	US 12,134,784 B2
	Method for preparing intestinal tract cell layer derived from pluripotent stem cell
Tamihide Matsunaga, Nagoya-chi (JP); Takahiro Iwao, Nagoya-chi (JP); Daichi Onozato, Nagoya-chi (JP); and Isamu Ogawa, Nagoya-chi (JP)
Assigned to PUBLIC UNIVERSITY CORPORATION NAGOYA CITY UNIVERSITY, Nagoya (JP)
Appl. No. 17/290,446
Filed by PUBLIC UNIVERSITY CORPORATION NAGOYA CITY UNIVERSITY, Nagoya (JP)
PCT Filed Oct. 31, 2019, PCT No. PCT/JP2019/042938 § 371(c)(1), (2) Date Apr. 30, 2021, PCT Pub. No. WO2020/091020, PCT Pub. Date May 7, 2020.
Claims priority of application No. 2018-207108 (JP), filed on Nov. 2, 2018; and application No. 2019-092336 (JP), filed on May 15, 2019.
Prior Publication US 2022/0033780 A1, Feb. 3, 2022
Int. Cl. C12N 5/071 (2010.01); A61L 27/38 (2006.01); G01N 33/50 (2006.01)

CPC C12N 5/0679 (2013.01) [A61L 27/3804 (2013.01); G01N 33/5014 (2013.01); G01N 33/5044 (2013.01); C12N 2501/01 (2013.01); C12N 2501/11 (2013.01); C12N 2501/115 (2013.01); C12N 2501/119 (2013.01); C12N 2501/15 (2013.01); C12N 2501/155 (2013.01); C12N 2501/415 (2013.01); C12N 2501/727 (2013.01); C12N 2506/45 (2013.01); C12N 2533/52 (2013.01)]

6 Claims

1. A preparation method for an intestinal tract cell layer from a pluripotent stem cell, comprising the following steps (1) to (6):

(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;

(5) differentiating the spheroid formed in step (4) to form a small intestinal organoid, wherein the differentiation comprises culturing in the presence of an epidermal growth factor, a BMP inhibitor, and a Wnt signal activator; and

(6) subjecting the cells constituting the small intestinal organoid formed in step (5) to plane culture carried out in the presence of an epidermal growth factor and a TGF β receptor inhibitor, wherein at least part of the plane culture of step (6) is air-liquid interface culture.