). Overall, 25.9% of respondents reported auditory symptoms following classes. Analysis in the context of dBA and dBC produced congruent conclusions and interpretations. CONCLUSIONS: Sound levels in many fitness classes remain dangerously high. However, music level can be lowered without a significant impact on perceived exercise intensity and many participants prefer lower sound levels than current levels.

Cell Type-Specific Expression Analysis of the Inner Ear: A Technical Report.

OBJECTIVE: The cellular diversity of the inner ear has presented a technical challenge in obtaining molecular insight into its development and function. The application of technological advancements in cell type-specific expression enable clinicians and researchers to leap forward from classic genetics to obtaining mechanistic understanding of congenital and acquired hearing loss. This understanding is essential for development of therapeutics to prevent and reverse diseases of the inner ear, including hearing loss. The objective of this study is to describe and compare the available tools for cell type-specific analysis of the ear, as a means to support decision making in study design. STUDY DESIGN: Three major approaches for cell type-specific analysis of the ear including fluorescence-activated cell sorting (FACS), ribosomal and RNA pulldown techniques, and single cell RNA-seq (scRNA-seq) are compared and contrasted using both published and original data. RESULTS: We demonstrate the strength and weaknesses of these approaches leading to the inevitable conclusion that to maximize the utility of these approaches, it is important to match the experimental approach with the tissue of origin, cell type of interest, and the biological question. Often, a combined approach (eg, cell sorting and scRNA-seq or expression analysis using 2 separate approaches) is required. Finally, new tools for visualization and analysis of complex expression data, such as the gEAR platform (umgear.org), collate cell type-specific gene expression from the ear field and provide unprecedented access to both clinicians and researchers. LEVEL OF EVIDENCE: N/A Laryngoscope, 131:S1-S16, 2021.