Enhanced Inner-Ear Organoid Formation from Mouse Embryonic Stem Cells by Photobiomodulation.
ABSTRACT: 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.
Project description:Stem cell-derived inner ear sensory epithelia are a promising source of tissues for treating patients with hearing loss and dizziness. We recently demonstrated how to generate inner ear sensory epithelia, designated as inner ear organoids, from mouse embryonic stem cells (ESCs) in a self-organizing 3D culture. Here we improve the efficiency of this culture system by elucidating how Wnt signaling activity can drive the induction of otic tissue. We found that a carefully timed treatment with the potent Wnt agonist CHIR99021 promotes induction of otic vesicles-a process that was previously self-organized by unknown mechanisms. The resulting otic-like vesicles have a larger lumen size and contain a greater number of Pax8/Pax2-positive otic progenitor cells than organoids derived without the Wnt agonist. Additionally, these otic-like vesicles give rise to large inner ear organoids with hair cells whose morphological, biochemical and functional properties are indistinguishable from those of vestibular hair cells in the postnatal mouse inner ear. We conclude that Wnt signaling plays a similar role during inner ear organoid formation as it does during inner ear development in the embryo.
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.
Project description:Age-related neurosensory deficit of the inner ear is mostly due to a loss of hair cells (HCs). Development of stem cell-based therapy requires a better understanding of factors and signals that drive stem cells into otic sensory progenitor cells (OSPCs) to replace lost HCs. Human induced pluripotent stem cells (hiPSCs) theoretically represent an unlimited supply for the generation of human OSPCs in vitro. In this study, we developed a monolayer-based differentiation system to generate an enriched population of OSPCs via a stepwise differentiation of hiPSCs. Gene and protein expression analyses revealed the efficient induction of a comprehensive panel of otic/placodal and late otic markers over the course of the differentiation. Furthermore, whole transcriptome analysis confirmed a developmental path of OSPC differentiation from hiPSCs. We found that modulation of WNT and transforming growth factor-? (TGF-?) signaling combined with fibroblast growth factor 3 (FGF3) and FGF10 treatment over a 6-day period drives the expression of early otic/placodal markers followed by late otic sensory markers within 13 days, indicative of a differentiation into embryonic-like HCs. In summary, we report a rapid and efficient strategy to generate an enriched population of OSPCs from hiPSCs, thereby establishing the value of this approach for disease modeling and cell-based therapies of the inner ear.
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.
Project description:The development of therapeutic interventions for hearing loss requires fundamental knowledge about the signaling pathways controlling tissue development as well as the establishment of human cell-based assays to validate therapeutic strategies ex vivo Recent advances in the field of stem cell biology and organoid culture systems allow the expansion and differentiation of tissue-specific progenitors and pluripotent stem cells in vitro into functional hair cells and otic-like neurons. We discuss how inner ear organoids have been developed and how they offer for the first time the opportunity to validate drug-based therapies, gene-targeting approaches and cell replacement strategies.
Project description:In mammals, the permanence of many forms of hearing loss is the result of the inner ear's inability to replace lost sensory hair cells. Here, we apply a differentiation strategy to guide human embryonic stem cells (hESCs) into cells of the otic lineage using chemically defined attached-substrate conditions. The generation of human otic progenitor cells was dependent on fibroblast growth factor (FGF) signaling, and protracted culture led to the upregulation of markers indicative of differentiated inner ear sensory epithelia. Using a transgenic ESC reporter line based on a murine Atoh1 enhancer, we show that differentiated hair cell-like cells express multiple hair cell markers simultaneously. Hair cell-like cells displayed protrusions reminiscent of stereociliary bundles, but failed to fully mature into cells with typical hair cell cytoarchitecture. We conclude that optimized defined conditions can be used in vitro to attain otic progenitor specification and sensory cell differentiation.
Project description:Inner ear sensory epithelia contain mechanosensitive hair cells that transmit information to the brain through innervation with bipolar neurons. Mammalian hair cells do not regenerate and are limited in number. Here we investigate the potential to generate mechanosensitive hair cells from mouse embryonic stem cells in a three-dimensional (3D) culture system. The system faithfully recapitulates mouse inner ear induction followed by self-guided development into organoids that morphologically resemble inner ear vestibular organs. We find that organoid hair cells acquire mechanosensitivity equivalent to functionally mature hair cells in postnatal mice. The organoid hair cells also progress through a similar dynamic developmental pattern of ion channel expression, reminiscent of two subtypes of native vestibular hair cells. We conclude that our 3D culture system can generate large numbers of fully functional sensory cells which could be used to investigate mechanisms of inner ear development and disease as well as regenerative mechanisms for inner ear repair.
Project description:Use of human induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC) for cell replacement therapies holds great promise. Several limitations including low yields and heterogeneous populations of differentiated cells hinder the progress of stem cell therapies. A fate restricted immortalized multipotent otic progenitor (iMOP) cell line was generated to facilitate efficient differentiation of large numbers of functional hair cells and spiral ganglion neurons (SGN) for inner ear cell replacement therapies. Starting from dissociated cultures of single iMOP cells, protocols that promote cell cycle exit and differentiation by basic fibroblast growth factor (bFGF) withdrawal were described. A significant decrease in proliferating cells after bFGF withdrawal was confirmed using an EdU cell proliferation assay. Concomitant with a decrease in proliferation, successful differentiation resulted in expression of molecular markers and morphological changes. Immunostaining of Cdkn1b (p27(KIP)) and Cdh1 (E-cadherin) in iMOP-derived otospheres was used as an indicator for differentiation into inner ear sensory epithelia while immunostaining of Cdkn1b and Tubb3 (neuronal β-tubulin) was used to identify iMOP-derived neurons. Use of iMOP cells provides an important tool for understanding cell fate decisions made by inner ear neurosensory progenitors and will help develop protocols for generating large numbers of iPSC or ESC-derived hair cells and SGNs. These methods will accelerate efforts for generating otic cells for replacement therapies.
Project description:The mammalian cochlea is a highly specialized organ within the inner ear. Sensory hair cells (HC) in the cochlea detect and transduce sound waves into electrical impulses that are sent to the brain. Studies of the molecular pathways regulating HC formation are hindered by the very sparse nature of HCs, where only ~3300 are found within an entire mouse cochlea. Current cell lines mimic certain aspects of HCs but lack terminal HC marker expression. Here we successfully "pseudo-immortalized" cochlear progenitor cells using the "conditional reprogramming" technique. These cells, termed "Conditionally Reprogrammed Otic Stem Cells" (CR-OSC), are able to bypass the senescence inherent to cochlear progenitor cells without genetic alterations, allowing for the generation of over 15 million cells from a single cochlea. These cells can be differentiated and up-regulate both early and terminal differentiation genes associated with HCs, including the terminal HC differentiation marker prestin. CR-OSCs also respond to known HC cues, including upregulation of HC genes in response to Atoh1 overexpression, and upregulation of prestin expression after thyroid hormone application. Overall, we describe the creation of a HC line capable of regulated expression of HC genes that can easily be recreated in any laboratory from any mouse of interest.
Project description:Sensorineural hearing loss is caused by the loss of sensory hair cells and neurons of the inner ear. Once lost, these cell types are not replaced. Two genes expressed in the developing inner ear are c-Myc and Sox2. We created immortalized multipotent otic progenitor (iMOP) cells, a fate-restricted cell type, by transient expression of C-MYC in SOX2-expressing otic progenitor cells. This activated the endogenous C-MYC and amplified existing SOX2-dependent transcripts to promote self-renewal. RNA-seq and ChIP-seq analyses revealed that C-MYC and SOX2 occupy over 85% of the same promoters. C-MYC and SOX2 target genes include cyclin-dependent kinases that regulate cell-cycle progression. iMOP cells continually divide but retain the ability to differentiate into functional hair cells and neurons. We propose that SOX2 and C-MYC regulate cell-cycle progression of these cells and that downregulation of C-MYC expression after growth factor withdrawal serves as a molecular switch for differentiation.