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

Scientific Reports, Dec 2015

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

Abstract 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. Introduction Auditory hair cells (HCs) are mechanosensory cells in the cochlea that are critical for audition. HCs are highly specialized cells that are present in relatively low abundance with approximately 3300 HCs per mouse cochlea1. Two types of HCs exist within the cochlea, the inner hair cells, which are primarily responsible for the detection and transduction of sound into neuronal signaling, and the outer hair cells (OHCs), which are electromotile and act as a cochlear amplifier2,3,4. Electromotility of OHCs is controlled by the non-traditional motor protein prestin5, which is coded for by the Slc26a5 gene, and is a unique protein expressed in OHCs. Without the amplification provided by prestin/OHCs, mice suffer a substantial loss of hearing3,4 demonstrating the importance of this protein for auditory function. Despite the crucial role for prestin in the cochlea, relatively little is known about the transcriptional regulation of prestin. Thyroid hormone (TH) was the first factor discovered to regulate prestin expression based on observations that hypothyroidism can result in hearing abnormalities6,7,8. It was later demonstrated that TH binds directly to and activates prestin9,10,11. Later studies correlated transcription factors such as Pou4f3 with prestin expression11, but these studies have been unable to further clarify the mechanisms underlying these correlations. One of the major limiting factors for the study of prestin regulation is the lack of an appropriate system to analyze. Most studies to date have been performed in cochlear explants, vastly limiting the material available, the speed at which experiments can be done, and dramatically increasing the cost of the experiment. Indeed, this is true for investigations into the regulation of any genes or proteins expressed specifically in HCs. To bridge this gap, multiple cell lines have been developed to aid in the study of HC development or to be used as screening tools for the prevention of ototoxicity. Many of these cell lines were created from the “immorto-mouse”12,13,14 and exhibit several aspects of HCs15,16. These cell lines have been used to identify dozens of compounds, and pathways that ameliorate ototoxic effects of cisplatin or aminoglycoside antibiotic treatment17,18,19. 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 death20,21. Lineage restricted auditory progenitor cells, often called otic spheres, or otic stem cells, can be isolated from embryonic and postnatal cochleae22,23,24, and differentiated into cells which bear many hallmarks of a HC22,23,24,25, including the ability to express the terminal HC gene, prestin, under differentiating conditions25. While promising, these cells can only be grown for a few generations, yielding only thousands of cells25, limiting their use for large scale studies. In other epithelial tissues, such as breast and prostate, the passage number limitations inherent to lineage restricted progenitor cells can be overcome by “conditional reprogramming” (CR) of the cells26,27,28. This procedure involves growing lineage restricted progenitor cells on a layer of feeder cells, while treating the cells with an inhibitor of rho associated kinases (ROCK). Amazingly, this procedure allowed for the unrestricted growth of the breast and prostate progenitor cells, without compromising their ability to differenti (...truncated)


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Brandon J. Walters, Shiyong Diao, Fei Zheng, Bradley J. Walters, Wanda S. Layman, Jian Zuo. Pseudo-immortalization of postnatal cochlear progenitor cells yields a scalable cell line capable of transcriptionally regulating mature hair cell genes, Scientific Reports, 2015, Issue: 5, DOI: 10.1038/srep17792