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US 12,734,196 B2Granted
Methods of differentiating neural cells and related compositions and methods of use
AssigneeAspen Neuroscience, Inc.
InventorsAndres Bratt-Leal, Jeanne Loring, Ha Tran, Roy Williams, Jim Mossman
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Abstract
The present disclosure provides methods of lineage specific differentiation of pluripotent stem cells, including induced pluripotent stem cells, into floor plate midbrain progenitor cells, determined dopamine (DA) neuron progenitor cells, and/or DA neurons. Also provided are compositions uses thereof, such as for treating neurodegenerative diseases and conditions, including Parkinson's disease.
Claims29
A method of differentiating neural cells, the method comprising:
- (a) performing a first incubation comprising culturing pluripotent stem cells in a non-adherent culture vessel under conditions to produce a cellular spheroid, wherein beginning at the initiation of the first incubation the cells are exposed to (i) an inhibitor of TGF-β/activin-Nodal signaling;
- (ii) at least one activator of Sonic Hedgehog (SHH) signaling;
- (iii) an inhibitor of bone morphogenetic protein (BMP) signaling;
- and (iv) an inhibitor of glycogen synthase kinase 3β (GSK3β) signaling, wherein day 0 is the first day on which the pluripotent stem cells are exposed to the inhibitor of TGF-β/activin-Nodal signaling and the inhibitor of bone morphogenetic protein (BMP) signaling;
- and
- (b) performing a second incubation comprising culturing cells of the spheroid in a substrate-coated culture vessel under conditions to neurally differentiate the cells.
3 more elements
A method of differentiating neural cells, the method comprising:
- (a) performing a first incubation comprising culturing pluripotent stem cells in a culture vessel that is coated with a substrate selected from one or more of laminin, collagen, entactin, heparin sulfate proteoglycans, and combinations thereof, wherein beginning at the initiation of the first incubation the cells are exposed to (i) an inhibitor of TGF-β/activin-Nodal signaling;
- (ii) at least one activator of Sonic Hedgehog (SHH) signaling;
- (iii) an inhibitor of bone morphogenetic protein (BMP) signaling;
- and (iv) an inhibitor of glycogen synthase kinase 3β (GSK3β) signaling, wherein day 0 is the first day on which the pluripotent stem cells are exposed to the inhibitor of TGF-β/activin-Nodal signaling and the inhibitor of bone morphogenetic protein (BMP) signaling, wherein the method does not comprise exposing the cells to fibroblast growth factor 8 (FGF8);
- and
- (b) performing a second incubation comprising culturing cells obtained in step (a) in a substrate-coated culture vessel under conditions to neurally differentiate the cells.
3 more elements
A method of differentiating neural cells, the method comprising:
- (a) performing a first incubation comprising culturing pluripotent stem cells in a culture vessel that is coated with a substrate selected from one or more of laminin, collagen, entactin, heparin sulfate proteoglycans, and combinations thereof, wherein beginning at the initiation of the first incubation the cells are exposed to (i) an inhibitor of TGF-β/activin-Nodal signaling;
- (ii) at least one activator of Sonic Hedgehog (SHH) signaling;
- (iii) an inhibitor of bone morphogenetic protein (BMP) signaling comprising LDN193189;
- and (iv) an inhibitor of glycogen synthase kinase 3β (GSK3β) signaling, wherein day 0 is the first day on which the pluripotent stem cells are exposed to the inhibitor of TGF-β/activin-Nodal signaling and the inhibitor of bone morphogenetic protein (BMP) signaling;
- and
- (b) performing a second incubation comprising culturing cells obtained in step (a) in a substrate-coated culture vessel under conditions to neurally differentiate the cells.
3 more elements
Dependencies are inferred from the claim text.