RESUMO
We have established a model for the in-vitro differentiation of mouse cochlear hair cells and have used it to explore the influence of retinoic acid on proliferation, cytoskeletal proteins and voltage-gated potassium conductances. The model is based on the conditionally immortal cell line University of Sheffield/ventral otocyst-epithelial cell line clone 36 (US/VOT-E36), derived from ventral otic epithelial cells of the mouse at embryonic day 10.5 and transfected with a reporter for myosin VIIa. Retinoic acid did not increase cell proliferation but led to up-regulation of myosin VIIa and formation of prominent actin rings that gave rise to numerous large, linear actin bundles. Cells expressing myosin VIIa had larger potassium conductances and did not express the cyclin-dependent kinase inhibitor p27(kip1). US/VOT-E36 endogenously expressed the voltage-gated potassium channel alpha-subunits Kv1.3 and Kv2.1, which we subsequently identified in embryonic and neonatal hair cells in both auditory and vestibular sensory epithelia in vivo. These subunits could underlie the embryonic and neonatal delayed-rectifiers recorded in nascent hair cells in vivo. Kv2.1 was particularly prominent on the basolateral membrane of cochlear inner hair cells. Kv1.3 was distributed throughout all hair cells but tended to be localized to the cuticular plates. US/VOT-E36 recapitulates a coherent pattern of cell differentiation under the influence of retinoic acid and will provide a convenient model for screening the effects of other extrinsic factors on the differentiation of cochlear epithelial cell types in vitro.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Cóclea/citologia , Proteínas do Citoesqueleto/metabolismo , Células Epiteliais/efeitos dos fármacos , Potássio/metabolismo , Tretinoína/farmacologia , Animais , Proteína Morfogenética Óssea 4 , Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Proteínas Morfogenéticas Ósseas/farmacologia , Contagem de Células , Linhagem Celular , Meios de Cultura Livres de Soro/farmacologia , Interações Medicamentosas , Embrião de Mamíferos , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Modelos Animais , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Transfecção/métodosRESUMO
The auditory neuroblast cell line US/VOT-N33 (N33), which is conditionally immortal, was studied as an in vitro model for the differentiation of spiral ganglion neurons (SGNs) and as a candidate for cell transplantation in rodents. It expresses numerous molecular markers characteristic of auditory neuroblasts, including the transcription factors GATA3, NeuroD, Brn3a and Islet1, as well as the neuronal cytoskeletal protein beta3-tubulin. It displays active migratory behaviour in vitro and in vivo. In the presence of the fibroblast growth factors FGF1 or FGF2 it differentiates bipolar morphologies similar to those of native SGNs. In coculture with neonatal cochlear tissue it is repelled from epithelial surfaces but not from native SGNs, alongside which it extends parallel neuronal processes. When injected into the retina in vivo, EGFP-labelled N33 cells were traced for 1-2 weeks and migrated rapidly within the subretinal space. Cells that found their way into the retinal ganglion cell layer extended multiple processes but did not express beta3-tubulin. The ability of N33 to migrate, to differentiate, to localize with native SGNs in vitro and to survive in vivo suggests that they provide an effective model for SGN differentiation and for cell transplantation into the ear.
