Modulation of Wnt Signaling Enhances Inner Ear Organoid Development in 3D Culture.
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ABSTRACT:
SUBMITTER: DeJonge RE
PROVIDER: S-EPMC5015985 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Inner ear development requires the coordination of cell types from distinct epithelial, mesenchymal and neuronal lineages. Although we have learned much from animal models, many details about human inner ear development remain elusive. We recently developed an in vitro model of human inner ear organogenesis using pluripotent stem cells in a 3D culture, fostering the growth of a sensorineural circuit, including hair cells and neurons. Despite previously characterizing some cell types, many remain undefined. This study aimed to chart the in vitro development timeline of the inner ear organoid to understand the mechanisms at play. Using single-cell RNA sequencing at ten stages during the first 36 days of differentiation, we tracked the evolution from pluripotency to various ear cell types after exposure to specific signaling modulators. Our findings showcase gene expression that influences differentiation, identifying a plethora of ectodermal and mesenchymal cell types. We also discern aspects of the organoid model consistent with in vivo development, while highlighting potential discrepancies. Our study establishes the Inner Ear Organoid Developmental Atlas (IODA), offering deeper insights into human biology and improving inner ear tissue differentiation.
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Project description:This protocol describes a culture system in which inner-ear sensory tissue is produced from mouse embryonic stem (ES) cells under chemically defined conditions. This model is amenable to basic and translational investigations into inner ear biology and regeneration. In this protocol, mouse ES cells are aggregated in 96-well plates in medium containing extracellular matrix proteins to promote epithelialization. During the first 14 d, a series of precisely timed protein and small-molecule treatments sequentially induce epithelia that represent the mouse embryonic non-neural ectoderm, preplacodal ectoderm and otic vesicle epithelia. Ultimately, these tissues develop into cysts with a pseudostratified epithelium containing inner ear hair cells and supporting cells after 16-20 d. Concurrently, sensory-like neurons generate synapse-like structures with the derived hair cells. We have designated the stem cell-derived epithelia harboring hair cells, supporting cells and sensory-like neurons as inner ear organoids. This method provides a reproducible and scalable means to generate inner ear sensory tissue in vitro.
| S-EPMC6453571 | biostudies-literature
Project description:The inner ear contains sensory epithelia that detect head movements, gravity and sound. It is unclear how to develop these sensory epithelia from pluripotent stem cells, a process that will be critical for modelling inner ear disorders or developing cell-based therapies for profound hearing loss and balance disorders. So far, attempts to derive inner ear mechanosensitive hair cells and sensory neurons have resulted in inefficient or incomplete phenotypic conversion of stem cells into inner-ear-like cells. A key insight lacking from these previous studies is the importance of the non-neural and preplacodal ectoderm, two critical precursors during inner ear development. Here we report the stepwise differentiation of inner ear sensory epithelia from mouse embryonic stem cells (ESCs) in three-dimensional culture. We show that by recapitulating in vivo development with precise temporal control of signalling pathways, ESC aggregates transform sequentially into non-neural, preplacodal and otic-placode-like epithelia. Notably, in a self-organized process that mimics normal development, vesicles containing prosensory cells emerge from the presumptive otic placodes and give rise to hair cells bearing stereocilia bundles and a kinocilium. Moreover, these stem-cell-derived hair cells exhibit functional properties of native mechanosensitive hair cells and form specialized synapses with sensory neurons that have also arisen from ESCs in the culture. Finally, we demonstrate how these vesicles are structurally and biochemically comparable to developing vestibular end organs. Our data thus establish a new in vitro model of inner ear differentiation that can be used to gain deeper insight into inner ear development and disorder.
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Project description:We report the application of single cell RNA sequencing for determining cell type and quantity in developing inner ear organoids derived from human pluripotent stem cells. By obtaining samples from multiple time points throughout differentiation, we developed a roadmap at single cell resolution of the multiple cell lineages which give rise to in vitro inner ear tissue as well as 'off-target' tissue types. We find that the cells in the day 0 sample contained primarily a homogenous population of pluripotent stem cells. On day 3, after treatment with BMP4, we find that the cells have differentiated into a split between surface ectoderm, the progenitor of the inner ear tissue, and neuroectoderm. On day 6 and 8, we observed the emergence of cranial placode tissue, both anterior and posterior. Then, after treatment starting on day 8 with a WNT agonist, CHIR, we observed the maturation of inner ear tissue into hair cells and neurons, starting on day 18 and increasing through the last time point collected, day 36. This study provides an atlas of inner ear organoid differentiation from human pluripotent stem cells.
2021-11-23 | GSE188936 | GEO
Project description:The inner ear is a complex organ containing highly specialised cell types and structures that are critical for sensing sound and movement. In vivo, the inner ear is difficult to study due to the osseous nature of the otic capsule and its encapsulation within an intricate bony labyrinth. As such, mammalian inner ear explants are an invaluable tool for the study and manipulation of the complex intercellular connections, structures, and cell types within this specialised organ. The greatest strength of this technique is that the complete organ of Corti, or peripheral vestibular organs including hair cells, supporting cells and accompanying neurons, is maintained in its in situ form. The greatest weakness of in vitro hair cell preparations is the short time frame in which the explanted tissue remains viable. Yet, cochlear explants have proven to be an excellent experimental model for understanding the fundamental aspects of auditory biology, substantiated by their use for over 40 years. In this protocol, we present a modernised inner ear explant technique that employs organotypic cell culture inserts and serum free media. This approach decreases the likelihood of explant damage by eliminating the need for adhesive substances. Serum free media also restricts excessive cellular outgrowth and inter-experimental variability, both of which are side effects of exogenous serum addition to cell cultures. The protocol described can be applied to culture both cochlear and vestibular explants from various mammals. Example outcomes are demonstrated by immunohistochemistry, hair cell quantification, and electrophysiological recordings to validate the versatility and viability of the protocol.
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Project description:A forward genetic screen of N-ethyl-N-nitrosourea mutagenized Xenopus tropicalis has identified an inner ear mutant named eclipse (ecl). Mutants developed enlarged otic vesicles and various defects of otoconia development; they also showed abnormal circular and inverted swimming patterns. Positional cloning identified specificity protein 8 (sp8), which was previously found to regulate limb and brain development. Two different loss-of-function approaches using transcription activator-like effector nucleases and morpholino oligonucleotides confirmed that the ecl mutant phenotype is caused by down-regulation of sp8. Depletion of sp8 resulted in otic dysmorphogenesis, such as uncompartmentalized and enlarged otic vesicles, epithelial dilation with abnormal sensory end organs. When overexpressed, sp8 was sufficient to induce ectopic otic vesicles possessing sensory hair cells, neurofilament innervation in a thickened sensory epithelium, and otoconia, all of which are found in the endogenous otic vesicle. We propose that sp8 is an important factor for initiation and elaboration of inner ear development.
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Project description:Photobiomodulation (PBM) stimulates different types of stem cells to migrate, proliferate, and differentiate in vitro and in vivo. However, little is known about the effects of PBM on the differentiation of embryonic stem cells (ESCs) toward the otic lineage. Only a few reports have documented the in vitro differentiation of ESCs into inner-ear hair cells (HCs) due to the complexity of HCs compared with other target cell types. In this study, we determined the optimal condition to differentiate the ESCs into the otic organoid using different culture techniques and PBM parameters. The efficiency of organoid formation within the embryoid body (EB) was dependent on the cell density of the hanging drop. PBM, using 630 nm wavelength light-emitting diodes (LEDs), further improved the differentiation of inner-ear hair cell-like cells coupled with reactive oxygen species (ROS) overexpression. Transcriptome analysis showed the factors that are responsible for the effect of PBM in the formation of otic organoids, notably, the downregulation of neural development-associated genes and the hairy and enhancer of split 5 (Hes5) gene, which inhibits the differentiation of prosensory cells to hair cells. These data enrich the current differentiation protocols for generating inner-ear hair cells.
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Project description:Mutations in leucine-rich repeat kinase 2 (lrrk2) are the most common genetic cause of Parkinson's disease. Difficulty in elucidating the pathogenic mechanisms resulting from disease-associated Lrrk2 variants stems from the complexity of Lrrk2 function and activities. Lrrk2 contains multiple protein-protein interacting domains, a GTPase domain, and a kinase domain. Lrrk2 is implicated in many cellular processes including vesicular trafficking, autophagy, cytoskeleton dynamics, and Wnt signaling. Here, we generated a zebrafish lrrk2 allelic series to study the requirements for Lrrk2 during development and to dissect the importance of its various domains. The alleles are predicted to encode proteins that either lack all functional domains (lrrk2sbu304 ), the GTPase, and kinase domains (lrrk2sbu71 ) or the kinase domain (lrrk2sbu96 ). All three lrrk2 mutants are viable, morphologically normal, and display wild-type-like locomotion. Because Lrrk2 modulates Wnt signaling in some contexts, we assessed Wnt signaling in all three mutant lines. Analysis of Wnt signaling by studying the expression of target genes using whole mount RNA in situ hybridization and a transgenic Wnt reporter revealed wild-type domains of Wnt activity in each of the mutants. However, we found that Wnt pathway activation is attenuated in lrrk2sbu304/sbu304 , which lacks both scaffolding and catalytic domains, but not in the other alleles during late embryogenesis. This supports a model in which Lrrk2 scaffolding functions are key to a context-dependent role in promoting canonical Wnt signaling.
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Project description:Many systems biology studies lack context-relevant data and as a consequence the predictive capabilities can be limited in developing targeted cancer therapeutics. Production of colon crypt in vitro is ideal for studying colon systems biology. This report presents the first production of, to our knowledge, physiologically-shaped, functional colon crypts in vitro (i.e. single crypts with cells expressing Mucin 2 and Chromogranin A). Time-lapsed monitoring of crypt formation revealed an increased frequency of single-crypt formation in the absence of noggin. Using quantitative 3D immunofluorescence of ?-catenin and E-cadherin, spatial-temporal dynamics of these proteins in normal colon crypt cells stimulated with Wnt3A or inhibited by cycloheximide has been measured. Colon adenoma cultures established from APC(min/+) mouse have developmental differences and ?-catenin spatial localization compared to normal crypts. Quantitative data describing the effects of signalling pathways and proteins dynamics for both normal and adenomatous colon crypts is now within reach to inform a systems approach to colon crypt biology.
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Project description:Producing hair cells of the inner ear is the major goal of ongoing research that combines advances in developmental and stem cell biology. The recent advent of an inner ear organoid protocol-resulting in three-dimensional stem cell-derived tissues resembling vestibular sensory epithelia-has sparked interest in applications such as regeneration, drug discovery, and disease modeling. In this study, we adapted this protocol for a novel mouse embryonic stem cell line with a fluorescent reporter for Pax2 expression. We used Pax2EGFP/+ organoid formation to model otic induction, the pivotal developmental event when preplacodal tissue adopts otic fate. We found upregulation of Pax2 and activation of ERK downstream of fibroblast growth factor signaling in organoid formation as in embryonic inner ear development. Pax2 expression was evident from the EGFP reporter beginning at the vesicle formation stage and persisting through generation of the sensory epithelium. The native ventralizing signal sonic hedgehog was largely absent from the cell aggregates as otic vesicles began to form, confirming the dorsal vestibular organoid fate. Nonetheless, cochlear- or vestibular-like neurons appeared to delaminate from the derived otic vesicles and formed synaptic contacts with hair cells in the organoids. Cell lines with transcriptional reporters such as Pax2EGFP/+ facilitate direct evaluation of morphological changes during organoid production, a major asset when establishing and validating the culture protocol.
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