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US 12,577,530 B2Granted
Generation of a population of hindbrain cells and hindbrain-like organoids from pluripotent stem cells
AssigneesThe J. David Gladstone Institutes, a testamentary trust established under the Will of J. David Gladstone, The Regents of the University of California
InventorsJessica C. Butts, Todd C. McDevitt
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Abstract
Provided herein are methods of generating hindbrain cells, including respiratory hindbrain cells, from pluripotent stem cells. Also provided are methods of generating a three-dimensional organoid comprising a population of hindbrain cells including a heterogeneous population of interneurons.
Claims33
A method of generating a three-dimensional organoid comprising a population of hindbrain cells comprising a heterogeneous population of interneurons, the method comprising:
- a) treating a population of mammalian pluripotent stem cells (PSCs) in vitro with a Wingless-Int (WNT) signaling pathway activator;
- b) culturing the cells of a) in a first neural induction medium comprising a retinoic acid signaling pathway activator;
- and
- c) culturing the cells of b) in a second neural induction medium comprising the retinoic acid signaling pathway activator, a sonic hedgehog (Shh) signaling pathway activator;
- and a Notch signaling pathway inhibitor;
- wherein the culturing results in generation of the population of hindbrain cells comprising the heterogeneous population of interneurons, wherein the heterogeneous population of interneurons comprises V2a interneurons, V0 interneurons, chemosensing interneurons, or a combination thereof, and generation of the three-dimensional organoid,
- wherein said culturing the population of PSCs is performed on a three-dimensional substrate or a pyramidal mold.
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