Project description:In this study, we generated a human inner ear atlas containing three stages of inner ear development. This atlas was used to evaluate the differentiation approach of human pluripotent stem cells in to complex inner ear tissue, known as inner ear organoids. The primary goal of this single-nucleus RNA-sequencing analysis was to capture the cell type diversity of the human inner ear at different stages of development. The secondary goal was to define the similarity of organoid-derived inner ear cell types with the atlas-derived human inner ear cell types and to determine the developmental stage of the organoid-derived inner ear cell types.
Project description:In this study, we used a differentiation approach for guiding human pluripotent stem cells in to complex inner ear tissue, known as inner ear organoids. The primary goal of this single-cell and single-nucleus RNA-sequencing analyses was to define the cellular composition of the inner ear organoids, which we have shown and confirmed by histology. The secondary goal was to determine whether the inner ear organoids generated using different cell lines (WA01 hESCs, two reporter lines from the parental line GM25256, and LUMC0004iCtrl10 hiPSCs) contain similar cell types.
Project description:The inner ear in mammals is derived from a simple ectodermal thickening called the otic placode. Through a series of complex morphological changes, the placode forms the mature inner ear comprising of the auditory organ (cochlea) and the vestibular/balance organs (utricle, saccule, and three semi-circular canals). The vast majority of genes known to be involved during inner ear development have been found through mutational screens or by chance. To identify genes that can serve as novel candidates required for inner ear development, and also candidate genes for uncloned human deafnesses, inner ear tissues from mouse embryos from E9 to E15 were microdissected and expression-profiled at half-day intervals. Also profiled was the non-inner ear mesenchymal tissue surrounding the inner ear tissue. Various patterns of gene expression were identified, and significant biological pathways that these genes represented were identified. Also identified were mouse genes whose human orthologs are located within uncloned non-syndromic deafness intervals, thus serving as candidates for sequence analysis. Experiment Overall Design: Inner ear tissues from E9 to E15 were microdissected at half-day intervals. E9 is the earliest stage when the otic placode is clearly visible and able to be microdissected cleanly. E15 is the stage when all the organs of the inner ear have become established, as have the sensory hair and non-sensory support cells within those organs. For each of the stages from E9 to E10, whole inner ears were profiled. For each of the stages from E10.5 to E12, the primordial cochlear and vestibular organs were profiled separately. For each of the stages from E12.5 to E15, the cochlea and the saccule were profiled separately, whereas the utricle and the three ampullae were combined and profiled together. Any given tissue from any given stage was a collection of anywhere between 4 to 17 identical tissues, and was obtained in duplicate (i.e. from different litters). Hence, a total of 58 inner ear samples were obtained. Moreover, non-inner ear tissue found in the immediate vicinity of inner ear tissue was also obtained and profiled. Specifically, all non-inner ear tissue from E9 was profiled in duplicate. Non-inner ear tissue from E9.5 to E10.5 was pooled and profiled together (in duplicate), whereas that from E11 to E15 was pooled and profiled together (also in duplicate). Therefore, a total of 6 non-inner samples were obtained.
Project description:We have employed a novel approach for the identification of functionally important microRNA (miRNA)-target interactions using integrated miRNA, transcriptome and proteome profiles with advanced in silico analysis. By looking at both the transcript and protein levels of expression, a thorough coverage of miRNA regulation was obtained. Microdissected auditory and vestibular sensory epithelia were used as the model system, thus being the first time such a comparison was carried out in a neuroepithelial system. Moreover, this is one of only a few studies employing proteome screening for the identification of miRNA targets. Notably, this approach can be employed for the study of other tissues and organs. We detected the expression of 157 miRNAs in the inner ear sensory epithelia, with 53 miRNAs differentially expressed between the cochlea and vestibule. By searching for enrichment and depletion of miRNA targets in the transcript and protein datasets with a reciprocal or similar expression, respectively, as the regulatory miRNA, we identified functionally important miRNAs. Finally, the interaction between miR-135b and PSIP1-P75, a transcriptional coacitvator previously unknown in the inner ear, was identified and validated experimentally. We suggest that miR-135b may serve as a cellular effector, involved in regulating some of the differences between the cochlear and vestibular hair cells. We investigated the mRNA expression profile of the cochlear and vestibular sensory epithelia from inner ears of postnatal day 2 mice using the Affymetrix GeneChip® 430 2Mouse Genome array. Cochlear and vestibular sensory epithelia were dissected from wild type C3H mice and collected separately. The vestibular epithelia consisted of the saccule, utricle and the lateral and anterior cristae. Both the cochlear and vestibular sensory epithelia were dissected with their underlying mesenchyma. Altogether three pools, three biological replicates, of each tissue type were collected consisting of cochlear or vestibular sensory epithelia dissected from 10 to 12 inner ears.
Project description:Inner ear disorders are among the most common congenital abnormalities; however, current tissue culture models lack the cell type diversity to study these disorders and normal otic development. Here, we demonstrate the robustness of human pluripotent stem cell-derived inner ear organoids (IEOs) and evaluate cell type heterogeneity by single-cell transcriptomics. To validate our findings, we construct a single-cell atlas of human fetal and adult inner ear tissue. Our study identifies various cell types in the IEOs including periotic mesenchyme, type I and type II vestibular hair cells, and developing vestibular and cochlear epithelium. Many genes linked to congenital inner ear dysfunction are confirmed to be expressed in these cell types. Additional cell-cell communication analysis within IEOs and fetal tissue highlights the role of endothelial cells on the developing sensory epithelium. These findings provide insights into this organoid model and its potential applications in studying inner ear development and disorders.
Project description:Inner ear organoids recapitulate development and are intended to generate cell types of the otic lineage for applications such as basic science research and cell replacement strategies. Here, we use single-cell sequencing to study the cellular heterogeneity of late-stage mouse inner ear organoid sensory epithelia, which we validated by comparison with data sets of the mouse cochlea and vestibular epithelia. We resolved supporting cell sub-types, cochlear like hair cells, and vestibular Type I and Type II like hair cells. While cochlear like hair cells aligned best with an outer hair cell trajectory, vestibular like hair cells followed developmental trajectories similar to in vivo programs branching into Type II and then Type I extrastriolar hair cells. These results highlight the transcriptional accuracy of the organoid developmental program but will also inform future strategies to improve synaptic connectivity and regional specification.
Project description:Inner ear auditory and vestibular tissues differ in their responses to mechanical stimuli. Chick cochlea and utricle sensory epithelia were microdissected at E20-E21. RNA was extracted and cRNA hybridized to Affymetrix microarrays.
Project description:The inner ear in mammals is derived from a simple ectodermal thickening called the otic placode. Through a series of complex morphological changes, the placode forms the mature inner ear comprising of the auditory organ (cochlea) and the vestibular/balance organs (utricle, saccule, and three semi-circular canals). The vast majority of genes known to be involved during inner ear development have been found through mutational screens or by chance. To identify genes that can serve as novel candidates required for inner ear development, and also candidate genes for uncloned human deafnesses, inner ear tissues from mouse embryos from E9 to E15 were microdissected and expression-profiled at half-day intervals. Also profiled was the non-inner ear mesenchymal tissue surrounding the inner ear tissue. Various patterns of gene expression were identified, and significant biological pathways that these genes represented were identified. Also identified were mouse genes whose human orthologs are located within uncloned non-syndromic deafness intervals, thus serving as candidates for sequence analysis. Keywords: Developmental timecourse
Project description:The inner ear continues to grow and develop until the auditory and vestibular systems reach full maturity and all of the genes involved in this process have yet to be identified. Previous gene based analysis have primarily focused on the early developmental stages following induction and initial formation of the inner ear. The aim of this study is to identify new candidate genes for inner ear development. Microarrays were used to produce expression profiles from larval stages 56,57,58 of the Xenopus laevis inner ear. The data produced from this work represent an annotated resource that can be utilized by the Xenopus community to provide candidates for further functional analysis. Xenopus inner ears were isolated from larval animals for RNA extraction and hybridization to Affymetrix GeneChip microarrays.