Method for inducing differentiation of alveolar epithelial cells

Abstract

This invention provides a method for stably producing type II alveolar epithelial cells from pluripotent stem cells. Specifically, the invention relates to a method for producing type II alveolar epithelial cells from pluripotent stem cells comprising steps of: (1) culturing pluripotent stem cells in a medium containing activin A and a GSK3β inhibitor; (2) culturing the cells obtained in Step (1) in a medium containing a BMP inhibitor and a TGFβ inhibitor; (3) culturing the cells obtained in Step (2) in a medium containing BMP4, retinoic acid, and a GSK3β inhibitor; (4) culturing the ventral anterior foregut cells obtained in Step (3) in a medium containing a GSK3β inhibitor, FGF10, KGF, and a NOTCH signal inhibitor; and (5) subjecting the alveolar epithelial progenitor cells obtained in Step (4) to three-dimensional culture in a medium containing a steroid drug, a cAMP derivative, a phosphodiesterase inhibitor, and KGF.

1 . A method for type II alveolar epithelial cell culture comprising a step of subjecting type II alveolar epithelial cells to three-dimensional culture in a medium containing a steroid drug, a cAMP derivative, a phosphodiesterase inhibitor, and keratinocyte growth factor (KGF). 
     
     
         2 . The method according to  claim 1 , wherein the steroid drug is dexamethasone, the cAMP derivative is 8-Br-cAMP, and the phosphodiesterase inhibitor is 3-isobutyl-1-methylxanthine (IBMX). 
     
     
         3 . The method according to  claim 1 , which comprises subjecting type II alveolar epithelial cells to three-dimensional culture in a medium further supplemented with a Rho kinase (ROCK) inhibitor. 
     
     
         4 . The method according to  claim 3 , wherein the ROCK inhibitor is Y-27632. 
     
     
         5 . The method according to  claim 1 , which comprises subjecting type II alveolar epithelial cells to three-dimensional culture in a medium further supplemented with a WNT signal inhibitor and/or insulin like growth factor 2 (IGF2). 
     
     
         6 . The method according to  claim 5 , wherein the WNT signal inhibitor is WNT inhibitory factor 1 (WIF1). 
     
     
         7 . The method according to  claim 1 , wherein the type II alveolar epithelial cells are produced by a method for producing a cell population comprising type II alveolar epithelial cells from pluripotent stem cells comprising Steps (1) to (5) below:
 (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 morphogenetic protein (BMP) inhibitor and a transforming growth factor beta (TGFβ) inhibitor;   (3) culturing the cells obtained in Step (2) in a medium containing bone morphogenetic protein 4 (BMP4), retinoic acid, and a GSK3β inhibitor;   (4) culturing the ventral anterior foregut cells obtained in Step (3) in a medium containing a GSK3β inhibitor, Fibroblast Growth Factor 10 (FGF10), keratinocyte growth factor (KGF), and a NOTCH signal inhibitor for a duration of time until alveolar epithelial progenitor cells are induced; followed by a step of isolating carboxypeptidase M-positive (CPM-positive) cells as alveolar epithelial progenitor cells; and   (5) subjecting the induced alveolar epithelial progenitor cells obtained in Step (4) to three-dimensional culture in a basal medium supplemented with additives consisting of a steroid drug, a cAMP derivative, a phosphodiesterase inhibitor, and KGF;   following Step (5), a further step of isolating cells positive for one or more type II alveolar epithelial cell markers selected from the group consisting of surfactant protein C (SFTPC), epithelial cell adhesion molecule (EpCAM), and carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) as type II alveolar epithelial cells,   wherein cells positive for staining of acidic fractions for live cells are isolated as type II alveolar epithelial cells;   thereby obtaining a cell population, wherein the type II alveolar epithelial cells are at least 50% of the cell population, relative to total epithelial cells.