US 12,492,373 B2
Production method for nerve tissue
Atsushi Kuwahara, Kobe (JP); Suguru Yamasaki, Kobe (JP); Yasushi Hiramine, Kobe (JP); Yoshiki Sasai, Wako (JP); and Masayo Takahashi, Wako (JP)
Assigned to RACTHERA CO., LTD., Tokyo (JP); and RIKEN, Wako (JP)
Filed by Racthera Co., Ltd., Osaka (JP); and RIKEN, Wako (JP)
Filed on Dec. 22, 2021, as Appl. No. 17/559,458.
Application 17/559,458 is a continuation of application No. 15/521,334, granted, now 11,214,771, previously published as PCT/JP2015/080016, filed on Oct. 23, 2015.
Claims priority of application No. 2014-217867 (JP), filed on Oct. 24, 2014.
Prior Publication US 2022/0112457 A1, Apr. 14, 2022
Int. Cl. C12N 5/0793 (2010.01); A61K 35/12 (2015.01); A61K 35/30 (2015.01); A61L 27/00 (2006.01); A61L 27/38 (2006.01); C12N 5/079 (2010.01); C12N 5/0797 (2010.01); C12Q 1/02 (2006.01); G01N 33/50 (2006.01)
CPC C12N 5/062 (2013.01) [A61K 35/12 (2013.01); A61K 35/30 (2013.01); A61L 27/00 (2013.01); A61L 27/383 (2013.01); A61L 27/3895 (2013.01); C12N 5/0618 (2013.01); C12N 5/0619 (2013.01); C12N 5/0621 (2013.01); C12N 5/0623 (2013.01); C12Q 1/02 (2013.01); G01N 33/5014 (2013.01); C12N 2500/99 (2013.01); C12N 2501/115 (2013.01); C12N 2501/15 (2013.01); C12N 2501/155 (2013.01); C12N 2501/16 (2013.01); C12N 2501/40 (2013.01); C12N 2501/41 (2013.01); C12N 2501/415 (2013.01); C12N 2501/727 (2013.01); C12N 2506/45 (2013.01); C12N 2533/52 (2013.01)] 22 Claims
 
1. A method for producing cerebral cells or a cerebral tissue, comprising the following steps (1)-(3):
(1) a first step of culturing pluripotent stem cells in the absence of feeder cells and in a medium comprising (a) a Sonic hedgehog signal transduction pathway activating substance or a combination of a TGFβ family signal transduction pathway inhibiting substance and the Sonic hedgehog signal transduction pathway activating substance, and (b) a factor for maintaining an undifferentiated state of the pluripotent stem cells for a period of 0.5 hr-144 hr,
(2) a second step of culturing the cells obtained in the first step in suspension to form a cell aggregate, and
(3) a third step of culturing the aggregate obtained in the second step in suspension in the presence of a TGFβ family signal transduction pathway inhibiting substance and/or a Wnt signal transduction pathway inhibiting substance to obtain an aggregate containing cerebral cells or a cerebral tissue,
wherein the factor for maintaining an undifferentiated state of the pluripotent stem cells comprises a FGF signal transduction pathway activating substance and insulin,
wherein the FGF signal transduction pathway activating substance is bFGF,
wherein the TGFβ family signal transduction pathway inhibiting substance is Lefty, SB431542, A-83-01 or LDN193189,
wherein the Sonic hedgehog signal transduction pathway activating substance is Shh, SAG or Purmorphamine,
and wherein the Wnt signal transduction pathway inhibiting substance is IWR-1-endo.