Public University Corporation Nagoya City University

Fujifilm Corporation

Takahiro Iwao

Tamihide Matsunaga

Tomoki Kabeya

Shinji Mima

Isamu Ogawa

Daichi Onozato

Satoshi Kondo

Shota Mizuno

Toshihide Miyashita

An object is to prepare an intestinal organoid having a characteristic close to the small intestine of a living body, from a pluripotent stem cell. An intestinal organoid is prepared from a pluripotent stem cell, by the following steps of: (1) differentiating the pluripotent stem cell into an endoderm-like cell; (2) differentiating the endoderm-like cell obtained in step (1) into an intestinal stem cell-like cell; (3) culturing the intestinal stem cell-like cell obtained in step (2) in the presence of an epidermal growth factor, a fibroblast growth factor, a TGF β receptor inhibitor, a GSK-3 β inhibitor, and a ROCK inhibitor; (4) culturing the cell obtained in step (3) to form a spheroid; and (5) differentiating the spheroid formed in step (4) to form an intestinal organoid, wherein the differentiation includes culturing in the presence of an epidermal growth factor, a BMP inhibitor, and a Wnt signal activator. Also, a plane culture system is prepared by subjecting the cells constituting the intestinal organoid formed in step (5) to plane culture in the presence of an epidermal growth factor and a TGF β receptor inhibitor. A highly functional evaluation system having the villi structure is constructed by using air-liquid interface culture in the plane culture.

Method for preparing intestinal tract cell layer derived from pluripotent stem cell

An object of the present invention is to provide a new means capable of easily and efficiently preparing cells showing a function more similar to the function of intestinal epithelial cells of a living body. According to the present invention, pluripotent stem cells are induced to differentiate into intestinal stem cell-like cells by a method for inducing differentiation of pluripotent stem cells into intestinal epithelial cells, the method including a step (1) of differentiating pluripotent stem, cells into intestinal stem cell-like cells and a step (2) of differentiating the intestinal stem cell-like cells obtained in the step (1) into intestinal epithelial cell-like cells by using an MEK1 inhibitor, a DNA methylation inhibitor, a TGFβ receptor inhibitor, EGF, and a cAMP activator in combination.

Method for inducing differentiation of pluripotent stem cells into intestinal epithelial cells

An object of the present invention is to prepare a functional intestinal organoid from pluripotent stem cells. An intestinal organoid is prepared from pluripotent stem cells, by the following steps (1) to (4): (1) differentiating pluripotent stem cells into endoderm-like cells; (2) differentiating the endoderm-like cells obtained in step (1) into intestinal stem cell-like cells; (3) culturing the intestinal stem cell-like cells obtained in step (2) to form spheroids; and (4) differentiating the spheroids formed in step (3) to form an intestinal organoid, the step including culture in the presence of a MEK1/2 inhibitor, a DNA methylation inhibitor, a TGF-β receptor inhibitor, and a γ-secretase inhibitor, in addition to an epidermal growth factor, a BMP inhibitor, and a Wnt signal activator.

Method for producing intestinal organoid derived from pluripotent stem cells

It is an object to provide a culture method which is capable of maintaining and/or culturing iPS cell-derived intestinal stem cells, while maintaining the properties of intestinal stem cells. The induced pluripotent stem cell-derived intestinal stem cell-like cells are cultured in the presence of a GSK-3&#x3b2; inhibitor, a histone deacetylation inhibitor, and a serum replacement, or in the presence of a GSK-3&#x3b2; inhibitor and a serum replacement. Preferably, the culture is carried out under conditions in which one or more compounds selected from the group consisting of an epidermal growth factor, a TGF&#x3b2; receptor inhibitor and a fibroblast growth factor are further present.

Maintenance culture of induced pluripotent stem cell-derived intestinal stem cells

An object of the present invention is to provide a novel method which enables convenient preparation of cells exhibiting functions close to that of intestinal epithelial cells of living bodies, and use of the method. The differentiation of induced pluripotent stem cells into intestinal epithelial cells is induced by step of differentiating induced pluripotent stem cells into endoderm-like cells; step of differentiating the endoderm-like cells obtained in step into intestinal stem cell-like cells; and step of differentiating the intestinal stem cell-like cells obtained in step into intestinal epithelial cell-like cells, wherein step includes culture in the presence of a MEK1 inhibitor, a DNA methyltransferase inhibitor, a TGF-&#x3b2; receptor inhibitor, and EGF and under the condition that cAMP is supplied to the cells.

Growing community of inventors

Nagoya, Japan

Takahiro Iwao