MYC Gene Delivery to Adult Mouse Utricles Stimulates Proliferation of Postmitotic Supporting Cells In Vitro

Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, North Carolina, United States of America.
PLoS ONE (Impact Factor: 3.23). 10/2012; 7(10):e48704. DOI: 10.1371/journal.pone.0048704
Source: PubMed


The inner ears of adult humans and other mammals possess a limited capacity for regenerating sensory hair cells, which can lead to permanent auditory and vestibular deficits. During development and regeneration, undifferentiated supporting cells within inner ear sensory epithelia can self-renew and give rise to new hair cells; however, these otic progenitors become depleted postnatally. Therefore, reprogramming differentiated supporting cells into otic progenitors is a potential strategy for restoring regenerative potential to the ear. Transient expression of the induced pluripotency transcription factors, Oct3/4, Klf4, Sox2, and c-Myc reprograms fibroblasts into neural progenitors under neural-promoting culture conditions, so as a first step, we explored whether ectopic expression of these factors can reverse supporting cell quiescence in whole organ cultures of adult mouse utricles. Co-infection of utricles with adenoviral vectors separately encoding Oct3/4, Klf4, Sox2, and the degradation-resistant T58A mutant of c-Myc (c-MycT58A) triggered significant levels of supporting cell S-phase entry as assessed by continuous BrdU labeling. Of the four factors, c-MycT58A alone was both necessary and sufficient for the proliferative response. The number of BrdU-labeled cells plateaued between 5-7 days after infection, and then decreased ∼60% by 3 weeks, as many cycling cells appeared to enter apoptosis. Switching to differentiation-promoting culture medium at 5 days after ectopic expression of c-MycT58A temporarily attenuated the loss of BrdU-labeled cells and accompanied a very modest but significant expansion of the sensory epithelium. A small number of the proliferating cells in these cultures labeled for the hair cell marker, myosin VIIA, suggesting they had begun differentiating towards a hair cell fate. The results indicate that ectopic expression of c-MycT58A in combination with methods for promoting cell survival and differentiation may restore regenerative potential to supporting cells within the adult mammalian inner ear.

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    • "Therefore, cell cycle re-entry by supporting cells is not sufficient for the regeneration of hair cells, suggesting that some other characteristics of supporting cells also need to be altered. Recently, several challenges for the induction of the dedifferentiation of supporting cells have been described (Burns et al., 2012; Lou et al., 2013). In those studies, transcription factors for the generation of inducible pluripotent stem (iPS) cells were utilized for the induction of the dedifferentiation of inner ear cells. "
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    ABSTRACT: Sensorineural hearing loss (SNHL) is a common disability in the world; however, at present, options for the pharmacological treatment of SNHL are very limited. Previous studies involving human temporal bone analyses have revealed that the degeneration of the cochlea is a common mechanism of SNHL. A major problem for the development of novel pharmacotherapy for SNHL has been the limited regeneration capacity in mammalian cochlear cells. However, recent progress in basic studies has led to several effective strategies for the induction of regeneration in the mammalian cochlea, in accordance with the stage of degeneration. In addition, recent advances in the identification of human deafness genes and their characterization in mouse models have elucidated cellular and/or molecular mechanisms of SNHL, which will contribute to clarify molecular targets of pharmacotherapy for treatment of SNHL.
    Frontiers in Pharmacology 09/2014; 5:206. DOI:10.3389/fphar.2014.00206 · 3.80 Impact Factor
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    • "Further, the findings of adult SCs with intense, pan-nuclear γH2AX that were positive for cleaved caspase-3 and showed only negligible Rad51 expression suggested that incomplete DNA repair could lead to apoptosis. These results are consistent with prior data showing signs of apoptosis of cell cycle reactivated SCs of the adult utricle upon c-Myc overexpression [38]. What might be the mechanisms underlying slower kinetics of DNA damage signaling in adult SCs? Cellular differentiation is accompanied by an increase in heterochromatinization that is known to affect DNA repair [39]. "
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    ABSTRACT: Supporting cells (SCs) of the cochlear (auditory) and vestibular (balance) organs hold promise as a platform for therapeutic regeneration of the sensory hair cells. Prior data have shown proliferative restrictions of adult SCs forced to re-enter the cell cycle. By comparing juvenile and adult SCs in explant cultures, we have here studied how proliferative restrictions are linked with DNA damage signaling. Cyclin D1 overexpression, used to stimulate cell cycle re-entry, triggered higher proliferative activity of juvenile SCs. Phosphorylated form of histone H2AX (γH2AX) and p53 binding protein 1 (53BP1) were induced in a foci-like pattern in SCs of both ages as an indication of DNA double-strand break formation and activated DNA damage response. Compared to juvenile SCs, γH2AX and the repair protein Rad51 were resolved with slower kinetics in adult SCs, accompanied by increased apoptosis. Consistent with thein vitro data, in a Rb mutant mouse model in vivo, cell cycle re-entry of SCs was associated with γH2AX foci induction. In contrast to cell cycle reactivation, pharmacological stimulation of SC-to-hair-cell transdifferentiation in vitro did not trigger γH2AX. Thus, DNA damage and its prolonged resolution are critical barriers in the efforts to stimulate proliferation of the adult inner ear SCs.
    Aging 06/2014; 6(6):496-510. · 6.43 Impact Factor
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    • "Burns et al. examined the effects of iPSC transcription factors on cell cycle re-entry of supporting cells in mouse vestibular epithelia, and demonstrated that c-Myc alone is necessary and sufficient to promote the proliferation of supporting cells (Burns et al., 2012). However, detailed assessments of reprogramming in supporting cells have not been performed. "
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    ABSTRACT: Abstract As an initial step for using technology derived from induced pluripotent stem cells (iPSCs) in the field of inner ear therapeutics, we examined the potential of four transcription factors, Oct3/4, Sox2, Klf4, and c-Myc, which are employed in the generation of iPSCs, for dedifferentiating cochlear epithelial cells. Otospheres, which are sphere-forming cells derived from dissociated cochlear epithelial cells of neonatal mice, were used as a cell source. The four transcription factors were introduced into otospheres using retroviral vectors. Virally transduced otospheres formed embryonic stem cell-like colonies that expressed markers for pluripotent stem cells and were capable of differentiating into the three germ layers in vivo and in vitro. These findings illustrate that viral transduction of four transcription factors can lead to reprogramming of cochlear epithelial cells, which may contribute to future studies of dedifferentiation of cochlear epithelial cells in tissue and identification of key molecules for otic induction.
    11/2013; 15(6):514-519. DOI:10.1089/cell.2013.0020
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