Generating High-Fidelity Cochlear Organoids from Human Pluripotent Stem Cells

Moore, Stephen T.; Nakamura, Takashi; Nie, Jing; Solivais, Alexander J.; Aristizabal-Ramirez, Isabel; Ueda, Yoshitomo; Manikandan, Mayakannan; Vummidi, Shweta Reddy; Romano, Daniel R.; Perrin, Benjamin J.; Nelson, Rick F.; Frolenkov, Gregory I.; Lopes, Susana M. Chuva de Sousa; Hashino, Eri

doi:10.2139/ssrn.4170188

Cited by 5 publications

(11 citation statements)

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“…To investigate the mechanisms underlying the failure in hair cell and supporting cell generation in d70 CHD7 KO/KO organoids, we performed transcriptome profiling in the seemingly normal d20 CHD7 KO/KO otic progenitor cells using scRNA-seq. We previously generated a PAX2−2a-nGFP (PAX2 nG ) reporter hESC line to label the otic progenitor cells with nuclear GFP 26 , and all of our four CHD7 mutant lines were built on this PAX2 nG genetic background (Supplementary Fig. 6).…”

Section: Loss Of Chd7 Causes Dysregulation Of Otic Development Genesmentioning

confidence: 99%

“…We first labeled the hair cells with a highly specific POU4F3−2a-ntdTomato (POU4F3 nT ) fluorescence reporter in the WT and the CHD7 KO/+ genetic backgrounds (Supplementary Fig. 10) 26 . We FACSseparated the POU4F3 nT+ and POU4F3 nTcells from micro-dissected WT and CHD7 KO/+ d70 organoids (Supplementary Fig.…”

Section: Deafness Genes Are Dysregulated In Chd7 Ko/+ Hair Cellsmentioning

confidence: 99%

“…Ribonuclease protein (RNP) complex 61 targeting the CHD7 stop codon locus was assembled by incubating high fidelity Cas9 protein (HiFi Cas9 nuclease 3NLS, IDT) with crRNA:tracrRNA duplex (CHD7 crRNA: 5'-ACTTGAACTGGAACTGGTAC -3', IDT). The RNP complex, the donor plasmid, as well as an electroporation enhancer (IDT) were transfected into the WA25-based PAX2−2A-nGFP reporter hES cell line 26 with 4D Nucleofector (Lonza) using the P3 Primary Cell 4D-Nucleofector X kit (Lonza) and Program CB-150. After nucleofection, cells were plated in RevitaCell-supplemented E8fn medium for one day for improved cell survival rate, followed by treatment with 1 µM Scr7 (Xcessbio) on the second day for enhanced HDR efficiency 62 .…”

Section: Crispr Genomic Editingmentioning

confidence: 99%

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CHD7 regulates otic lineage specification and hair cell differentiation in human inner ear organoids

et al. 2022

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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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Section: Loss Of Chd7 Causes Dysregulation Of Otic Development Genesmentioning

confidence: 99%

Section: Deafness Genes Are Dysregulated In Chd7 Ko/+ Hair Cellsmentioning

confidence: 99%

Section: Crispr Genomic Editingmentioning

confidence: 99%

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CHD7 regulates otic lineage specification and hair cell differentiation in human inner ear organoids

et al. 2022

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“…IEO are a relatively new addition to the catalogue of in vitro derived organ models. Our work, together with other recent unpublished studies, aim at providing a robust foundation for broader application of IEOs (Moore et al, 2022;Steinhart et al;van der Valk et al, 2022).…”

Section: Benchmarking Human Ipsc Derived Inner Ear Organoidsmentioning

confidence: 83%

“…Additional activation of SHH signaling and inhibition of WNT during otic vesicle maturation seem to promote cochlear fate. This becomes however apparent only in long term cultures >140 days (Moore et al, 2022).…”

Section: Hair Cell Maturationmentioning

confidence: 96%

Human pluripotent stem cells-derived inner ear organoids recapitulate otic developmentin vitro

Doda

Jimenez

Rehrauer

et al. 2023

Preprint

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Our molecular understanding of the early stages of human inner ear development has been limited by the difficulty in accessing fetal samples at early gestational stages. As an alternative, previous studies have shown that inner ear morphogenesis can be partially recapitulated using induced pluripotent stem cells (iPSCs) directed to differentiate into Inner Ear Organoids (IEOs). Once validated and benchmarked, these systems could represent unique tools to complement and refine our understanding of human otic differentiation and model developmental defects. Here, we provide the first direct comparisons of the early human embryonic otocyst and human iPSC-derived IEOs. We use multiplexed immunostaining, and single-cell RNA sequencing to characterize IEOs at three key developmental steps, providing a new and unique signature of in vitro derived otic -placode, -epithelium, -neuroblasts, and -sensory epithelia. In parallel, we evaluate the expression and localization of critical markers at these equivalent stages in human embryos. We show that the placode derived in vitro (days 8-12) has similar marker expression to the developing otic placode of Carnegie Stage (CS) 11 embryos and subsequently (days 20-40) this gives rise to otic epithelia and neuroblasts comparable to the CS13 embryonic stage. Differentiation of sensory epithelia, including supporting cells and hair cells starts in vitro at days 50-60 of culture. The maturity of these cells is equivalent to vestibular sensory epithelia at week 10 or cochlear tissue at week 12 of development, before functional onset. Together, our data indicate that the current state-of-the-art protocol enables the specification of bona fide otic tissue, supporting the further application of IEOs to inform inner ear biology and disease.

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Modeling, applications and challenges of inner ear organoid

Qi,

Zhang,

Wang

et al. 2024

Smart Medicine

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More than 6% of the world's population is suffering from hearing loss and balance disorders. The inner ear is the organ that senses sound and balance. Although inner ear disorders are common, there are limited ways to intervene and restore its sensory and balance functions. The development and establishment of biologically therapeutic interventions for auditory disorders require clarification of the basics of signaling pathways that control inner ear development and the establishment of endogenous or exogenous cell‐based therapeutic methods. In vitro models of the inner ear, such as organoid systems, can help identify new protective or regenerative drugs, develop new gene therapies, and be considered as potential tools for future clinical applications. Advances in stem cell technology and organoid culture offer unique opportunities for modeling inner ear diseases and developing personalized therapies for hearing loss. Here, we review and discuss the mechanisms for the establishment and the potential applications of inner ear organoids.

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Generating High-Fidelity Cochlear Organoids from Human Pluripotent Stem Cells

Cited by 5 publications

References 0 publications

CHD7 regulates otic lineage specification and hair cell differentiation in human inner ear organoids

CHD7 regulates otic lineage specification and hair cell differentiation in human inner ear organoids

Human pluripotent stem cells-derived inner ear organoids recapitulate otic developmentin vitro

Modeling, applications and challenges of inner ear organoid

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