US 12,404,489 B2
Establishing topographic organization in three-dimensional tissue culture
Lorenz Studer, New York, NY (US); and Gustav Cederquist, New York, NY (US)
Assigned to MEMORIAL SLOAN-KETTERING CANCER CENTER, New York, NY (US)
Filed by MEMORIAL SLOAN-KETTERING CANCER CENTER, New York, NY (US)
Filed on Jan. 27, 2020, as Appl. No. 16/773,341.
Application 16/773,341 is a continuation of application No. PCT/US2018/044296, filed on Jul. 30, 2018.
Claims priority of provisional application 62/538,350, filed on Jul. 28, 2017.
Prior Publication US 2020/0239841 A1, Jul. 30, 2020
Int. Cl. C12N 5/079 (2010.01); G01N 33/50 (2006.01)
CPC C12N 5/0618 (2013.01) [G01N 33/5082 (2013.01); C12N 2501/15 (2013.01); C12N 2501/155 (2013.01); C12N 2501/16 (2013.01); C12N 2501/41 (2013.01); C12N 2501/415 (2013.01); C12N 2503/02 (2013.01); C12N 2506/02 (2013.01); C12N 2513/00 (2013.01)] 17 Claims
OG exemplary drawing
 
1. A method of producing a topographically organized brain organoid, comprising:
(i) providing an organizer that is a source of a diffusible organizing agent;
(ii) placing the organizer in apposition to an aggregate of undifferentiated or partially differentiated stem cells to form an organizer/aggregate complex;
(iii) contacting the organizer/aggregate complex with at least one inhibitor selected from the group consisting of bone morphogenetic protein (BMP) inhibitors, transforming growth factor beta (TGFβ)/Activin-Nodal inhibitors, and Wingless (Wnt) inhibitors for at least 1 day; and
(iv) culturing the organizer/aggregate complex in vitro for at least 20 days to form the topographically organized brain organoid;
wherein the organizer comprises one or more genetically modified cells that express the organizing agent or one or more beads that release the organizing agent;
wherein the organizer releases the organizing agent to form a gradient concentration of the organizing agent effective in generating cells, in the organoid, having different phenotypes at different distances from the organizer for at least 10 days;
wherein the organizing agent is selected from the group consisting of SHH signaling agonists, molecules that induce SHH protein expression, molecules that promote SHH activity, and a combination thereof;
wherein the stem cells are selected from the group consisting of embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and a combination thereof;
wherein said brain organoid comprises:
(i) a cortex-like region comprising cells expressing FOXG1 and/or PAX6;
(ii) a lateral ganglionic eminence (LGE)-like region comprising cells expressing GSH2 and contiguous to a region expressing PAX6;
(iii) a medial ganglionic eminence (MGE)-like region comprising cells expressing NKX2.1 and FOXG1;
(iv) a hypothalamus-like region comprising cells expressing NKX2.2 and NKX2.1;
(v) an anterior hypothalamic-like region comprising cells expressing NKX2.2; and
(vi) a ventro-posterior hypothalamic-like region comprising cells expressing NKX2.1 but not FOXG1.