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

Jackson JD (2012) MYC Gene Delivery to Adult Mouse Utricles Stimulates Proliferation of Postmitotic Supporting Cells In Vitro. PLoS ONE 7(10): e48704. doi:10.1371/journal.pone.0048704 MYC Gene Delivery to Adult Mouse Utricles Stimulates Proliferation of Postmitotic Supporting Cells In Vitro Joseph C. Burns 0 James J. Yoo 0 Anthony Atala 0 John D. Jackson 0 Domingos Henrique, Instituto de Medicina Molecular, Portugal 0 Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine , Winston-Salem, North Carolina , United States of America 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. - Competing Interests: The Wake Forest Institute for Regenerative Medicine has filed a U. S. Provisional Patent Application on the technology described herein (submitted March 7, 2012). This does not alter the authors adherence to all the PLOS ONE policies on sharing data and materials. The sensory epithelia within the inner ears of adult mammals and humans are highly differentiated, postmitotic, and regeneration deficient. Thus, the loss of sound- and acceleration-detecting hair cells from auditory or vestibular sensory epithelia leads to permanent hearing or balance impairments, respectively. In contrast, the less differentiated sensory epithelia within the inner ears of developing mice and non-mammals of all ages are capable of more significant hair cell regeneration after damage, and nonmammals can recover sensory function [15]. During sensory epithelial development and regeneration, cells that morphologically resemble supporting cells act as otic progenitors that can self-renew and give rise to new hair cells. In vitro and in vivo evidence suggests that the progressive, postnatal depletion of these progenitors, likely via terminal differentiation, limits regeneration in mammals [4,619]. Ectopic, long-term expression of the four transcription factors, Oct3/4, Sox2, Klf4, and c-Myc reprograms isolated somatic cells into induced pluripotent stem cells (iPSCs) [2023]. The initial stages of the reprogramming process result in a partially dedifferentiated, pre-iPSC state, and transient expression of the iPSC factors has recently been utilized to directly reprogram somatic cells into lineage-restricted, multipotent progenitor/stem cells [2429]. Therefore, applying the iPSC reprogramming technology typically used with isolated somatic cells to intact inner ear organs may be a novel approach for dedifferentiating adult mammalian supporting cells while they remain in situ. Here, we show that adenoviral-mediated expression of a degradation-resistant mutant form of the iPSC factor, c-Myc, induces robust S-phase entry of supporting cells in cultured utricles from adult mice. In contrast, supporting cells remained postmitotic after ectopic expression of the three other iPSC factors, Oct3/4, Klf4, and Sox2. We present evidence that at least a portion of the cells were able to progress into M-phase, and a small number of cells replicating their DNA were found 21 days post-virus (DPV); however, many of the cycling cells appeared to enter apoptosis between 7 and 14 DPV. Switching from growth medium to serumfree differentiation medium could prevent the loss of cycling cells, but the protective effect of serum deprivation was temporary and subsided by 14 DPV. Within the protective time window, a modest but significant increase in the area of the sensory epithelium was detected at 10 DPV, and a very small number of cells that had replicated their DNA labeled with antibodies to the hair cell marker myosin VIIA. These results provide evidence that ectopic expression of c-MycT58A in inner ear organs may restore proliferative plasticity to postmitotic supporting cells. Materials and Methods Animals and dissection of utricles All animal work was approved by the Animal Care and Use Committee of Wake Forest University (protocol number: A11 222). Swiss Webster mice, adults of either sex (.6 weeks old) and timed-pregnant females, were obtained from Charles River (Wilmington, MA). Labyrinths were dissected from temporal bones in ice-cold DMEM/F-12 (Invitrogen, Carlsbad, CA), the utricles were isolated, and the roof, otoconia, and nerve were mechanically removed under aseptic conditions. The dissected organs contained the entire sensory epithelium, a small portion of the surrounding non-sensory epithelium, and the underlying stromal tissue. Organ culture and infection with adenoviral vectors Adenoviruses containing vectors encoding Oct3/4, Klf4, Sox2, c-MycT58A, or GFP under the control of a cytomegalovirus (CMV) promoter were obtained from Stemgent (Cambridge, MA). To construct its adenoviruses, Stemgent uses the AdEasy adenoviral vector system, which allows for insertion of a promoter and gene of interest into the E1-, E3-deleted backbone of adenovirus serotype 5. The cDNA plasmids cloned into the viral genome are described in Stadfeldt et al. [30] and can be obtained from Addgene. The Addgene plasmid ID (...truncated)