Establishing topographic organization in three-dimensional tissue culture

Inventors

Studer, Lorenz • Cederquist, Gustav

Assignees

Memorial Sloan Kettering Cancer Center

Abstract

The present disclosure relates to methods and compositions for generating topographically organized tissues in vitro, for the resulting cultured tissue and components thereof, and for uses of such cultured tissue and its components in drug discovery, toxicology studies, and therapy.

Core Innovation

The invention relates to producing a topographically organized brain organoid in vitro by providing an organizer that is a source of a diffusible organizing agent and placing the organizer in apposition to an aggregate of undifferentiated or partially differentiated stem cells to form an organizer/aggregate complex. The organizer/aggregate complex is contacted with at least one inhibitor selected from BMP inhibitors, TGFβ/Activin-Nodal inhibitors, and Wnt inhibitors for at least 1 day, and then cultured in vitro for at least 20 days to form the topographically organized brain organoid.

The organizer releases the organizing agent to form a gradient concentration effective in generating cells in the organoid having different phenotypes at different distances from the organizer for at least 10 days. The organizing agent is selected from SHH signaling agonists, molecules that induce SHH protein expression, molecules that promote SHH activity, and a combination thereof. In the resulting organoid, distinct regions are defined by expression of FOXG1 and/or PAX6, GSH2 and contiguous PAX6, NKX2.1 and FOXG1, NKX2.2 and NKX2.1, NKX2.2, and NKX2.1 but not FOXG1.

In specific embodiments, the organizer comprises one or more genetically modified cells that express the organizing agent or one or more beads that release the organizing agent. In another embodiment, the organizer is comprised of human stem cells engineered to express hSHH and releases hSHH to form a gradient concentration effective in generating the brain organoid comprising at least distinct forebrain and posterior regions for at least 10 days, while the aggregate has a spheroid shape.

The document further includes region-defining transcription-factor markers used to identify cortex-like, LGE-like, MGE-like, and hypothalamus-like regions with anterior and ventro-posterior distinctions.

Claims Coverage

The partial content provides two independent claims covering methods for producing topographically organized brain organoids using an organizer that releases a diffusible SHH-related organizing agent gradient while inhibiting BMP, TGFβ/Activin-Nodal, and Wnt signaling for at least 1 day and culturing for at least 20 days, yielding regionally patterned organoids defined by specific transcription-factor markers.

Across the independent claims, the scope centers on an organizer/aggregate complex in which the organizer provides a diffusible SHH-related organizing agent and establishes an organizing-agent gradient effective for at least 10 days, while the complex is contacted with BMP/TGFβ-Activin-Nodal/Wnt inhibitors for at least 1 day and cultured for at least 20 days to yield topographically organized brain organoids containing multiple named region-like areas defined by specific transcription-factor marker combinations.

Stated Advantages

Documented Applications