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

Abstract

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.

DNA damage signaling regulates age-dependent proliferative capacity of quiescent inner ear supporting cells

“…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?…”

Cotransfection of Pax2 and Math1 promote in situ cochlear hair cell regeneration after neomycin insult

“…This indicated that overexpression of Pax2 could stimulate the proliferation of supporting cells but was not enough to regenerate cochlear hair cells. Similarly, another study37 reported that co-infection of utricles with adenoviral vectors encoding the degradation-resistant T58A mutant of c-Myc (c-MycT58A) triggered significant levels of supporting cell S-phase entry in adult vestibular sensory epithelium, but only a small number of the proliferating cells differentiated into hair cells. These results suggest that inducing hair cell differentiation in addition to promoting residual supporting cell proliferation is necessary for hair cell regeneration.…”

Development and regeneration of vestibular hair cells in mammals

“…CyclinD1 expression declines as hair cells and supporting cells exit the cell cycle, and forced expression of cyclin D1 in neonatal and adult mouse utricles triggers cell cycle reentry of hair cells and/or supporting cells [177, 178]. Conditional deletion of N-Myc in the embryonic ear reduces growth of the sensory epithelium by inhibiting proliferation [179, 180], and overexpression of N-Myc (J. Burns, unpublished results) or c-Myc, a closely related family member, results in robust proliferation of supporting cells in the adult mouse utricle in vitro [181]. Neither cyclin D1 nor c-Myc overexpression is able to induce re-growth of the sensory epithelium, as proliferating cells either become arrested in G2 or undergo apoptosis after passing through M phase, apparently due to accumulation of DNA damage [182].…”