Methods for Generating Inner Ear Cells in Vitro

Abstract

Methods, compositions and kits are provided for generating inner ear cells in vitro. These methods find use a number of applications, such as in preparing inner ear cells for in vitro screening for agents that are toxic to inner ear cells, for in vitro screening for agents that prevent against, mitigate, or reverse the toxic effects of such agents, and for in vitro screening for agents that promote otoregeneration.

1 . A method of generating inner ear cells from pluripotent stem cells in vitro, the method comprising:
 a. culturing pluripotent stem cells in the presence of at least one factor that suppresses the formation of endoderm and mesoderm and at least one ectoderm rostralizing factor, wherein a population comprising preplacodal ectodermal cells is produced;   b. culturing the population comprising preplacodal ectodermal cells under adherent conditions in the presence of at least one FGF, wherein a population comprising otic progenitor cells is produced; and   c. culturing the population comprising otic progenitor cells under adherent conditions, wherein a population comprising inner ear cells is produced.   
     
     
         2 . The method according to  claim 1 , wherein the at least one factor that suppresses the formation of endodermal and mesoderm is selected from an inhibitor of Wnt signaling and an inhibitor of TGFB signaling. 
     
     
         3 . The method according to  claim 2 , wherein the at least one factors includes at least one inhibitor of Wnt signaling and at least one inhibitor of TGFB signaling. 
     
     
         4 . The method according to  claim 1 , wherein the at least one ectoderm rostralizing factor is a factor that activates IGF signaling. 
     
     
         5 . The method according to  claim 1 , wherein culturing the population comprising preplacodal ectodermal cells comprises:
 i. culturing the preplacodal ectodermal cells under adherent conditions in the presence of at least one FGF and one or more additional factors that promote the induction of otic progenitor cells; and   ii. culturing the induced otic progenitor cells under adherent conditions in the presence of FGF growth factors and one or more additional factors that promote the stabilization of otic progenitor cells.   
     
     
         6 . The method according to  claim 5 , wherein the one or more additional factors that promote the induction of otic progenitor cells is selected from the group consisting of an inhibitor of BMP signaling, an activator of Wnt signaling, and FGF19. 
     
     
         7 . The method according to  claim 6 , wherein the one or more additional factors that promote the stabilization of otic progenitor cells is selected from the group consisting of an activator of BMP signaling and FGF19. 
     
     
         8 . The method according to  claim 1 , wherein the population comprising preplacodal ectodermal cells is mechanically enriched for preplacodal ectodermal cells prior to culturing. 
     
     
         9 . The method according to  claim 1 , wherein the population comprising otic progenitor cells is mechanically enriched for otic progenitor cells prior to step (c). 
     
     
         10 . The method according to  claim 1 , wherein the otic progenitor cells are expanded prior to step (c). 
     
     
         11 . The method according to  claim 1 , wherein the pluripotent stem cells are from human, and culturing the otic progenitor cells comprises culturing in the absence of feeder cells. 
     
     
         12 . The method according to  claim 1 , wherein the pluripotent stem cells are from rodent, and culturing the otic progenitor cells comprises culturing in the presence of feeder cells. 
     
     
         13 . A method of generating human inner ear cells in vitro, the method comprising:
 culturing a population of cells that are enriched for otic progenitor cells under adherent conditions in the absence of feeder cells to form inner ear cells.   
     
     
         14 . The method according to  claim 13 , wherein the population of cells that is enriched for otic progenitor cells is mechanically enriched for otic progenitor cells and/or is enriched by culturing pluripotent stem cells under conditions that promote the formation of otic progenitor cells. 
     
     
         15 . The method according to  claim 13 , wherein the population of cells enriched for otic progenitor cells is enriched by:
 a. culturing pluripotent stem cells in the presence of at least one factor that suppresses the formation of endoderm and mesoderm and at least one ectoderm rostralizing factor, wherein a population comprising preplacodal ectodermal cells is produced;   b. culturing the population comprising preplacodal ectodermal cells under conditions that induce the formation of otic progenitor cells; and   c. culturing the population in which the formation of otic progenitor cells has been induced under conditions that promote the stabilization of otic progenitor cells;   wherein the population of cells that is formed is a population that is enriched for otic progenitor cells.   
     
     
         16 . The method according to  claim 13 , wherein the population comprising preplacodal ectodermal cells is mechanically enriched for preplacodal ectodermal cells. 
     
     
         17 .- 20 . (canceled) 
     
     
         21 . The method according to  claim 1 , wherein step (b) comprises culturing the population comprising preplacodal ectodermal cells under adherent conditions in the presence of an inhibitor of BMP signaling and the at least one FGF. 
     
     
         22 . The method of  claim 5 , wherein the step i comprises culturing the preplacodal ectodermal cells under adherent conditions in the presence of the at least one FGF, an inhibitor of BMP signaling, and an activator of Wnt signaling. 
     
     
         23 . The method of  claim 22 , wherein the step ii comprises culturing the induced otic progenitor cells under adherent conditions in the presence of a FGF, an activator of BMP signaling and FGF19. 
     
     
         24 . The method of  claim 11 , wherein the method comprises:
 culturing the human pluripotent stem cells in the presence of an inhibitor of Wnt signaling, an inhibitor of TGFB signaling, and an activator of IGF signaling, wherein a population comprising preplacodal ectodermal cells is produced;   culturing the preplacodal ectodermal cells under adherent conditions in the presence of a FGF and an inhibitor of BMP signaling to promote the induction of otic progenitor cells;   culturing the induced otic progenitor cells under adherent conditions in the presence of a FGF and an activator of BMP signaling to promote the stabilization of otic progenitor cells; and   culturing the otic progenitor cells under adherent conditions in the absence of feeder cells, wherein a population comprising inner ear cells is produced.