Methods of generating human inner ear sensory epithelia and sensory neurons

Abstract

Provided herein are methods for directing differentiation of human pluripotent stem cells into inner ear sensory epithelia and sensory neurons. More particularly, provided herein are methods for obtaining three-dimensional cultures comprising human pluripotent stem cell-derived pre-otic epithelium, otic vesicles, and inner ear sensory epithelia containing hair cells, sensory neurons, and supporting cells.

We claim: 
     
         1 . A method of obtaining human pre-otic epithelial cells, comprising the steps of:
 (a) culturing human pluripotent stem cell aggregates in a culture medium comprising a Bone Morphogenetic Protein (BMP) and an inhibitor of Transforming Growth Factor Beta (TGFβ) signaling for about eight to about ten days;   (b) further culturing the cultured aggregates of (a) in the presence of a Fibroblast Growth Factor (FGF) and an inhibitor of BMP signaling for about 4 days; and   (b) contacting the further cultured aggregates of (b) to a Wnt agonist for about 4 days;   whereby cells within the contacted aggregates differentiate into pre-otic epithelial cells.   
     
     
         2 . The method of  claim 1 , wherein the FGF is FGF-2. 
     
     
         3 . The method of  claim 1 , wherein the BMP is selected from the group consisting of BMP2, BMP4, and BMP7. 
     
     
         4 . The method of  claim 1 , wherein the inhibitor of BMP signaling is LDN-193189. 
     
     
         5 . The method of  claim 1 , wherein the inhibitor of TGFβ1-mediated signaling is selected from the group consisting of SB431542 and A-83-01. 
     
     
         6 . The method of  claim 1 , wherein the Wnt agonist is an inhibitor of GSK3. 
     
     
         7 . The method of  claim 6 , wherein the inhibitor of GSK3 is selected from the group consisting of CHIR99021, lithium chloride (LiCl), and 6-bromoindirubin-3′-oxime (BIO). 
     
     
         8 . A method of obtaining a three-dimensional composition comprising human inner ear sensory tissue, the method comprising the steps of
 (a) embedding human pre-otic epithelial cells obtained according to the method of  claim 1  in a semi-solid culture medium comprising extracellular matrix protein;   (b) culturing the embedded pre-otic epithelial cells in the presence of a Wnt agonist for about 40 to about 60 days under conditions that promote self-assembly of embedded pre-otic epithelial cells into otic vesicles, whereby a three-dimensional composition comprising human inner ear sensory tissue is obtained.   
     
     
         9 . The method of  claim 8 , wherein the Wnt agonist is an inhibitor of GSK3. 
     
     
         10 . The method of  claim 9 , wherein the inhibitor of GSK3 is selected from the group consisting of CHIR99021, lithium chloride (LiCl), and 6-bromoindirubin-3′-oxime (BIO). 
     
     
         11 . The method of  claim 8 , wherein the extracellular matrix is a basement membrane extract (BME). 
     
     
         12 . The method of  claim 8 , wherein the three-dimensional composition comprises one or more mechanosensory cells. 
     
     
         13 . The method of  claim 8 , wherein the three-dimensional composition comprises one or more sensory neuron cells. 
     
     
         14 . The method of  claim 8 , wherein the three-dimensional composition comprises one or more sensory neuron cells that form synaptic connections with mechanosensory cells.