Generation of inner ear auditory hair cell
Methods for generating cells of the inner ear, e.g., hair cells and supporting cells, from stem cells, e.g., mesenchymal stem cells, are provided, as well as compositions including the inner ear cells. Methods for the therapeutic use of the inner ear cells for the treatment of hearing loss are also described.
1. An in vitro method of generating a differentiated inner ear auditory hair cell, the method comprising:
(i) providing a population of mesenchymal stem cells obtained from mammalian bone marrow;
(ii) culturing the mesenchymal stem cell in serum-free medium containing insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF);
(iii) inducing the stem cell by maintaining said cell in medium comprising neurotrophin-3 (NT-3) and brain derived neurotrophic factor (BDNF) for time sufficient to differentiate into a progenitor cell that expresses Sox2, Pax6, nestin, and musashih; and
(iv) culturing the progenitor cell in the presence of a gamma secretase inhibitor in an amount and for a time sufficient to produce a differentiated inner ear auditory hair cell that expresses myosin VIIa, and espin and optionally expresses one or both of atonal homolog 1 (Atoh1) or jagged 2,
thereby producing a differentiated inner ear auditory hair cell, wherein the gamma secretase inhibitor is selected from the group consisting of an arylsulfonamide, a dibenzazepine, a benzodiazepine, N—[N-(3,5-difluorophenacetyl)-L-alanyl]-(S)-phenylglycine t-butyl ester (DAPT), L-685,458, and MK0752.
2. The method of claim 1 , wherein the mesenchymal stem cell is obtained from a subject who has sensorineural hair cell loss.
3. The method of claim 1 , wherein the inner ear auditory hair cell expresses Atoh1, myosin7a, and epsin.