Browsing by Author "Ueda, Yoshitomo"

Now showing 1 - 4 of 4
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    CHD7 regulates otic lineage specification and hair cell differentiation in human inner ear organoid
    (Springer Nature, 2022-11-17) Nie, Jing; Ueda, Yoshitomo; Solivais, Alexander J.; Hashino, Eri; Otolaryngology -- Head and Neck Surgery, School of Medicine
    Mutations in CHD7 cause CHARGE syndrome, affecting multiple organs including the inner ear in humans. We investigate how CHD7 mutations affect inner ear development using human pluripotent stem cell-derived organoids as a model system. We find that loss of CHD7 or its chromatin remodeling activity leads to complete absence of hair cells and supporting cells, which can be explained by dysregulation of key otic development-associated genes in mutant otic progenitors. Further analysis of the mutant otic progenitors suggests that CHD7 can regulate otic genes through a chromatin remodeling-independent mechanism. Results from transcriptome profiling of hair cells reveal disruption of deafness gene expression as a potential underlying mechanism of CHARGE-associated sensorineural hearing loss. Notably, co-differentiating CHD7 knockout and wild-type cells in chimeric organoids partially rescues mutant phenotypes by restoring otherwise severely dysregulated otic genes. Taken together, our results suggest that CHD7 plays a critical role in regulating human otic lineage specification and hair cell differentiation.
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    Defining developmental trajectories of prosensory cells in human inner ear organoids at single-cell resolution
    (The Company of Biologists, 2023) Ueda, Yoshitomo; Nakamura, Takashi; Nie, Jing; Solivais, Alexander J.; Hoffman, John R.; Daye, Becca J.; Hashino, Eri; Otolaryngology -- Head and Neck Surgery, School of Medicine
    The inner ear sensory epithelia contain mechanosensitive hair cells and supporting cells. Both cell types arise from SOX2-expressing prosensory cells, but the mechanisms underlying the diversification of these cell lineages remain unclear. To determine the transcriptional trajectory of prosensory cells, we established a SOX2-2A-ntdTomato human embryonic stem cell line using CRISPR/Cas9, and performed single-cell RNA-sequencing analyses with SOX2-positive cells isolated from inner ear organoids at various time points between differentiation days 20 and 60. Our pseudotime analysis suggests that vestibular type II hair cells arise primarily from supporting cells, rather than bi-fated prosensory cells in organoids. Moreover, ion channel- and ion-transporter-related gene sets were enriched in supporting cells versus prosensory cells, whereas Wnt signaling-related gene sets were enriched in hair cells versus supporting cells. These findings provide valuable insights into how prosensory cells give rise to hair cells and supporting cells during human inner ear development, and may provide a clue to promote hair cell regeneration from resident supporting cells in individuals with hearing loss or balance disorders.
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    Generating high-fidelity cochlear organoids from human pluripotent stem cells
    (Elsevier, 2023) Moore, Stephen T.; Nakamura, Takashi; Nie, Jing; Solivais, Alexander J.; Aristizábal-Ramírez, Isabel; Ueda, Yoshitomo; Manikandan, Mayakannan; Reddy, V. Shweta; Romano, Daniel R.; Hoffman, John R.; Perrin, Benjamin J.; Nelson, Rick F.; Frolenkov, Gregory I.; Chuva de Sousa Lopes, Susana M.; Hashino, Eri; Otolaryngology -- Head and Neck Surgery, School of Medicine
    Mechanosensitive hair cells in the cochlea are responsible for hearing but are vulnerable to damage by genetic mutations and environmental insults. The paucity of human cochlear tissues makes it difficult to study cochlear hair cells. Organoids offer a compelling platform to study scarce tissues in vitro; however, derivation of cochlear cell types has proven non-trivial. Here, using 3D cultures of human pluripotent stem cells, we sought to replicate key differentiation cues of cochlear specification. We found that timed modulations of Sonic Hedgehog and WNT signaling promote ventral gene expression in otic progenitors. Ventralized otic progenitors subsequently give rise to elaborately patterned epithelia containing hair cells with morphology, marker expression, and functional properties consistent with both outer and inner hair cells in the cochlea. These results suggest that early morphogenic cues are sufficient to drive cochlear induction and establish an unprecedented system to model the human auditory organ.
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    Photo-click hydrogels for 3D in situ differentiation of pancreatic progenitors from induced pluripotent stem cells
    (BMC, 2023-08-30) Arkenberg, Matthew R.; Ueda, Yoshitomo; Hashino, Eri; Lin, Chien‑Chi; Otolaryngology -- Head and Neck Surgery, School of Medicine
    Background: Induced pluripotent stem cells (iPSC) can be differentiated to cells in all three germ layers, as well as cells in the extraembryonic tissues. Efforts in iPSC differentiation into pancreatic progenitors in vitro have largely been focused on optimizing soluble growth cues in conventional two-dimensional (2D) culture, whereas the impact of three-dimensional (3D) matrix properties on the morphogenesis of iPSC remains elusive. Methods: In this work, we employ gelatin-based thiol-norbornene photo-click hydrogels for in situ 3D differentiation of human iPSCs into pancreatic progenitors (PP). Molecular analysis and single-cell RNA-sequencing were utilized to elucidate on the distinct identities of subpopulations within the 2D and 3D differentiated cells. Results: We found that, while established soluble cues led to predominately PP cells in 2D culture, differentiation of iPSCs using the same soluble factors led to prominent branching morphogenesis, ductal network formation, and generation of diverse endoderm populations. Through single-cell RNA-sequencing, we found that 3D differentiation resulted in enrichments of pan-endodermal cells and ductal cells. We further noted the emergence of a group of extraembryonic cells in 3D, which was absent in 2D differentiation. The unexpected emergence of extraembryonic cells in 3D was found to be associated with enrichment of Wnt and BMP signaling pathways, which may have contributed to the emergence of diverse cell populations. The expressions of PP signature genes PDX1 and NKX6.1 were restored through inhibition of Wnt signaling at the beginning of the posterior foregut stage. Conclusions: To our knowledge, this work established the first 3D hydrogel system for in situ differentiation of human iPSCs into PPs.