In certain embodiments, the cells are further contacted with DA neuron lineage activators and inhibitors, for example, brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), Cyclic adenosine monophosphate (cAMP), Transforming growth factor beta (TGFβ, for example, TGFβ3), ascorbic acid (AA), and DAPT (which is also known as, N-L-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester LY-374973, N-S-phenylglycine t-butyl ester or N-S-phenylglycine t-butyl ester). In certain embodiments, the presently disclosed methods provide for advantages over methods of differentiating stem cells into midbrain DA cells that do not comprise an increase of a Wnt activating compound, for example, by reducing the expression of markers of immature progenitor cells, for example, PAX6, and producing cells that differentiate into functional Tyrosine Hydroxylase expressing mDA cells following engraftment. The presently disclosed subject matter relates to the discovery that midbrain dopamine (DA) neurons, and precursors thereof, can be differentiated from human stem cells by dual inhibition of SMAD signaling (for example, by inhibition of TGFβ/Activin-Nodal signaling and BMP signaling), along with the activation of Sonic Hedgehog (SHH) signaling, and activation of wingless (Wnt) signaling, wherein the concentration of a Wnt activating compound is increased about 4 days after initial contact of the cells to the SMAD inhibitors, SHH activator, and Wnt activator. The presently disclosed subject matter relates to midbrain dopamine (DA) neurons (mDA), and precursors thereof, derived from human stem cells at least in part by in vitro differentiation. Therefore there is a need for compositions and methods for differentiating neuronal progenitor cells to be used in treating neurodegenerative disorders such as Parkinson's disease.
However the results of these studies showed little in vivo capability to restore neuronal function and often resulted in the growth of unwanted tumors in the patients. Research and technological developments relating to directed differentiation of embryonic and somatic stem cells has taken place in the field of diseases of the central nervous system (CNS), such as for Huntington's, Alzheimer's, Parkinson's, and multiple sclerosis.
Previously, embryonic and somatic stem cells were used as therapeutics and model systems for neurodegenerative diseases. The presently disclosed subject matter relates to midbrain dopamine (DA) neurons, and precursors thereof, derived from human stem cells, and uses thereof for cell-based treatment of neurological disorders. 1, 2015, priority to each of which is claimed, and the contents of each of which are incorporated by reference in their entireties herein. This application is a Continuation of International Patent Application No.
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