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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.

Titel:	METHOD FOR INDUCING DIFFERENTIATION OF ALVEOLAR EPITHELIAL CELLS
Link:	View record in USPTO Patent Applications (Volltext)
Veröffentlichung:	2022
Medientyp:	Patent
Sonstiges:	Nachgewiesen in: USPTO Patent Applications Sprachen: English Document Number: 20220186189 Publication Date: June 16, 2022 Appl. No: 17/653185 Application Filed: March 02, 2022 Assignees: KYOTO UNIVERSITY (Kyoto, JP) Claim: 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). Claim: 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). Claim: 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. Claim: 4. The method according to claim 3, wherein the ROCK inhibitor is Y-27632. Claim: 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). Claim: 6. The method according to claim 5, wherein the WNT signal inhibitor is WNT inhibitory factor 1 (WIF1). Claim: 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. Current International Class: 12

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METHOD FOR INDUCING DIFFERENTIATION OF ALVEOLAR EPITHELIAL CELLS