Loss of aminoglycoside sensitivity in HEI-OC1 cells?

Cederroth, Christopher R.

doi:10.1016/j.heares.2012.08.011

Cited by 8 publications

(5 citation statements)

References 5 publications

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“…Nevertheless, we found that there were pockets of cells within the cultures that appeared to differentiate amongst unchanged cells supporting the notion that the cultures had mixed cell phenotypes. Others have noted this heterogeneity amongst different batches of HEI-OC1 cells (Cederroth, 2012). In our studies, we did find that the ablation of Cx43 drove a higher expression of myosin V11a and calsequestrin suggesting that Cx43 mediated GJIC may be acting as a negative regulator of some genes important in cell differentiation.…”

Section: Discussionsupporting

confidence: 56%

GJB2 Mutations Linked to Hearing Loss Exhibit Differential Trafficking and Functional Defects as Revealed in Cochlear-Relevant Cells

Beach

Abitbol

Allman

et al. 2020

Front. Cell Dev. Biol.

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GJB2 gene (that encodes Cx26) mutations are causal of hearing loss highlighting the importance of Cx26-based channel signaling amongst the supporting cells in the organ of Corti. While the majority of these GJB2 mutations are inherited in an autosomal recessive manner, others are inherited in an autosomal dominant manner and lead to syndromic hearing loss as well as skin diseases. To assess if common or divergent mechanisms are at the root of GJB2-linked hearing loss, we expressed several mutants in cochlear-relevant HEI-OC1 cells derived from the developing organ of Corti. Since supporting cells of the mature mammalian organ of Corti have negligible Cx43, but HEI-OC1 cells are rich in Cx43, we first used CRISPR-Cas9 to ablate endogenous Cx43, thus establishing a connexin-deficient platform for controlled reintroduction of hearing-relevant connexins and Cx26 mutants. We found three distinct outcomes and cellular phenotypes when hearing loss-linked Cx26 mutants were expressed in cochlear-relevant cells. The dominant syndromic Cx26 mutant N54K had trafficking defects and did not fully prevent wild-type Cx26 gap junction plaque formation but surprisingly formed gap junctions when co-expressed with Cx30. In contrast, the dominant syndromic S183F mutant formed gap junctions incapable of transferring dye and, as expected, co-localized in the same gap junctions as wild-type Cx26 and Cx30, but also gained the capacity to intermix with Cx43 within gap junctions. Both recessive non-syndromic Cx26 mutants (R32H and R184P) were retained in intracellular vesicles including early endosomes and did not co-localize with Cx30. As might be predicted, none of the Cx26 mutants prevented Cx43 gap junction plaque formation in Cx43-rich HEI-OC1 cells while Cx43-ablation had little effect on the expression of reference genes linked to auditory cell differentiation. We conclude from our studies in cochlear-relevant cells that the selected Cx26 mutants likely evoke hearing loss via three unique connexin defects that are independent of Cx43 status.

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Section: Discussionsupporting

confidence: 56%

GJB2 Mutations Linked to Hearing Loss Exhibit Differential Trafficking and Functional Defects as Revealed in Cochlear-Relevant Cells

Beach

Abitbol

Allman

et al. 2020

Front. Cell Dev. Biol.

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“…The simplest explanation for this observation is that genetic drift has caused HEI-OC1 cells to lose prestin expression over the numerous generations that have passed since their development. In fact, drift has previously been reported for some HEI-OC1 populations in regard to aminoglycoside sensitivity 20 21 . While CR-OSCs are not exempt from genetic drift, e.g.…”

Section: Discussionmentioning

confidence: 88%

“…Although these cell lines have proven useful for ototoxic screening studies, they have not been ideal for studying terminal HC differentiation. Additionally, studies have shown that some of these cell lines have begun to show significant phenotypic drift and are no longer sensitive to aminoglycoside induced cell death 20 21 .…”

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confidence: 99%

Pseudo-immortalization of postnatal cochlear progenitor cells yields a scalable cell line capable of transcriptionally regulating mature hair cell genes

Walters

Diao

Zheng

et al. 2015

Sci Rep

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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.

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“…Drug responses vary depending on cell culture conditions and genetic drift has been problematic in the culture of HEI-OC1 (Kalinec et al, 2016). Over time, HEI-OC1 cells have developed drug resistance and have lost some key hair cell characteristics, such as prestin expression which is normally present in cochlear outer hair cells (Cederroth, 2012; Walters et al, 2015; Kalinec et al, 2016). As such, fundamental research in the field of auditory biology relies extensively on the use of mammalian inner ear explant preparations.…”

Section: Introductionmentioning

confidence: 99%

Organotypic Culture of Neonatal Murine Inner Ear Explants

Ogier

Burt

Drury

et al. 2019

Front. Cell. Neurosci.

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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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Loss of aminoglycoside sensitivity in HEI-OC1 cells?

Cited by 8 publications

References 5 publications

GJB2 Mutations Linked to Hearing Loss Exhibit Differential Trafficking and Functional Defects as Revealed in Cochlear-Relevant Cells

GJB2 Mutations Linked to Hearing Loss Exhibit Differential Trafficking and Functional Defects as Revealed in Cochlear-Relevant Cells

Pseudo-immortalization of postnatal cochlear progenitor cells yields a scalable cell line capable of transcriptionally regulating mature hair cell genes

Organotypic Culture of Neonatal Murine Inner Ear Explants

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