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.
References
[1]
Corwin JT, Cotanche DA (1988) Regeneration of sensory hair cells after acoustic trauma. Science 240: 1772–1774.
[2]
Ryals BM, Rubel EW (1988) Hair cell regeneration after acoustic trauma in adult Coturnix quail. Science 240: 1774–1776.
[3]
Warchol ME (2011) Sensory regeneration in the vertebrate inner ear: differences at the levels of cells and species. Hearing research 273: 72–79.
[4]
Burns JC, Cox BC, Thiede BR, Zuo J, Corwin JT (2012) In vivo proliferative regeneration of balance hair cells in newborn mice. J Neurosci 32: 6570–6577.
[5]
Kelley MW, Talreja DR, Corwin JT (1995) Replacement of hair cells after laser microbeam irradiation in cultured organs of corti from embryonic and neonatal mice. J Neurosci 15: 3013–3026.
[6]
Li H, Liu H, Heller S (2003) Pluripotent stem cells from the adult mouse inner ear. Nat Med 9: 1293–1299.
[7]
Oshima K, Grimm CM, Corrales CE, Senn P, Martinez Monedero R, et al. (2007) Differential distribution of stem cells in the auditory and vestibular organs of the inner ear. J Assoc Res Otolaryngol 8: 18–31.
[8]
White PM, Doetzlhofer A, Lee YS, Groves AK, Segil N (2006) Mammalian cochlear supporting cells can divide and trans-differentiate into hair cells. Nature 441: 984–987.
[9]
Zhai S, Shi L, Wang BE, Zheng G, Song W, et al. (2005) Isolation and culture of hair cell progenitors from postnatal rat cochleae. Journal of neurobiology 65: 282–293.
[10]
Collado MS, Burns JC, Meyers JR, Corwin JT (2011) Variations in shape-sensitive restriction points mirror differences in the regeneration capacities of avian and mammalian ears. PloS One 6: e23861.
[11]
Davies D, Magnus C, Corwin JT (2007) Developmental changes in cell-extracellular matrix interactions limit proliferation in the mammalian inner ear. Eur J Neurosci 25: 985–998.
[12]
Gu R, Montcouquiol M, Marchionni M, Corwin JT (2007) Proliferative responses to growth factors decline rapidly during postnatal maturation of mammalian hair cell epithelia. Eur J Neurosci 25: 1363–1372.
[13]
Hume CR, Kirkegaard M, Oesterle EC (2003) ErbB expression: the mouse inner ear and maturation of the mitogenic response to heregulin. J Assoc Res Otolaryngol 4: 422–443.
[14]
Lu Z, Corwin JT (2008) The influence of glycogen synthase kinase 3 in limiting cell addition in the mammalian ear. Dev Neurobiol 68: 1059–1075.
[15]
Meyers JR, Corwin JT (2007) Shape change controls supporting cell proliferation in lesioned mammalian balance epithelium. J Neurosci 27: 4313–4325.
[16]
Zheng JL, Helbig C, Gao WQ (1997) Induction of cell proliferation by fibroblast and insulin-like growth factors in pure rat inner ear epithelial cell cultures. J Neurosci 17: 216–226.
[17]
Burns JC, Christophel JJ, Collado MS, Magnus C, Carfrae M, et al. (2008) Reinforcement of cell junctions correlates with the absence of hair cell regeneration in mammals and its occurrence in birds. The Journal of comparative neurology 511: 396–414.
[18]
Burns JC, On D, Baker W, Collado MS, Corwin JT (2012) Over Half the Hair Cells in the Mouse Utricle First Appear After Birth, with Significant Numbers Originating from Early Postnatal Mitotic Production in Peripheral and Striolar Growth Zones. Journal of the Association for Research in Otolaryngology: JARO.
[19]
Ruben RJ (1967) Development of the inner ear of the mouse: a radioautographic study of terminal mitoses. Acta Otolaryngol: Suppl 220: 221–244.
[20]
Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, et al. (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131: 861–872.
[21]
Ho R, Chronis C, Plath K (2011) Mechanistic insights into reprogramming to induced pluripotency. Journal of cellular physiology 226: 868–878.
[22]
Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126: 663–676.
[23]
Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, et al. (2007) Induced pluripotent stem cell lines derived from human somatic cells. Science 318: 1917–1920.
[24]
Kim J, Efe JA, Zhu S, Talantova M, Yuan X, et al. (2011) Direct reprogramming of mouse fibroblasts to neural progenitors. Proceedings of the National Academy of Sciences of the United States of America 108: 7838–7843.
[25]
Efe JA, Hilcove S, Kim J, Zhou H, Ouyang K, et al. (2011) Conversion of mouse fibroblasts into cardiomyocytes using a direct reprogramming strategy. Nature cell biology 13: 215–222.
[26]
Meissner A, Wernig M, Jaenisch R (2007) Direct reprogramming of genetically unmodified fibroblasts into pluripotent stem cells. Nature biotechnology 25: 1177–1181.
[27]
Mikkelsen TS, Hanna J, Zhang X, Ku M, Wernig M, et al. (2008) Dissecting direct reprogramming through integrative genomic analysis. Nature 454: 49–55.
[28]
Silva J, Barrandon O, Nichols J, Kawaguchi J, Theunissen TW, et al. (2008) Promotion of reprogramming to ground state pluripotency by signal inhibition. PLoS biology 6: e253.
[29]
Sridharan R, Tchieu J, Mason MJ, Yachechko R, Kuoy E, et al. (2009) Role of the murine reprogramming factors in the induction of pluripotency. Cell 136: 364–377.
[30]
Stadtfeld M, Nagaya M, Utikal J, Weir G, Hochedlinger K (2008) Induced pluripotent stem cells generated without viral integration. Science 322: 945–949.
[31]
Montcouquiol M, Kelley MW (2003) Planar and vertical signals control cellular differentiation and patterning in the mammalian cochlea. The Journal of neuroscience: the official journal of the Society for Neuroscience 23: 9469–9478.
[32]
Kirkegaard M, Nyengaard JR (2005) Stereological study of postnatal development in the mouse utricular macula. J Comp Neurol 492: 132–144.
[33]
Jones JM, Montcouquiol M, Dabdoub A, Woods C, Kelley MW (2006) Inhibitors of differentiation and DNA binding (Ids) regulate Math1 and hair cell formation during the development of the organ of Corti. J Neurosci 26: 550–558.
[34]
Woods C, Montcouquiol M, Kelley MW (2004) Math1 regulates development of the sensory epithelium in the mammalian cochlea. Nat Neurosci 7: 1310–1318.
[35]
Driver EC, Kelley MW (2010) Transfection of mouse cochlear explants by electroporation. Current protocols in neuroscience/editorial board, Jacqueline N Crawley [et al] Chapter 4: Unit 4 34 31–10.
[36]
Holt JR, Johns DC, Wang S, Chen ZY, Dunn RJ, et al. (1999) Functional expression of exogenous proteins in mammalian sensory hair cells infected with adenoviral vectors. J Neurophysiol 81: 1881–1888.
[37]
Luebke AE, Foster PK, Muller CD, Peel AL (2001) Cochlear function and transgene expression in the guinea pig cochlea, using adenovirus- and adeno-associated virus-directed gene transfer. Human gene therapy 12: 773–781.
[38]
Lin V, Golub JS, Nguyen TB, Hume CR, Oesterle EC, et al. (2011) Inhibition of notch activity promotes nonmitotic regeneration of hair cells in the adult mouse utricles. The Journal of neuroscience: the official journal of the Society for Neuroscience 31: 15329–15339.
[39]
Loponen H, Ylikoski J, Albrecht JH, Pirvola U (2011) Restrictions in cell cycle progression of adult vestibular supporting cells in response to ectopic cyclin d1 expression. PloS One 6: e27360.
[40]
Brandon CS, Voelkel-Johnson C, May LA, Cunningham LL (2012) Dissection of adult mouse utricle and adenovirus-mediated supporting-cell infection. Journal of visualized experiments: JoVE.
[41]
Laine H, Sulg M, Kirjavainen A, Pirvola U (2010) Cell cycle regulation in the inner ear sensory epithelia: role of cyclin D1 and cyclin-dependent kinase inhibitors. Dev Biol 337: 134–146.
[42]
Chang DW, Claassen GF, Hann SR, Cole MD (2000) The c-Myc transactivation domain is a direct modulator of apoptotic versus proliferative signals. Molecular and cellular biology 20: 4309–4319.
[43]
Gregory MA, Hann SR (2000) c-Myc proteolysis by the ubiquitin-proteasome pathway: stabilization of c-Myc in Burkitt's lymphoma cells. Molecular and cellular biology 20: 2423–2435.
[44]
Salghetti SE, Kim SY, Tansey WP (1999) Destruction of Myc by ubiquitin-mediated proteolysis: cancer-associated and transforming mutations stabilize Myc. The EMBO journal 18: 717–726.
[45]
Sears R, Nuckolls F, Haura E, Taya Y, Tamai K, et al. (2000) Multiple Ras-dependent phosphorylation pathways regulate Myc protein stability. Genes & development 14: 2501–2514.
[46]
Kawamoto K, Oh SH, Kanzaki S, Brown N, Raphael Y (2001) The functional and structural outcome of inner ear gene transfer via the vestibular and cochlear fluids in mice. Molecular therapy: the journal of the American Society of Gene Therapy 4: 575–585.
[47]
Oesterle EC, Campbell S, Taylor RR, Forge A, Hume CR (2008) Sox2 and JAGGED1 expression in normal and drug-damaged adult mouse inner ear. J Assoc Res Otolaryngol 9: 65–89.
[48]
Kee N, Sivalingam S, Boonstra R, Wojtowicz JM (2002) The utility of Ki-67 and BrdU as proliferative markers of adult neurogenesis. Journal of neuroscience methods 115: 97–105.
[49]
Webster M, Witkin KL, Cohen-Fix O (2009) Sizing up the nucleus: nuclear shape, size and nuclear-envelope assembly. Journal of cell science 122: 1477–1486.
[50]
Hendzel MJ, Wei Y, Mancini MA, Van Hooser A, Ranalli T, et al. (1997) Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation. Chromosoma 106: 348–360.
[51]
Gordon RE, Lane BP (1980) Duration of cell cycle and its phases measured in synchronized cells of squamous cell carcinoma of rat trachea. Cancer research 40: 4467–4472.
[52]
Dover R, Potten CS (1988) Heterogeneity and cell cycle analyses from time-lapse studies of human keratinocytes in vitro. Journal of cell science 89 ( Pt 3): 359–364.
[53]
Morgan DO (2007) The cell cycle: principles of control. Sunderland, MA: New Science Press. xxvii, 297 p.
[54]
Lowenheim H, Furness DN, Kil J, Zinn C, Gultig K, et al. (1999) Gene disruption of p27(Kip1) allows cell proliferation in the postnatal and adult organ of corti. Proc Natl Acad Sci U S A 96: 4084–4088.
[55]
Sage C, Huang M, Karimi K, Gutierrez G, Vollrath MA, et al. (2005) Proliferation of functional hair cells in vivo in the absence of the retinoblastoma protein. Science 307: 1114–1118.
[56]
Sage C, Huang M, Vollrath MA, Brown MC, Hinds PW, et al. (2006) Essential role of retinoblastoma protein in mammalian hair cell development and hearing. Proc Natl Acad Sci U S A 103: 7345–7350.
[57]
Yu Y, Weber T, Yamashita T, Liu Z, Valentine MB, et al. (2010) In vivo proliferation of postmitotic cochlear supporting cells by acute ablation of the retinoblastoma protein in neonatal mice. J Neurosci 30: 5927–5936.
[58]
Laine H, Doetzlhofer A, Mantela J, Ylikoski J, Laiho M, et al. (2007) p19(Ink4d) and p21(Cip1) collaborate to maintain the postmitotic state of auditory hair cells, their codeletion leading to DNA damage and p53-mediated apoptosis. J Neurosci 27: 1434–1444.
[59]
Mantela J, Jiang Z, Ylikoski J, Fritzsch B, Zacksenhaus E, et al. (2005) The retinoblastoma gene pathway regulates the postmitotic state of hair cells of the mouse inner ear. Development 132: 2377–2388.
[60]
Chen P, Zindy F, Abdala C, Liu F, Li X, et al. (2003) Progressive hearing loss in mice lacking the cyclin-dependent kinase inhibitor Ink4d. Nat Cell Biol 5: 422–426.
[61]
Kanzaki S, Beyer LA, Swiderski DL, Izumikawa M, Stover T, et al. (2006) p27(Kip1) deficiency causes organ of Corti pathology and hearing loss. Hearing research 214: 28–36.
[62]
Weber T, Corbett MK, Chow LM, Valentine MB, Baker SJ, et al. (2008) Rapid cell-cycle reentry and cell death after acute inactivation of the retinoblastoma gene product in postnatal cochlear hair cells. Proc Natl Acad Sci U S A 105: 781–785.
[63]
Hu Z, Corwin JT (2007) Inner ear hair cells produced in vitro by a mesenchymal-to-epithelial transition. Proc Natl Acad Sci U S A 104: 16675–16680.
[64]
Oshima K, Shin K, Diensthuber M, Peng AW, Ricci AJ, et al. (2010) Mechanosensitive hair cell-like cells from embryonic and induced pluripotent stem cells. Cell 141: 704–716.
[65]
Dang CV (2012) MYC on the path to cancer. Cell 149: 22–35.
[66]
Karn J, Watson JV, Lowe AD, Green SM, Vedeckis W (1989) Regulation of cell cycle duration by c-myc levels. Oncogene 4: 773–787.
[67]
Chen ZY, Wang X, Zhou Y, Offner G, Tseng CC (2005) Destabilization of Kruppel-like factor 4 protein in response to serum stimulation involves the ubiquitin-proteasome pathway. Cancer research 65: 10394–10400.
[68]
Tian Y, Luo A, Cai Y, Su Q, Ding F, et al. (2010) MicroRNA-10b promotes migration and invasion through KLF4 in human esophageal cancer cell lines. The Journal of biological chemistry 285: 7986–7994.
[69]
Saxe JP, Tomilin A, Scholer HR, Plath K, Huang J (2009) Post-translational regulation of Oct4 transcriptional activity. PloS one 4: e4467.
[70]
Meyer N, Penn LZ (2008) Reflecting on 25 years with MYC. Nature reviews Cancer 8: 976–990.
[71]
Pocsfalvi G, Votta G, De Vincenzo A, Fiume I, Raj DA, et al. (2011) Analysis of secretome changes uncovers an autocrine/paracrine component in the modulation of cell proliferation and motility by c-Myc. Journal of proteome research 10: 5326–5337.
[72]
Collado MS, Thiede BR, Baker W, Askew C, Igbani LM, et al. (2011) The postnatal accumulation of junctional E-cadherin is inversely correlated with the capacity for supporting cells to convert directly into sensory hair cells in mammalian balance organs. The Journal of neuroscience: the official journal of the Society for Neuroscience 31: 11855–11866.
[73]
Batsche E, Cremisi C (1999) Opposite transcriptional activity between the wild type c-myc gene coding for c-Myc1 and c-Myc2 proteins and c-Myc1 and c-Myc2 separately. Oncogene 18: 5662–5671.
[74]
Gottardi CJ, Wong E, Gumbiner BM (2001) E-cadherin suppresses cellular transformation by inhibiting beta-catenin signaling in an adhesion-independent manner. The Journal of cell biology 153: 1049–1060.
Luscher B, Vervoorts J (2012) Regulation of gene transcription by the oncoprotein MYC. Gene 494: 145–160.
[77]
Dominguez-Frutos E, Lopez-Hernandez I, Vendrell V, Neves J, Gallozzi M, et al. (2011) N-myc controls proliferation, morphogenesis, and patterning of the inner ear. The Journal of neuroscience: the official journal of the Society for Neuroscience 31: 7178–7189.
[78]
Kopecky B, Santi P, Johnson S, Schmitz H, Fritzsch B (2011) Conditional deletion of N-Myc disrupts neurosensory and non-sensory development of the ear. Developmental dynamics: an official publication of the American Association of Anatomists 240: 1373–1390.
[79]
Yamanaka S (2009) A fresh look at iPS cells. Cell 137: 13–17.
[80]
De Filippis L, Ferrari D, Rota Nodari L, Amati B, Snyder E, et al. (2008) Immortalization of human neural stem cells with the c-myc mutant T58A. PloS one 3: e3310.
[81]
Yeh E, Cunningham M, Arnold H, Chasse D, Monteith T, et al. (2004) A signalling pathway controlling c-Myc degradation that impacts oncogenic transformation of human cells. Nature cell biology 6: 308–318.
[82]
Alexiades MR, Cepko C (1996) Quantitative analysis of proliferation and cell cycle length during development of the rat retina. Developmental dynamics: an official publication of the American Association of Anatomists 205: 293–307.
[83]
Sulg M, Kirjavainen A, Pajusola K, Bueler H, Ylikoski J, et al. (2010) Differential sensitivity of the inner ear sensory cell populations to forced cell cycle re-entry and p53 induction. Journal of neurochemistry 112: 1513–1526.
[84]
Efe JA, Yuan X, Jiang K, Ding S (2011) Development unchained: how cellular reprogramming is redefining our view of cell fate and identity. Science progress 94: 298–322.
[85]
Qian L, Huang Y, Spencer CI, Foley A, Vedantham V, et al. (2012) In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes. Nature 485: 593–598.
[86]
Bermingham NA, Hassan BA, Price SD, Vollrath MA, Ben-Arie N, et al. (1999) Math1: an essential gene for the generation of inner ear hair cells. Science 284: 1837–1841.
[87]
Zheng JL, Gao WQ (2000) Overexpression of Math1 induces robust production of extra hair cells in postnatal rat inner ears. Nat Neurosci 3: 580–586.
[88]
Izumikawa M, Minoda R, Kawamoto K, Abrashkin KA, Swiderski DL, et al. (2005) Auditory hair cell replacement and hearing improvement by Atoh1 gene therapy in deaf mammals. Nat Med 11: 271–276.
[89]
Shou J, Zheng JL, Gao WQ (2003) Robust generation of new hair cells in the mature mammalian inner ear by adenoviral expression of Hath1. Mol Cell Neurosci 23: 169–179.
[90]
Staecker H, Praetorius M, Baker K, Brough DE (2007) Vestibular hair cell regeneration and restoration of balance function induced by math1 gene transfer. Otol Neurotol 28: 223–231.
[91]
Schlecker C, Praetorius M, Brough DE, Presler RG Jr., Hsu C, et al. (2011) Selective atonal gene delivery improves balance function in a mouse model of vestibular disease. Gene therapy.
[92]
Kelly MC, Chang Q, Pan A, Lin X, Chen P (2012) Atoh1 directs the formation of sensory mosaics and induces cell proliferation in the postnatal mammalian cochlea in vivo. The Journal of neuroscience: the official journal of the Society for Neuroscience 32: 6699–6710.
[93]
Liu Z, Dearman JA, Cox BC, Walters BJ, Zhang L, et al. (2012) Age-dependent in vivo conversion of mouse cochlear pillar and deiters' cells to immature hair cells by atoh1 ectopic expression. The Journal of neuroscience: the official journal of the Society for Neuroscience 32: 6600–6610.
[94]
Oesterle EC, Chien WM, Campbell S, Nellimarla P, Fero ML (2011) p27(Kip1) is required to maintain proliferative quiescence in the adult cochlea and pituitary. Cell cycle 10: 1237–1248.
[95]
Chen P, Segil N (1999) p27(Kip1) links cell proliferation to morphogenesis in the developing organ of Corti. Development 126: 1581–1590.
[96]
Ono K, Nakagawa T, Kojima K, Matsumoto M, Kawauchi T, et al. (2009) Silencing p27 reverses post-mitotic state of supporting cells in neonatal mouse cochleae. Molecular and cellular neurosciences 42: 391–398.
[97]
Rocha-Sanchez SM, Scheetz LR, Contreras M, Weston MD, Korte M, et al. (2011) Mature mice lacking Rbl2/p130 gene have supernumerary inner ear hair cells and supporting cells. The Journal of neuroscience: the official journal of the Society for Neuroscience 31: 8883–8893.
[98]
Huang M, Sage C, Tang Y, Lee SG, Petrillo M, et al. (2011) Overlapping and distinct pRb pathways in the mammalian auditory and vestibular organs. Cell Cycle 10: 337–351.
