Stem cell-derived midbrain dopamine neurons, midbrain neurons and uses thereof

Abstract

The present disclosure provides methods for generating midbrain dopamine neurons (mD As) and precursors thereof, mDAs and precursors thereof generated by such methods and compositions comprising such cells, and uses thereof for preventing and / or treating neurological disorders. The present disclosure further provides methods of preventing, modeling, and / or treating at least one symptom in a subject having a neurological disorder.

Description

[0001] 072734.1886 PATENT

[0002] STEM CELL-DERIVED MIDBRAIN DOPAMINE NEURONS, MIDBRAIN NEURONS AND USES THEREOF

[0003] 1. CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority to U.S. Provisional Application No. 63 / 735,067, filed December 17, 2024, the content of which is hereby incorporated by reference in its entirety, and to which priority is claimed.

[0005] 2. GRANT FUNDING

[0006] This invention was made with government support under HD112917 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0007] 3. TECHNICAL FIELD

[0008] The present disclosure provides methods for generating midbrain dopamine (mDA) neurons and precursors thereof, as well as the mDA neurons and precursors thereof generated by such methods and compositions comprising such neurons and precursors. The present disclosure also provides uses of the mDA neurons and precursors as well as compositions comprising such neurons and precursors for preventing, modeling, and / or treating neurological disorders.

[0009] 4. BACKGROUND

[0010] Parkinson’s disease (PD) is characterized by the loss of mDA neurons that lead to well-known motor symptoms such as tremor, rigor, and bradykinesia (Lees, et al. Lancet 373, 2055-2066 (2009)). While other cell types such as enteric, olfactory or cortical neurons are also affected (Del Tredici, et al. Neuropathol Appl Neurobiol 42, 33-50 (2016)), mDA neurons remain the key focus for developing novel cell-based treatments (Barker, etal. Nature reviews Neurology 11, 492-503 (2015); Tabar, etal. Nat Rev Genet 15, 82-92 (2014)) and for PD disease modeling (Sanchez-Danes, etal. EMBO Mol Med 4, 380-395 (2012); Miller, etal. Cell stem cell 13, 691-705 (2013); Chung, et al. Stem Cell Reports 7, 664-677 (2016); Reinhardt, etal. Cell stem cell 12, 354-367 (2013); Chung, et al. Science 342, 983-987 (2013); Cooper, et al. Sci Transl Med 4, 141ral90 (2012)). Human pluripotent stem cells (hPSCs), comprising both human ES and iPS cells, have become the cell type of choice for deriving mDA neurons in vitro. Despite progress in human mDA derivation, there remains need for 072734.1886 PATENT improved methods for generating midbrain dopamine neurons that are suitable for treating neurodegenerative disorders such as Parkinson’s disease.

[0011] 5. SUMMARY

[0012] The present disclosure provides in vitro methods for inducing differentiation of stem cells. In certain embodiments, the in vitro methods for inducing differentiation comprise: contacting the stem cells with at least one inhibitor of wingless (Wnt) signaling for up to about 2 days, contacting the stem cells with at least one inhibitor of Small Mothers Against Decapentaplegic (SMAD) signaling and at least one activator of Sonic hedgehog (SHH) signaling; and contacting the stem cells with at least one activator of Wnt signaling to obtain a population of differentiated cells expressing at least one marker indicating a midbrain dopamine neuron or a precursor thereof. In certain embodiments, the stem cells are contacted with the at least one inhibitor of Wnt signaling for about 1 day or about 2 days. In certain embodiments, the cells are contacted with the at least one inhibitor of SMAD signaling for at least about 5 days. In certain embodiments, the cells are contacted with the at least one inhibitor of SMAD signaling for about 5 days, about 6 days, or about 7 days. In certain embodiments, the cells are contacted with the at least one activator of SHH signaling for at least about 5 days. In certain embodiments, the cells are contacted with the at least one activator of SHH signaling for about 5 days, about 6 days, or about 7 days. In certain embodiments, the cells are contacted with the at least one activator of Wnt signaling for at least about 5 days. In certain embodiments, the cells are contacted with the at least one activator of Wnt signaling for about 7 days, about 8 days, or about 9 days. In certain embodiments, the initial contact of the cells with the at least one activator of Wnt signaling is at least about 2 days from the initial contact of the cells with the at least one SMAD inhibitor. In certain embodiments, the initial contact of the cells with the at least one activator of Wnt signaling is about 2 days from the initial contact of the cells with the at least one SMAD inhibitor. In certain embodiments, the at least one inhibitor of SMAD signaling comprises an inhibitor of TGFp / Activin-Nodal signaling, an inhibitor of BMP signaling, or a combination thereof. In certain embodiments, the at least one inhibitor of TGFp / Activin- Nodal signaling comprises an inhibitor of ALK5. In certain embodiments, the at least one inhibitor of TGFp / Activin-Nodal signaling is selected from the group consisting of SB431542, derivatives of SB431542, and combinations thereof. In certain embodiments, the derivative of SB431542 comprises A83-01. In certain embodiments, the at least one inhibitor of TGFp / Activin-Nodal signaling comprises SB431542. In certain embodiments, the at least 072734.1886

[0013] PATENT one inhibitor of BMP signaling is selected from the group consisting of LDN193189, Noggin, dorsomorphin, derivatives of LDN193189, derivatives of Noggin, derivatives of dorsomorphin, and combinations thereof. In certain embodiments, the at least one inhibitor of BMP comprises LDN-193189. In certain embodiments, the at least one activator of Wnt signaling comprises an inhibitor of glycogen synthase kinase 3p (GSK3P) signaling. In certain embodiments, the at least one activator of Wnt signaling is selected from the group consisting of CHIR99021, CHIR98014, AMBMP hydrochloride, LP 922056, Lithium, deoxycholic acid, BIO, SB-216763, Wnt3A, Wntl, Wnt5a, derivatives thereof, and combinations thereof. In certain embodiments, the at least one activator of Wnt signaling comprises CHIR99021. In certain embodiments, the at least one activator of SHH signaling is selected from the group consisting of SHH proteins, Smoothened agonists (SAG), and combinations thereof. In certain embodiments, the SHH protein is selected from the group consisting of recombinant SHHs, modified N-terminal SHHs, and combinations thereof. In certain embodiments, the modified N-terminal SHH comprises two Isoleucines at the N- terminus. In certain embodiments, the modified N-terminal SHH has at least about 90% sequence identity to an un-modified N-terminal SHH. In certain embodiments, the unmodified N-terminal SHH is a un-modified mouse N-terminal SHH or a un-modified human N-terminal SHH. In certain embodiments, the modified N-terminal SHH comprises SHH C25II. In certain embodiments, the SAG comprises purmorphamine. In certain embodiments, the at least one marker indicating a midbrain dopamine neuron or a precursor thereof is selected from the group consisting of EN1, OTX2, TH, NURR1, FOXA2, PITX3, LMX1A, LM03, SNCA, ADCAP1, CHRNA4, SOX6, DAT, VMAT2, WNT1, GIRK2, and combinations thereof. In certain embodiments, the differentiated cells do no express at least one marker selected from the group consisting of PAX6, EMX2, LHX2, SMA, SIX1, PITX2, SIM1, POU4F1, PHOX2A, BARHL1, BARHL2, GBX2, HOXA1, HOXA2, HOXB1, HOXB2, POU5F1, NANOG, and combinations thereof.

[0014] In certain embodiments, the methods further comprise isolating cells that express at least one positive marker and do not express at least one negative marker. In certain embodiments, the at least one positive marker is selected from the group consisting of PITX3, ALDH1A1, TH, and combinations thereof. In certain embodiments, the at least one positive marker comprises ALDH1A1. In certain embodiments, the at least one negative marker is selected from CD49e, CD340, and combination thereof. In certain embodiments, the stem cells are pluripotent stem cells. In certain embodiments, the stem cells are selected from the group consisting of nonembryonic stem cells, embryonic stem cells, induced pluripotent stem 072734.1886 PATENT cells, and combinations thereof. In certain embodiments, the stem cells are human stem cells, non-human primate stem cells, or rodent stem cells. In certain embodiments, the stem cells are human stem cells.

[0015] The present disclosure further provides cell populations of in vitro differentiated cells, wherein the in vitro differentiated cells are obtained by a method disclosed herein.

[0016] The present disclosure further provides compositions comprising the cell populations disclosed herein. In certain embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

[0017] The present disclosure further provides kits for inducing differentiation of stem cells to midbrain dopamine neurons or precursors thereof, comprising: at least one inhibitor of Wnt signaling, at least one inhibitor of SMAD signaling, at least one activator of SHH signaling, and at least one activator of Wnt signaling. In certain embodiments, the kit further comprises instructions for inducing differentiation of the stem cells into a population of differentiated cells that express at least one marker indicating a midbrain dopamine neuron or a precursor thereof.

[0018] The present disclosure further provides methods of preventing, modeling, and / or treating at least one symptom in a subject having a neurological disorder, comprising administering to the subject an effective amount of one of the followings: a cell population disclosed herein, or a composition disclosed herein. In certain embodiments, the neurological disorder is characterized by reduction of midbrain dopamine neuron function. In certain embodiments, the reduction of midbrain dopamine neuron function is age related. In certain embodiments, the neurological disorder is selected from the group consisting of Parkinsonism, Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, multiple sclerosis, and combinations thereof. In certain embodiments, the neurological disorder is selected from the group consisting of Parkinsonism, Parkinson’s disease, and combinations thereof. In certain embodiments, the symptom for a neurological disorder is selected from the group consisting of tremor, bradykinesia, flexed posture, postural instability, rigidity, dysphagia, and dementia. In certain embodiments, the cell populations or compositions disclosed herein are for use in preventing, modeling, and / or treating at least one symptom in a subj ect having a neurological disorder in a subj ect. In certain embodiments, the neurological disorder is characterized by reduction of midbrain dopamine neuron function. In certain embodiments, the reduction of midbrain dopamine neuron function is age related. In certain embodiments, the neurological disorder is selected from the group consisting of Parkinsonism, Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, multiple 072734.1886

[0019] PATENT sclerosis, and combinations thereof. In certain embodiments, the neurological disorder is selected from the group consisting of Parkinsonism, Parkinson’s disease, and combinations thereof. In certain embodiments, the symptom for a neurological disorder is selected from the group consisting of tremor, bradykinesia, flexed posture, postural instability, rigidity, dysphagia, and dementia.

[0020] 6. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG. 1 shows schematic illustrating protocols for differentiating stem cells to mDA neurons or precursors. Inhibition of Wnt signaling is applied on days 0-2 as an “inverted bump”.

[0022] FIGS. 2A-2C show day 11 immunocytochemistry (ICC) of cultures obtained using the “inverted bump” protocol. FIG. 2A shows day 11 ICC from a first experiment showing cultures stained for EN 1 and F OXA2 at D 11 with either the original “boost” or new “inverted bump” protocols. FIGS. 2B-2C show day 11 ICC from a second experiment showing cultures stained for OTX2, EN1 and Islet 1 (FIG. 2B) or GBX2 and FOXA2 (FIG. 2C) with either the original “boost” or new “inverted bump” protocols.

[0023] FIGS. 3A-3E show the “inverted bump” lock-in approach for the generation of midbrain dopamine (mDA) neurons. FIG. 3A illustrates schematics showing the original “boost” protocol for mDA generation (upper panel), which utilizes Wnt activation with 1.0 pM CHIR99021 DO-4 followed by 6.0 pM CHIR99021 D4-D10, and the new “inverted bump” protocol for mDA generation (lower panel), which utilizes Wnt inhibition (e.g., 2.0 pM XAV939) DO-2 followed by Wnt activation with 6.0 pM CHIR99021 D2-10. FIG. 3B shows day 11 immunocytochemistry (ICC) showing cultures stained for midbrain markers OTX2, EN1 and FOXA2 after reseeding at Dl l with either the original “boost” or new “inverted bump” protocols. FIG. 3C shows day 25 flow cytometry data showing induction of PITX3+CD99+ mDA precursors. FIG. 3D shows D30 ICC showing mDA neurons positive for PITX3, TH and FOXA2. FIG. 3E shows quanitification of DI 1 ICC.

[0024] 7. DETAILED DESCRIPTION

[0025] The present disclosure provides methods for generating mDA neurons and precursors thereof, as well as mDA neurons and precursors thereof generated by such methods, and compositions comprising such neurons and precursors. The present disclosure also provides uses of the mDA neurons and precursors as well as compositions comprising such neurons and precursors for preventing, modeling, and / or treating neurological disorders. For example, 072734.1886

[0026] PATENT but not by way of limitation, the present disclosure provides improved protocols for neural induction and mDA neuron differentiation from stem cells (e.g., human pluripotent stem cells (hPSCs)), including clinical grade protocols. Having access to improved mDA neuron differentiation protocols enables the field to use lower cell numbers, achieve more complete mDA neuron restoration and reduce potential side effects. Accordingly, the presently disclosed protocols can improve efficiency and / or safety by increasing the purity of potential grafts, as contaminating cell types in grafts can be a potential source of inefficiency and / or safety risk. Finally, the presently disclosed protocols enhance the precision and reproducibility of mDA neurons in modeling human disease in a dish. The presently disclosed protocols improve faithfulness and robustness of mDA differentiation. The presently disclosed protocols can be widely adapted and can be of broad use.

[0027] Non-limiting embodiments of the presently disclosed subject matter are described by the present specification and Examples.

[0028] For purposes of clarity of disclosure and not by way of limitation, the detailed description is divided into the following subsections:

[0029] 7.1. Definitions;

[0030] 7.2. Methods of Differentiating Stem Cells;

[0031] 7.3. Cell Populations and Compositions;

[0032] 7.4. Methods of Preventing, Modeling, and / or Treating Neurological Disorders;

[0033] 7.5 Methods of Identifying Therapeutic Compounds; and

[0034] 7.6. Kits.

[0035] 7.1. Definitions

[0036] The terms used in this specification generally have their ordinary meanings in the art, within the context of the present disclosure and in the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the compositions and methods of the present disclosure and how to make and use them.

[0037] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, e.g., up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or 072734.1886 PATENT processes, the term can mean within an order of magnitude, e.g., within 5 -fold, or within 2- fold, of a value.

[0038] As used herein, the term “signaling” in reference to a “signal transduction protein” refers to a protein that is activated or otherwise affected by ligand binding to a membrane receptor protein or some other stimulus. Examples of signal transduction protein include, but are not limited to, a SMAD, a Wingless (Wnt) complex protein, including beta-catenin, NOTCH, transforming growth factor beta (TGFP), Activin, Nodal, glycogen synthase kinase 3P (GSK3P) proteins, bone morphogenetic proteins (BMP) and fibroblast growth factors (FGF). For many cell surface receptors or internal receptor proteins, ligand-receptor interactions are not directly linked to the cell’ s response. The ligand activated receptor can first interact with other proteins inside the cell before the ultimate physiological effect of the ligand on the cell’s behavior is produced. Often, the behavior of a chain of several interacting cell proteins is altered following receptor activation or inhibition. The entire set of cell changes induced by receptor activation is called a signal transduction mechanism or signaling pathway.

[0039] As used herein, the term “signals” refer to internal and external factors that control changes in cell structure and function. They can be chemical or physical in nature.

[0040] As used herein, the term “ligands” refers to molecules and proteins that bind to receptors, e.g., transforming growth factor-beta (TFGP), Activin, Nodal, bone morphogenic proteins (BMPs), etc.

[0041] “Inhibitor” as used herein, refers to a compound or molecule (e.g., small molecule, peptide, peptidomimetic, natural compound, siRNA, anti-sense nucleic acid, aptamer, or antibody) that interferes with (e.g., reduces, decreases, suppresses, eliminates, or blocks) the signaling function of the molecule or pathway (e.g., Wnt signaling pathway, and SMAD signaling). An inhibitor can be any compound or molecule that changes any activity of a named protein (signaling molecule, any molecule involved with the named signaling molecule, a named associated molecule, such as a glycogen synthase kinase 3p (GSK3P)). (e.g., including, but not limited to, the signaling molecules described herein). For example, an inhibitor of SMAD signaling can function, for example, via directly contacting SMAD, contacting SMAD mRNA, causing conformational changes of SMAD, decreasing SMAD protein levels, or interfering with SMAD interactions with signaling partners, and affecting the expression of SMAD target genes.

[0042] Inhibitors also include molecules that indirectly regulate biological activity, for example, SMAD biological activity, by intercepting upstream signaling molecules (e.g., 072734.1886 PATENT within the extracellular domain, examples of a signaling molecule and an effect include: Noggin which sequesters bone morphogenic proteins, inhibiting activation of ALK receptors 1,2,3, and 6, thus preventing downstream SMAD activation. Likewise, Chordin, Cerberus, Follistatin, similarly sequester extracellular activators of SMAD signaling. Bambi, a transmembrane protein, also acts as a pseudo-receptor to sequester extracellular TGFp signaling molecules). Antibodies that block upstream or downstream proteins are contemplated for use to neutralize extracellular activators of protein signaling, and the like. Although the foregoing example relates to SMAD signaling inhibition, similar or analogous mechanisms can be used to inhibit other signaling molecules. Examples of inhibitors include, but are not limited to: LDN193189 (LDN) and SB431542 (SB) (LSB) for SMAD signaling inhibition, and IWP2 for Wnt inhibition. Inhibitors are described in terms of competitive inhibition (binds to the active site in a manner as to exclude or reduce the binding of another known binding compound) and allosteric inhibition (binds to a protein in a manner to change the protein conformation in a manner which interferes with binding of a compound to that protein’ s active site) in addition to inhibition induced by binding to and affecting a molecule upstream from the named signaling molecule that in turn causes inhibition of the named molecule. An inhibitor can be a “direct inhibitor” that inhibits a signaling target or a signaling target pathway by actually contacting the signaling target.

[0043] “Activators,” as used herein, refer to compounds that increase, induce, stimulate, activate, facilitate, or enhance activation the signaling function of the molecule or pathway, e.g., Wnt signaling, SHH signaling, etc.

[0044] As used herein, the term “Wnt” or “wingless” in reference to a ligand refers to a group of secreted proteins (e.g., integration 1 in humans) that are capable of interacting with a Wnt receptor, such as a receptor in the Frizzled and LRPDerailed / RYK receptor family. As used herein, the term “a Wnt or wingless signaling pathway refers to a signaling pathway composed of Wnt family ligands and Wnt family receptors, such as Frizzled and LRPDerailed / RYK receptors, mediated with or without P-catenin. The Wnt signaling pathway include canonical Wnt signaling (e.g., mediation by P-catenin) and non-canonical Wnt signaling (mediation without P-catenin).

[0045] As used herein, the term “derivative” refers to a chemical compound with a similar core structure.

[0046] As used herein, the term “a population of cells” or “a cell population” refers to a group of at least two cells. In non-limiting examples, a cell population can include at least about 10, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, 072734.1886

[0047] PATENT at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000 cells. The population can be a pure population comprising one cell type, such as a population of midbrain DA precursors, or a population of undifferentiated stem cells, e.g., a population of A9 subtype midbrain dopamine neurons. Alternatively, the population can comprise more than one cell type, for example a mixed cell population, e.g., a cell population mixed of A9 subtype midbrain dopamine neurons and A10 subtype midbrain dopamine neurons.

[0048] As used herein, the term “stem cell” refers to a cell with the ability to divide for indefinite periods in culture and to give rise to specialized cells.

[0049] As used herein, the term “embryonic stem cell” and “ESC” refer to a primitive (undifferentiated) cell that is derived from preimplantation-stage embryo, capable of dividing without differentiating for a prolonged period in culture, and are known to develop into cells and tissues of the three primary germ layers. A human embryonic stem cell refers to an embryonic stem cell that is from a human embryo. As used herein, the term “human embryonic stem cell” or “hESC” refers to a type of pluripotent stem cells derived from early stage human embryos, up to and including the blastocyst stage, that is capable of dividing without differentiating for a prolonged period in culture, and are known to develop into cells and tissues of the three primary germ layers.

[0050] As used herein, the term “embryonic stem cell line” refers to a population of embryonic stem cells which have been cultured under in vitro conditions that allow proliferation without differentiation for up to days, months to years.

[0051] As used herein, the term “pluripotent” refers to an ability to develop into the three developmental germ layers of the organism including endoderm, mesoderm, and ectoderm.

[0052] As used herein, the term “totipotent” refers to an ability to give rise to all the cell types of the body plus all of the cell types that make up the extraembryonic tissues such as the placenta.

[0053] As used herein, the term “multipotent” refers to an ability to develop into more than one cell type of the body.

[0054] As used herein, the term “induced pluripotent stem cell” or “iPSC” refers to a type of pluripotent stem cell formed by the introduction of certain embryonic genes (such as but not limited to OCT4, SOX2, and KLF4 transgenes) (see, for example, Takahashi and Yamanaka Cell 126, 663-676 (2006), herein incorporated by reference) into a somatic cell.

[0055] As used herein, the term “neuron” refers to a nerve cell, the principal functional units of the nervous system. A neuron consists of a cell body and its processes - an axon and at 072734.1886 PATENT least one dendrite. Neurons transmit information to other neurons or cells by releasing neurotransmitters at synapses.

[0056] As used herein, the term “differentiation” refers to a process whereby an unspecialized embryonic cell acquires the features of a specialized cell such as a neuron, heart, liver, or muscle cell. Differentiation is controlled by the interaction of a cell’s genes with the physical and chemical conditions outside the cell, usually through signaling pathways involving proteins embedded in the cell surface.

[0057] As used herein, the term “directed differentiation” refers to a manipulation of stem cell culture conditions to induce differentiation into a particular (for example, desired) cell type, such as midbrain dopamine neurons or precursors thereof. In references to a stem cell, “directed differentiation” refers to the use of small molecules, growth factor proteins, and other growth conditions to promote the transition of a stem cell from the pluripotent state into a more mature or specialized cell fate.

[0058] As used herein, the term “inducing differentiation” in reference to a cell refers to changing the default cell type (genotype and / or phenotype) to a non-default cell type (genotype and / or phenotype). Thus, “inducing differentiation in a stem cell” refers to inducing the stem cell (e.g., human stem cell) to divide into progeny cells with characteristics that are different from the stem cell, such as genotype (e.g., change in gene expression as determined by genetic analysis such as a microarray) and / or phenotype (e.g., change in expression of a protein marker of mDA neurons or precursors thereof, such as EN1, OTX2, TH, NURR1, FOXA2, LMX1A, PITX3, LM03, SNCA, ADCAP1, CHRNA4, ALDH1A1, SOX6, WNT1, DAT, VMAT2, and GIRK2).

[0059] As used herein, the term “cell culture” refers to a growth of cells in vitro in an artificial medium for research or medical treatment.

[0060] As used herein, the term “culture medium” refers to a liquid that covers cells in a culture vessel, such as a Petri plate, a multi-well plate, and the like, and contains nutrients to nourish and support the cells. Culture medium can also include growth factors added to produce desired changes in the cells.

[0061] As used herein, the term “contacting” a cell or cells with a compound (e.g., at least one inhibitor, activator, and / or inducer) refers to providing the compound in a location that permits the cell or cells access to the compound. The contacting can be accomplished using any suitable method. For example, contacting can be accomplished by adding the compound, in concentrated form, to a cell or population of cells, for example in the context of a cell 072734.1886

[0062] PATENT culture, to achieve the desired concentration. Contacting can also be accomplished by including the compound as a component of a formulated culture medium.

[0063] As used herein, the term “zzz vitro" refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments exemplified, but are not limited to, test tubes and cell cultures.

[0064] As used herein, the term “zzz vivo" refers to the natural environment (e.g., an animal or a cell) and to processes or reactions that occur within a natural environment, such as embryonic development, cell differentiation, neural tube formation, etc.

[0065] As used herein, the term “expressing” in relation to a gene or protein refers to making an mRNA or protein which can be observed using assays such as microarray assays, antibody staining assays, and the like.

[0066] As used herein, the term “marker” or “cell marker” refers to gene or protein that identifies a particular cell or cell type. A marker for a cell may not be limited to one marker, markers can refer to a “pattern” of markers such that a designated group of markers can identity a cell or cell type from another cell or cell type.

[0067] As used herein, the term “derived from” or “established from” or “differentiated from” when made in reference to any cell disclosed herein refers to a cell that was obtained from (e.g., isolated, purified, etc.) an ultimate parent cell in a cell line, tissue (such as a dissociated embryo, or fluids using any manipulation, such as, without limitation, single cell isolation, culture in vitro, treatment and / or mutagenesis using for example proteins, chemicals, radiation, infection with virus, transfection with DNA sequences, such as with a morphogen, etc., selection (such as by serial culture) of any cell that is contained in cultured parent cells. A derived cell can be selected from a mixed population by virtue of response to a growth factor, cytokine, selected progression of cytokine treatments, adhesiveness, lack of adhesiveness, sorting procedure, and the like.

[0068] An “individual” or “subject” herein is a vertebrate, such as a human or non-human animal, for example, a mammal. Mammals include, but are not limited to, humans, non- human primates, farm animals, sport animals, rodents and pets. Non-limiting examples of non-human animal subjects include rodents such as mice, rats, hamsters, and guinea pigs; rabbits; dogs; cats; sheep; pigs; goats; cattle; horses; and non-human primates such as apes and monkeys.

[0069] As used herein, the term “disease” refers to any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. 072734.1886

[0070] PATENT

[0071] As used herein, the term “treating” or “treatment” refers to clinical intervention in an attempt to alter the disease course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Therapeutic effects of treatment include, without limitation, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastases, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. By preventing progression of a disease or disorder, a treatment can prevent deterioration due to a disorder in an affected or diagnosed subject or a subject suspected of having the disorder, but also a treatment can prevent the onset of the disorder or a symptom of the disorder in a subject at risk for the disorder or suspected of having the disorder.

[0072] 7.2. Method of Differentiating Stem Cells

[0073] The present disclosure provides methods for inducing differentiation of stem cells, comprising contacting stem cells with at least one inhibitor of Small Mothers Against Decapentaplegic (SMAD) signaling (referred to as “SMAD inhibitor”), at least one inhibitor of wingless (Wnt) signaling (referred to as “Wnt inhibitor”), at least one activator of Sonic hedgehog (SHH) signaling (referred to as “SHH activator”), and at least one activator of Wnt signaling (referred to as “Wnt activator”), to obtain a cell population comprising differentiated cells expressing at least one marker indicating a mDA neuron or a precursor thereof.

[0074] 7.2.1. Stem Cells

[0075] The presently disclosed subject matter provides in vitro methods for inducing differentiation of stem cells to produce mDA neurons and precursors thereof. In certain embodiments, the stem cells are pluripotent stem cells. In certain embodiments, the pluripotent stem cells are selected from embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and combinations thereof. In certain embodiments, the stem cells are multipotent stem cells. Non-limiting examples of stem cells that can be used with the presently disclosed methods include nonembryonic stem cells, embryonic stem cells, induced nonembryonic pluripotent cells, and engineered pluripotent cells. In certain embodiments, the stem cells are human stem cells. Non-limiting examples of human stem cells include human embryonic stem cells (hESC), human pluripotent stem cell (hPSC), human induced pluripotent stem cells (hiPSC), human parthenogenetic stem cells, primordial germ cell-like pluripotent stem cells, epiblast stem cells, F-class pluripotent stem cells, somatic stem cells, cancer stem cells, or any other cell capable of lineage specific differentiation. In certain 072734.1886

[0076] PATENT embodiments, the stem cell is a human embryonic stem cell (hESC). In certain embodiments, the stem cell is a human induced pluripotent stem cell (hiPSC). In certain embodiments, the stem cells are non-human stem cells. In certain embodiments, the stem cell is a nonhuman primate stem cell. In certain embodiments, the stem cell is a rodent stem cell.

[0077] In certain embodiments, the stem cell or a progeny cell thereof contains an introduced heterologous nucleic acid, where said nucleic acid may encode a desired nucleic acid or protein product or have informational value (see, for example, U.S. Patent No. 6,312,911, which is incorporated by reference in its entirety). Non-limiting examples of protein products include markers detectable via in vivo imaging studies, for example receptors or other cell membrane proteins. Non-limiting examples of markers include fluorescent proteins (such as green fluorescent protein (GFP), blue fluorescent protein (EBFP, EBFP2, Azurite, mKalamal), cyan fluorescent protein (ECFP, Cerulean, CyPet, mTurquoise2), and yellow fluorescent protein derivatives (YFP, Citrine, Venus, YPet, EYFP)), P-galactosidase (LacZ), chloramphenicol acetyltransferase (cat), neomycin phosphotransferase (neo), enzymes (such as oxidases and peroxidases), and antigenic molecules. As used herein, the terms “reporter gene” or “reporter construct” refer to genetic constructs comprising a nucleic acid encoding a protein that is easily detectable or easily assayable, such as a colored protein, fluorescent protein such as GFP or an enzyme such as beta-galactosidase (lacZ gene). In certain embodiments, the reporter can be driven by a recombinant promoter of a premature postmitotic mDA neuron marker gene, for example, NURR1.

[0078] 7.2.2. SMAD Inhibitors

[0079] Non-limiting examples of SMAD inhibitors include inhibitors of transforming growth factor beta (TGFP) / Activin-Nodal signaling (referred to as “TGFp / Activin-Nodal inhibitor”), and inhibitors of bone morphogenetic proteins (BMP) signaling. In certain embodiments, the methods disclosed herein comprise contacting a population of stem cells (e.g., human stem cells) with an effective amount(s) of one or more inhibitor of transforming growth factor beta (TGFP) / Activin-Nodal signaling. In certain embodiments, the inhibitor of TGFp / Activin- Nodal signaling neutralizes the ligands including TGFPs, bone morphogenetic protines (BMPs), Nodal, and activins, and / or block their signal pathways through blocking the receptors and downstream effectors. Non-limiting examples of TGFp / Activin-Nodal inhibitors include those disclosed in WO / 2010 / 096496, WO / 2011 / 149762, WO / 2013 / 067362, WO / 2014 / 176606, WO / 2015 / 077648, Chambers et al., Nat Biotechnol. 2009 Mar;27(3):275-80, Kriks et al., Nature. 2011 Nov 6;480(7378):547-51, and Chambers et al., Nat Biotechnol 2012 Jul l;30(7):715-20 (2012), all of which are incorporated by 072734.1886

[0080] PATENT reference in their entireties herein for all purposes. In certain embodiments, the at least one TGFp / Activin-Nodal inhibitor is selected from inhibitors of ALK5, inhibitors of ALKA, inhibitors of ALK7, and combinations thereof). In certain embodiments, the TGFp / Activin- Nodal inhibitor comprises an inhibitor of ALK5. In certain embodiments, the TGFp / Activin- Nodal inhibitor stimulates SMAD phosphorylation of Smad2 and Smad3. In certain embodiments, the inhibitor of TGFp / Activin-Nodal signaling is a small molecule selected from SB431542, derivatives thereof, and mixtures thereof. “SB431542” refers to a molecule with a number CAS 301836-41-9, a molecular formula of C22H18N4O3, and a name of 4-[4- (l,3-benzodioxol-5-yl)-5-(2-pyridinyl)-lH-imidazol-2-yl]-benzamide, for example, see structure below:

[0081] In certain embodiments, the at least one inhibitor of SMAD signaling comprises at least one inhibitor of TGFp / Activin-Nodal signaling. In certain embodiments, the TGFp / Activin-Nodal inhibitor comprises SB431542. In certain embodiments, the TGFp / Activin-Nodal inhibitor comprises a derivative of SB431542. In certain embodiments, the derivative of SB431542 is A83-01.

[0082] The stem cells can be contacted with the one or more inhibitor of TGFp / Activin-Nodal signaling for at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, at least about 21 days, at least about 22 days, at least about 23 days, at least about 24 days, at least about 25 days, at least about 26 days, at least about 27 days, at least about 28 days, at least about 29 days, or at least about 30 days, to produce a population of mDA neurons or precursors thereof. In certain embodiments, the stem cells are contacted with the one or more inhibitor of TGFp / Activin-Nodal signaling for up to about 3 days, up to about 4 days, up to about 5 days, up to about 6 days, up to about 7 days, up to about 8 days, up to about 9 days, up to about 10 days, up to about 11 days, up to about 12 days, up to about 13 days, up to about 14 days, up to about 15 days, up to about 16 072734.1886

[0083] PATENT days, up to about 17 days, up to about 18 days, up to about 19 days, up to about 20 days, up to about 21 days, up to about 22 days, up to about 23 days, up to about 24 days, up to about 25 days, up to about 26 days, up to about 27 days, up to about 28 days, up to about 29 days, or up to about 30 days, to produce a population of mDA neurons or precursors thereof. In certain embodiments, the stem cells are contacted with the one or more inhibitor of TGFp / Activin-Nodal signaling for between about 4 days and about 30 days, between about 4 days to about 27 days, between about 4 days and about 26 days, between about 4 days and about 25 days, between about 4 days and about 24 days, between about 4 days and about 20 days, between about 4 days and about 15 days, between about 4 days and about 10 days, between about 5 days and about 15 days, between about 5 days and about 10 days, between about 10 days and about 15 days, between about 15 days and about 20 days, between about 10 days and about 20 days, between about 20 days and about 25 days, or between about 25 days and about 30 days, to produce a population of mDA neurons or precursors thereof. In certain embodiments, the stem cells are contacted with the one or more inhibitor of TGFp / Activin-Nodal signaling for between 10 days and about 15 days, to produce a population of mDA neurons or precursors thereof. In certain embodiments, the stem cells are contacted with the one or more inhibitor of TGFp / Activin-Nodal signaling for about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, or about 30 day, to produce a population of mDA neurons or precursors thereof. In certain embodiments, the stem cells are contacted with the one or more inhibitor of TGFp / Activin-Nodal signaling for about 11 days, to produce a population of mDA neurons or precursors thereof.

[0084] In certain embodiments, the stem cells are contacted with the one or more inhibitor of TGFp / Activin-Nodal signaling in a concentration of from about 1 nM to about 300 nM, from about 5 nM to about 250 nM, from about 10 nM to about 200 nM, from about 10 nM to about 50 nM, from about 50 nM to about 150 nM, from about 80 nM to about 120 nM, from about 90 nM to about 110 nM, from about 50 nM to about 100 nM, from about 100 nM to about 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM, or from about 250 nM to about 300 nM. In certain embodiments, the stem cells are contacted with the one or more inhibitor of TGFp / Activin-Nodal signaling in a concentration of from about 80 nM to about 120 nM. In certain embodiments, the stem cells are contacted with the one 072734.1886

[0085] PATENT or more inhibitor of TGFp / Activin-Nodal signaling in a concentration of about 100 nM. In certain embodiments, the stem cells are contacted with the one or more inhibitor of TGFp / Activin-Nodal signaling in any one of the above-described concentrations daily. In certain embodiments, the stem cells are contacted with the one or more inhibitor of TGFp / Activin-Nodal signaling in a concentration of about 100 nM daily.

[0086] In certain embodiments, the at least one SMAD inhibitor comprises an inhibitor of BMP signaling (referred to as “BMP inhibitor”). Non-limiting examples of BMP inhibitors include those disclosed in WO2011 / 149762, Chambers et al., Nat Biotechnol. 2009 Mar;27(3):275-80, Kriks et al., Nature. 2011 Nov 6;480(7378):547-51, and Chambers et al., Nat Biotechnol . 2012 Jul l;30(7):715-20, all of which are incorporated by reference in their entireties. In certain embodiments, the BMP inhibitor is a small molecule selected from LDN193189, Noggin, dorsomorphin, derivatives thereof, and mixtures thereof. “LDN193189” refers to a small molecule DM-3189, IUPAC name 4-(6-(4-(piperazin-l- yl)phenyl)pyrazolo[l,5-a]pyrimidin-3-yl)quinoline, with a chemical formula of C25H22N6 with the following formula.

[0087] LDN193189 is capable of functioning as a SMAD signaling inhibitor. LDN193189 is also highly potent small-molecule inhibitor of ALK2, ALK3, and ALK6, protein tyrosine kinases (PTK), inhibiting signaling of members of the ALK1 and ALK3 families of type I TGFP receptors, resulting in the inhibition of the transmission of multiple biological signals, including the bone morphogenetic proteins (BMP) BMP2, BMP4, BMP6, BMP7, and Activin cytokine signals and subsequently SMAD phosphorylation of Smadl, Smad5, and Smad8 (Yu et al. (2008) Nat Med 14: 1363-1369; Cuny et al. (2008) Bioorg. Med. Chem. Lett. 18: 4388-4392, herein incorporated by reference).

[0088] In certain embodiments, the BMP inhibitor comprises LDN193189. In certain embodiments, the BMP inhibitor comprises Noggin.

[0089] In certain embodiments, the stem cells are exposed to one SMAD inhibitor, e.g., one TGFp / Activin-Nodal inhibitor. In certain embodiments, the TGFp / Activin-Nodal inhibitor 072734.1886

[0090] PATENT is SB431542. In certain embodiments, the TGFp / Activin-Nodal inhibitor is a derivative of SB431542. In certain embodiments, the TGFp / Activin-Nodal inhibitor is A83-01.

[0091] In certain embodiments, the stem cells are exposed to two SMAD inhibitors. In certain embodiments, the two SMAD inhibitors are a TGFp / Activin-Nodal inhibitor and a BMP inhibitor. In certain embodiments, the stem cells are exposed to SB431542 or A83-01, and LDN193189 or Noggin. In certain embodiments, the stem cells are exposed to SB431542 and LDN193189. In certain embodiments, the stem cells are exposed to A83-01 and LDN193189. In certain embodiments, the stem cells are exposed to SB431542 and Noggin. In certain embodiments, the stem cells are exposed to A83-01 and Noggin.

[0092] In certain embodiments, the stem cells are exposed to or contacted with at least one SMAD inhibitor for at least about 5 days, or at least about 10 days. In certain embodiments, the stem cells are contacted with or exposed to the at least one SMAD inhibitor for up to about 5 days, or up to about 10 days. In certain embodiments, the stem cells are contacted with or exposed to the at least one SMAD inhibitor for between about 5 days and about 10 days. In certain embodiments, the stem cells are contacted with or exposed to the at least one SMAD inhibitor for about 5 days. In certain embodiments, the stem cells are contacted with or exposed to the at least one SMAD inhibitor for about 6 days. In certain embodiments, the stem cells are contacted with or exposed to the at least one SMAD inhibitor for about 7 days. In certain embodiments, the cells are contacted with or exposed to the at least one SMAD inhibitor from day 0 through day 5. In certain embodiments, the cells are contacted with or exposed to the at least one SMAD inhibitor from day 0 through day 6. In certain embodiments, the cells are contacted with or exposed to the at least one SMAD inhibitor from day 0 through day 7. In certain embodiments, the at least one SMAD inhibitor is added every day or every other day to a cell culture medium comprising the stem cells from day 0 through day 5. In certain embodiments, the at least one SMAD inhibitor is added every day or every other day to a cell culture medium comprising the stem cells from day 0 through day 6. In certain embodiments, the at least one SMAD inhibitor is added every day or every other day to a cell culture medium comprising the stem cells from day 0 through day 7. In certain embodiments, the at least one SMAD inhibitor is added every day (daily) to a cell culture medium comprising the stem cells from day 0 to day 5. In certain embodiments, the at least one SMAD inhibitor is added every day (daily) to a cell culture medium comprising the stem cells from day 0 to day 6. In certain embodiments, the at least one SMAD inhibitor is added every day (daily) to a cell culture medium comprising the stem cells from day 0 to day 7. 072734.1886

[0093] PATENT

[0094] In certain embodiments, the cells are contacted with or exposed to a TGFp / Activin- Nodal inhibitor. In certain embodiments, the concentration of the TGFp / Activin-Nodal inhibitor contacted with or exposed to the cells is between about 1 pM and about 20 pM, between about 1 pM and about 10 pM, between about 1 pM and about 15 pM, between about 10 pM and about 15 pM, between about 5 pM and about 10 pM, between about 5 pM and about 15 pM, between about 5 pM and about 20 pM, or between about 15 pM and about 20 pM. In certain embodiments, the concentration of the TGFp / Activin-Nodal inhibitor contacted with or exposed to the cells is between about 1 pM and about 10 pM. In certain embodiments, the concentration of the TGFp / Activin-Nodal inhibitor contacted with or exposed to the cells is about 5 pM. about 10 pM. In certain embodiments, the concentration of the TGFp / Activin-Nodal inhibitor contacted with or exposed to the cells is about 10 pM. In certain embodiments, the TGFp / Activin-Nodal inhibitor comprises SB431542 or a derivative thereof (e.g., A83-01). In certain embodiments, the TGFp / Activin-Nodal inhibitor comprises SB431542.

[0095] In certain embodiments, the cells are contacted with or exposed to a BMP inhibitor. In certain embodiments, the concentration of the BMP inhibitor contacted with or exposed to the cells is between about 50 nM and about 500 nM, or between about 100 nM and about 500 nM, or between about 200 nM and about 500 nM, or between about 200 and about 300 nM, or between about 200 nM and about 400 nM, or between about 100 nM and about 250 nM, or between about 100 nM and about 250 nM, or between about 200 nM and about 250 nM, or between about 250 nM and about 300 nM. In certain embodiments, the concentration of the BMP inhibitor contacted with or exposed to the cells is between about 200 nM and about 300 mM. In certain embodiments, the concentration of the BMP inhibitor contacted with or exposed to the cells is about 150 nM, about 200 nM, about 250 nM, about 300 nM, or about 350 nM. In certain embodiments, the concentration of the BMP inhibitor contacted with or exposed to the cells is about 250 nM. In certain embodiments, the BMP inhibitor comprises LDN193189 or a derivative thereof. In certain embodiments, the BMP inhibitor comprises LDN193189.

[0096] In certain embodiments, the cells are contacted with or exposed to the TGFp / Activin- Nodal inhibitor and the BMP inhibitor simultaneously. In certain embodiments, the stem cells are contacted with or exposed to the TGFp / Activin-Nodal inhibitor and the BMP inhibitor for about 5 days. In certain embodiments, the stem cells are contacted with or exposed to the TGFp / Activin-Nodal inhibitor and the BMP inhibitor for about 6 days. In certain embodiments, the stem cells are contacted with or exposed to the TGFp / Activin-Nodal 072734.1886

[0097] PATENT inhibitor and the BMP inhibitor for about 7 days. In certain embodiments, the cells are contacted with or exposed to the TGFp / Activin-Nodal inhibitor and the BMP inhibitor from day 0 through day 5. In certain embodiments, the cells are contacted with or exposed to the TGFp / Activin-Nodal inhibitor and the BMP inhibitor from day 0 through day 6. In certain embodiments, the cells are contacted with or exposed to the TGFp / Activin-Nodal inhibitor and the BMP inhibitor from day 0 through day 7. In certain embodiments, the TGFp / Activin- Nodal inhibitor and the BMP inhibitor are added every day or every other day to a cell culture medium comprising the stem cells from day 0 through day 5. In certain embodiments, the TGFp / Activin-Nodal inhibitor and the BMP inhibitor are added every day or every other day to a cell culture medium comprising the stem cells from day 0 through day 6. In certain embodiments, the TGFp / Activin-Nodal inhibitor and the BMP inhibitor are added every day or every other day to a cell culture medium comprising the stem cells from day 0 through day 7. In certain embodiments, the TGFp / Activin-Nodal inhibitor and the BMP inhibitor are added every day (daily) to a cell culture medium comprising the stem cells from day 0 to day

[0098] 5. In certain embodiments, the TGFp / Activin-Nodal inhibitor and the BMP inhibitor are added every day (daily) to a cell culture medium comprising the stem cells from day 0 to day

[0099] 6. In certain embodiments, the TGFp / Activin-Nodal inhibitor and the BMP inhibitor are added every day (daily) to a cell culture medium comprising the stem cells from day 0 to day

[0100] 7.

[0101] 7.2.3. Wnt A ctivators

[0102] In certain embodiments, the method of in vitro inducing differentiation of stem cells to into mDA neurons or precursors thereof comprises contacting the cells with an effective amount(s) of at least one activator of wingless (Wnt) signaling (also referred to as “Wnt activators”). As used herein, the term “WNT” or “wingless” in reference to a ligand refers to a group of secreted proteins (i.e. Inti (integration 1) in humans) capable of interacting with a WNT receptor, such as a receptor in the Frizzled and LRPDerailed / RYK receptor family. As used herein, the term “WNT” or “wingless” in reference to a signaling pathway refers to a signal pathway composed of Wnt family ligands and Wnt family receptors, such as Frizzled and LRPDerailed / RYK receptors, mediated with or without P-catenin.

[0103] In certain embodiments, the at least one Wnt activator lowers GSK3P for activation of Wnt signaling. Thus, in certain embodiments, the Wnt activator is a GSK3P inhibitor. A GSK3P inhibitor is capable of activating a WNT signaling pathway, see e.g., Cadigan, et al., J Cell Sci. 2006;119:395-402; Kikuchi, et al., Cell Signaling. 2007;19:659-671, which are incorporated by reference herein in their entireties. As used herein, the term “glycogen 072734.1886

[0104] PATENT synthase kinase 3p inhibitor” or “GSK3P inhibitor” refers to a compound that inhibits a glycogen synthase kinase 3p enzyme, for example, see Doble, et al., J Cell Sci. 2003;116: 1175-1186, which is incorporated by reference herein in its entirety. Non-limiting examples of GSK3P inhibitors include CHIR99021, BIO ((3E)-6-bromo-3-[3- (hydroxyamino)indol-2-ylidene]-lH-indol-2-one), AMBMP hydrochloride, LP 922056, SB- 216763, CHIR98014, Lithium, 3F8, deoxycholic acid, and those disclosed in WO201 1 / 149762, WO13 / 067362, Chambers etal., Nat Biotechnol. 2012 Jul l;30(7):715-20, Kriks et al., Nature. 2011 Nov 6;480(7378):547-51, and Calder et al., J Neurosci. 2015 Aug 19;35(33): 11462-81, all of which are incorporated by reference in their entireties.

[0105] Non-limiting examples of Wnt activators include CHIR99021, Wnt3A, Wntl, Wnt5a, BIO ((3E)-6-bromo-3-[3-(hydroxyamino)indol-2-ylidene]-lH-indol -2-one), AMBMP hydrochloride, LP 922056, SB-216763, CHIR98014, Lithium, 3F8, deoxycholic acid, and those disclosed in WO2011 / 149762, WO13 / 067362, Chambers et al., Nat Biotechnol. 2012 Jul l;30(7):715-20, Kriks et al., Nature. 2011 Nov 6;480(7378):547-51, and Calder et al., J Neurosci. 2015 Aug 19;35(33): 11462-81, all of which are incorporated by reference in their entireties. In certain embodiments, the at least one Wnt activator is a small molecule selected from CHIR99021, Wnt3A, Wntl, Wnt5a, BIO, CHIR98014, Lithium, 3F8, deoxycholic acid, derivatives thereof, and mixtures thereof.

[0106] In certain embodiments, the at least one Wnt activator comprises CHIR99021 or a derivative thereof. In certain embodiments, the at least one Wnt activator comprises CHIR9902L “CHIR99021” (also known as “aminopyrimidine” or “3-[3-(2-Carboxyethyl)- 4-methylpyrrol-2-methylidenyl]-2-indolinone”) refers to IUPAC name 6-(2-(4-(2,4- dichlorophenyl)-5-(4-methyl-lH-imidazol-2-yl)pyrimidin-2-ylamino) ethylamino)nicotinonitrile with the following formula.

[0107] CHIR99021 is highly selective, showing nearly thousand-fold selectivity against a panel of related and unrelated kinases, with an IC50=6.7 nM against human GSK3P and nanomolar IC50 values against rodent GSK3P homologs. 072734.1886

[0108] PATENT

[0109] In certain embodiments, the cells are contacted with or exposed to the at least one Wnt activator for at least about 5 days, at least about 10 days, at least about 15 days, or at least about 20 days. In certain embodiments, the cells are contacted with or exposed to the at least one Wnt activator for up to about 5 days, up to about 10 days, up to about 15 days, or up to about 20 days. In certain embodiments, the cells are contacted with or exposed to the at least one Wnt activator for between about 5 days and about 20 days, between about 5 days and about 15 days, between about 10 days and about 20 days, between about 5 days and about 15 days, or between about 10 days and about 15 days. In certain embodiments, the cells are contacted with the at least one Wnt activator for between about 10 days and about 20 days. In certain embodiments, the cells are contacted with the at least one Wnt activator for about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, or about 17 days.

[0110] In certain embodiments, the cells are contacted with the at least one Wnt activator from day 2 through day 12. In certain embodiments, the cells are contacted with the at least one Wnt activator from day 2 through day 11. In certain embodiments, the cells are contacted with the at least one Wnt activator from day 2 through day 10. In certain embodiments, the cells are contacted with the at least one Wnt activator from day 1 through day 12. In certain embodiments, the cells are contacted with the at least one Wnt activator from day 1 through day 11. In certain embodiments, the cells are contacted with the at least one Wnt activator from day 1 through day 10. In certain embodiments, the cells are contacted with the at least one Wnt activator from day 0 through day 16. In certain embodiments, the cells are contacted with the at least one Wnt activator from day 2 through day 16. In certain embodiments, the at least one Wnt activator is added every day or every other day to a cell culture medium comprising the cells from day 0 through day 16.

[0111] In certain embodiments, the at least one Wnt activator is added every day or every other day to a cell culture medium comprising the cells from day 2 through day 12. In certain embodiments, the at least one Wnt activator is added every day (daily) to a cell culture medium comprising the cells from day 2 through day 12. In certain embodiments, the at least one Wnt activator is added every day or every other day to a cell culture medium comprising the cells from day 2 through day 11. In certain embodiments, the at least one Wnt activator is added every day (daily) to a cell culture medium comprising the cells from day 2 through day 11. In certain embodiments, the at least one Wnt activator is added every day or every other day to a cell culture medium comprising the cells from day 2 through day 10. In certain embodiments, the at least one Wnt activator is added every day (daily) to a cell culture 072734.1886

[0112] PATENT medium comprising the cells from day 2 through day 10. In certain embodiments, the at least one Wnt activator is added every day or every other day to a cell culture medium comprising the cells from day 1 through day 12. In certain embodiments, the at least one Wnt activator is added every day (daily) to a cell culture medium comprising the cells from day 1 through day 12. In certain embodiments, the at least one Wnt activator is added every day or every other day to a cell culture medium comprising the cells from day 1 through day 11. In certain embodiments, the at least one Wnt activator is added every day (daily) to a cell culture medium comprising the cells from day 1 through day 11. In certain embodiments, the at least one Wnt activator is added every day or every other day to a cell culture medium comprising the cells from day 1 through day 10. In certain embodiments, the at least one Wnt activator is added every day (daily) to a cell culture medium comprising the cells from day 1 through day 10. In certain embodiments, the at least one Wnt activator is added every day (daily) to a cell culture medium comprising the cells from day 0 through day 16. In certain embodiments, the at least one Wnt activator is added every day or every other day to a cell culture medium comprising the cells from day 2 through day 16. In certain embodiments, the at least one Wnt activator is added every day (daily) to a cell culture medium comprising the cells from day 2 through day 16.

[0113] In certain embodiments, the cells are contacted with or exposed to the at least one Wnt activator at least about 1 day, or at least about 2 days, or at least about 3 days, or at least about 4 days from the initial contact of the cells with the at least one SMAD inhibitor. In certain embodiments, the cells are contacted with or exposed to the at least one Wnt activator about 1 day, or about 2 days, or about 3 days, or about 4 days from the initial contact of the cells with the at least one SMAD inhibitor. In certain embodiments, the cells are contacted with or exposed to the at least one Wnt activator about 2 days from the initial contact of the cells with the at least one SMAD inhibitor.

[0114] In certain embodiments, the cells are contacted with or exposed to the at least one Wnt activator at least about 1 day, or at least about 2 days, or at least about 3 days, or at least about 4 days from the initial contact of the cells with the at least one SMAD inhibitor, and the cells are contacted with or exposed to the at least one Wnt activator for about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, or about 17 days.

[0115] In certain embodiments, the at least one Wnt activator comprises a GSK3P inhibitor. In certain embodiments, the at least one Wnt activator comprises CHIR99021 or a derivative thereof. In certain embodiments, the at least one Wnt activator comprises CHIR99021. 072734.1886

[0116] PATENT

[0117] 7.2.4. SHH Activators

[0118] As used herein, the term “Sonic hedgehog,” “SHH,” or “Shh” refers to a protein that is one of at least three proteins in the mammalian signaling pathway family called hedgehog, another is desert hedgehog (DHH) wile a third is Indian hedgehog (UTH). SHH interacts with at least two transmembrane proteins by interacting with transmembrane molecules Patched (PTC) and Smoothened (SMO). SHH typically binds to PTC, which then allows the activation of SMO as a signal transducer. In the absence of SHH, PTC typically inhibits SMO, which in turn activates a transcriptional repressor so transcription of certain genes does not occur. When SHH is present and binds to PTC, PTC cannot interfere with the functioning of SMO. With SMO uninhibited, certain proteins are able to enter the nucleus and act as transcription factors allowing certain genes to be activated (see Gilbert, 2000 Developmental Biology (Sunderland, Mass., Sinauer Associates, Inc., Publishers). In certain embodiments, an SHH activator refers to any molecule or compound that is capable of activating a SHH signaling pathway, including a molecule or compound that is capable of binding to PTC or a SMO. In certain embodiments, the at least one SHH activator is selected from the group consisting of molecules that bind to PCT, molecules that bind to SMO, and combinations thereof. Nonlimiting examples of SHH activators include those disclosed in WO 10 / 096496, WO13 / 067362, Chambers et al., Nat Biotechnol. 2009 Mar;27(3):275-80, and Kriks et al., Nature. 2011 Nov 6;480(7378):547-51. In certain embodiments, the at least one SHH activator is selected from the group consisting of a SHH protein, a SMO agonist, or a combination thereof. In certain embodiments, the SHH protein is selected from the group consisting of a recombinant SHH, a modified N-terminal SHH, or a combination thereof. In certain embodiments, the recombinant SHH comprises a N-terminal fragment and a C- terminal fragment. In certain embodiments, the modified N-terminal SHH comprises two Isoleucines at the N-terminus. In certain embodiments, the modified N-terminal SHH has at least about 80%, about 85%, about 90%, about 95%, or about 99% sequence identity to an un-modified N-terminal SHH. In certain embodiments, the modified N-terminal SHH has at least about 80%, about 85%, about 90%, about 95%, or about 99% sequence identity to an un-modified human N-terminal SHH. In certain embodiments, the modified N-terminal SHH has at least about 80%, about 85%, about 90%, about 95%, or about 99% sequence identity to an un-modified mouse N-terminal SHH. In certain embodiments, the modified N-terminal SHH comprises SHH C25II. In certain embodiments, the modified N-terminal SHH comprises SHH C24II. 072734.1886

[0119] PATENT

[0120] Non-limiting examples of SMO agonists (SAGs) include purmorphamine, GSA10, and 20(S)- hydroxy Cholesterol. In certain embodiments, the SAG comprises purmorphamine.

[0121] In certain embodiments, the cells are contacted with or exposed to the at least one SHH activator for at least about 5 days, or at least about 10 days. In certain embodiments, the cells are contacted with or exposed to the at least one SHH activator for up to about 5 days, or up to about 10 days. In certain embodiments, the cells are contacted with or exposed to the at least one SHH activator for between about 5 days and about 10 days. In certain embodiments, the cells are contacted with or exposed to the at least one SHH activator for about 5 days. In certain embodiments, the cells are contacted with or exposed to the at least one SHH activator for 6 days. In certain embodiments, the cells are contacted with or exposed to the at least one SHH activator from day 0 through day 6. In certain embodiments, the at least one SHH activator is added every day or every other day to a cell culture medium comprising the cells from day 0 through day 6. In certain embodiments, the at least one SHH activator is added every day (daily) to a cell culture medium comprising the cells from day 0 through day 6. In certain embodiments, the cells are contacted with or exposed to the at least one SHH activator for 7 days. In certain embodiments, the cells are contacted with or exposed to the at least one SHH activator from day 0 through day 7. In certain embodiments, the at least one SHH activator is added every day or every other day to a cell culture medium comprising the cells from day 0 through day 7. In certain embodiments, the at least one SHH activator is added every day (daily) to a cell culture medium comprising the cells from day 0 through day 7.

[0122] In certain embodiments, the concentration of the at least one SHH activator contacted with or exposed to the cells is between about 50 ng / mL and about 1000 ng / mL, between about 100 ng / mL and about 1000 ng / mL, between about 20 ng / mL and about 1000 ng / mL, between about 300 ng / mL and about 1000 ng / mL, between about 400 ng / mL and about 1000 ng / mL, between about 500 ng / mL and about 1000 ng / mL, between about 400 ng / mL and about 800 ng / mL, between about 400 ng / mL and about 700 ng / mL, between about 400 ng / mL and about 600 ng / mL, or between about 500 ng / mL and about 600 ng / mL. In certain embodiments, the concentration of the at least one SHH activator contacted with or exposed to the cells is between about 400 ng / mL and about 600 ng / mL. In certain embodiments, the concentration of the at least one SHH activator contacted with or exposed to the cells is about 400 ng / mL, about 450 ng / mL, about 500 ng / mL, about 550 ng / mL, or about 600 ng / mL. In certain 072734.1886

[0123] PATENT embodiments, the concentration of the at least one SHH activator contacted with or exposed to the cells is about 500 ng / mL.

[0124] In certain embodiments, the at least one activator of SHH signaling comprises SHH C25IL

[0125] 7.2.5. Wnt Inhibitors

[0126] Wnt signaling includes canonical Wnt signaling and non-canonical Wnt signaling. In certain embodiments, the at least one Wnt inhibitor is capable of inhibiting canonical Wnt signaling. In certain embodiments, the at least one Wnt inhibitor is capable of inhibiting both canonical Wnt signaling and non-canonical Wnt signaling. Non-limiting examples of Wnt inhibitors that are capable of inhibiting both canonical Wnt signaling and non-canonical Wnt signaling include IWP2, IWRl-endo, IWP-01, Wnt-C59, IWP-L6, IWP12, LGK-974, IWR- 1, ETC-159, iCRT3, IWP-4, Salinomycin, Pyrvinium Pamoate, iCRT14, FH535, CCT251545, Wogonin, NCB-0846, Hexachrorophene, KY02111, SO3031 (KY01-I), SO2031 (KY02-I), BC2059, PKF115-584, Quercetin, NSC668036, G007-LK, and derivatives thereof. In certain embodiments, the at least one Wnt inhibitor is selected from the group consisting of IWP2, IWRl-endo, XAV939, IWP-01, Wnt-C59, IWP-L6, LGK- 974, IWR-1, Wnt-C59, ETC-159, iCRT3, IWP-4, ICG-001, Salinomycin, Pyrvinium Pamoate, iCRT14, FH535, CCT251545, KYA1797K, Wogonin, NCB-0846,

[0127] Hexachrorophene, PNU-74654, KY02111, SO3031 (KY01-I), SO2031 (KY02-I), Triptonide, IWP12, BC2059, PKF115-584, Quercetin, NSC668036, G007-LK, MSAB, LF3, JW55, Isoquercitrin, WIKI4 (Wnt Inhibitor Kinase Inhibitor 4), derivatives thereof, and combinations thereof. In certain embodiments, the at least one inhibitor of Wnt signaling is selected from the group consisting of IWP2, IWRl-endo, IWP-01, IWP12, Wnt-C59, IWP- L6, LGK-974, IWR-1, ETC-159, iCRT3, IWP-4, Salinomycin, Pyrvinium Pamoate, iCRT14, FH535, CCT251545, Wogonin, NCB-0846, Hexachrorophene, KY02111, SO3031 (KY01- I), SO2031 (KY02-I), BC2059, PKF115-584, Quercetin, NSC668036, G007-LK, derivatives thereof, and combinations thereof. In certain embodiments, the at least one inhibitor of Wnt signaling is selected from the group consisting of XAV939, ICG-001, PNU-74654, Triptonide, KYA1797K, MSAB, LF3, JW55, Isoquercitrin, WIKI4, derivatives thereof, and combinations thereof. In certain embodiments, the at least one Wnt inhibitor comprises IWP2 or a derivative thereof.

[0128] In certain embodiments, the cells are contacted with or exposed to the at least one Wnt inhibitor for at least about 1 day or at least about 2 days. In certain embodiments, the cells are contacted with or exposed to the at least one Wnt inhibitor for up to about 1 day or up to 072734.1886

[0129] PATENT about 2 days. In certain embodiments, the cells are contacted with or exposed to the at least one Wnt inhibitor for between about 1 days and about 2 days. In certain embodiments, the cells are contacted with or exposed to the at least one Wnt inhibitor for about 1 day. In certain embodiments, the cells are contacted with or exposed to the at least one Wnt inhibitor for about 2 days. In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one Wnt inhibitor is initiated at least about 1 day, or at least about 2 days, or at least about 3 days, or at least about 4 days, or at least about 5 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor. In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one Wnt inhibitor is initiated concurrently with the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor.

[0130] In certain embodiments, the cells are further contacted with or exposed to the at least one Wnt inhibitor about 10 days, about 11 days, about 12 days, or about 13 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor, and the cells are contacted with or exposed to the at least one Wnt inhibitor for about 5 days or about 6 days.

[0131] In certain embodiments, the at least one Wnt inhibitor is added every day or every other day to a cell culture medium comprising the cells for about 2 days. In certain embodiments, the at least one Wnt inhibitor is added every day (daily) to a cell culture medium comprising the cells for about 2 days.

[0132] In certain embodiments, the concentration of the at least one Wnt inhibitor contacted with or exposed to the cells is between about 0.5 pM and about 20 pM, between about 0.5 pM and about 10 pM, between about 0.5 pM and about 5 pM, between about 0.5 pM and about 1 pM, between about 0.5 pM and about 2 pM, between about 5 pM and about 10 pM, between about 10 pM and about 20 pM, between about 1 pM and about 2 pM, or between about 1 pM and about 5 pM. In certain embodiments, the concentration of the at least one Wnt inhibitor contacted with or exposed to the cells is between about 0.5 pM and about 2 pM. In certain embodiments, the concentration of the at least one Wnt inhibitor contacted with or exposed to the cells is about 2 pM.

[0133] In certain embodiments, the at least one Wnt inhibitor comprises XAV939, IWP2, or combination thereof.

[0134] 7.2.6. FGF A ctivators

[0135] FGF family includes secreted signaling proteins (secreted FGFs) that signal to receptor tyrosine kinases. Phylogenetic analysis suggests that 22 FGF genes can be arranged 072734.1886 PATENT into seven subfamilies containing two to four members each. Branch lengths are proportional to the evolutionary distance between each gene.

[0136] In certain embodiments, the at least one FGF activator is selected from the group consisting of FGF8a, FGF17, FGF18, FGF8b, FGF2, FGF4, and derivatives thereof. In certain embodiments, the at least one FGF activator is selected from the group consisting of FGF8a, FGF17, FGF18, FGF2, FGF4, and derivatives thereof. In certain embodiments, the at least one FGF activator is selected from the group consisting of FGF8a, FGF17, and FGF18.

[0137] The FGF8 subfamily is comprised of FGF8a, FGF8b, FGF17, and FGF18. Early patterning of the vertebrate midbrain and cerebellum is regulated by a mid / hindbrain organizer that produces FGF8a, FGF8b, FGF 17 and FGF 18. It has been shown that FGF8b functions differently from FGF8a, FGF17, and FGF18 (Liu et al, Development. 2003 Dec;130(25):6175-85). FGF8b is the only protein that can induce the gene Gbx2 and strongly activate the pathway inhibitors Spry 1 / 2, as well as repress the midbrain gene Otx2 (Liu 2003). Moreover, FGF8b extends the organizer along the junction between the induced Gbx2 domain and the remaining Otx2 region in the midbrain, correlating with cerebellum development (Liu 2003). By contrast, FGF8a, FGF17, and FGF18 cause expansion of the midbrain and upregulating midbrain gene expression (Liu 2003).

[0138] In certain embodiments, the at least one FGF activator is capable of causing expansion of the midbrain and upregulating midbrain gene expression. In certain embodiments, the at least one FGF activator is capable of promoting midbrain development. In certain embodiments, the at least one FGF activator is selected from the group consisting of FGF8a, FGF 17, FGF 18, FGF2, FGF4, derivatives thereof, and combinations thereof. In certain embodiments, the at least one FGF activator is selected from the group consisting of FGF8a, FGF 17, FGF 18, and combinations thereof. In certain embodiments, the at least one FGF activator comprises or is FGF 18.

[0139] In certain embodiments, the cells are contacted with or exposed to the at least one FGF activator for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 8 days, or at least about 10 days. In certain embodiments, the cells are contacted with or exposed to the at least one FGF activator for at least about 4 days. In certain embodiments, the cells are contacted with or exposed to the at least one FGF activator for at least about 5 days. In certain embodiments, the cells are contacted with or exposed to the at least one FGF activator for at least 6 days. In certain embodiments, the cells are contacted with or exposed to the at least one FGF activator 072734.1886 PATENT for up to about 5 days (e.g., up to 5 days, up to 6 days, or up to 7 days), or up to about 10 days (e.g., up to 8 days, up to 9 days, up to 10 days, up to 11 days, up to 12 days), or up to about 15 days, or up to about 20 days. In certain embodiments, the cells are contacted with or exposed to the at least one FGF activator for at least about 4 days and / or for up to about 7 days. In certain embodiments, the cells are contacted with or exposed to the at least one FGF activator for between about 1 days and about 20 days, between about 1 day and about 15 days, between about 1 day and about 5 days, between about 5 days and about 20 days, between about 5 days and about 15 days, or between about 5 days and about 10 days, between about 10 days and about 20 days.

[0140] In certain embodiments, the cells are contacted with or exposed to the at least one FGF activator for between about 1 day and about 10 days. In certain embodiments, the cells are contacted with or exposed to the at least one FGF activator for about 3 days, about 4 days, about 5 days, about 6 days, or about 8 days. In certain embodiments, the cells are contacted with or exposed to the at least one FGF activator for between about 1 days and about 6 days. In certain embodiments, the cells are contacted with or exposed to the at least one FGF activator for about 4 days. In certain embodiments, the cells are contacted with or exposed to the at least one FGF activator for about 5 days. In certain embodiments, the cells are contacted with or exposed to the at least one FGF activator for about 6 days. In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one FGF activator is initiated at least about 5 days, or at least about 10 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor. In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one FGF activator is initiated no later than about 15 days or no later than about 20 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor. In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one FGF activator is initiated no later than 18 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor. In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one FGF activator is initiated between about 5 days and about 20 days, between about 5 days and about 20 days, between about 10 days and about 15 days, between about 10 days and 18 days, between about 5 days and about 15 days, or between about 10 days and about 20 days, from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor. In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one FGF activator is initiated between about 5 days and about 10 days from the initial contact 072734.1886 PATENT of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor. In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one FGF activator is initiated about 10 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor. In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one FGF activator is initiated 11 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor. In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one FGF activator is initiated 12 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor. In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one FGF activator is initiated 13 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor.

[0141] In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one FGF activator is initiated about 10 days, about 11 days, about 12 days, or about 13 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor, and the cells are contacted with the at least one FGF activator for about 3 days, or about 4 days, or about 5 days. In certain embodiments, the cells are contacted with or exposed to the at least one FGF activator from day 10 through day 16. In certain embodiments, the cells are contacted with or exposed to the at least one FGF activator from day 11 through day 16. In certain embodiments, the cells are contacted with or exposed to the at least one FGF activator from day 12 through day 16. In certain embodiments, the at least one FGF activator is added every day or every other day to a cell culture medium comprising the cells from day 10 through day 16. In certain embodiments, the at least one FGF activator is added every day (daily) to a cell culture medium comprising the cells from day 10 through day 16. In certain embodiments, the at least one FGF activator is added every day or every other day to a cell culture medium comprising the cells from day 11 through day 16. In certain embodiments, the at least one FGF activator is added every day (daily) to a cell culture medium comprising the cells from day 11 through day 16. In certain embodiments, the at least one FGF activator is added every day or every other day to a cell culture medium comprising the cells from day 12 through day 16. In certain embodiments, the at least one FGF activator is added every day (daily) to a cell culture medium comprising the cells from day 12 through day 16.

[0142] In certain embodiments, the concentration of the at least one FGF activator contacted with or exposed to the cells is between about 10 ng / mL and about 500 ng / mL, between about 072734.1886

[0143] PATENT

[0144] 50 ng / mL and about 500 ng / mL, between about 100 ng / mL and about 500 ng / mL, between about 100 ng / mL and about 400 ng / mL, between about 100 ng / mL and about 300 ng / mL, between about 100 ng / mL and about 200 ng / mL, or between about 100 ng / mL and about 250 ng / mL. In certain embodiments, the concentration of the at least one FGF activator contacted with or exposed to the cells is between about 100 ng / mL and about 200 ng / mL. In certain embodiments, the concentration of the at least one FGF activator contacted with or exposed to the cells is about 100 ng / mL. In certain embodiments, concentration of the at least one FGF activator contacted with or exposed to the cells is about 200 ng / mL.

[0145] In certain embodiments, the at least one FGF activator comprises FGF18.

[0146] 7.2. 7. BMP Activators

[0147] In certain embodiments, the cells are contacted with effective amounts of one or more inhibitor of TGFp / Activin-Nodal signaling and effective amounts of one or more activator of BMP signaling.

[0148] In certain embodiments, the BMP activator is contacted to the cells for at least 2 days, at least 3 days, at least 4 days, or at least 5 days, or for up to about 2 days, up to about 3 days, up to about 4 days, up to about 5 days or more. In a specific embodiment, the BMP active agent is contacted to the cells for at least about 2 days.

[0149] In certain embodiments, the activator of BMP signaling is selected from the group consisting of BMP2, BMP4, BMP6, BMP7, derivatives thereof, and mixtures thereof. In certain embodiments, the activator of BMP signaling is contacted to the cells at a concentration of between about 0.01 and 5 ng / ml, between about 0.1 and 2 ng / mL, or between about 1 and 1.5 ng / mL. In a specific embodiment the activator of BMP signaling is contacted to the cells at a concentration of about 1 ng / mL.

[0150] 7.2.8. Exemplary Methods

[0151] The present disclosure provides methods for inducing differentiation of stem cells. In certain embodiments, the stem cells are contacted with or exposed to at least one inhibitor of Wnt signaling. In certain embodiments, the cells are further contacted with at least one inhibitor of SMAD signaling. In certain embodiments, the cells are further contacted with at least one activator of SHH signaling. In certain embodiments, the cells are further contacted with at least one activator of Wnt signaling.

[0152] In certain embodiments, the methods comprise contacting the stem cells with at least one inhibitor of Wnt signaling, contacting the stem cells with at least one inhibitor of SMAD signaling and at least one activator of SHH signaling, and contacting the stem cells with at 072734.1886 PATENT least one activator of Wnt signaling to obtain a population of differentiated cells expressing at least one marker indicating a midbrain dopamine neuron or a precursor thereof.

[0153] In certain embodiments, the cells are contacted or exposed to the at least one inhibitor of Wnt signaling for up to about 2 days, or for about 1 day or about 2 days.

[0154] In certain embodiments, the cells are contacted with or exposed to the at least one inhibitor of SMAD signaling for at least about 5 days, or for about 5 days, about 6 days, or about 7 days. In certain embodiments, the at least one inhibitor of SMAD signaling comprises an inhibitor of TGFp / Activin-Nodal signaling, an inhibitor of BMP signaling, or a combination thereof. In certain embodiments, the at least one inhibitor of SMAD signaling comprises an inhibitor of TGFp / Activin-Nodal signaling. In certain embodiments, the at least one inhibitor of TGFp / Activin-Nodal signaling comprises an inhibitor of ALK5. In certain embodiments, the at least one inhibitor of TGFp / Activin-Nodal signaling is selected from the group consisting of SB431542, derivatives of SB431542 (e.g., A83-01), and combinations thereof. In certain embodiments, the at least one inhibitor of BMP signaling is selected from the group consisting of LDN193189, Noggin, dorsomorphin, derivatives of LDN193189, derivatives of Noggin, derivatives of dorsomorphin, and combinations thereof.

[0155] In certain embodiments, the cells are contacted with or exposed to the at least one activator of SHH signaling for at least about 5 days, or for about 5 days, about 6 days, or about 7 days. In certain embodiments, the at least one activator of SHH signaling is selected from the group consisting of SHH proteins, Smoothened agonists (SAG) (e.g., purmorphamine), and combinations thereof. In certain embodiments, the SHH protein is selected from the group consisting of recombinant SHHs, modified N-terminal SHHs, and combinations thereof. In certain embodiments, the modified N-terminal SHH comprises two Isoleucines at the N-terminus. In certain embodiments, the modified N-terminal SHH has at least about 90% sequence identity to an un-modified N-terminal SHH. In certain embodiments, the un-modified N-terminal SHH is a un-modified mouse N-terminal SHH or a un-modified human N-terminal SHH. In certain embodiments, the modified N-terminal SHH comprises SHH C25II.

[0156] In certain embodiments, the cells are contacted with or exposed to the at least one activator of Wnt signaling for at least about 5 days or for about 7 days, about 8 days, or about 9 days. In certain embodiments, the initial contact of the cells with the at least one activator of Wnt signaling is at least about 2 days from the initial contact of the cells with the at least one SMAD inhibitor. In certain embodiments, the initial contact of the cells with the at least one activator of Wnt signaling is about 2 days from the initial contact of the cells with the at 072734.1886 PATENT least one SMAD inhibitor. In certain embodiments, the at least one activator of Wnt signaling comprises an inhibitor of glycogen synthase kinase 3p (GSK3P) signaling. In certain embodiments, the at least one activator of Wnt signaling is selected from the group consisting of CHIR99021, CHIR98014, AMBMP hydrochloride, LP 922056, Lithium, deoxycholic acid, BIO, SB-216763, Wnt3A, Wntl, Wnt5a, derivatives thereof, and combinations thereof.

[0157] In certain embodiments, said stem cells are human stem cells. In certain embodiments, said human stem cells are selected from the group consisting of human embryonic stem cells, human induced pluripotent stem cells, human parthenogenetic stem cells, primoridal germ cell-like pluripotent stem cells, epiblast stem cells, and F-class pluripotent stem cells.

[0158] In certain embodiments, the methods further comprise subjecting the population of differentiated cells to conditions favoring differentiation of midbrain dopamine neuron precursors to midbrain dopamine neurons. In certain embodiments, the conditions comprise exposing the cells to at least one of brain-derived neurotrophic factor (BDNF), glial cell- derived neurotrophic factor (GDNF), Cyclic adenosine monophosphate (cAMP), Transforming growth factor beta 3 (TGFP3), ascorbic acid (AA), and DAPT.

[0159] In certain embodiments, the at least one marker indicating a midbrain dopamine neuron or a precursor thereof is selected from the group consisting of EN1, OTX2, TH, NURR1, FOXA2, PITX3, LMX1A, LM03, SNCA, ADCAP1, CHRNA4, SOX6, DAT, VMAT2, WNT1, GIRK2, or combinations thereof.

[0160] In certain embodiments, the stem cells are contacted with or exposed to at least one TGFp / Activin-Nodal inhibitor, at least one BMP inhibitor, and at least one SHH activator for about 5 days, and the cells are contacted with the at least one Wnt activator, and at a concentration of about 6 pM for about 5 days, and at a concentration of about 3 pM for about 5 days. The cells are contacted with or exposed to the at least one Wnt inhibitor, wherein the contact of the cells with the at least one Wnt inhibitor is initiated concurrently with the initial contact of the cells with the at least one SMAD inhibitor; the stem cells are contacted with the at least one Wnt inhibitor for about 2 days; and the contact of the cells with the at least one Wnt activator is initiated about 2 days from the initial contact of the cells with the at least one SMAD inhibitor.

[0161] In certain embodiments, the stem cells are contacted with or exposed to at least one TGFp / Activin-Nodal inhibitor, at least one BMP inhibitor, and at least one SHH activator for about 5 days, and the cells are contacted with the at least one Wnt activator, and at a concentration of about 6 pM for about 5 days, and at a concentration of about 3 pM for about 072734.1886

[0162] PATENT

[0163] 5 days; wherein the contact of the cells with the at least one Wnt activator is initiated about 2 days from the initial contact of the cells with the at least one SMAD inhibitor.

[0164] In certain embodiments, the stem cells are contacted with or exposed to at least one TGFp / Activin-Nodal inhibitor, at least one BMP activator, and at least one SHH activator for about 5 days, and the cells are contacted with the at least one Wnt activator, and at a concentration of about 6 pM for about 5 days, and at a concentration of about 3 pM for about 5 days. The cells are contacted with or exposed to the at least one Wnt inhibitor, wherein the contact of the cells with the at least one Wnt inhibitor is initiated concurrently with the initial contact of the cells with the at least one SMAD inhibitor; the stem cells are contacted with the at least one Wnt inhibitor for about 2 days; and the contact of the cells with the at least one Wnt activator is initiated about 2 days from the initial contact of the cells with the at least one SMAD inhibitor.

[0165] In certain embodiments, the stem cells are contacted with or exposed to at least one TGFp / Activin-Nodal inhibitor, at least one BMP activator, and at least one SHH activator for about 5 days, and the cells are contacted with the at least one Wnt activator, and at a concentration of about 6 pM for about 5 days, and at a concentration of about 3 pM for about 5 days; wherein the contact of the cells with the at least one Wnt activator is initiated about 2 days from the initial contact of the cells with the at least one SMAD inhibitor.

[0166] 7.2.9. Cell Culture Media

[0167] In certain embodiments, the above-described inhibitors and activators are added to a cell culture medium comprising the cells. Suitable cell culture media include, but are not limited to, Knockout® Serum Replacement (“KSR”) medium, Neurobasal® medium (NB), N2 medium, B-27 medium, and Essential 8® / Essential 6® (“E8 / E6”) medium, and combinations thereof. KSR medium, NB medium, N2 medium, B-27 medium, and E8ZE6 medium are commercially available. KSR medium is a defined, serum-free formulation optimized to grow and maintain undifferentiated hESCs in culture.

[0168] In certain embodiments, the cell culture medium is a KSR medium. The components of a KSR medium are disclosed in WO2011 / 149762. In certain embodiments, a KSR medium comprises Knockout DMEM, Knockout Serum Replacement, L-Glutamine, Pen / Strep, MEM, and 13 -mercaptoethanol. In certain embodiments, 1 liter of KSR medium comprises 820 mL of Knockout DMEM, 150 mL of Knockout Serum Replacement, 10 mL of 200 mM L-Glutamine, 10 mL of Pen / Strep, 10 mL of 10 mM MEM, and 55 pM of 13- mercaptoethanol. 072734.1886

[0169] PATENT

[0170] In certain embodiments, the cell culture medium is an E8ZE6 medium. E8ZE6 medium is a feeder-free and xeno-free medium that supports the growth and expansion of human pluripotent stem cells. E8 / E6 medium has been proven to support somatic cell reprogramming. In addition, E8ZE6 medium can be used as a base for the formulation of custom media for the culture of PSCs. One example E8 / E6 medium is described in Chen et al., Nat Methods 2011 May;8(5):424-9, which is incorporated by reference in its entirety. One example E8ZE6 medium is disclosed in WO 15 / 077648, which is incorporated by reference in its entirety. In certain embodiments, an E8ZE6 cell culture medium comprises DMEM / F12, ascorbic acid, selenium, insulin, NaHCOs, transferrin, FGF2 and TGFp. The E8ZE6 medium differs from a KSR medium in that E8ZE6 medium does not include an active BMP ingredient. Thus, in certain embodiments, when an E8ZE6 medium is used to culture the presently disclosed stem cells to differentiate into mDA neurons or precursors thereof, at least one BMP inhibitor is not required to be added to the E8ZE6 medium. In certain embodiments, when an E8ZE6 medium is used to culture the presently disclosed stem cells to differentiate into mDA neurons or precursors thereof, at least one BMP inhibitor is added to the E8ZE6 medium.

[0171] 7.2.10. Differentiated Cells

[0172] In certain embodiments, the method comprises obtaining a cell population of the differenced cells, wherein at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the differentiated cells express at least one marker indicating a mDA neuron or a precursor thereof. Non-limiting examples of markers indicating a mDA neuron or a precursor thereof include engrailed- 1 (EN1), orthodenticle homeobox 2 (OTX2), tyrosine hydroxylase (TH), nuclear receptor related-1 protein (NURR1), forkhead box protein A2 (FOXA2), and LIM homeobox transcription factor 1 alpha (LMX1 A), PITX3, LM03, SNCA, ADCAP1, CHRNA4, ALDH1A1, DAT, VMAT1, SOX6, WNT1, and GIRK2.

[0173] In certain embodiments, the differentiated cells express the at least one marker indicating a mDA neuron or a precursor thereof at least about 10 days (e.g., about 15 days, about 20 days, about 30 days, about 40 days, or about 50 days) from the initial contact of the cells with the at least one SMAD inhibitor. In certain embodiments, the differentiated cells express the at least one marker indicating a mDA neuron or a precursor thereof about 15 days (e.g., 15 days, 16 days, or 17 days) from the initial contact of the cells with the at least one SMAD inhibitor. 072734.1886

[0174] PATENT

[0175] In certain embodiments, the mDA neurons or precursors generated by the methods disclosed herein have a detectable expression level of at least one mDA neuron marker at least about 2 weeks, at least about 3 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years post in vivo transplantation. In certain embodiments, the mDA neurons or precursors thereof generated by the methods disclosed herein have a detectable expression level of at least one mDA neuron marker at least about 2 weeks post in vivo transplantation. In certain embodiments, the mDA neurons or precursors thereof generated by the methods disclosed herein can have a detectable expression level of at least one mDA neuron marker up to about 1 month, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 1 year, up to about 2 years, up to about 3 years, up to about 4 years, or up to about 5 years post in vivo transplantation. In certain embodiments, the mDA neurons or precursors thereof generated by the methods disclosed herein have a detectable expression level of at least one mDA neuron marker about

[0176] 1 month post in vivo transplantation. In certain embodiments, the mDA neurons or precursors thereof generated by the methods disclosed herein have a detectable expression level of at least one mDA neuron marker about 2 months post in vivo transplantation. In certain embodiments, the mDA neurons or precursors thereof generated by the methods disclosed herein have a detectable expression level of at least one marker selected from the group consisting of TH, EN1, NURR1, and ALDH1A1 at least about 1 month post in vivo transplantation. In certain embodiments, the mDA neurons or precursors thereof generated by the methods disclosed herein have a detectable expression level of at least one marker selected from the group consisting of TH, EN1, NURR1, and ALDH1 Al about 2 months post in vivo transplantation. In certain embodiments, the mDA neurons or precursors thereof generated by the methods disclosed herein have a detectable expression level of at least one marker selected from the group consisting of TH, EN1, NURR1, and ALDH1 Al at least about

[0177] 2 months post in vivo transplantation.

[0178] In certain embodiments, the differentiated cells derived from the presently disclosed methods do not express or have a low expression of at least one marker selected from PAX6, EMX2, LHX2, SMA, SIX1, PITX2, SIM1, POU4F1, PHOX2A, BARHL1, BARHL2, GBX2, H0XA1, H0XA2, HOXB1, HOXB2, POU5F1, NANOG, and combinations thereof.

[0179] In certain embodiments, the cells are contacted with the activators and inhibitors described herein at a concentration and time effective to decrease expression of SMA, SIX1, 072734.1886

[0180] PATENT

[0181] PITX2, SIM1, POU4F1, and / or PHOX2A. In certain embodiments, the cells are contacted with the activators and inhibitors described herein at a concentration and time effective to decrease expression of PAX6, BARHL1, and / or BARHL2.

[0182] In certain embodiments, at least about 80% of the differentiated cells express FOXA2 and EN1 about 15 days from the initial contact of the stem cells with the at least one inhibitor of SMAD signaling. In certain embodiments, greater than about 80% (e.g., greater than about 85% or greater than about 90%) of the differentiated cells express FOXA2 and EN1 16 days from the initial contact of the stem cells with the at least one inhibitor of SMAD signaling. 7.2.11. Sorting Methods

[0183] In certain embodiments, the differentiation methods disclosed herein further comprise isolating mDA neurons and precursors thereof based on at least one or at least two surface markers. In certain embodiments, the surface marker is a negative surface marker, wherein the cells do not express a detectable level of the negative surface marker. In certain embodiments, the surface marker is a positive surface marker, wherein the cells express a detectable level of the positive surface marker.

[0184] In certain embodiments, the differentiation methods disclosed herein further comprise isolating cells that do not express a detectable level of at least one negative surface marker. In certain embodiments, the differentiation methods disclosed herein further comprise isolating cells that express a detectable level of at least one positive surface marker. In certain embodiments, the differentiation methods disclosed herein further comprise isolating cells that do not express a detectable level of at least one negative surface marker and express a detectable level of at least one positive surface marker.

[0185] In certain embodiments, the at least one negative surface marker is selected from the group consisting of CD49e, CD99, CD340, and combinations thereof. In certain embodiments, the at least one negative surface marker comprises CD49e. In certain embodiments, the at least one positive surface marker is selected from the group consisting of CD171, CD 184, and combinations thereof. In certain embodiments, the at least one positive surface marker comprises CD 184.

[0186] In certain embodiments, the differentiation methods disclosed herein further comprise isolating cells that do not express a detectable level of CD49e and express a detectable level of CD 184.

[0187] Any surface-marker based cell isolation technology known in the art can be used in the presently disclosed methods. In certain embodiments, flow cytometry is used to the presently disclosed isolation methods. 072734.1886

[0188] PATENT

[0189] 7.2.72. Differentiation of mDA Precursors to mDA neurons

[0190] In certain embodiments, the cells (e.g., mDA precursors) are further contacted with DA neuron lineage specific activators and inhibitors, for example, L-glutamine, brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), Cyclic adenosine monophosphate (cAMP), Transforming growth factor beta (TGFP, for example, TGFP3), ascorbic acid (AA), and DAPT (which is also known as, N-[(3,5-Difluorophenyl)acetyl]-L- alanyl-2-phenyl]glycine-l,l-dimethylethyl ester; LY-374973, N-[N-(3,5-

[0191] Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester; or N-[N- (3,5- difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester). In certain embodiments, the cells are contacted with the foregoing DA neuron lineage specific activators and inhibitors for at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, or at least about 10 or more days, for example, between about 2 days and about 20 days, between about 3 days and about 19 days, between about 4 days and about 18 days, between about 5 days and about 17 days, between about 6 days and about 16 days, between about 7 days and about 15 days, between about 8 days and about 15 days, between about 9 days and about 14 days, or between about 10 days and about 13 days. In certain embodiments, the cells are contacted with the foregoing DA neuron lineage specific activators and inhibitors for up to about 2, up to about 3, up to about 4, up to about 5, up to about 6, up to about 7, up to about 8, up to about 9, or up to about 10 days or more days. In certain embodiments, the cells are contacted with the foregoing DA neuron lineage specific activators and inhibitors for about 4 days, about 5 days, about 6 days, about 7 days, or about 8 days.

[0192] In certain embodiments, the cells are contacted with L-glutamine at a concentration of between about 0.5 mM and about 5 mM, or between about 1 mM and about 5 mM, or between about 1.5 mM and about 2.5 mM, or between about 1 mM and about 2 mM. In certain embodiments, the cells are contacted with L-glutamine at a concentration of about 2 mM.

[0193] In certain embodiments, the cells are contacted with BDNF at a concentration of between about 5 ng / ml and about 50 ng / mL, or between about 10 ng / ml and about 50 ng / mL, or between about 10 ng / ml and about 40 ng / mL, or between about 20 ng / ml and about 50 ng / mL, or between about 20 ng / ml and about 40 ng / mL, or between about 10 ng / ml and about 30 ng / mL, or between about 10 ng / ml and about 20 ng / mL, or between about 20 ng / ml and about 30 ng / mL. In certain embodiments, the cells are contacted with BDNF at a concentration of about 20 ng / mL. 072734.1886

[0194] PATENT

[0195] In certain embodiments, the cells are contacted with ascorbic acid (AA) at a concentration of between about 50 nM and about 500 nM, or between about 100 nM and about 500 nM, or between about 100 nM and about 400 nM, or between about 200 nM and about 400 nM, or between about 200 nM and about 300 nM, or between about 100 nM and about 300 nM. In certain embodiments, the cells are contacted with AA at a concentration of about 200 nM.

[0196] In certain embodiments, the cells are contacted with GDNF at a concentration of between about 5 ng / ml and about 50 ng / mL, or between about 10 ng / ml and about 50 ng / mL, or between about 10 ng / ml and about 40 ng / mL, or between about 20 ng / ml and about 50 ng / mL, or between about 20 ng / ml and about 40 ng / mL, or between about 10 ng / ml and about 30 ng / mL, or between about 10 ng / ml and about 20 ng / mL, or between about 20 ng / ml and about 30 ng / mL. In certain embodiments, the cells are contacted with GDNF at a concentration of about 20 ng / mL.

[0197] In certain embodiments, the cells are contacted with cAMP at a concentration of between about 200 nM and about 800 nM, or between about 200 nM and about 700 nM, or between about 300 nM and about 700 nM, or between about 300 nM and about 600 nM, or between about 400 nM and about 600 nM, or between about 450 nM and about 550 nM. In certain embodiments, the cells are contacted with cAMP at a concentration of about 500 nM.

[0198] In certain embodiments, the cells are contacted with TGFP3 at a concentration of between about 0.01 ng / ml and about 5 ng / mL, or between about 0.1 ng / ml and about 4 ng / mL, or between about 0.5 ng / ml and about 5 ng / mL, or between about 1 ng / ml and about 3 ng / mL, or between about 1 ng / ml and about 2 ng / mL. In certain embodiments, the cells are contacted with TGFP3 at a concentration of about 1 ng / mL.

[0199] In certain embodiments, the cells are contacted with DAPT at a concentration of between about 1 nM and about 50 nM, or between about 5 nM and about 50 nM, or between about 1 nM and about 20 nM, or between about 5 nM and about 20 nM, or between about 1 nM and about 10 nM, or between about 5 nM and about 10 nM, or between about 5 nM and about 15 nM, or between about 10 nM and about 20 nM, or between about 10 nM and about 30 nM, or between about 30 nM and about 50 nM,. In certain embodiments, the cells are contacted with DAPT at a concentration of about 10 nM.

[0200] In certain embodiments, the differentiated midbrain DA precursors are further cultured as described by U.S. Publication No. 2015 / 0010514, which is incorporated by reference in its entirety.

[0201] 7.3. Cell Populations and Compositions 072734.1886

[0202] PATENT

[0203] The presently disclosure provides a cell population of in vitro differentiated cells obtained by the methods disclosed herein, for example, in Section 5.2.

[0204] The presently disclosure provides a cell population of in vitro differentiated cells, wherein at least about 50% (e.g., at least about 55%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%) of the cells express at least one marker indicating a mDA neuron or a precursor thereof. Non-limiting examples of markers indicating a mDA neuron or a precursor thereof include EN1, OTX2, TH, NURR1, FOXA2, LMX1A, PITX3, LM03, SNCA, ADCAP1, CHRNA4, SOX6, ALDH1A1, WNT1, DAT, VMAT1, and GIRK2. The presently disclosure also provides compositions comprising such cell populations. In certain embodiments, the in vitro differentiated cells are obtained by the differentiation methods described herewith, for example, in Section 5.2.

[0205] In certain embodiments, less than about 50% (e.g., less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, or less than about 0.1%) of the differentiated cells express at least one marker selected from PAX6, EMX2, LHX2, SMA, SIX1, PITX2, SIM1, POU4F1, PHOX2A, BARHL1, BARHL2, GBX2, H0XA1, H0XA2, HOXB1, HOXB2, POU5F1, NANOG, and combinations thereof.

[0206] In addition, the present disclosure provides compositions comprising any of the cell populations disclosed herein.

[0207] In certain embodiments, the cells are comprised in a composition that further comprises a biocompatible scaffold or matrix, for example, a biocompatible three- dimensional scaffold that facilitates tissue regeneration when the cells are implanted or grafted to a subject. In certain embodiments, the biocompatible scaffold comprises extracellular matrix material, synthetic polymers, cytokines, collagen, polypeptides or proteins, polysaccharides including fibronectin, laminin, keratin, fibrin, fibrinogen, hyaluronic acid, heparin sulfate, chondroitin sulfate, agarose or gelatin, and / or hydrogel. (See, e.g., U.S. Publication Nos. 2015 / 0159135, 2011 / 0296542, 2009 / 0123433, and 2008 / 0268019, the contents of each of which are incorporated by reference in their entireties). In certain embodiments, the composition further comprises growth factors for promoting maturation of the implanted / grafted cells into midbrain DA cells.

[0208] In certain embodiments, the composition comprises a cell population of from about 1 x 104to about 1 x 1010, from about 1 x 104to about 1 x 105, from about 1 x 105to about 1 x 072734.1886 PATENT 109, from about 1 x 105to about 1 x 106, from about 1 x 105to about 1 x 107, from about 1 x 106to about 1 x 107, from about 1 x 106to about 1 x 108, from about 1 x 107to about 1 x 108, from about 1 x 108to about 1 x 109, from about 1 x 108to about 1 x IO10, or from about 1 x 109to about 1 x IO10the cells are administered to a subject. In certain embodiments, from about 1 x 105to about 1 x 107the cells thereof are administered to a subject.

[0209] In certain embodiments, said composition is frozen. In certain embodiments, said composition further comprises at least one cryoprotectant, for example, but not limited to, dimethylsulfoxide (DMSO), glycerol, polyethylene glycol, sucrose, trehalose, dextrose, or a combination thereof.

[0210] In certain embodiments, the composition further comprises a biocompatible scaffold or matrix, for example, a biocompatible three-dimensional scaffold that facilitates tissue regeneration when the cells are implanted or grafted to a subject. In certain embodiments, the biocompatible scaffold comprises extracellular matrix material, synthetic polymers, cytokines, collagen, polypeptides or proteins, polysaccharides including fibronectin, laminin, keratin, fibrin, fibrinogen, hyaluronic acid, heparin sulfate, chondroitin sulfate, agarose or gelatin, and / or hydrogel. (See, e.g., U.S. Publication Nos. 2015 / 0159135, 2011 / 0296542, 2009 / 0123433, and 2008 / 0268019, the contents of each of which are incorporated by reference in their entireties).

[0211] In certain embodiments, the composition is a pharmaceutical composition that comprises a pharmaceutically acceptable carrier. The compositions can be used for preventing and / or treating a neurodegenerative disorder include Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, and multiple sclerosis.

[0212] The presently disclosed subject matter also provides a device comprising the differentiated cells or the composition comprising thereof, as disclosed herein. Non-limiting examples of devices include syringes, fine glass tubes, stereotactic needles and cannulas.

[0213] 7.4. Method of Preventins, Modeling, and / or Treatins Neurological Disorders

[0214] The cell populations and compositions disclosed herein (e.g., those disclosed in Section 5.3) can be used for preventing, modeling, and / or treating at least a symptom in a subject having a neurological disorder. The presently disclosed subject matter provides for methods of preventing, modeling, and / or treating at least a symptom in a subject having a neurological disorder. In certain embodiments, the method comprises administering an effective amount of the presently disclosed stem-cell-derived mDA neurons or a composition comprising thereof into a subject suffering from a neurological disorder. In certain 072734.1886 PATENT embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

[0215] In certain embodiments, the neurological disorder is characterized by reduction of midbrain dopamine neuron function. The reduction of midbrain dopamine neuron function can be age related.

[0216] In certain embodiments, the symptom for a neurological disorder is selected from the group consisting of tremor, bradykinesia, flexed posture, postural instability, rigidity, dysphagia, and dementia.

[0217] Non-limiting examples of neurological disorders include Parkinsonism, Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, and multiple sclerosis. In certain embodiments, the neurological disorder is Parkinsonism or Parkinson’s disease.

[0218] In certain embodiments, the neurological disorder is Parkinson’s disease. Primary motor signs of Parkinson’s disease include, for example, but not limited to, tremor of the hands, arms, legs, jaw and face, bradykinesia or slowness of movement, rigidity or stiffness of the limbs and trunk and postural instability or impaired balance and coordination.

[0219] In certain embodiments, the neurological disorder is a parkinsonism disease, which refers to diseases that are linked to an insufficiency of dopamine in the basal ganglia, which is a part of the brain that controls movement. Symptoms include tremor, bradykinesia (extreme slowness of movement), flexed posture, postural instability, and rigidity. Nonlimiting examples of parkinsonism diseases include corticobasal degeneration, Lewy body dementia, multiple systematrophy, and progressive supranuclear palsy.

[0220] The cells or compositions can be administered or provided systemically or directly to a subject for preventing, modeling, and / or treating a neurological disorder. In certain embodiments, the cells or compositions are directly injected into an organ of interest (e.g., the central nervous system (CNS)). In certain embodiments, the cells or compositions are directly injected into the striatum.

[0221] The cells or compositions can be administered in any physiologically acceptable vehicle. The cells or compositions can be administered via localized injection, orthotopic (OT) injection, systemic injection, intravenous injection, or parenteral administration. In certain embodiments, the cells or compositions are administered to a subject suffering from a neurodegenerative disorder via orthotopic (OT) injection.

[0222] The cells or compositions can be conveniently provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which can be buffered to a selected pH. Liquid preparations are normally 072734.1886

[0223] PATENT easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like) and suitable mixtures thereof. Sterile injectable solutions can be prepared by incorporating the compositions of the presently disclosed subject matter, e.g., a composition comprising the presently disclosed stem-cell-derived precursors, in the required amount of the appropriate solvent with various amounts of the other ingredients, as desired. Such compositions can be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can also be lyophilized. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as “REMINGTON’S PHARMACEUTICAL SCIENCE”, 17th edition, 1985, incorporated herein by reference, can be consulted to prepare suitable preparations, without undue experimentation.

[0224] Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, alum inurn monostearate and gelatin.

[0225] Viscosity of the compositions, if desired, can be maintained at the selected level using a pharmaceutically acceptable thickening agent. Methylcellulose can be used because it is readily and economically available and is easy to work with. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like. The concentration of the thickener can depend upon the agent selected. The important point is to use an amount that will achieve the selected viscosity. The choice of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, e.g., liquid dosage form (e.g., 072734.1886

[0226] PATENT whether the composition is to be formulated into a solution, a suspension, gel or another liquid form, such as a time release form or liquid-filled form).

[0227] Those skilled in the art will recognize that the components of the compositions should be selected to be chemically inert and will not affect the viability or efficacy of the presently disclosed stem-cell-derived precursors. This will present no problem to those skilled in chemical and pharmaceutical principles, or problems can be readily avoided by reference to standard texts or by simple experiments (not involving undue experimentation), from this disclosure and the documents cited herein.

[0228] One consideration concerning the therapeutic use of the cells is the quantity of cells necessary to achieve an optimal effect. An optimal effect includes, but is not limited to, repopulation of CNS regions of a subject suffering from a neurodegenerative disorder, and / or improved function of the subject’s CNS.

[0229] An “effective amount” (or “therapeutically effective amount”) is an amount sufficient to affect a beneficial or desired clinical result upon treatment. An effective amount can be administered to a subject in at least one doses. In terms of treatment, an effective amount is an amount that is sufficient to palliate, ameliorate, stabilize, reverse or slow the progression of the neurodegenerative disorder, or otherwise reduce the pathological consequences of the neurodegenerative disorder. The effective amount is generally determined by the physician on a case-by-case basis and is within the skill of one in the art. Several factors are typically taken into account when determining an appropriate dosage to achieve an effective amount. These factors include age, sex and weight of the subject, the condition being treated, the severity of the condition and the form and effective concentration of the cells administered.

[0230] In certain embodiments, an effective amount of the cells is an amount that is sufficient to repopulate a CNS region of a subject suffering from a neurological disorder. In certain embodiments, an effective amount of the cells is an amount that is sufficient to improve the function of the CNS of a subject suffering from a neurodegenerative disorder, e.g., the improved function can be about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99% or about 100% of the function of a normal person’s CNS.

[0231] The quantity of cells to be administered will vary for the subject being treated. In certain embodiments, from about 1 x 104to about 1 x 1010, from about 1 x 104to about 1 x 105, from about 1 x 105to about 1 x io9, from about 1 x 105to about 1 x io6, from about 1 x 105to about 1 x io7, from about 1 x 106to about 1 x io7, from about 1 x 106to about 1 x 108, from about 1 x 107to about 1 x 108, from about 1 x 108to about 1 x 109, from about 1 x 072734.1886

[0232] PATENT

[0233] 108to about 1 x IO10, or from about 1 x 109to about 1 x IO10of the cells are administered to a subject. In certain embodiments, from about 1 x 105to about 1 x 107of the cells are administered to a subject suffering from a neurological disorder. In certain embodiments, from about 1 x io6to about 1 x 107of the cells are administered to a subject suffering from a neurological disorder. In certain embodiments, from about 1 x 106to about 4 x 106of the cells are administered to a subject suffering from a neurological disorder. The precise determination of what would be considered an effective dose can be based on factors individual to each subject, including their size, age, sex, weight, and condition of the particular subject. Dosages can be readily ascertained by those skilled in the art from this disclosure and the knowledge in the art.

[0234] 7.5 Methods of Identifying Therapeutic Compounds

[0235] The presently disclosed subject matter provides for in vitro methods of identifying or screening compounds suitable for preventing and / or treating a neurological disorder. In certain embodiments, the method comprises identifying a compound that is capable of rescuing at least one symptom presented by a population of cells, wherein the population of cells are selected from the group consisting of presently disclosed mDA neurons or precursors derived from stem cells (e.g., pluripotent stem cells) or a combination or mixture thereof.

[0236] In certain embodiments, the method comprise: (a) providing (i) a population of cells, wherein the population of cells are selected from the group consisting of presently disclosed mDA neurons or precursors derived from stem cells (e.g., pluripotent stem cells), and a combination or mixture thereof, and (ii) a test compound; (b) contacting the population of cells with the test compound; and (c) measuring the behavior of the population of cells. In certain embodiments, the population of cells (e.g., mDA neurons or precursors) are contacted with the test compound for at least about 6 hours, about 12 hours, about 24 hours (1 day), e.g., about 24 hours (1 day), about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days.

[0237] In certain embodiments, the population of cells are mDA neurons or precursors derived from stem cells. In certain embodiments, the stem cells are obtained from a subject having a neurological disorder. In certain embodiments, the neurological disorder is selected from the group consisting of Parkinsonism, Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, multiple sclerosis, and combinations thereof. In certain embodiments, the neurological disorder is selected from the group consisting of Parkinsonism, Parkinson’s disease, and combinations thereof 072734.1886

[0238] PATENT

[0239] In certain embodiments, the method further comprises determining a causal genetic mutation of a neurological disorder. Methods for determining a causal genetic mutation are known in the art, including, but not limited to, genetic lineage analyses among familial patient groups, forward and reverse genetics using mouse model.

[0240] 7.6. Kits

[0241] The presently disclosed subject matter provides kits for inducing differentiation of stem cells to mDA neurons or precursors thereof. In certain embodiments, the kit comprises (a) at least one inhibitor of SMAD signaling, (b) at least one activator of Wnt signaling, (c) at least one activator of SHH signaling, and (d) at least one inhibitor of Wnt signaling. In certain embodiments, the kit further comprises (e) instructions for inducing differentiation of the stem cells into a population of differentiated cells that express at least one marker indicating a mDA neuron or a precursor thereof.

[0242] In certain embodiments, the instructions comprise contacting the stem cells with the inhibitor(s) and activator(s) in a specific sequence. The sequence of contacting the inhibitor(s) and activator(s) can be determined by the cell culture medium used for culturing the stem cells.

[0243] In certain embodiments, the instructions comprise contacting the stem cells with the inhibitor(s) and activator(s) as described by the methods of the present disclosure (see Section 5.2).

[0244] In certain embodiments, the present disclosure provides kits comprising an effective amount of a cell population or a composition disclosed herein in unit dosage form. In certain embodiments, the kit comprises a sterile container which contains the therapeutic composition; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.

[0245] In certain embodiments, the kit comprises instructions for administering the cell population or composition to a subject suffering from a neurological disorder. The instructions can comprise information about the use of the cells or composition for preventing, modeling, and / or treating at least a symptom in a subject having a neurological disorder. In certain embodiments, the instructions comprise at least one of the following: description of the therapeutic agent; dosage schedule and administration for preventing, modeling, and / or treating at least a symptom in a subject having a neurological disorder or symptoms thereof; precautions; warnings; indications; counter-indications; over dosage 072734.1886

[0246] PATENT information; adverse reactions; animal pharmacology; clinical studies; and / or references. The instructions can be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.

[0247] In certain embodiments, the instructions comprise directions to contact or expose the stem cells with at least one inhibitor of Wnt signaling. In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one inhibitor of SMAD signaling. In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one activator of SHH signaling. In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one activator of Wnt signaling.

[0248] In certain embodiments, the instructions comprise directions to contact or expose the stem cells with at least one inhibitor of Wnt signaling, at least one inhibitor of SMAD signaling, at least one activator of SHH signaling, and at least one activator of Wnt signaling to obtain a population of differentiated cells expressing at least one marker indicating a midbrain dopamine neuron or a precursor thereof.

[0249] In certain embodiments, the at least one inhibitor of SMAD signaling comprises one or more inhibitor of TGFp / Activin-Nodal signaling. In certain embodiments, the instructions comprise directions to contact the stem cells with the one or more inhibitor of TGFp / Activin- Nodal signaling for at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, at least about 21 days, at least about 22 days, at least about 23 days, at least about 24 days, at least about 25 days, at least about 26 days, at least about 27 days, at least about 28 days, at least about 29 days, or at least about 30 days, to produce a population of mDA neurons or precursors thereof. In certain embodiments, the instructions comprise directions to contact the stem cells with the one or more inhibitor of TGFp / Activin-Nodal signaling for up to about 3 days, up to about 4 days, up to about 5 days, up to about 6 days, up to about 7 days, up to about 8 days, up to about 9 days, up to about 10 days, up to about 11 days, up to about 12 days, up to about 13 days, up to about 14 days, up to about 15 days, up to about 16 days, up to about 17 days, up to about 18 days, up to about 19 days, up to about 20 days, up to about 21 days, up to about 22 days, up to about 23 days, up to about 24 days, up to about 25 days, up to about 26 days, up to about 27 days, 072734.1886

[0250] PATENT up to about 28 days, up to about 29 days, or up to about 30 days, to produce a population of mDA neurons or precursors thereof. In certain embodiments, the instructions comprise directions to contact the stem cells with the one or more inhibitor of TGFp / Activin-Nodal signaling for between about 4 days and about 30 days, between about 4 days to about 27 days, between about 4 days and about 26 days, between about 4 days and about 25 days, between about 4 days and about 24 days, between about 4 days and about 20 days, between about 4 days and about 15 days, between about 4 days and about 10 days, between about 5 days and about 15 days, between about 5 days and about 10 days, between about 10 days and about 15 days, between about 15 days and about 20 days, between about 10 days and about 20 days, between about 20 days and about 25 days, or between about 25 days and about 30 days, to produce a population of mDA neurons or precursors thereof. In certain embodiments, the instructions comprise directions to contact the stem cells with the one or more inhibitor of TGFp / Activin-Nodal signaling for between 10 days and about 15 days, to produce a population of mDA neurons or precursors thereof. In certain embodiments, the instructions comprise directions to contact the stem cells with the one or more inhibitor of TGFp / Activin- Nodal signaling for about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, or about 30 day, to produce a population of mDA neurons or precursors thereof. In certain embodiments, the instructions comprise directions to contact the stem cells with the one or more inhibitor of TGFp / Activin-Nodal signaling for about 11 days, to produce a population of mDA neurons or precursors thereof.

[0251] In certain embodiments, the instructions comprise directions to contact the stem cells with the one or more inhibitor of SMAD signaling for at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, at least about 21 days, at least about 22 days, at least about 23 days, at least about 24 days, at least about 25 days, at least about 26 days, at least about 27 days, at least about 28 days, at least about 29 days, or at least about 30 days, to produce a population of mDA neurons or precursors thereof. In certain embodiments, the instructions comprise directions to contact the stem cells with the one or more inhibitor of SMAD signaling for up to about 3 days, up 072734.1886

[0252] PATENT to about 4 days, up to about 5 days, up to about 6 days, up to about 7 days, up to about 8 days, up to about 9 days, up to about 10 days, up to about 11 days, up to about 12 days, up to about 13 days, up to about 14 days, up to about 15 days, up to about 16 days, up to about 17 days, up to about 18 days, up to about 19 days, up to about 20 days, up to about 21 days, up to about 22 days, up to about 23 days, up to about 24 days, up to about 25 days, up to about 26 days, up to about 27 days, up to about 28 days, up to about 29 days, or up to about 30 days, to produce a population of mDA neurons or precursors thereof. In certain embodiments, the instructions comprise directions to contact the stem cells with the one or more inhibitor of SMAD signaling for between about 4 days and about 30 days, between about 4 days to about 27 days, between about 4 days and about 26 days, between about 4 days and about 25 days, between about 4 days and about 24 days, between about 4 days and about 20 days, between about 4 days and about 15 days, between about 4 days and about 10 days, between about 5 days and about 15 days, between about 5 days and about 10 days, between about 10 days and about 15 days, between about 15 days and about 20 days, between about 10 days and about 20 days, between about 20 days and about 25 days, or between about 25 days and about 30 days, to produce a population of mDA neurons or precursors thereof. In certain embodiments, the instructions comprise directions to contact the stem cells with the one or more inhibitor of SMAD signaling for between 10 days and about 15 days, to produce a population of mDA neurons or precursors thereof. In certain embodiments, the instructions comprise directions to contact the stem cells with the one or more inhibitor of SMAD signaling for about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, or about 30 day, to produce a population of mDA neurons or precursors thereof. In certain embodiments, the instructions comprise directions to contact the stem cells with the one or more inhibitor of SMAD signaling for about 11 days, to produce a population of mDA neurons or precursors thereof. In certain embodiments, the stem cells are exposed to two SMAD inhibitors. In certain embodiments, the two SMAD inhibitors are a TGFp / Activin-Nodal inhibitor and a BMP inhibitor.

[0253] In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one inhibitor of SMAD signaling for at least about 5 days, or for about 5 days, about 6 days, or about 7 days. In certain embodiments, the at least one inhibitor of SMAD signaling comprises an inhibitor of TGFp / Activin-Nodal signaling, an 072734.1886 PATENT inhibitor of BMP signaling, or a combination thereof. In certain embodiments, the at least one inhibitor of SMAD signaling comprises an inhibitor of TGFp / Activin-Nodal signaling. In certain embodiments, the at least one inhibitor of TGFp / Activin-Nodal signaling comprises an inhibitor of ALK5. In certain embodiments, the at least one inhibitor of TGFp / Activin- Nodal signaling is selected from the group consisting of SB431542, derivatives of SB431542 (e.g., A83-01), and combinations thereof. In certain embodiments, the at least one inhibitor of BMP signaling is selected from the group consisting of LDN193189, Noggin, dorsomorphin, derivatives of LDN193189, derivatives of Noggin, derivatives of dorsomorphin, and combinations thereof.

[0254] In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one Wnt inhibitor for at least about 1 day or at least about 2 days. In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one Wnt inhibitor for up to about 1 day or up to about 2 days. In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one Wnt inhibitor for between about 1 days and about 2 days. In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one Wnt inhibitor for about 1 day. In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one Wnt inhibitor for about 2 days. In certain embodiments, the instructions further comprise directions to initially contact or expose the stem cells with at least one Wnt inhibitor at least about 1 day, or at least about 2 days, or at least about 3 days, or at least about 4 days, or at least about 5 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor. In certain embodiments, the instructions further comprise directions to initially contact or expose the stem cells with at least one Wnt inhibitor concurrently with the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor.

[0255] In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one activator of SHH signaling for at least about 5 days, or for about 5 days, about 6 days, or about 7 days. In certain embodiments, the at least one activator of SHH signaling is selected from the group consisting of SHH proteins, Smoothened agonists (SAG) (e.g., purmorphamine), and combinations thereof. In certain embodiments, the SHH protein is selected from the group consisting of recombinant SHHs, modified N-terminal SHHs, and combinations thereof. In certain embodiments, the modified N-terminal SHH comprises two Isoleucines at the N-terminus. In certain embodiments, the 072734.1886

[0256] PATENT modified N-terminal SHH has at least about 90% sequence identity to an un-modified N- terminal SHH. In certain embodiments, the un-modified N-terminal SHH is a un-modified mouse N-terminal SHH or a un-modified human N-terminal SHH. In certain embodiments, the modified N-terminal SHH comprises SHH C25II. In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one activator of SHH signaling for at least about 5 days, or at least about 10 days. In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one activator of SHH signaling for up to about 5 days, or up to about 10 days. In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one activator of SHH signaling for between about 5 days and about 10 days. In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one activator of SHH signaling for about 5 days. In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one activator of SHH signaling for about 6 days. In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one activator of SHH signaling from day 0 through day 6. In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one activator of SHH signaling for every day or every other day from day 0 through day 6. In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one activator of SHH signaling for every day (daily) from day 0 through day 6. In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one activator of SHH signaling for 7 days. In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one activator of SHH signaling from day 0 through day 7. In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one activator of SHH signaling every day or every other day from day 0 through day 7. In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one activator of SHH signaling every day (daily) from day 0 through day 7.

[0257] In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one activator of Wnt signaling for at least about 5 days, at least about 10 days, at least about 15 days, or at least about 20 days. In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one activator of Wnt signaling for up to about 5 days, up to about 10 days, up to about 15 072734.1886

[0258] PATENT days, or up to about 20 days In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one activator of Wnt signaling for between about 5 days and about 20 days, between about 5 days and about 15 days, between about 10 days and about 20 days, between about 5 days and about 15 days, or between about 10 days and about 15 days. In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one activator of Wnt signaling for between about 10 days and about 20 days. In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one activator of Wnt signaling for about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, or about 17 days. In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one activator of Wnt signaling from day 2 through day 12. In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one activator of Wnt signaling from day 2 through day 11. In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one activator of Wnt signaling for day 2 through day 10. In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one activator of Wnt signaling from day 1 through day 12. In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one activator of Wnt signaling from day 1 through day 11. In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one activator of Wnt signaling from day 1 through day 10. In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one activator of Wnt signaling from day 0 through day 16. In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one activator of Wnt signaling from day 2 through day 16. In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one activator of Wnt signaling every day or every other day from day 0 through day 16. In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one activator of Wnt signaling every day or every other day from day 2 through day 12. In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one activator of Wnt signaling every day (daily) from day 2 through day 12. In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one activator of Wnt 072734.1886

[0259] PATENT signaling every day or every other day from day 2 through day 11 In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one activator of Wnt signaling every day (daily) from day 2 through day 11. In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one activator of Wnt signaling every day or every other day from day 2 through day 10. In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one activator of Wnt signaling every day (daily) from day 2 through day 10. In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one activator of Wnt signaling every day or every other day from day 1 through day 12 In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one activator of Wnt signaling every day (daily) from day 1 through day 12. In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one activator of Wnt signaling for every day or every other day from day 1 through day 11. In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one activator of Wnt signaling every day (daily) from day 1 through day 11. In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one activator of Wnt signaling every day or every other day from day 1 through day 10. In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one activator of Wnt signaling for every day (daily) from day 1 through day 10. In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one activator of Wnt signaling every day (daily) from day 0 through day 16. In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one activator of Wnt signaling every day or every other day from day 2 through day 16. In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one activator of Wnt signaling every day (daily) from day 2 through day 16. n certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one activator of Wnt signaling at least about 1 day, or at least about 2 days, or at least about 3 days, or at least about 4 days from the initial contact of the cells with the at least one SMAD inhibitor. In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one activator of Wnt signaling about 1 day, or about 2 days, or about 3 days, or about 4 days from the initial contact of the cells with the at least one SMAD inhibitor. In certain embodiments, the instructions further 072734.1886

[0260] PATENT comprise directions to contact or expose the stem cells with at least one activator of Wnt signaling about 2 days from the initial contact of the cells with the at least one SMAD inhibitor. In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one activator of Wnt signaling at least about 1 day, or at least about 2 days, or at least about 3 days, or at least about 4 days from the initial contact of the cells with the at least one SMAD inhibitor, and for about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, or about 17 days. In certain embodiments, the instructions further comprise directions to contact or expose the stem cells with at least one activator of Wnt signaling for at least about 5 days or for about 7 days, about 8 days, or about 9 days. In certain embodiments, the instructions further comprise directions to initially contact or expose the stem cells with at least one activator of Wnt signaling at least about 2 days from the initial contact of the cells with the at least one SMAD inhibitor. In certain embodiments, the instructions further comprise directions to initially contact or expose the stem cells with at least one activator of Wnt signaling about 2 days from the initial contact of the cells with the at least one SMAD inhibitor. In certain embodiments, the at least one activator of Wnt signaling comprises an inhibitor of glycogen synthase kinase 3p (GSK3P) signaling. In certain embodiments, the at least one activator of Wnt signaling is selected from the group consisting of CHIR99021, CHIR98014, AMBMP hydrochloride, LP 922056, Lithium, deoxycholic acid, BIO, SB-216763, Wnt3A, Wntl, Wnt5a, derivatives thereof, and combinations thereof.

[0261] In certain embodiments, the at least one marker indicating a midbrain dopamine neuron or a precursor thereof is selected from the group consisting of EN1, OTX2, TH, NURR1, FOXA2, PITX3, LMX1A, LM03, SNCA, ADCAP1, CHRNA4, SOX6, DAT, VMAT2, WNT1, GIRK2, or combinations thereof.

[0262] In certain embodiments, the instructions further comprise directions to contact the cells (e.g., mDA precursors) with DA neuron lineage specific activators and inhibitors, for example, L-glutamine, brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), Cyclic adenosine monophosphate (cAMP), Transforming growth factor beta (TGFP, for example, TGFP3), ascorbic acid (AA), and DAPT (which is also known as, N-[(3,5-Difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-l,l-dimethylethyl ester; LY-374973, N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester; or N-[N- (3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester). In certain embodiments, the instructions further comprise directions to contact the cells with the 072734.1886

[0263] PATENT foregoing DA neuron lineage specific activators and inhibitors for at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, or at least about 10 or more days, for example, between about 2 days and about 20 days, between about 3 days and about 19 days, between about 4 days and about 18 days, between about 5 days and about 17 days, between about 6 days and about 16 days, between about 7 days and about 15 days, between about 8 days and about 15 days, between about 9 days and about 14 days, or between about 10 days and about 13 days. In certain embodiments, the instructions further comprise directions to contact the cells with the foregoing DA neuron lineage specific activators and inhibitors for up to about 2, up to about 3, up to about 4, up to about 5, up to about 6, up to about 7, up to about 8, up to about 9, or up to about 10 days or more days. In certain embodiments, the instructions further comprise directions to contact the cells with the foregoing DA neuron lineage specific activators and inhibitors for about 4 days, about 5 days, about 6 days, about 7 days, or about 8 days.

[0264] In certain embodiments, the kit comprises instructions for administering a population of the presently disclosed stem-cell-derived mDA neurons or a composition comprising thereof into a subject suffering from a neurological disorder. In certain embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier. In certain embodiments, the neurological disorder is characterized by reduction of midbrain dopamine neuron function. The reduction of midbrain dopamine neuron function can be age related. In certain embodiments, the symptom for a neurological disorder is selected from the group consisting of tremor, bradykinesia, flexed posture, postural instability, rigidity, dysphagia, and dementia. Non-limiting examples of neurological disorders include Parkinsonism, Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, and multiple sclerosis. In certain embodiments, the neurological disorder is Parkinsonism or Parkinson’s disease. In certain embodiments, the neurological disorder is Parkinson’s disease. Primary motor signs of Parkinson’s disease include, for example, but not limited to, tremor of the hands, arms, legs, jaw and face, bradykinesia or slowness of movement, rigidity or stiffness of the limbs and trunk and postural instability or impaired balance and coordination. In certain embodiments, the neurological disorder is a parkinsonism disease, which refers to diseases that are linked to an insufficiency of dopamine in the basal ganglia, which is a part of the brain that controls movement. Symptoms include tremor, bradykinesia (extreme slowness of movement), flexed posture, postural instability, and rigidity. Non-limiting examples of parkinsonism diseases include corticobasal 072734.1886 PATENT degeneration, Lewy body dementia, multiple systematrophy, and progressive supranuclear palsy.

[0265] 8. EXAMPLES

[0266] Example 1: “inverted bump” in D A protocol

[0267] Midbrain dopaminergic neuron (mDA) differentiation is thought to require low levels of WNT activation within the first two days of hPSC differentiation. The rationale for this approach is the caudalization of neural progenitors away from forebrain fates and toward midbrain fates. However, using this strategy can sometimes result in generation of hindbrain derivatives in unpredictable amounts, as well as other off-target lineages. The presence of off-target cell types in these cultures is of particular concern in the generation of hPSC- derived mDA neurons for the treatment of Parkinson’s disease, a strategy that has recently shown promise in Phase 1 clinical trials.

[0268] The competency window for transition of hPSC-derived neural progenitors to posterior fates closes at 48 hrs (FIG. 3). Any WNT activation before this point has the chance to induce hindbrain fate, while WNT activation afterwards can caudalize within the anterior cranial compartment (i.e. forebrain / midbrain lineages). The previously described mDA differentiation protocol was modified such that WNT signaling was inhibited for the first 48hrs (using 2uM XAV; “inverted bump”) before being activated (6uM CHIR99021 onset at D2 instead of D4; FIG. 3 A). Using this strategy, it was still possible to generate EN1+ FOXA2+ OTX2+ mDA progenitors at DI 1 (FIGS. 2A-2B), and there was an increase in EN1 efficiency from -40% to 70% (FIGS. 3B and 3E). The proportion of CD56 and PITX3 positive cells at D25 was equivalent to controls (FIG. 3C) and the efficient generation of TH+ FOXA2+ PITX3+ dopaminergic neurons was possible by D30 (FIG. 3D). Additionally, hindbrain contaminating cells (indicated by GBX2 expression) are at the lowest in the WNT inhibition condition (XAV treatment). These results provide a platform for increased robustness of mDA neuron generation and also confirm the primary axis patterning principles identified for differentiating hPSCs in this study.

[0269] 9. REFERENCES

[0270] 1. Xiong, M. et al. Human Stem Cell-Derived Neurons Repair Circuits and Restore Neural Function. Cell Stem Cell 28, 112-126. e6 (2021). 072734.1886

[0271] PATENT

[0272] 2. Xi, J. et al. Specification of Midbrain Dopamine Neurons from Primate Pluripotent Stem Cells. Stem Cells 30, 1655-1663 (2012).

[0273] 3. Gantner, C. W. et al. Viral Delivery of GDNF Promotes Functional Integration of Human Stem Cell Grafts in Parkinson’s Disease. Cell Stem Cell 26, 511-526. e5 (2020). 4. Kim, T. W. et al. Biphasic Activation of WNT Signaling Facilitates the Derivation of

[0274] Midbrain Dopamine Neurons from hESCs for Translational Use. Cell Stem Cell 28, 343- 355. e5 (2021).

[0275] 5. Kirkeby, A. et al. Generation of Regionally Specified Neural Progenitors and Functional Neurons from Human Embryonic Stem Cells under Defined Conditions. Cell Rep. 1, 703-714 (2012).

Claims

072734.1886PATENTWHAT IS CLAIMED IS:

1. An in vitro method for inducing differentiation of stem cells, comprising:(a) contacting the stem cells with at least one inhibitor of wingless (Wnt) signaling for up to about 2 days;(b) contacting the stem cells with at least one inhibitor of Small Mothers Against Decapentaplegic (SMAD) signaling and at least one activator of Sonic hedgehog (SHH) signaling; and(c) contacting the stem cells with at least one activator of Wnt signaling to obtain a population of differentiated cells expressing at least one marker indicating a midbrain dopamine neuron or a precursor thereof.

2. The method of claim 1, wherein the stem cells are contacted with the at least one inhibitor of Wnt signaling for about 1 day or about 2 days.

3. The method of claim 1 or claim 2, wherein the cells are contacted with the at least one inhibitor of SMAD signaling for at least about 5 days.

4. The method of any one of claims 1-3, wherein the cells are contacted with the at least one inhibitor of SMAD signaling for about 5 days, about 6 days, or about 7 days.

5. The method of any one of claims 1-4, wherein the cells are contacted with the at least one activator of SHH signaling for at least about 5 days.

6. The method of any one of claims 1-5, wherein the cells are contacted with the at least one activator of SHH signaling for about 5 days, about 6 days, or about 7 days.

7. The method of any one of claims 1-6, wherein the cells are contacted with the at least one activator of Wnt signaling for at least about 5 days.

8. The method of any one of claims 1-7, wherein the cells are contacted with the at least one activator of Wnt signaling for about 7 days, about 8 days, or about 9 days.

9. The method any one of claims 1-8, wherein the initial contact of the cells with the at least one activator of Wnt signaling is at least about 2 days from the initial contact of the cells with the at least one SMAD inhibitor.072734.1886PATENT10. The method any one of claims 1-9, wherein the initial contact of the cells with the at least one activator of Wnt signaling is about 2 days from the initial contact of the cells with the at least one SMAD inhibitor.

11. The method of any one of claims 1-10, wherein the at least one inhibitor of SMAD signaling comprises an inhibitor of TGFp / Activin-Nodal signaling, an inhibitor of BMP signaling, or a combination thereof.

12. The method of claim 11, wherein the at least one inhibitor of TGFp / Activin-Nodal signaling comprises an inhibitor of ALK5.

13. The method of claim 11 or 12, wherein the at least one inhibitor of TGFp / Activin- Nodal signaling is selected from the group consisting of SB431542, derivatives of SB431542, and combinations thereof.

14. The method of claim 13, wherein the derivative of SB431542 comprises A83-01.

15. The method of any one of claims 11-14, wherein the at least one inhibitor of TGFp / Activin-Nodal signaling comprises SB431542.

16. The method of claim 11, wherein the at least one inhibitor of BMP signaling is selected from the group consisting of LDN193189, Noggin, dorsomorphin, derivatives of LDN193189, derivatives of Noggin, derivatives of dorsomorphin, and combinations thereof.

17. The method of claim 11 or 16, wherein the at least one inhibitor of BMP comprises LDN-193189.

18. The method of any one of claims 1-17, wherein the at least one activator of Wnt signaling comprises an inhibitor of glycogen synthase kinase 3p (GSK3P) signaling.

19. The method of any one of claims 1-18, wherein the at least one activator of Wnt signaling is selected from the group consisting of CHIR99021, CHIR98014, AMBMP hydrochloride, LP 922056, Lithium, deoxycholic acid, BIO, SB-216763, Wnt3A, Wntl, Wnt5a, derivatives thereof, and combinations thereof.

20. The method of any one of claims 1-19, wherein the at least one activator of Wnt signaling comprises CHIR99021.072734.1886PATENT21. The method of any one of claims 1-20, wherein the at least one activator of SHH signaling is selected from the group consisting of SHH proteins, Smoothened agonists (SAG), and combinations thereof.

22. The method of claim 21, wherein the SHH protein is selected from the group consisting of recombinant SHHs, modified N-terminal SHHs, and combinations thereof.

23. The method of claim 22, wherein the modified N-terminal SHH comprises two Isoleucines at the N-terminus.

24. The method of claim 22 or 23, wherein the modified N-terminal SHH has at least about 90% sequence identity to an un-modified N-terminal SHH.

25. The method of claim 24, wherein the un-modified N-terminal SHH is a un-modified mouse N-terminal SHH or a un-modified human N-terminal SHH.

26. The method of any one of claims 22-25, wherein modified N-terminal SHH comprises SHH C25II.

27. The method of claim 21, wherein the SAG comprises purmorphamine.

28. The method of any one of claims 1-27, wherein the at least one marker indicating a midbrain dopamine neuron or a precursor thereof is selected from the group consisting of EN1, OTX2, TH, NURR1, FOXA2, PITX3, LMX1A, LM03, SNCA, ADCAP1, CHRNA4, SOX6, DAT, VMAT2, WNT1, GIRK2, and combinations thereof.

29. The method of any one of claims 1-28, wherein the differentiated cells do no express at least one marker selected from the group consisting of PAX6, EMX2, LHX2, SMA, SIX1, PITX2, SIM1, POU4F1, PHOX2A, BARHL1, BARHL2, GBX2, H0XA1, HOXA2, HOXB1, HOXB2, POU5F1, NANOG, and combinations thereof.

30. The method of any one of claims 1-29, further comprising isolating cells that express at least one positive marker and do not express at least one negative marker.

31. The method of claim 30, wherein the at least one positive marker is selected from the group consisting of PITX3, ALDH1A1, TH, and combinations thereof.072734.1886PATENT32. The method of claim 30 or 31, wherein the at least one positive marker comprises ALDH1A1.

33. The method of any one of claims 30-32, wherein the at least one negative marker is selected from CD49e, CD340, and combination thereof.

34. The method of any one of claims 1-33, further comprising subjecting the population of differentiated cells to conditions favoring differentiation of midbrain dopamine neuron precursors to midbrain dopamine neurons.

35. The method of claim 34, wherein the conditions comprise exposing the cells to at least one of brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), Cyclic adenosine monophosphate (cAMP), Transforming growth factor beta 3 (TGFP3), ascorbic acid (AA), and DAPT.

36. The method of any one of claims 1-35, wherein the stem cells are pluripotent stem cells.

37. The method of any one of claims 1-36, wherein the stem cells are selected from the group consisting of nonembryonic stem cells, embryonic stem cells, induced pluripotent stem cells, and combinations thereof.

38. The method of any one of claims 1-37, wherein the stem cells are human stem cells, non-human primate stem cells, or rodent stem cells.

39. The method of any one of claims 1-38, wherein the stem cells are human stem cells.

40. A cell population of in vitro differentiated cells, wherein the in vitro differentiated cells are obtained by a method of any one of claims 1-39.

41. A composition comprising the cell population of claim 40.

42. The composition of claim 41, which is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

43. A kit for inducing differentiation of stem cells to midbrain dopamine neurons or precursors thereof, comprising:(a) at least one inhibitor of Wnt signaling;072734.1886PATENT(b) at least one inhibitor of SMAD signaling;(c) at least one activator of SHH signaling; and(d) at least one activator of Wnt signaling.

44. The kit of claim 43, further comprising (f) instructions for inducing differentiation of the stem cells into a population of differentiated cells that express at least one marker indicating a midbrain dopamine neuron or a precursor thereof.

45. A method of preventing, modeling, and / or treating at least one symptom in a subject having a neurological disorder, comprising administering to the subject an effective amount of one of the followings:(a) the cell population of claim 40; or(b) the composition of claim 41 or 42.

46. The method of claim 45, wherein the neurological disorder is characterized by reduction of midbrain dopamine neuron function.

47. The method of claim 46, wherein the reduction of midbrain dopamine neuron function is age related.

48. The method of any one of claims 45-47, wherein the neurological disorder is selected from the group consisting of Parkinsonism, Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, multiple sclerosis, and combinations thereof.

49. The method of any one of claims 45-48, wherein the neurological disorder is selected from the group consisting of Parkinsonism, Parkinson’s disease, and combinations thereof.

50. The method of any one of claims 45-49, wherein the symptom for a neurological disorder is selected from the group consisting of tremor, bradykinesia, flexed posture, postural instability, rigidity, dysphagia, and dementia.

51. The cell population of claim 40 or the composition of claim 41 or 42 for use in preventing, modeling, and / or treating at least one symptom in a subject having a neurological disorder in a subject.

52. The cell population or composition for use of claim 51, wherein the neurological disorder is characterized by reduction of midbrain dopamine neuron function.072734.1886PATENT53. The cell population or composition for use of claim 52, wherein the reduction of midbrain dopamine neuron function is age related.

54. The cell population or composition for use of any one of claims 51-53, wherein the neurological disorder is selected from the group consisting of Parkinsonism, Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, multiple sclerosis, and combinations thereof.

55. The cell population or composition for use of any one of claims 51-54, wherein the neurological disorder is selected from the group consisting of Parkinsonism, Parkinson’s disease, and combinations thereof.

56. The cell population or composition for use of any one of claims 51-55, wherein the symptom for a neurological disorder is selected from the group consisting of tremor, bradykinesia, flexed posture, postural instability, rigidity, dysphagia, and dementia.

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