Compositions and methods for obtaining stem cell derived lung tissue, and related uses therof

Abstract

The invention disclosed herein generally relates to methods and systems for converting stem cells into specific tissue(s) or organ(s) through directed differentiation. In particular, the invention disclosed herein relates to methods and systems for promoting definitive endoderm formation from pluripotent stem cells. The invention disclosed herein further relates to methods and systems for promoting ventral-anterior foregut spheroid tissue formation, 3-dimensional lung tissue formation, and lung organoid tissue formation produced in vitro from the described methods

What is claimed is: 
     
         1 . A method, comprising:
 culturing definitive endoderm cells in vitro, wherein the culturing results in differentiation of the definitive endoderm cells into tissue comprising ventral-anterior foregut spheroid tissue, wherein the culturing comprises activating and/or inhibiting one or more signaling pathways within the definitive endoderm cells, wherein the one or more signaling pathways are selected from the group consisting of the Wnt signaling pathway, the FGF signaling pathway, the BMP signaling pathway, and the TGFβ signaling pathway; and   obtaining ventral-anterior foregut spheroid tissue from the cultured definitive endoderm cells.   
     
     
         2 . The method of  claim 1 ,
 wherein the activating and/or inhibiting one or more signaling pathways within the definitive endoderm cells comprises activating the Wnt and FGF signaling pathways and inhibiting the BMP and TGFβ signaling pathways,   wherein activating the Wnt signaling pathway within the definitive endoderm cells comprises culturing the definitive endoderm cells with a small molecule or agonist that activates the Wnt signaling pathway,   wherein activating the FGF signaling pathway within the definitive endoderm cells comprises culturing the definitive endoderm cells with a small molecule or agonist that activates the FGF signaling pathway,   wherein inhibiting the BMP signaling pathway within the definitive endoderm cells comprises culturing the definitive endoderm cells with a small molecule or antagonist that inhibits the BMP signaling pathway,   wherein inhibiting the TGFβ signaling pathway within the definitive endoderm cells comprises culturing the definitive endoderm cells with a small molecule or antagonist that inhibits the TGFβ signaling pathway.   
     
     
         3 . The method of  claim 2 ,
 wherein the small molecule or agonist that activates the Wnt signaling pathway is CHIR99021,   wherein the small molecule or agonist that activates the FGF signaling pathway is selected from FGF1, FGF2, FGF3, FGF4, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23,   wherein the small molecule or antagonist that inhibits the BMP signaling pathway is Noggin,   wherein the small molecule or antagonist that inhibits the TGFβ signaling pathway is SB431542.   
     
     
         4 . The method of  claim 1 ,
 wherein activating the Wnt signaling pathway occurs through culturing the definitive endoderm cells with one or more molecules configured to activate a Wnt protein, wherein the Wnt protein is selected from the group consisting of Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, and Wnt16.   
     
     
         5 . The method of  claim 1 , comprising at least one of the following:
 wherein the activating and/or inhibiting one or more signaling pathways within the definitive endoderm cells occurs over a specified temporal period,   wherein the activating and/or inhibiting one or more signaling pathways within the definitive endoderm cells occurs comprises activating and/or inhibiting two or more signaling pathways,   wherein the activating and/or inhibiting two or more signaling pathways occurs simultaneously,   wherein the activating and/or inhibiting two or more signaling pathways does not occur simultaneously.   
     
     
         6 . The method of  claim 1 , wherein the definitive endoderm cells are derived from pluripotent stem cells, wherein the pluripotent stem cells are embryonic stem cells and/or induced pluripotent stem cells. 
     
     
         7 . The method of  claim 6 , wherein the definitive endoderm cells are derived by contacting the pluripotent stem cell with Activin A. 
     
     
         8 . The method of  claim 1 , further comprising:
 culturing the obtained ventral-anterior foregut spheroid tissue in vitro, wherein the culturing results in differentiation of the obtained ventral-anterior foregut spheroid tissue into tissue comprising 3-dimensional lung tissue, wherein the culturing comprising activating the Hedgehog signaling pathway; and   obtaining 3-dimensional lung tissue from the cultured tissue comprising ventral-anterior foregut spheroid tissue.   
     
     
         9 . The method of  claim 8 , wherein culturing the obtained ventral-anterior foregut spheroid tissue through activating the Hedgehog signaling pathway comprises culturing the ventral-anterior foregut spheroid tissue with a small molecule or agonist that activates the Hedgehog signaling pathway. 
     
     
         10 . The method of  claim 9 , wherein the small molecule or agonist that activates the Hedgehog signaling pathway is smoothened agonist (SAG). 
     
     
         11 . The method of  claim 8 , further comprising:
 culturing the obtained 3-dimensional lung tissue in vitro, wherein the culturing results in differentiation of the obtained tissue comprising 3-dimensional lung tissue into tissue lung organoid tissue, wherein the culturing comprising activating the FGF signaling pathway; and   obtaining lung organoid tissue from the cultured 3-dimensional lung tissue.   
     
     
         12 . The method of  claim 11 , wherein culturing the obtained 3-dimensional lung tissue through activating the FGF signaling pathway comprises culturing the obtained 3-dimensional lung tissue with a small molecule or agonist that activates the FGF signaling pathway. 
     
     
         13 . The method of  claim 12 , wherein the small molecule or agonist that activates the FGF signaling pathway is selected from FGF1, FGF2, FGF3, FGF4, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23. 
     
     
         14 . The method of  claim 12 , wherein the small molecule or agonist that activates the FGF signaling pathway is FGF10. 
     
     
         15 . The method of  claim 11 , wherein the obtained lung organoid tissue comprises one or more of upper airway-like epithelium with basal cells, immature ciliated cells surrounded by smooth muscle and myofibroblasts, and alveolar-like cells. 
     
     
         16 . The method of  claim 1 , wherein the culturing and obtaining steps are conducted in vitro. 
     
     
         17 . A composition comprising one or more of ventral-anterior foregut spheroid tissue, 3-dimensional lung tissue, and lung organoid tissue. 
     
     
         18 . A kit comprising one or more of ventral-anterior foregut spheroid tissue, 3-dimensional lung tissue, and lung organoid tissue.