Generation of inner ear cells

Abstract

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 . A method of treating a subject experiencing or at risk for developing hearing loss or vestibular dysfunction, the method comprising:
 contacting a progenitor cell that expresses one or more of nestin, sox2, musashih, or Pax2, with a gamma secretase inhibitor in an amount and for a time sufficient to induce the progenitor cell to express one or more of atonal homolog 1 (Atoh1), jagged 2, p27 Kip , neurogenin 1 (Ngn1), NeuroD, myosin VIIa, or epsin, thereby producing a more differentiated cell; and   administering one or more of the more differentiated cells to the subject's inner ear, thereby treating the hearing loss or vestibular dysfunction.   
     
     
         2 . The method of  claim 1 , wherein the progenitor cell is obtained by a method comprising:
 providing a stem cell; and   culturing the stem cell under conditions sufficient to induce the stem cell to differentiate into the progenitor cell.   
     
     
         3 . The method of  claim 2 , wherein the stem cell is a mesenchymal stem cell. 
     
     
         4 . The method of  claim 2 , wherein the stem cell is obtained from bone marrow. 
     
     
         5 . The method of  claim 2 , wherein the stem cell is obtained from the subject. 
     
     
         6 . The method of  claim 2 , wherein culturing the stem cell comprises maintaining the stem cell in medium comprising an epidermal growth factor, a fibroblast growth factor, or an insulin-like growth factor. 
     
     
         7 . The method of  claim 2 , wherein culturing the stem cell comprises maintaining the stem cell in medium comprising neurotrophin-3 or a brain derived neurotrophic factor. 
     
     
         8 . The method of  claim 1 , wherein the gamma secretase inhibitor is an arylsulfonamide, a dibenzazepine, a benzodiazepine, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-(S)-phenylglycine t-butyl ester (DAPT), L-685,458, or MK0752. 
     
     
         9 . The method of  claim 1 , wherein the gamma secretase inhibitor is a nucleic acid that selectively inhibits the expression of gamma secretase. 
     
     
         10 . The method of  claim 9 , wherein the nucleic acid is a small interfering RNA, an antisense oligonucleotide, or a morpholino oligo. 
     
     
         11 . The method of  claim 1 , wherein the more differentiated cell is an inner ear auditory hair cell or an inner ear support cell. 
     
     
         12 . The method of  claim 1 , wherein the subject has or is at risk for developing a vestibular dysfunction that results in dizziness, imbalance, or vertigo. 
     
     
         13 . The method of  claim 1 , wherein the subject has or is at risk for developing sensorineural hearing loss, auditory neuropathy, or both. 
     
     
         14 . A method of providing an auditory hair cell, the method comprising:
 obtaining a stem cell;   culturing the stem cell under conditions sufficient to induce the stem cell to differentiate into a progenitor cell that expresses one or more of nestin, sox2, musashih, Pax2;   contacting the progenitor cell with a gamma secretase inhibitor in an amount and for a time sufficient to induce the progenitor cell to express one or more of Atoh1 jagged 2, p27 Kip , neurogenin 1 (Ngn1), NeuroD, myosin VIIa, or espin, thereby producing an auditory hair cell.   
     
     
         15 . The method of  claim 14 , wherein the stem cell is a mesenchymal stem cell. 
     
     
         16 . The method of  claim 14 , wherein the stem cell is obtained from bone marrow. 
     
     
         17 . The method of  claim 14 , wherein culturing the stem cell comprises maintaining the stem cell in medium comprising an epidermal growth factor, a fibroblast growth factor, or an insulin-like growth factor. 
     
     
         18 . The method of  claim 14 , wherein culturing the stem cell comprises maintaining the stem cell in medium comprising neurotrophin-3 or a brain derived neurotrophic factor. 
     
     
         19 . The method of  claim 14 , wherein the gamma secretase inhibitor is an arylsulfonamide, a dibenzazepine, a benzodiazepine, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-(S)-phenylglycine t-butyl ester (DAPT), L-685,458, or MK0752. 
     
     
         20 . The method of  claim 14 , wherein the gamma secretase inhibitor is a nucleic acid that selectively inhibits the expression of gamma secretase. 
     
     
         21 . The method of  claim 20 , wherein the nucleic acid is a small interfering RNA, an antisense oligonucleotide, or a morpholino oligo. 
     
     
         22 . The method of  claim 14 , wherein the progenitor cell is isolated. 
     
     
         23 . The method of  claim 14 , wherein the auditory hair cell is isolated. 
     
     
         24 . The method of  claim 23 , wherein the isolated auditory hair cell is administered to the inner ear of a subject experiencing or at risk for developing hearing loss or vestibular dysfunction, thereby treating the hearing loss or vestibular dysfunction. 
     
     
         25 . An isolated auditory hair cell made by the method of  claim 14 . 
     
     
         26 . A method of providing an auditory support cell, the method comprising:
 obtaining a stem cell;   culturing the stem cell under conditions sufficient to induce the stem cell to differentiate into a progenitor cell that expresses one or more of nestin, sox2, musashih, Pax2;   contacting the progenitor cell with a gamma secretase inhibitor in an amount and for a time sufficient to induce the progenitor cell to express one or more of claudin14, connexin 26, p75Trk, Notch 1, or S100A, thereby providing an auditory support cell.   
     
     
         27 . The method of  claim 26 , wherein the stem cell is a mesenchymal stem cell. 
     
     
         28 . The method of  claim 26 , wherein the stem cell is obtained from bone marrow. 
     
     
         29 . The method of  claim 26 , wherein culturing the stem cell comprises maintaining the stem cell in medium comprising an epidermal growth factor, a fibroblast growth factor, or an insulin-like growth factor. 
     
     
         30 . The method of  claim 26 , wherein culturing the stem cell comprises maintaining the stem cell in medium comprising neurotrophin-3 or a brain derived neurotrophic factor. 
     
     
         31 . The method of  claim 26 , wherein the gamma secretase inhibitor is an arylsulfonamide, a dibenzazepine, a benzodiazepine, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-(S)-phenylglycine t-butyl ester (DAPT), L-685,458, or MK0752. 
     
     
         32 . The method of  claim 26 , wherein the gamma secretase inhibitor is a nucleic acid that selectively inhibits the expression of gamma secretase. 
     
     
         33 . The method of  claim 32 , wherein the nucleic acid is a small interfering RNA, an antisense oligonucleotide, or a morpholino oligo. 
     
     
         34 . The method of  claim 26 , wherein the progenitor cell is isolated. 
     
     
         35 . The method of  claim 26 , wherein the auditory support cell is isolated. 
     
     
         36 . The method of  claim 35 , wherein the isolated auditory support cell is administered to the inner ear of a subject experiencing or at risk for developing hearing loss or vestibular dysfunction, thereby treating the hearing loss or vestibular dysfunction 
     
     
         37 . An isolated auditory support cell made by the method of  claim 26 .