Method for inducing alveolar epithelial progenitor cells

Abstract

This invention provides a method for stably producing alveolar epithelial progenitor cells from pluripotent stem cells, including steps of culturing pluripotent stem cells in (1) a medium containing activin A and a GSK3β inhibitor, (2) a medium containing a BMP inhibitor and a TGFβ inhibitor, and (3) a medium containing BMP4, retinoic acid, and a GSK3β inhibitor.

1 . A cell population predominantly comprising carboxypeptidase M (CPM)-positive cells as alveolar epithelial progenitor cells or alveolar epithelial cells. 
     
     
         2 . The cell population according to  claim 1 , wherein the cell population is produced by extracting CPM-positive cells, as alveolar epithelial progenitor cells, from a cell population comprising alveolar epithelial progenitor cells using CPM as a marker, thereby obtaining a cell population predominantly comprising CPM-positive cells. 
     
     
         3 . A method for producing a cell population predominantly comprising CPM-positive cells as alveolar epithelial progenitor cells comprising extracting CPM-positive cells as alveolar epithelial progenitor cells using CPM as a marker, from a cell population comprising alveolar epithelial progenitor cells, thereby obtaining a cell population predominantly comprising CPM-positive cells. 
     
     
         4 . The cell population according to  claim 1 , wherein the cell population is produced by a method for producing alveolar epithelial progenitor cells from pluripotent stem cells comprising Steps (1) to (6):
 (1) culturing pluripotent stem cells in a medium containing activin A and a glycogen synthase kinase 3β (GSK3β) inhibitor;   (2) culturing the cells obtained in Step (1) in a medium containing a bone morphogenic protein (BMP) inhibitor and a transforming growth factor β (TGFβ) inhibitor;   (3) culturing the cells obtained in Step (2) in a medium containing BMP4, retinoic acid, and a GSK3β inhibitor;   (4) culturing the cells obtained from Step (3) in a medium containing a fibroblast growth factor  10  (FGF10);   (5) culturing the cells obtained from Step (4) in a medium containing a steroid drug, a cyclic adenosine monophosphate (cAMP) derivative, a phosphodiesterase inhibitor, and a keratinocyte growth factor (KGF); and   (6) extracting CPM-positive cells as alveolar epithelial progenitor cells using the CPM as a marker following Step (5);   thereby obtaining a cell population predominantly comprising CPM-positive cells.   
     
     
         5 . The cell population according to  claim 4 , wherein the medium of Step (1) further comprises a Rho kinase (ROCK) inhibitor and/or a histone deacetylase (HDAC) inhibitor. 
     
     
         6 . The cell population according to  claim 4 , wherein Step (1) comprises culturing for 6 days or longer, Step (2) comprises culturing for 4 days or longer, Step (3) comprises culturing for 4 days or longer, Step (4) comprises culturing for 7 days or longer and Step (5) comprises culturing for 4 days or longer. 
     
     
         7 . The cell population according to  claim 4 , wherein the GSK3β inhibitor is CHIR99021, the BMP inhibitor is Noggin, the TGFβ inhibitor is SB431542, the steroid drug is dexamethasone, the cAMP derivative is 8Br-cAMP, and the phosphodiesterase inhibitor is 3-isobutyl-1-methylxanthine (IBMX). 
     
     
         8 . The cell population according to  claim 1 , wherein the cell population is produced by a method for producing alveolar epithelial progenitor cells from pluripotent stem cells comprising Steps (1) to (3b):
 (1) culturing pluripotent stem cells in a medium containing activin A and a glycogen synthase kinase 3β (GSK 3 β) inhibitor;   (2) culturing the cells obtained in Step (1) in a medium containing a bone morphogenic protein (BMP) inhibitor and a transforming growth factor β (TGFβ) inhibitor;   (3a) culturing the cells obtained in Step (2) in a medium containing BMP4, retinoic acid, and a GSK3β inhibitor; and   (3b) extracting CPM-positive cells as alveolar epithelial progenitor cells using the CPM as a marker following Step (3a);   thereby obtaining a cell population predominantly comprising CPM-positive cells.   
     
     
         9 . The cell population according to  claim 8 , wherein the medium of Step (1) in the method further comprises a Rho kinase (ROCK) inhibitor and/or a histone deacetylase (HDAC) inhibitor. 
     
     
         10 . The cell population according to  claim 8 , wherein Step (1) comprises culturing for 6 days or longer, Step (2) comprises culturing for 4 days or longer, and Step (3a) comprises culturing for 4 days or longer. 
     
     
         11 . The cell population according to  claim 8 , wherein the GSK3β inhibitor is CHIR99021, the BMP inhibitor is Noggin, and the TGFβ inhibitor is SB431542. 
     
     
         12 . The cell population according to  claim 1 , wherein the cell population is produced by a method for producing alveolar epithelial cells from pluripotent stem cells comprising Steps (1) to (3c):
 (1) culturing pluripotent stem cells in a medium containing activin A and a glycogen synthase kinase 3β (GSK3β) inhibitor;   (2) culturing the cells obtained in Step (1) in a medium containing a bone morphogenic protein (BMP) inhibitor and a transforming growth factor β (TGFβ) inhibitor;   (3a) culturing the cells obtained in Step (2) in a medium containing BMP4, retinoic acid, and a GSK3β inhibitor;   (3b) extracting CPM-positive cells as alveolar epithelial progenitor cells using the CPM as a marker following Step (3a); and   (3c) co-culturing the extracted alveolar epithelial progenitor cells together with human fetal pulmonary fibroblasts, following Step (3b);   thereby obtaining a cell population predominantly comprising CPM-positive cells.   
     
     
         13 . The cell population according to  claim 12 , wherein the medium of Step (1) further comprises a Rho kinase (ROCK) inhibitor and/or a histone deacetylase (HDAC) inhibitor. 
     
     
         14 . The cell population according to  claim 12 , wherein Step (1) comprises culturing for 6 days or longer, Step (2) comprises culturing for 4 days or longer, and Step (3a) comprises culturing for 4 days or longer. 
     
     
         15 . The cell population according to  claim 12 , wherein the GSK3β inhibitor is CHIR99021, the BMP inhibitor is Noggin, and the TGFβ inhibitor is SB431542. 
     
     
         16 . The cell population according to  claim 12 , wherein step (3c) is conducted by co-culturing in three-dimensional cell culture. 
     
     
         17 . The cell population according to  claim 1 , wherein the CPM-positive cells are human CPM-positive cells. 
     
     
         18 . The cell population according to  claim 1 , wherein the proportion of CPM-positive cells relative to total cells in the cell population is at least 50%. 
     
     
         19 . The method according to  claim 3 , wherein the CPM-positive cells are human CPM-positive cells. 
     
     
         20 . The method according to  claim 3 , wherein the proportion of CPM-positive cells relative to total cells in the obtained cell population is at least 50%. 
     
     
         21 . An agent for treatment of a pulmonary alveolar disease comprising the cell population according to  claim 1 . 
     
     
         22 . A method for treating a pulmonary alveolar disease comprising a step of administering the cell population according to  claim 1  to a patient afflicted with a disease that destroy pulmonary alveolus. 
     
     
         23 . The method according to  claim 22 , wherein a sheet comprising the cell population is applied to alveolar epithelium of the patient. 
     
     
         24 . The method according to  claim 22 , wherein a suspension comprising the cell population is implanted in pulmonary alveolus of the patient.