US 12,378,518 B2
Differentiation induction from human pluripotent stem cells into hypothalamic neurons
Hidetaka Suga, Nagoya (JP); Koichiro Ogawa, Nagoya (JP); Takatoshi Kasai, Nagoya (JP); and Hiroshi Arima, Nagoya (JP)
Assigned to National University Corporation Nagoya University, Nagoya (JP)
Appl. No. 16/068,929
Filed by National University Corporation Nagoya University, Nagoya (JP)
PCT Filed Jan. 18, 2017, PCT No. PCT/JP2017/001544
§ 371(c)(1), (2) Date Jul. 10, 2018,
PCT Pub. No. WO2017/126551, PCT Pub. Date Jul. 27, 2017.
Claims priority of application No. 2016-010940 (JP), filed on Jan. 22, 2016.
Prior Publication US 2019/0010452 A1, Jan. 10, 2019
Int. Cl. C12N 5/0793 (2010.01); C12N 1/00 (2006.01); C12N 5/071 (2010.01)
CPC C12N 5/0619 (2013.01) [C12N 1/00 (2013.01); C12N 5/0616 (2013.01); C12N 2500/02 (2013.01); C12N 2501/13 (2013.01); C12N 2501/155 (2013.01); C12N 2501/41 (2013.01); C12N 2501/727 (2013.01); C12N 2501/999 (2013.01); C12N 2502/076 (2013.01); C12N 2502/081 (2013.01); C12N 2506/02 (2013.01); C12N 2506/03 (2013.01); C12N 2506/45 (2013.01); C12N 2527/00 (2013.01); C12N 2533/32 (2013.01); C12N 2533/52 (2013.01)] 7 Claims
 
1. A method for producing a cellular structure comprising dorsal hypothalamic tissue, the method comprising the steps of:
(1) culturing an aggregate of human pluripotent stem cells in suspension in a medium containing a bone morphogenetic protein signal transduction pathway activating substance and a substance acting on the Shh signaling pathway for 10 to 26 days,
(2) further culturing the cell aggregate obtained in the step (1) in a medium containing a substance acting on the Shh signaling pathway, but not containing a bone morphogenetic protein signal transduction pathway activating substance for 20 to 100 days, and
(3) dissociating the cell aggregate obtained in the step (2) into single cells and subjecting the dissociated single cells to dissociation culture for 40 to 110 days, in a medium comprising a ciliary neurotrophic factor (CNTF), a brain-derived neurotrophic factor (BDNF) or LM22A-4, a neurotrophin 3 (NT-3), a fetal bovine serum or a serum alternative, an N2 supplement, and a B27 supplement, and thereby obtaining a dorsal hypothalamic tissue comprising one or more neurons selected from the group consisting of vasopressin neurons secreting vasopressin, oxytocin neurons secreting oxytocin, thyrotropin releasing hormone neurons secreting thyrotropin releasing hormone, corticotropin releasing hormone neurons secreting corticotropin releasing hormone and neuropeptide Y neurons secreting neuropeptide Y; wherein the dissociation culture is performed under an oxygen partial pressure about between 35-60%;
wherein the bone morphogenetic protein signal transduction pathway activating substance in the step (1) is BMP4, and the concentration thereof in the medium is 0.1 nM to 2.0 nM,
wherein the substance acting on the Shh signaling pathway in the steps (1) and (2) is SAG, and the concentration thereof in the medium is 0.1 μM to 1.0 μM.