Use of stem cells to generate inner ear cells

Abstract

This invention relates generally to methods and compositions for inducing stem cell or progenitor cell differentiation, and more particularly to methods and compositions for inducing differentiation of stem cells and/or progenitor cells into cells that function within the inner ear.

1 . A method of producing a population of neural progenitor cells, the method comprising:
 providing a first population of cells comprising stem cells that have undetectable levels of Sox1 expression;   culturing the first population of cells in suspension in the absence of serum and feeder cells, under conditions and for a time period sufficient to induce differentiation of said first population of cells into neural progenitor cells, thereby obtaining a second population of cells comprising neural progenitor cells.   
     
     
         2 . The method of  claim 1 , further comprising assaying the levels of Sox1 in the first and second population of cells, wherein a higher level of Sox1 expression in the second population of cells as compared to the first population of cells indicates that the second population of cells comprises neural progenitor cells. 
     
     
         3 . The method of  claim 1 , further comprising assaying expression of one or more of nestin, Pax2, Math1, NeuroD, and GFAP in the second population of cells, wherein expression one or more of nestin, Pax2, Math1, NeuroD, and GFAP indicates that the second population of cells comprises neural progenitor cells. 
     
     
         4 . The method of  claim 1 , wherein the second population of cells express one or more of nestin, Pax2, Math1, NeuroD, GFAP, BMP4, BMP7, Notch1, Jag1, and Jag2. 
     
     
         5 . The method of  claim 1 , wherein the stem cells comprise embryonic stem cells. 
     
     
         6 . The method of  claim 5 , wherein the embryonic stem cells comprise murine embryonic stem cells. 
     
     
         7 . The method of  claim 5 , wherein the embryonic stem cells comprise human embryonic stem cells. 
     
     
         8 . The method of  claim 1 , wherein the first and second populations of cells comprise one or more of a reporter under the control of a beta-actin promoter or a reporter under the control of a Sox1 promoter. 
     
     
         9 . The method of  claim 8 , wherein the reporter is a fluorescent protein. 
     
     
         10 . The method of  claim 1 , wherein following differentiation of the first population of cells, the method further comprises:
 disrupting clusters of two or more neural progenitor cells to yield individual neural progenitor cells;   culturing the individual neural progenitor cells for a time and under conditions sufficient to promote attachment of the cells to a surface; and   maintaining the attached cells in the presence of one or more growth factors for a time and under conditions sufficient to promote cell proliferation, thereby increasing the number of neural progenitor cells.   
     
     
         11 . The method of  claim 10 , wherein the attached cells are cultured in the presence of 10% serum. 
     
     
         12 . The method of  claim 10 , wherein the second and third population of cells express one or more of Sox1, nestin, Pax2, and Math1. 
     
     
         13 . The method of  claim 10 , wherein the growth factor is basic fibroblast growth factor. 
     
     
         14 . The method of  claim 10 , further comprising culturing the neural progenitor cells in the absence of growth factor for a time sufficient to promote differentiation of the neural progenitor cells into sensory neural cells. 
     
     
         15 . The method of  claim 1 , further comprising culturing the neural progenitor cells in the absence of growth factor for a time sufficient to promote differentiation of the neural progenitor cells into sensory neural cells. 
     
     
         16 . The method of  claim 14 , wherein the sensory neural cells comprise one or more of neurons, glial cells, and oligodendrocytes. 
     
     
         17 . The method of  claim 14 , wherein the sensory neural cells express one or both of (i) less Sox1 than the neural progenitor cells and (ii) higher levels of one or more of TrkC and Map2 than the neural progenitor cells. 
     
     
         18 . The method of  claim 14 , wherein one or more of β-III tubulin and GFAP is detectable in the sensory neural cells. 
     
     
         19 . The method of  claim 1 , further comprising culturing the neural progenitor cells in the presence of an effective amount of bone morphogenetic protein 4 (BMP4) for a time period sufficient to promote differentiation of the cells into sensory neural cells. 
     
     
         20 . The method of  claim 19 , wherein the sensory neural cells express elevated levels of GATA3, TrkB, and TrkC when compared to the neural progenitor cells. 
     
     
         21 . A method of producing a population of sensory neural cells, the method comprising:
 providing a population of stem cells having undetectable levels of Sox1 expression;   culturing the stem cells in the absence of serum for a time period sufficient to promote differentiation of the stem cells into a population of cells comprising neural progenitor cells;   contacting the neural progenitor cells with an effective amount of one or both of (i) bone morphogenetic protein 4 (BMP4) or (ii) retinoic acid for a time period sufficient to promote differentiation of the neural progenitor cells into sensory neural cells, thereby producing a population of sensory neural cells.   
     
     
         22 . The method of  claim 21 , wherein the neural progenitor cells express detectable levels of Sox1. 
     
     
         23 . The method of  claim 21 , wherein the stem cells comprise embryonic stem cells. 
     
     
         24 . The method of  claim 21 , wherein the stem cells are human stem cells or murine stem cells. 
     
     
         25 . The method of  claim 21 , wherein the stem cells are cultured in the presence of human leukemia inhibitory factor (LIF). 
     
     
         26 . The method of  claim 21 , wherein one or both of the stem cells and neural progenitor cells are cultured in the presence of one or more growth factors. 
     
     
         27 . The method of  claim 21 , wherein the sensory neural cells have elevated levels of GATA3, TrkB, and TrkC as compared to the stem cells. 
     
     
         28 . The method of  claim 1 , further comprising culturing the neural progenitor cells in the presence of an effective amount of retinoic acid for a time period sufficient to promote differentiation of the cells into sensory neural cells. 
     
     
         29 . A method of producing a population of sensory neural cells, the method comprising:
 providing a population of cells comprising inner ear stem cells;   culturing the inner ear stem cells in the absence of serum and in the presence of one or more of (i) an effective amount of retinoic acid or (ii) bone morphogenetic protein 4 (BMP4) for a time sufficient to promote differentiation of the inner ear stem cells into sensory neural cells.   
     
     
         30 . The method of  claim 29 , wherein the inner ear stem cells are cultured in the presence of one or more growth factors. 
     
     
         31 . The method of  claim 29 , wherein the inner ear stem cells express detectable levels of Sox1. 
     
     
         32 . The method of  claim 29 , wherein the inner ear stem cells express undetectable levels of β-III tubulin, GFAP, and myosin VIIa. 
     
     
         33 . The method of  claim 29 , wherein the sensory neural cells express higher levels of Pax2 as compared to the inner ear stem cells. 
     
     
         34 . The method of  claim 33 , further comprising, prior to culturing the inner ear cells in the presence of retinoic acid or BMP4:
 (a) culturing the inner ear stem cells in suspension in the absence of serum for a time period sufficient for the cells to form clusters of cells comprising two or more cells;   (b) disrupting the cluster of cells to yield a population of individual cells;   (c) culturing the individual cells in suspension in the absence of serum suspension for a time period sufficient for the cells to form clusters of cells comprising two or more cells; and   optionally repeating steps (a) to (c) until a desired number of inner ear stem cells is obtained, and then   culturing the inner ear stem cells in the presence of serum for a time sufficient to promote attachment of the cells to a surface.   
     
     
         35 . A method of treating, or preventing the progression of, sensorineural hearing loss in a subject, the method comprising:
 providing a population of neural progenitor cells obtained using the method of  claim 1 , and   administering said population of cells into the inner ear of a subject, thereby treating or preventing the development or progression of sensorineural hearing loss in the subject.   
     
     
         36 . The method of  claim 35 , wherein the cells are administered by injection into the luminae of the cochlea, into the auditory nerve trunk in the internal auditory meatus, or into the scala tympani. 
     
     
         37 . A method of treating, or preventing the development or progression of, sensorineural hearing loss in a subject, the method comprising:
 providing a population of sensory neural cells obtained using the method of  claim 14 , and   administering said population of sensory neural cells into the inner ear of a subject, thereby treating or preventing the progression of sensorineural hearing loss in the subject.   
     
     
         38 . The method of  claim 37 , wherein the cells are administered by injection into the luminae of the cochlea, into the auditory nerve trunk in the internal auditory meatus, or into the scala tympani. 
     
     
         39 . A method of treating, or preventing the progression of, sensorineural hearing loss in a subject, the method comprising:
 providing a population of sensory neural cells obtained using the method of  claim 15 , and   administering said population of sensory neural cells into the inner ear of a subject, thereby treating or preventing the progression of sensorineural hearing loss in the subject.   
     
     
         40 . The method of  claim 39 , wherein the cells are administered by injection into the luminae of the cochlea, into the auditory nerve trunk in the internal auditory meatus, or into the scala tympani. 
     
     
         41 . A method of treating, or preventing the development or progression of, sensorineural hearing loss in a subject, the method comprising:
 providing a population of cells obtained using the method of  claim 21 , and   administering said population of cells into the inner ear of a subject, thereby treating or preventing the progression of sensorineural hearing loss in the subject.   
     
     
         42 . The method of  claim 41 , wherein the cells are administered by injection into the luminae of the cochlea, into the auditory nerve trunk in the internal auditory meatus, or into the scala tympani. 
     
     
         43 . A method of treating, or preventing the development or progression of, sensorineural hearing loss in a subject, the method comprising:
 providing a population of cells obtained using the method of  claim 29 , and   administering said population of cells into the inner ear of a subject, thereby treating or preventing the progression of sensorineural hearing loss in the subject.   
     
     
         44 . The method of  claim 43 , wherein the cells are administered by injection into the luminae of the cochlea, into the auditory nerve trunk in the internal auditory meatus, or into the scala tympani. 
     
     
         45 . A method of identifying a candidate compound that promotes differentiation of cells into mature cells of the inner ear, the methods comprising:
 providing a cell expressing a reporter construct comprising a Math-1 regulatory region operably linked to a reporter gene;   contacting the cell with a test compound; and   detecting expression of the reporter gene;   wherein an increase in expression of the reporter gene in that presence of the test compound as compared to expression of the reporter gene in the absence of the test compound indicates that the test compound is a candidate compound that promotes differentiation of cells into mature cells of the inner ear.   
     
     
         46 . The method of  claim 45 , further comprising contacting a stem cell, inner ear stem cell, or neural progenitor cell with the candidate compound and evaluating the ability of the candidate compound to promote differentiation into a mature cell of the inner ear, and selecting a candidate compound that promotes differentiation into a mature cell of the inner ear. 
     
     
         47 . The method of  claim 45 , wherein the reporter gene is selected from the group consisting of a fluorescent protein, an enzymatically active protein, or a protein detectable in an antibody-based assay. 
     
     
         48 . The method of  claim 45 , wherein the mature cell of the inner ear is a hair cell or spiral ganglion neuron. 
     
     
         49 . The method of  claim 46 , further comprising administering the candidate compound to the inner ear of an animal model of sensorineural hearing loss, and evaluating the ability of the candidate compound to promote differentiation of inner ear stem cells into mature cells of the inner ear.