Methods for generating and isolating mesencephalic dopamine neurons

Abstract

To provide a method for generating and isolating midbrain dopamine neurons.SOLUTION: The present disclosure provides methods for producing midbrain dopamine neurons (mDA) and precursors thereof, mDA and precursors thereof produced 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 isolating mDA and their precursors from cell populations using the novel surface markers. In certain embodiments, the cells are contacted with the at least one activator of FGF signaling for at least about 1 day. In certain embodiments, the cells are contacted with the at least one activator of FGF signaling for up to about 15 days. In a particular embodiment, the cells are contacted with the at least one activator of FGF signaling for about 5 days.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 893,674, filed August 29, 2019, the contents of which are incorporated herein by reference in their entirety and from which priority is claimed.

[0002] 1. Introduction The present disclosure provides methods for generating midbrain dopamine neurons (mDA) and their precursors, mDA and their precursors generated by such methods, and compositions comprising such cells. The present disclosure also provides uses of mDA and compositions comprising same for preventing and / or treating neurological disorders. The present disclosure further provides methods for isolating mDA and its precursors from cell populations using novel surface markers. [Background technology]

[0003] 2.Background Previously, embryonic and somatic stem cells have been used as therapeutic agents and model systems for neurodegenerative diseases. Research and technological development on directed differentiation of embryonic and somatic stem cells has been conducted in the field of central nervous system (CNS) diseases, such as Huntington's disease, Alzheimer's disease, Parkinson's disease, and multiple sclerosis. However, the results of these studies have shown little in vivo ability to restore neuronal function and often resulted in undesirable tumor growth in patients.

[0004] Thus, there remains a need for improved methods for generating midbrain dopamine neurons suitable for treating neurodegenerative disorders such as Parkinson's disease. Summary of the Invention [Problem to be solved by the invention]

[0005] 3. Summary of the Invention The present disclosure provides midbrain dopamine neurons (mDA) and their precursors, methods for producing mDA and its precursors produced by such methods, compositions comprising such cells, and uses of such cells and compositions for preventing and / or treating neurological disorders. Additionally, the present disclosure provides methods for isolating mDA and its precursors from cell populations using novel surface markers. [Means for solving the problem]

[0006] In certain embodiments, the present disclosure provides an in vitro method for inducing differentiation of stem cells, the method comprising contacting the stem cells with at least one Small Mothers Against Decapentaplegic (SMAD) signaling inhibitor, at least one Sonic Hedgehog (SHH) signaling activator, and at least one Wingless (Wnt) signaling activator; and contacting the cells with at least one fibroblast growth factor (FGF) signaling activator to obtain a population of differentiated cells expressing at least one marker indicative of midbrain dopamine neurons (mDA) or precursors thereof, wherein the at least one FGF signaling activator is selected from FGF18, FGF17, FGF8a, and combinations thereof.

[0007] In certain embodiments, the present disclosure provides an in vitro method for inducing differentiation of stem cells, the method comprising contacting the stem cells with at least one Small Mothers Against Decapentaplegic (SMAD) signaling inhibitor, at least one Sonic Hedgehog (SHH) signaling activator, and at least one Wingless (Wnt) signaling activator; and contacting the cells with at least one fibroblast growth factor (FGF) signaling activator to obtain a population of differentiated cells expressing at least one marker indicative of midbrain dopamine neurons (mDA) or precursors thereof, wherein the initial contacting of the cells with the at least one FGF signaling activator is at least about 5 days after the initial contacting of the cells with the at least one SMAD signaling inhibitor.

[0008] In certain embodiments, the cells are contacted with at least one FGF signaling activator for at least about 1 day. In certain embodiments, the cells are contacted with at least one FGF signaling activator for up to about 15 days. In certain embodiments, the cells are contacted with at least one FGF signaling activator for about 5 days.

[0009] In certain embodiments, the initial contacting of the cells with the at least one activator of FGF signaling is at least about 5 days after the initial contacting of the cells with the at least one inhibitor of SMAD signaling.

[0010] In certain embodiments, the initial contact of the cells with the at least one activator of FGF signaling is about 10 days after the initial contact of the cells with the at least one inhibitor of SMAD signaling, hi certain embodiments, the initial contact of the cells with the at least one activator of FGF signaling is about 12 days after the initial contact of the cells with the at least one inhibitor of SMAD signaling.

[0011] In certain embodiments, the cells are contacted with at least one SMAD signaling inhibitor for about 5 days, hi certain embodiments, the cells are contacted with at least one SMAD signaling inhibitor for 7 days.

[0012] In certain embodiments, the cells are contacted with the at least one SHH signaling activator for about 5 days, hi certain embodiments, the cells are contacted with the at least one SHH signaling activator for 7 days.

[0013] In certain embodiments, the cells are contacted with the at least one activator of Wnt signaling for about 10 days. In certain embodiments, the cells are contacted with the at least one activator of Wnt signaling for 12 days.

[0014] In certain embodiments, the concentration of the at least one Wnt signaling activator is increased about 4 days after its initial contact with the stem cells. In certain embodiments, the concentration of the at least one Wnt signaling activator is increased by about 300% to about 1000% from the initial concentration of the at least one Wnt signaling activator. In certain embodiments, the concentration of the at least one Wnt signaling activator is increased to a concentration of about 3 μM to 10 μM. In certain embodiments, the concentration of the at least one Wnt signaling activator is increased to a concentration of about 3 μM. In certain embodiments, the concentration of the at least one Wnt signaling activator is increased to a concentration of about 7.5 μM.

[0015] In certain embodiments, the at least one FGF signaling activator comprises FGF18.

[0016] In certain embodiments, the at least one SMAD signaling inhibitor is selected from a TGFβ / Activin-Nodal signaling inhibitor, a bone morphogenetic protein (BMP) signaling inhibitor, and combinations thereof.

[0017] In certain embodiments, the at least one TGFβ / Activin-Nodal signaling inhibitor comprises an inhibitor of ALK5.

[0018] In certain embodiments, the at least one TGFβ / Activin-Nodal signaling inhibitor comprises SB431542, or a derivative or mixture thereof. In certain embodiments, the derivative of SB431542 is A83-01. In certain embodiments, the at least one TGFβ / Activin-Nodal signaling inhibitor comprises SB431542.

[0019] In certain embodiments, the at least one BMP signaling inhibitor comprises LDN193189, noggin, dorsomorphin, derivatives thereof, or mixtures thereof. In certain embodiments, the at least one BMP inhibitor comprises LDN-193189.

[0020] In certain embodiments, the at least one Wnt signaling activator comprises a glycogen synthase kinase 3β (GSK3β) signaling inhibitor.

[0021] In certain embodiments, the at least one activator of Wnt signaling is selected from CHIR99021, BIO, CHIR98014, lithium, 3F8, Wnt3A, Wnt1, Wnt5a, derivatives thereof, and mixtures thereof. In certain embodiments, the at least one activator of Wnt signaling comprises CHIR99021.

[0022] In certain embodiments, the at least one SHH signaling activator is selected from an SHH protein, a Smoothened agonist (SAG), a derivative thereof, and a mixture thereof. In certain embodiments, the SHH protein comprises recombinant SHH, purified SHH, or a combination thereof.

[0023] In certain embodiments, the recombinant SHH comprises a recombinant protein that is at least about 80% identical to an N-terminal fragment of mouse sonic hedgehog. In certain embodiments, the recombinant SHH comprises SHH C25II. In certain embodiments, the SAG comprises palmorfamine.

[0024] In certain embodiments, the at least one marker indicative of midbrain dopamine neurons or their precursors is selected from EN1, OTX2, TH, NURR1, FOXA2, PITX3, LMX1A, LMO3, SNCA, ADCAP1, CHRNA4, GIRK2, and combinations thereof.

[0025] In certain embodiments, the differentiated cells have a detectable level of expression of at least one marker indicative of midbrain dopamine neurons or precursors thereof at least about 10 days after initial contact of the stem cells with at least one SMAD signaling inhibitor.

[0026] In certain embodiments, the differentiated cells have a detectable level of expression of EN1 about 30 days after initial contact of the stem cell with at least one SMAD signaling inhibitor, hi certain embodiments, the differentiated cells have a detectable level of expression of EN1 about 40 days after initial contact of the stem cell with at least one SMAD signaling inhibitor.

[0027] In certain embodiments, the differentiated cells do not express at least one marker selected from PAX6, EMX2, LHX2, SMA, SIX1, PITX2, SIM1, POU4F1, PHOX2A, BARHL1, BARHL2, GBX2, HOXA2, HOXB2, POU5F1, NANOG, and combinations thereof.

[0028] In certain embodiments, the method further comprises subjecting the population of differentiated cells to conditions that favor differentiation of midbrain dopamine neuron precursors into midbrain dopamine neurons.

[0029] In certain embodiments, the conditions include exposing the cells to at least one of brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), cyclic adenosine monophosphate (cAMP), transforming growth factor beta 3 (TGFP3), ascorbic acid (AA), and DAPT.

[0030] In certain embodiments, the stem cells are selected from human, non-human primate, or rodent non-embryonic stem cells; human, non-human primate, or rodent embryonic stem cells; human, non-human primate, or rodent induced pluripotent stem cells; and human, non-human primate, or rodent recombinant pluripotent cells. In certain embodiments, the stem cells are human stem cells. In certain embodiments, the stem cells are pluripotent or multipotent stem cells. In certain embodiments, the stem cells are pluripotent stem cells. In certain embodiments, the pluripotent stem cells are selected from embryonic stem cells, induced pluripotent stem cells, and combinations thereof.

[0031] In another aspect, the disclosure provides a cell population of in vitro differentiated cells, said in vitro differentiated cells being obtained by any of the above methods.

[0032] In another aspect, the disclosure provides a cell population of in vitro differentiated cells, wherein at least about 50% of the cells express at least one marker indicative of midbrain dopamine neurons or precursors thereof, and less than about 50% of the differentiated cells express at least one marker selected from PAX6, EMX2, LHX2, SMA, SIX1, PITX2, SIM1, POU4F1, PHOX2A, BARHL1, BARHL2, GBX2, HOXA2, HOXB2, POU5F1, NANOG, and combinations thereof. In certain embodiments, the at least one marker indicative of midbrain dopamine neurons or precursors thereof is selected from EN1, OTX2, TH, NURR1, FOXA2, LMX1A, PITX3, LMO3, SNCA, ADCAP1, CHRNA4, and GIRK2.

[0033] In another aspect, the present disclosure provides a composition comprising the cell population disclosed herein. In certain embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

[0034] In another aspect, the disclosure provides a method for isolating midbrain dopamine neurons and their precursors from a population of cells, comprising isolating cells that do not express detectable levels of at least one negative surface marker and that express detectable levels of at least one positive surface marker.

[0035] In another aspect, the disclosure provides a method for isolating midbrain dopamine neurons and their precursors from a population of cells, the method comprising isolating cells that (a) do not express, or express a reduced level of, at least one negative surface marker at a detectable level relative to the average expression level of the at least one negative surface marker in the population of cells, and (b) have an elevated level of at least one positive surface marker relative to the average expression level of the at least one positive surface marker in the population of cells.

[0036] In certain embodiments, the at least one positive surface marker is selected from CD171, CD184, and combinations thereof. In certain embodiments, the at least one positive surface marker comprises CD184. In certain embodiments, the at least one negative surface marker is selected from CD49e, CD99, CD340, and combinations thereof. In certain embodiments, the at least one negative surface marker comprises CD49e. In certain embodiments, the method comprises isolating cells that do not express detectable levels of CD49e and that express detectable levels of CD184.

[0037] In certain embodiments, the methods comprise isolating cells that do not express or express reduced levels of CD49e relative to the average expression level of CD49e in the population of cells, and that express increased levels of CD184 relative to the average expression level of CD184 in the population of cells.

[0038] In another aspect, the present disclosure provides a cell population of in vitro differentiated cells, wherein at least about 50% of the cells express a detectable level of at least one positive surface marker and do not express a detectable level of at least one negative surface marker.

[0039] In another aspect, the disclosure provides a cell population of in vitro differentiated cells, wherein at least about 50% of the cells express an increased level of at least one positive surface marker compared to the average expression level of the at least one positive surface marker in the population of cells, and do not express a detectable level of, or express a reduced level of, at least one negative surface marker compared to the average expression level of the at least one negative surface marker in the population of cells.

[0040] In certain embodiments, the at least one positive surface marker is selected from CD171, CD184, and combinations thereof. In certain embodiments, the at least one positive surface marker comprises CD184. In certain embodiments, the at least one negative surface marker is selected from 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 comprises CD184 and the at least one negative surface marker comprises CD49e.

[0041] In another aspect, the present disclosure provides a composition comprising the cell population disclosed herein. In certain embodiments, the composition disclosed herein is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier. In another aspect, the present disclosure provides a kit for inducing differentiation of stem cells into midbrain dopamine neurons or their precursors, the kit comprising: (a) at least one SMAD signaling inhibitor; (b) at least one SHH signaling activator; (c) at least one Wnt signaling activator; and (d) at least one FGF signaling activator.

[0042] In certain embodiments, the kit further comprises (f) instructions for inducing differentiation of the stem cells into a population of differentiated cells that express at least one midbrain DA precursor marker.

[0043] In another aspect, the present disclosure provides a method of preventing and / or treating a neurodegenerative disorder in a subject, the method comprising administering to the subject an effective amount of (a) a cell population disclosed herein, or (b) one of the compositions disclosed herein.

[0044] In certain embodiments, the neurodegenerative disorder is Parkinson's disease, Huntington's disease, Alzheimer's disease, or multiple sclerosis.

[0045] In certain embodiments, the cell populations disclosed herein or the compositions disclosed herein are for use in preventing and / or treating a neurodegenerative disorder in a subject, hi certain embodiments, the neurodegenerative disorder is Parkinson's disease, Huntington's disease, Alzheimer's disease, or multiple sclerosis. 4. Brief description of the drawings [Brief explanation of the drawings]

[0046] [Figure 1] FIG. 1 shows EN1 expression in stem cell-derived mDA and progenitors from day 3 to day 30 using the Wnt-Boost protocol.

[0047] [Figure 2] FIG. 2 shows a protocol using FGF8b in combination with a Wnt-Boost protocol.

[0048] [Figure 3] Figure 3 shows that EN1 protein was maintained in differentiated cells in an FGF8b exposure duration-dependent manner. EN1-expressing cells also expressed FOXA2 and LMX1A.

[0049] [Figure 4-1] Figures 4A and 4B show the mRNA expression levels of differentiated cells measured by qRT-PCR on day 30. Figure 4A shows that the mRNA expression level of EN1 was maintained in an FGF8b exposure duration-dependent manner, while the expression levels of FOXA2, NURR1, and TH were comparable across all conditions. Figure 4B shows the mRNA expression levels of non-mDA markers, such as SMA and SIX1, that were induced in an FGF8b exposure duration-dependent manner. [Figure 4-2] Same as above.

[0050] [Figure 5] FIG. 5 shows SIX1 immunostaining of FGF8b-treated cells at day 30 of differentiation.

[0051] [Figure 6] FIG. 6 shows the mRNA expression of markers in FGF-treated cells on day 16 of differentiation.

[0052] [Figure 7] FIG. 7 shows immunostaining of markers in FGF8b and FGF18 treated cells on day 16 of differentiation.

[0053] [Figure 8] FIG. 8 shows RNA expression of markers in FGF8b- and FGF18-treated cells at days 27 and 40 of differentiation.

[0054] [Figure 9-1]FIG. 9A is a schematic diagram showing the donor vector structure for NURR1::GFP reporter hPSCs.

[0055] [Figure 9-2] FIG. 9B shows NURR1 mRNA levels in cells differentiated from hPSCs using the Wnt-Boost protocol.

[0056] [Figure 9-3] FIG. 9C shows the FACS results of midbrain DA neurons differentiated from H9-hPSCs and NURR1::GFP hPSCs on day 25 of differentiation.

[0057] [Figure 10-1] Figure 10A shows single-cell qRT-PCR results in NURR1:GFP-positive cells isolated at days 25 and 40 of differentiation.

[0058] [Figure 10-2] Figure 10B shows immunostaining of NURR1-sorted mDA expressing TH, FOXA2, and NURR1 at differentiation day 60. Cells were sorted on day 25 and then continuously cultured until day 60.

[0059] [Figure 11] FIG. 11 provides immunostaining images of in vivo transplanted NURR1::GFP-positive midbrain DA neurons 8 weeks after cells were injected into immunodeficient mice.

[0060] [Figure 12] Figure 12 provides immunostaining images of NURR1:GFP-positive cells cultured under the WNT-Boost protocol and the WNT-boost+FGF18 (days 12 to 16) protocol. Cells were sorted on day 25 and then continuously cultured until day 40.

[0061] [Figure 13]Figure 13A provides FOXA2, EN1, and NURR1 mRNA expression in NURR1:GFP-positive cells cultured under the WNT-Boost protocol and the WNT-boost+FGF18 (days 12-16) protocol. mRNA expression in both sorted and unsorted cells was compared.

[0062] Figure 13B shows that sorted NURR1:GFP-positive cells cultured under the WNT-Boost protocol and the WNT-Boost + FGF18 (days 12 to 16) protocol were transplanted into immunodeficient mice. Neurite outgrowth and the expression of TH and SC121 were detected in the lattice cells.

[0063] [Figure 14-1] Figure 14 shows that 390 surface markers were screened on mDA at day 25 of differentiation from NURR1::GFP hPSCs using the Wnt-Boost protocol. Antibodies were conjugated to PE, FITC, or APC. [Figure 14-2] Same as above. [Figure 14-3] Same as above. [Figure 14-4] Same as above. [Figure 14-5] Same as above.

[0064] [Figure 15-1] FIG. 15A shows that the positive surface markers CD171 and CD184 were enriched in NURR1+ cells.

[0065] [Figure 15-2] FIG. 15B provides RNA expression of CD171 and CD184 in differentiated cells using the Wnt-Boost protocol.

[0066] [Figure 16-1] FIG. 16A shows that the negative surface markers CD49e, CD99, and CD340 were enriched in NURR1+ cells.

[0067] [Figure 16-2] FIG. 16B provides RNA expression of CD49e, CD99, and CD340 in differentiated cells using the Wnt-Boost protocol.

[0068] [Figure 17] Figure 17 provides the morphology of cells sorted by CD49e low or CD49e high. Cells were sorted on day 25 of in vitro differentiation under the WNT-boost and WNT-boost+FGF18 protocols. After sorting, cells were cultured for an additional 15 days.

[0069] [Figure 18] FIG. 18 shows immunostaining images of sorted CD49e weak cells on day 40 of in vitro differentiation under the WNT-Boost protocol and the WNT-boost+FGF18 protocol.

[0070] [Figure 19] FIG. 19 shows the FACS results for the CD49e-based purification sorting of mDA derived from the hPSC cell line MEL1.

[0071] [Figure 20] Figure 20 shows the morphology of MEL1 hPSC-derived mDA cells sorted for CD49e low or CD49e high. Cells were sorted on day 25 of in vitro differentiation under the WNT-Boost protocol and the WNT-boost + FGF18 protocol. After sorting, cells were cultured for an additional 15 days.

[0072] [Figure 21] Figure 21 shows immunostaining images of CD49e weak mDA derived from MEL1 hPSCs on day 40 of in vitro differentiation under the WNT-Boost protocol and the WNT-boost+FGF18 protocol. CD49e weak cells were sorted on day 25.

[0073] [Figure 22]Figure 22 shows the relative mRNA expression in MEL1 hPSC-derived CD49e-poor mDA at day 40 of in vitro differentiation under the WNT-boost protocol and the WNT-boost+FGF18 protocol. CD49e-poor cells were sorted on day 25.

[0074] [Figure 23] Figure 23 shows the morphology of CD49e, CD99 or CD340 sorted cells under the WNT-Boost+FGF18 protocol at day 40 of in vitro differentiation. Cells were sorted at day 25 of in vitro differentiation.

[0075] [Figure 24] Figure 24 shows the relative mRNA expression of CD49e, CD99, or CD340 sorted cells under the WNT-Boost+FGF18 protocol at day 40 of in vitro differentiation. Cells were sorted at day 25 of in vitro differentiation.

[0076] [Figure 25] FIG. 25 shows the FACS sorting results of NURR1+ cells sorted by 49E (PE) and 171 (APC) on day 25 of in vitro differentiation under the WNT-Boost protocol.

[0077] [Figure 26] FIG. 26 shows the FACS sorting results of NURR1+ cells sorted by 49E (PE) and 184 (APC) on day 25 of in vitro differentiation under the WNT-Boost protocol.

[0078] [Figure 27] Figure 27 provides the morphology of cells sorted by CD49e, CD171, and CD188. Cells were sorted under the WNT-Boost protocol on day 25 of in vitro differentiation. After sorting, cells were cultured for an additional 2 days.

[0079] [Figure 28]Figure 28 shows the mRNA expression of CD49e, CD171, and CD188 sorted cells. Cells were sorted under the WNT-Boost protocol on day 25 of in vitro differentiation. After sorting, cells were cultured for an additional 2 days.

[0080] [Figure 29] FIG. 29 shows enrichment of the NURR1::GFP population in single CD49e weakly sorted cells at day 25 of in vitro differentiation.

[0081] [Figure 30] Figure 30 shows enrichment of the NURR1:GFP population in CD49e weak CD184 high double sorted cells at day 25 of in vitro differentiation.

[0082] [Figure 31] FIG. 31 shows that TH+ midbrain DA neurons co-expressed FOXA2 and GFP, implicating midbrain DA neuron identity in the enriched NURR1:GFP population in FIG.

[0083] [Figure 32] Figure 32 shows midbrain DA marker mRNA expression in cells sorted by CD49e and CD184 at day 25 of in vitro differentiation. After sorting, cells were cultured in vitro for an additional 15 days.

[0084] [Figure 33] Figure 33 shows non-mesencephalic DA mRNA expression in cells sorted by CD49e and CD184 on day 25 of in vitro differentiation. After sorting, cells were cultured in vitro for an additional 10 days.

[0085] [Figure 34-1]Figures 34A-34D show the in vivo survival of transplanted cells sorted with CD markers (CD49e depletion and CD184 enrichment) of the present disclosure after in vitro differentiation under the WNT-Boost protocol. Figure 34A shows robust survival of sorted cells compared to unsorted cells and enrichment of TH+ cells within the transplant. Figure 34B shows the reduction in the number of SOX2+ progenitor and KI67+ dividing cells one month after transplantation. Figure 34C shows the quantification of SOX2+ stained cells in Figure 34B. Figure 34D shows the quantification of Ki67+ cells in Figure 34B. [Figure 34-2] Same as above. [Figure 34-3] Same as above.

[0086] [Figure 35-1] Figures 35A-B show the in vivo survival and EN1 expression of cells differentiated under the WNT-boost and WNT-boost+FGF18 protocols. Figure 35A shows the percentage of cells expressing EN1. Figure 35B shows the appearance of striatal innervation with fibers at the transplant site. [Figure 35-2] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0087] 5. Detailed Description The present disclosure provides methods for producing mDA and its precursors, mDA and its precursors produced by such methods, compositions comprising such cells, and uses thereof for preventing and / or treating neurological disorders. Additionally, the present disclosure provides methods for isolating mDA and its precursors from cell populations using novel surface markers.

[0088] The present disclosure is based at least on the discovery that stem cell-derived mDA produced by the methods of the present disclosure have sustained expression of EN1, eg, expression of EN1 is maintained throughout the development and maturation of the mDA.

[0089] Non-limiting embodiments of the presently disclosed subject matter are illustrated herein and by example.

[0090] For purposes of clarity of disclosure, and not by way of limitation, the detailed description is divided into the subsections that follow. 5.1. Definition, 5.2. Methods for differentiating stem cells; 5.3. Methods for isolating midbrain DA neurons and their precursors 5.4. Compositions comprising midbrain DA neurons and their precursors; 5.5. Methods of Treating Neurodegenerative Disorders, and 5.6.Kit.

[0091] 5.1.Definition The terms used herein generally have their ordinary meaning in the art, within the context of this disclosure and within the specific context in which each term is used. Particular terms are discussed below or elsewhere herein to provide additional guidance to the practitioner in describing the compositions and methods of the present disclosure and how to make and use them.

[0092] The term "about" or "approximately" means within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which depends 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 standard deviations or more than 3 standard deviations, in accordance with 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 processes, the term can mean within an order of magnitude, e.g., within 5-fold or within 2-fold of a value.

[0093] As used herein, the term "signal" in reference to a "signaling 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 signaling proteins include, but are not limited to, SMADs, wingless (Wnt) complex proteins including β-catenin, NOTCH, transforming growth factor beta (TGFP), activin, Nodal, glycogen synthase kinase 3β (GSK3β) protein, bone morphogenetic proteins (BMPs), and fibroblast growth factors (FGFs). For many cell surface receptors or internal receptor proteins, the ligand-receptor interaction is not directly related to the cellular response. A ligand-activated receptor can first interact with other proteins within the cell before the ligand's ultimate physiological effect on cellular behavior occurs. Often, the behavior of several interacting cellular protein chains changes after receptor activation or inhibition. The entire series of cellular changes induced by receptor activation is called a signaling mechanism or pathway.

[0094] As used herein, the term "signals" refers to internal and external factors that control changes in cellular structure and function. They can be chemical or physical in nature.

[0095] As used herein, the term "ligand" refers to molecules and proteins that bind to receptors, such as transforming growth factor beta (TFGP), activin, Nodal, bone morphogenetic proteins (BMPs), and the like.

[0096] As used herein, "inhibitor" refers to a compound or molecule (e.g., a small molecule, peptide, peptidomimetic, natural compound, siRNA, antisense nucleic acid, aptamer, or antibody) that interferes with (e.g., reduces, diminishes, suppresses, eliminates, or blocks) the signaling function of a molecule or pathway. An inhibitor can be any compound or molecule that alters any activity of a specified protein (signaling molecule, any molecule involved in a specified signaling molecule, or a specified associated molecule, such as glycogen synthase kinase 3β (GSK3β)) (including, but not limited to, a signaling molecule described herein), for example, by directly contacting SMAD signaling, contacting SMAD mRNA, causing a conformational change in SMAD, reducing SMAD protein levels, or disrupting SMAD interaction with a signaling partner (e.g., including those described herein), and affecting the expression of a SMAD target gene (e.g., those described herein).

[0097] Inhibitors also include molecules that indirectly modulate the biological activity of upstream signaling molecules, e.g., SMAD biological activity (e.g., within their extracellular domains, examples of signaling molecules and effects include noggin, which sequesters bone morphogenetic protein and inhibits activation of ALK receptors 1, 2, 3, and 6, thus preventing downstream SMAD activation. Similarly, chordin, cerberus, and follistatin similarly sequester extracellular activators of SMAD signaling. The transmembrane protein bambi also acts as a pseudoreceptor to sequester extracellular TGFβ signaling molecules). Antibodies that block upstream or downstream proteins are contemplated for use to neutralize extracellular activators of protein signaling, etc. Inhibitors are described in terms of competitive inhibition (binding to the active site in a manner that precludes or reduces binding of another known binding compound) and allosteric inhibition (binding to the protein in a manner that alters the conformation of the protein in a manner that prevents binding of a compound to the active site of the protein), in addition to inhibition induced by binding to and affecting a molecule upstream of a specified signaling molecule, which in turn causes inhibition of the specified molecule. An inhibitor can be a "direct inhibitor," which inhibits a signaling target or a signaling target pathway by actually contacting the signaling target.

[0098] As used herein, "activator" refers to a compound that increases, induces, stimulates, activates, promotes or enhances the signaling function of a molecule or pathway, eg, Wnt signaling, SHH signaling, etc.

[0099] As used herein, the terms "WNT" or "wingless" with respect to a ligand refer to a group of secreted proteins (e.g., integration 1 in humans) that can interact with WNT receptors, such as receptors of the Frizzled and LRPDerailed / RYK receptor families. As used herein, the term "WNT or wingless signaling pathway" refers to a signaling pathway composed of a Wnt family ligand and a Wnt family receptor, e.g., Frizzled and LRPDerailed / RYK receptor, mediated with or without β-catenin. In certain embodiments, the WNT signaling pathway includes mediation by β-catenin, e.g., WNT / -catenin.

[0100] As used herein, the term "derivative" refers to a compound that has a similar core structure.

[0101] As used herein, the term "population of cells" or "cell population" refers to a group of at least two cells. In non-limiting examples, a cell population can contain at least about 10, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, or at least about 1000 cells. A population can be a pure population containing one cell type, such as a population of midbrain DA precursors or a population of undifferentiated stem cells. Alternatively, a population can contain more than one cell type, for example, a mixed cell population.

[0102] As used herein, the term "stem cell" refers to a cell that has the capacity to divide indefinitely in culture and give rise to specialized cells.

[0103] As used herein, the terms "embryonic stem cells" and "ESCs" refer to primitive (undifferentiated) cells derived from preimplantation stage embryos, capable of dividing in culture without differentiation for extended periods of time, and known to develop into cells and tissues of the three primary germ layers. Human embryonic stem cells refer to embryonic stem cells derived from a human embryo. As used herein, the terms "human embryonic stem cells" or "hESCs" refer to a type of pluripotent stem cell derived from an early stage human embryo up to and including the blastocyst stage, known to be capable of dividing in culture without differentiation for extended periods of time, and known to develop into cells and tissues of the three primary germ layers.

[0104] As used herein, the term "embryonic stem cell line" refers to a population of embryonic stem cells cultured under in vitro conditions that allow proliferation without differentiation for up to several days, months, or years.

[0105] As used herein, the term "totipotency" refers to the ability to give rise to all cell types of the body, as well as all cell types that make up extraembryonic tissues such as the placenta.

[0106] As used herein, the term "multipotency" refers to the ability to develop into more than one cell type of the body.

[0107] As used herein, the term "pluripotency" refers to the ability to develop into an organism's three developing germ layers, including endoderm, mesoderm, and ectoderm.

[0108] As used herein, the term "induced pluripotent stem cells" or "iPSCs" refers to a type of pluripotent stem cell formed by introducing certain embryonic genes (e.g., but not limited to, OCT4, SOX2, and KLF4 transgenes) into somatic cells (see, e.g., Takahashi and Yamanaka Cell 126, 663-676 (2006) which is incorporated herein by reference).

[0109] As used herein, the term "somatic cell" refers to any cell in the body other than a gamete (egg or sperm). Sometimes also called a "mature" cell.

[0110] As used herein, the term "somatic (adult) stem cells" refers to relatively rare undifferentiated cells found in many organs and differentiated tissues, which have limited capacity for both self-renewal (in the laboratory) and differentiation.

[0111] As used herein, the term "neuron" refers to a nerve cell, which is the primary functional unit of the nervous system. A neuron consists of a cell body and its processes—an axon and at least one dendrite. Neurons transmit information to other neurons or cells by releasing neurotransmitters at synapses.

[0112] As used herein, the term "proliferation" refers to an increase in cell number.

[0113] As used herein, the term "undifferentiated" refers to cells that have not yet developed into a specialized cell type.

[0114] As used herein, the term "differentiation" refers to the process by which unspecialized embryonic cells acquire the characteristics of specialized cells, such as neural, cardiac, hepatic, or muscle cells. Differentiation is typically controlled by the interaction of a cell's genes with extracellular physical and chemical conditions through signaling pathways involving proteins embedded in the cell surface.

[0115] As used herein, the term "directed differentiation" refers to the manipulation of stem cell culture conditions to induce differentiation into specific (e.g., desired) cell types, such as neural, neural crest, cranial placode, and non-neural ectodermal precursors. With respect to stem cells, "directed differentiation" refers to the use of small molecules, growth factor proteins, and other growth conditions to promote the transition of stem cells from a pluripotent state to a more mature or specialized cell fate.

[0116] As used herein, the term "inducing differentiation" with respect to a cell refers to changing a 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 a stem cell (e.g., a human stem cell) to divide into progeny cells with characteristics distinct from the stem cell, such as genotype (e.g., changes in gene expression determined by genetic analysis such as a microarray) and / or phenotype (e.g., changes in expression of protein markers of mDA or its precursors, such as EN1, OTX2, TH, NURR1, FOXA2, LMX1A, PITX3, LMO3, SNCA, ADCAP1, CHRNA4, and GIRK2).

[0117] As used herein, the term "cell culture" refers to the growth of cells in vitro in an artificial medium for research or medical treatment.

[0118] As used herein, the term "culture medium" refers to a liquid containing nutrients that covers, nourishes, and supports cells in a culture vessel, such as a Petri dish, multi-well plate, etc. Culture medium may also contain growth factors that are added to induce desired changes in the cells.

[0119] As used herein, the term "contacting" one or more cells with a compound (e.g., at least one inhibitor, activator, and / or inducer) refers to providing the compound in a location that allows the one or more cells to access the compound. Contacting can be achieved using any suitable method. For example, contacting can be achieved by adding a concentrated form of the compound to a cell or cell population, e.g., in the context of a cell culture, to achieve a desired concentration. Contacting can also be achieved by including the compound as a component of a formulated culture medium.

[0120] As used herein, the term "in vitro" refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments include, but are not limited to, test tubes and cell cultures.

[0121] As used herein, the term "in vivo" refers to the natural environment (eg, an animal or a cell) and to processes or reactions that occur within a natural environment, such as embryonic development, cell differentiation, neurulation, etc.

[0122] As used herein, the term "express" with respect to a gene or protein refers to making mRNA or protein that can be observed using an assay such as a microarray assay, an antibody staining assay, or the like.

[0123] As used herein, the term "marker" or "cell marker" refers to a gene or protein that identifies a particular cell or cell type. A cellular marker need not be limited to one marker; a marker can refer to a "pattern" of markers such that a specified group of markers can distinguish a cell or cell type from another cell or cell type.

[0124] As used herein, the terms "derived from," "established from," or "differentiated from," when made with reference to any cell disclosed herein, refer to cells obtained from an ultimate parent cell (e.g., isolated, purified, etc.) in a cell line, tissue (such as a dissociated embryo), or body fluid using any manipulation, including, but not limited to, single cell isolation, in vitro culture, treatment and / or mutagenesis with, for example, proteins, chemicals, radiation, infection with viruses, transfection with DNA sequences, e.g., morphogens, etc., and selection (such as by continuous culture) of any cells contained within the cultured parent cell. Derived cells may be selected from a mixed population by response to growth factors, cytokines, selected courses of cytokine treatment, adherence, lack of adherence, sorting procedures, etc.

[0125] As used herein, an "individual" or "subject" is a vertebrate, e.g., a human or a non-human animal, e.g., 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; cows; horses; and non-human primates, such as apes and monkeys.

[0126] 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.

[0127] As used herein, the term "treat" 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. The therapeutic effect of treatment includes, but is not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of direct or indirect pathological consequences of the disease, prevention of metastasis, slowing the rate of disease progression, remission or alleviation of the disease state, and remission or improvement of prognosis. By preventing the progression of a disease or disorder, treatment can prevent deterioration by the disorder in an affected or diagnosed subject or a subject suspected of having the disorder, but treatment can also prevent the onset of the disorder or symptoms of the disorder in a subject at risk of or suspected of having the disorder.

[0128] As used herein, the terms "negative," "weak," or "-" when used with respect to any surface marker disclosed herein refers to the surface marker (e.g., CD49e) not being expressed at a detectable level or being expressed at a reduced level on a cell compared to the average expression of the surface marker in the population of cells from which the cells are selected or sorted. As used herein, the terms "high," "strong," "+," or "positive" when used with respect to any surface marker disclosed herein refers to the surface marker (e.g., CD184) being expressed at a detectable level or being expressed at an increased level compared to the average expression of the surface marker in the population of cells.

[0129] In certain embodiments, cells are differentiated according to their surface marker expression levels based on readily discernible differences in staining intensity, as known to those skilled in the art. In certain embodiments, a cutoff for designating cells as surface marker "weak," "negative," or "-" cells can be set with respect to the staining intensity distribution (e.g., fluorescence intensity distribution) observed for all cells, where cells with less than about 50%, about 40%, about 30%, about 20%, about 10%, or about 5% of the staining intensity can be designated as surface marker "weak," "negative," or "-" cells. In certain embodiments, a cutoff for designating cells as surface marker "strong," "high," "+," or "positive" cells can be set with respect to the staining intensity distribution (e.g., fluorescence intensity distribution) observed for all cells, where cells with more than about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% of the staining intensity can be designated as surface marker "strong," "high," "+," or "positive" cells. In certain embodiments, a frequency distribution of surface marker staining is obtained for all cells, and population curves are fitted to cells assigned to the population they are most statistically likely to belong to given the higher and lower staining populations and statistical analysis of each population distribution.

[0130] 5.2. Methods for Differentiating Stem Cells The present disclosure provides an in vitro method for inducing differentiation of stem cells, the method comprising contacting the stem cells with at least one Small Mothers Against Decapentaplegic (SMAD) signaling inhibitor (referred to as a "SMAD inhibitor"), at least one Sonic Hedgehog (SHH) signaling activator (referred to as a "SHH activator"), and at least one Wingless (Wnt) signaling activator (referred to as a "Wnt activator"), and contacting the cells with at least one fibroblast growth factor (FGF) signaling activator (referred to as an "FGF activator") to obtain a cell population comprising differentiated cells expressing at least one marker indicative of mDA or mDA precursors.

[0131] 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 FGF8a, FGF17, FGF18, FGF2, and FGF4. In certain embodiments, the at least one FGF activator is selected from FGF8a, FGF17, and FGF18. In certain embodiments, the at least one FGF activator comprises FGF18.

[0132] In certain embodiments, the initial contact of the cells with the at least one FGF signaling activator occurs at least about 5 days after the initial contact of the cells with the at least one SMAD signaling inhibitor. In certain embodiments, the initial exposure of the cells to the at least one FGF activator occurs at least about 10 days after the initial exposure of the stem cells to the at least one SMAD inhibitor. In certain embodiments, the FGF activator is selected from FGF8a, FGF17, FGF18, FGF8b, FGF2, and FGF4. Exposure of the cells to the at least one FGF activator prolongs expression of EN1 by the differentiated cells.

[0133] In certain embodiments, the at least one marker indicative of mDA or an mDA precursor is selected from EN1, FOX1A, LMX1A, OTX2, NURR1, TH, PITX3, LMO3, SNCA, ADCAP1, CHRNA4, and GIRK2.

[0134] In certain embodiments, the concentration of the at least one Wnt activator is increased during exposure to the cells. In certain embodiments, the increase in the concentration of the at least one Wnt activator begins about 4 days after the initial exposure of the stem cells to the at least one SMAD inhibitor. In certain embodiments, the concentration of the at least one Wnt activator is increased by about 300% to about 1000%. In certain embodiments, the cells are exposed to the at least one Wnt activator at an increased concentration for at least about 7 days. In certain embodiments, at least one additional Wnt activator is added to increase the overall concentration of the Wnt activator(s).

[0135] In certain embodiments, the method further comprises contacting the cells with midbrain DA lineage-specific activators and inhibitors, such as BDNF, GDNF, cAMP, TGFβ, ascorbic acid (AA) and / or DAPT, to induce differentiation of mDA precursors into mDA.

[0136] 5.2.1. Stem cells The presently disclosed subject matter provides in vitro methods for inducing differentiation of stem cells to produce mDA and its precursors. 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 methods of the present disclosure include human, non-human primate, or rodent non-embryonic stem cells, embryonic stem cells, induced non-embryonic 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 (hESCs), human pluripotent stem cells (hPSCs), human induced pluripotent stem cells (hiPSCs), 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 cells capable of lineage-specific differentiation. In certain embodiments, the stem cells are human embryonic stem cells (hESCs). In certain embodiments, the stem cells are human induced pluripotent stem cells (hiPSCs).

[0137] In certain embodiments, stem cells or their progeny contain introduced heterologous nucleic acid, which may encode a desired nucleic acid or protein product or may be informative (see, e.g., U.S. Patent No. 6,312,911, incorporated by reference in its entirety). Non-limiting examples of protein products include markers detectable by in vivo imaging studies, such as receptors or other cell membrane proteins. Non-limiting examples of markers include fluorescent proteins (e.g., green fluorescent protein (GFP), blue fluorescent protein (EBFP, EBFP2, azurite, mKalama1), cyan fluorescent protein (ECFP, Cerulean, CyPet, mTurquoise2), and yellow fluorescent protein derivatives (YFP, citrine, Venus, YPet, EYFP)), β-galactosidase (LacZ), chloramphenicol acetyltransferase (cat), neomycin phosphotransferase (neo), enzymes (e.g., oxidases and peroxidases), and antigenic molecules. As used herein, the term "reporter gene" or "reporter construct" refers to a genetic construct comprising a nucleic acid encoding a readily detectable or readily assayable protein, such as a colored protein, a fluorescent protein such as GFP, or an enzyme such as β-galactosidase (lacZ gene). In certain embodiments, the reporter may be driven by a recombinant promoter of a premature, postmitotic midbrain DA neuron marker gene, e.g., NURR1.

[0138] SMAD inhibitors Non-limiting examples of SMAD inhibitors include transforming growth factor beta (TGFβ) / Activin-Nodal signaling inhibitors (referred to as "TGFβ / Activin-Nodal inhibitors") and bone morphogenetic protein (BMP) signaling inhibitors. In certain embodiments, TGFβ / Activin-Nodal inhibitors can neutralize ligands including TGFβ, BMP, Nodal, and activin and / or interrupt their signaling pathways by blocking receptors and downstream effectors. Non-limiting examples of TGFβ / Activin-Nodal inhibitors are described 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 1;30(7):715-20 (2012), all of which are incorporated by reference in their entirety for all purposes. In certain embodiments, the at least one TGFβ / Activin-Nodal inhibitor is selected from an inhibitor of ALK5, an inhibitor of ALK4, an inhibitor of ALK7, and combinations thereof. In certain embodiments, the TGFβ / Activin-Nodal inhibitor comprises an inhibitor of ALK5. In certain embodiments, the TGFβ / Activin-Nodal inhibitor is a small molecule selected from SB431542, its derivatives, and mixtures thereof. "SB431542" refers to the CAS number 301836-41-9, C 22 H 18 It refers to a molecule having the molecular formula N4O3 and the name 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide, see, for example, the structure below. [ka]

[0139] In certain embodiments, the TGFβ / Activin-Nodal inhibitor comprises SB431542. In certain embodiments, the TGFβ / Activin-Nodal inhibitor comprises a derivative of SB431542. In certain embodiments, the derivative of SB431542 is A83-01.

[0140] In certain embodiments, the at least one SMAD inhibitor comprises a BMP signaling inhibitor (referred to as a "BMP inhibitor"). Non-limiting examples of BMP inhibitors include those described in WO 2011 / 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 1;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 C 25 H 22 It refers to the small molecule DM-3189, which has the chemical formula N6 and the IUPAC name 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline. [ka]

[0141] LDN193189 can function as an SMAD signaling inhibitor. LDN193189 is also a highly potent small molecule inhibitor of ALK2, ALK3, and ALK6, protein tyrosine kinase (PTK), and inhibits the signaling of members of the ALK1 and ALK3 family of type I TGFβ receptors, resulting in the inhibition of the transmission of multiple biological signals, including bone morphogenetic protein (BMP) BMP2, BMP4, BMP6, BMP7, and activin cytokine signals, and the subsequent SMAD phosphorylation of Smad1, Smad5, and Smad8 (Yu et al. (2008) Nat Med 14:1363-1369; Cuny et al. (2008) Bioorg.Med.Chem.Lett.18:4388-4392, incorporated herein by reference).

[0142] In certain embodiments, the BMP inhibitor comprises LDN 193189. In certain embodiments, the BMP inhibitor comprises noggin.

[0143] In certain embodiments, stem cells are exposed to one SMAD inhibitor, for example, one TGFβ / Activin-Nodal inhibitor. In certain embodiments, the one TGFβ / Activin-Nodal inhibitor is SB431542 or A83-01. In certain embodiments, stem cells are exposed to two SMAD inhibitors. In certain embodiments, the two SMAD inhibitors are a TGFβ / Activin-Nodal inhibitor and a BMP inhibitor. In certain embodiments, stem cells are exposed to SB431542 or A83-01 and LDN193189 or noggin. In certain embodiments, stem cells are exposed to SB431542 and LDN193189.

[0144] In certain embodiments, 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, stem cells are contacted with or exposed to at least one SMAD inhibitor for up to about 5 days or up to about 10 days. In certain embodiments, stem cells are contacted with or exposed to at least one SMAD inhibitor for about 5 days to about 10 days. In certain embodiments, stem cells are contacted with or exposed to at least one SMAD inhibitor for about 5 days. In certain embodiments, stem cells are contacted with or exposed to at least one SMAD inhibitor for 7 days. In certain embodiments, cells are contacted with or exposed to at least one SMAD inhibitor from day 0 to day 6. In certain embodiments, at least one SMAD inhibitor is added to the cell culture medium containing the stem cells every day or every other day from day 0 to day 6. In certain embodiments, at least one SMAD inhibitor is added to the cell culture medium containing the stem cells every day (daily) from day 0 to day 6.

[0145] In certain embodiments, cells are contacted with or exposed to a TGFβ / Activin-Nodal inhibitor. In certain embodiments, the concentration of the TGFβ / Activin-Nodal inhibitor contacted with or exposed to the cells is about 1 μM to about 20 μM, about 1 μM to about 10 μM, about 1 μM to about 15 μM, about 10 μM to about 15 μM, about 5 μM to about 10 μM, about 5 μM to about 15 μM, about 5 μM to about 20 μM, or about 15 μM to about 20 μM. In certain embodiments, the concentration of the TGFβ / Activin-Nodal inhibitor contacted with or exposed to the cells is about 1 μM to about 10 μM. In certain embodiments, the concentration of the TGFβ / Activin-Nodal inhibitor contacted with or exposed to the cells is about 5 μM. About 10 μM. In certain embodiments, the concentration of the TGFβ / Activin-Nodal inhibitor contacted with or exposed to the cells is about 10 μM. In certain embodiments, the TGFβ / Activin-Nodal inhibitor comprises SB431542 or a derivative thereof (e.g., A83-01). In certain embodiments, the TGFβ / Activin-Nodal inhibitor comprises SB431542.

[0146] In certain embodiments, 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 about 50 nM to about 500 nM, or about 100 nM to about 500 nM, or about 200 nM to about 500 nM, or about 200 nM to about 300 nM, or about 200 nM to about 400 nM, or about 100 nM to about 250 nM, or about 100 nM to about 250 nM, or about 200 nM to about 250 nM, or about 250 nM to about 300 nM. In certain embodiments, the concentration of the BMP inhibitor contacted with or exposed to the cells is about 200 nM to 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.

[0147] In certain embodiments, cells are contacted with or exposed to a TGFβ / Activin-Nodal inhibitor and a BMP inhibitor simultaneously. In certain embodiments, stem cells are contacted with or exposed to a TGFβ / Activin-Nodal inhibitor and a BMP inhibitor for 7 days. In certain embodiments, cells are contacted with or exposed to a TGFβ / Activin-Nodal inhibitor and a BMP inhibitor from day 0 to day 6. In certain embodiments, a TGFβ / Activin-Nodal inhibitor and a BMP inhibitor are added daily or every other day to cell culture medium containing stem cells from day 0 to day 6. In certain embodiments, a TGFβ / Activin-Nodal inhibitor and a BMP inhibitor are added daily (every day) to cell culture medium containing stem cells from day 0 to day 6.

[0148] 5.2.3.Wnt activator In certain embodiments, at least one Wnt activator reduces GSK3β to activate Wnt signaling. Thus, in certain embodiments, the Wnt activator is a GSK3β inhibitor. GSK3P inhibitors can activate the WNT signaling pathway, see, for example, Cadigan et al., J Cell Sci. 2006;119:395-402; Kikuchi et al., Cell Signaling. 2007;19:659-671, which are incorporated herein by reference in their entirety. As used herein, the term "glycogen synthase kinase 3β inhibitor" or "GSK3β inhibitor" refers to a compound that inhibits the glycogen synthase kinase 3β enzyme, see, for example, Doble et al., J Cell Sci. 2003;116:1175-1186, which are incorporated herein by reference in their entirety.

[0149] Non-limiting examples of Wnt activators or GSK3β inhibitors include CHIR99021, Wnt3A, Wnt1, Wnt5a, BIO ((3E)-6-bromo-3-[3-(hydroxyamino)indol-2-ylidene]-1H-indol-2-one), CHIR98014, lithium, 3F8, WO 2011 / 149762, WO 13 / 067362, Chambers et al., Nat Biotechnol. 2012 Jul 1;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, Wnt1, Wnt5a, BIO, CHIR98014, lithium, 3F8, derivatives thereof, and mixtures thereof. 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. "CHIR99021" (also known as "aminopyrimidine" or "3-[3-(2-carboxyethyl)-4-methylpyrrole-2-methylidenyl]-2-indolinone") refers to the IUPAC name 6-(2-(4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-ylamino)ethylamino)nicotinonitrile, which has the formula: [ka]

[0150] CHIR99021 is highly selective, exhibiting nearly 1000-fold selectivity against a panel of related and unrelated kinases, with an IC50=6.7 nM against human GSK3β and nanomolar IC50 values ​​against rodent GSK3β homologues.

[0151] In certain embodiments, the cells are contacted with or exposed to 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 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 at least one Wnt activator for about 5 days to about 20 days, about 5 days to about 15 days, about 10 days to about 20 days, about 5 days to about 15 days, or about 10 days to about 15 days. In certain embodiments, the cells are contacted with at least one Wnt activator for about 10 days to about 15 days. In certain embodiments, the cells are contacted with at least one Wnt activator for about 10 days. In certain embodiments, the stem cells are contacted with at least one activator of Wnt signaling for 12 days. In certain embodiments, the cells are contacted with at least one activator of Wnt signaling from day 0 to day 11. In certain embodiments, the at least one Wnt activator is added to the cell culture medium containing the cells every day or every other day from day 0 to day 11. In certain embodiments, the at least one Wnt activator is added to the cell culture medium containing the cells every day (every day) from day 0 to day 11.

[0152] In certain embodiments, the concentration of at least one Wnt activator is increased during exposure to the cells (also referred to as a "Wnt Boost"). In certain embodiments, the increase or Wnt Boost begins at least about 2 days, at least about 4 days, or at least about 5 days after initial exposure of the cells to the at least one Wnt activator. In certain embodiments, the increase or Wnt Boost begins about 4 days after initial exposure of the cells to the at least one Wnt activator.

[0153] In certain embodiments, the cells are contacted with or exposed to increasing concentrations of at least one Wnt activator for at least about 5 days, or at least about 10 days. In certain embodiments, the cells are contacted with or exposed to increasing concentrations of at least one Wnt activator for at least about 5 days. In certain embodiments, the cells are contacted with increasing concentrations of at least one Wnt activator for up to about 5 days, up to about 10 days, or up to about 15 days. In certain embodiments, the cells are contacted with increasing concentrations of at least one Wnt activator for up to about 10 days.

[0154] In certain embodiments, cells are contacted with or exposed to increasing concentrations of at least one Wnt activator for about 5 days to about 15 days, or about 5 days to about 10 days, or about 10 days to about 15 days. In certain embodiments, cells are contacted with or exposed to increasing concentrations of at least one Wnt activator for about 5 days to about 10 days. In certain embodiments, cells are contacted with or exposed to increasing concentrations of at least one Wnt activator for about 5 days, about 10 days, or about 15 days. In certain embodiments, cells are contacted with or exposed to increasing concentrations of at least one Wnt activator for about 5 days. In certain embodiments, cells are contacted with or exposed to increasing concentrations of at least one Wnt activator for 6 days. In certain embodiments, cells are contacted with or exposed to increasing concentrations of at least one Wnt activator from day 4 to day 9. In certain embodiments, the cells are contacted with or exposed to increasing concentrations of at least one Wnt activator for about 10 days. In certain embodiments, the cells are contacted with or exposed to increasing concentrations of at least one Wnt activator for about 8 days. In certain embodiments, the cells are contacted with or exposed to increasing concentrations of at least one Wnt activator from day 4 to day 11.

[0155] In certain embodiments, the initial concentration of at least one Wnt activator contacted or exposed to the cells prior to the Wnt Boost is less than about 5 μM, less than about 3 μM, or less than about 1 μM, including, but not limited to, about 0.01 μM to about 5 μM, about 0.01 μM to about 3 μM, about 0.05 μM to about 3 μM, about 0.1 μM to about 3 μM, about 0.5 μM to about 3 μM, about 0.5 μM to about 2 μM, or about 0.5 μM to about 1 μM. In certain embodiments, the initial concentration of at least one Wnt activator contacted or exposed to the cells prior to the Wnt Boost is less than about 1 μM, e.g., about 0.1 μM, about 0.2 μM, about 0.3 μM, about 0.4 μM, about 0.5 μM, about 0.6 μM, about 0.7 μM, about 0.8 μM, about 0.9 μM, or about 1 μM. In certain embodiments, the initial concentration of at least one Wnt activator contacted or exposed to the cells prior to the Wnt Boost is about 0.5 μM. In certain embodiments, the initial concentration of at least one Wnt activator contacted or exposed to the cells prior to the Wnt Boost is about 0.7 μM.

[0156] In certain embodiments, the increased concentration of at least one Wnt activator after a Wnt Boost is about 3 μM or more, about 5 μM or more, about 10 μM or more, about 15 μM or more, or about 20 μM or more. In certain embodiments, the increased concentration of at least one Wnt activator after a Wnt Boost is about 3 μM to about 15 μM, about 3 μM to about 10 μM, or about 5 μM to about 10 μM. In certain embodiments, the increased concentration of at least one Wnt activator after a Wnt Boost is about 3 μM, about 3.5 μM, about 4 μM, about 4.5 μM, about 5 μM, about 5.5 μM, about 6 μM, about 6.5 μM, about 7 μM, about 7.5 μM, about 8 μM, about 8.5 μM, about 9 μM, about 9.5 μM, or about 10 μM. In certain embodiments, the increased concentration of at least one Wnt activator after a Wnt Boost is about 3 μM. In certain embodiments, the increased concentration of at least one Wnt activator after a Wnt Boost is about 7 μM. In certain embodiments, the increased concentration of at least one Wnt activator after a Wnt Boost is about 7.5 μM.

[0157] In certain embodiments, the concentration of at least one Wnt activator is increased by about 50% to about 2000%, or about 100% to about 1500%, or about 150% to about 1500%, or about 200% to about 1500%, or about 250% to about 1500%, or about 300% to about 1500%, or about 300% to about 1000%, or about 300% to about 400%, or about 500% to about 1000%, or about 800% to about 1000%, or about 900% to about 1000%, or about 950% to about 1000. In certain embodiments, the concentration of at least one Wnt activator is increased by about 300% to about 1000% from the initial concentration at which it is contacted or exposed to the cells. In certain embodiments, the concentration of the at least one Wnt activator is increased by about 300% to about 400% from the initial concentration at which it is contacted or exposed to the cells. In certain embodiments, the concentration of the at least one Wnt activator is increased by about 900% to about 1000% from the initial concentration at which it is contacted or exposed to the cells. In certain embodiments, the concentration of the at least one Wnt activator is increased by about 300%, about 350%, about 400%, about 450%, about 500%, about 550%, about 600%, 650%, about 700%, about 750%, about 800%, about 850%, about 900%, about 950%, about 1000%, about 1050%, or about 1100% from the initial concentration at which it is contacted or exposed to the cells. In certain embodiments, the concentration of the at least one Wnt activator is increased by about 300% from the initial concentration at which it is contacted or exposed to the cells. In certain embodiments, the concentration of the at least one Wnt activator is increased by about 350% from the initial concentration at which it is contacted or exposed to the cells. In certain embodiments, the concentration of the at least one Wnt activator is increased by about 950% from the initial concentration at which it is contacted or exposed to the cells. In certain embodiments, the concentration of the at least one Wnt activator is increased by about 1000% from the initial concentration at which it is contacted or exposed to the cells.

[0158] In certain embodiments, at least one Wnt activator comprises a GSK3β inhibitor. In certain embodiments, at least one Wnt activator comprises CHIR99021 or a derivative thereof. In certain embodiments, at least one Wnt activator comprises CHIR99021.

[0159] 5.2.4.SHH activators As used herein, the terms "Sonic hedgehog," "SHH," or "Shh" refer to a protein that is one of at least three proteins in the mammalian signaling pathway family called hedgehogs; another is Desert hedgehog (DHH) and the third is Indian hedgehog (IHH). SHH interacts with at least two transmembrane proteins by interacting with the transmembrane molecules Patched (PTC) and Smoothened (SMO). SHH typically binds to PTC and then activates SMO as a signal transduction molecule. In the absence of SHH, PTC typically inhibits SMO, which then activates a transcriptional repressor, preventing transcription of specific genes. When SHH is present and bound to PTC, PTC cannot prevent SMO from functioning. When SMO is not inhibited, certain proteins can 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 capable of activating the SHH signaling pathway, including a molecule or compound capable of binding to PCT or SMO. In certain embodiments, the SHH activator is selected from a molecule that binds to PCT, a molecule that binds to SMO, and combinations thereof. Non-limiting examples of SHH activators include those described in WO 10 / 096496, WO 13 / 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 SHH activator comprises an SHH protein, an SMO agonist, or a combination thereof. In certain embodiments, the SHH protein comprises recombinant SHH, purified SHH, or a combination thereof.In certain embodiments, the recombinant SHH comprises a recombinant protein that is at least about 80%, about 85%, about 90%, about 95%, or about 99% identical to an N-terminal fragment of mouse SHH. In certain embodiments, the recombinant SHH comprises SHH C25II. In certain embodiments, the SMO agonist comprises palmorfamine.

[0160] In certain embodiments, cells are contacted with or exposed to at least one SHH activator for at least about 5 days or at least about 10 days. In certain embodiments, cells are contacted with or exposed to at least one SHH activator for up to about 5 days or up to about 10 days. In certain embodiments, cells are contacted with or exposed to at least one SHH activator for about 5 to about 10 days. In certain embodiments, cells are contacted with or exposed to at least one SHH activator for about 5 days. In certain embodiments, cells are contacted with or exposed to at least one SHH activator for 7 days. In certain embodiments, cells are contacted with or exposed to at least one SHH activator from day 0 to day 6. In certain embodiments, at least one SHH activator is added to the cell culture medium containing the cells every day or every other day from day 0 to day 6. In certain embodiments, at least one SHH activator is added to the cell culture medium containing the cells every day (every day) from day 0 to day 6.

[0161] In certain embodiments, the concentration of at least one SHH activator contacted with or exposed to the cells is about 50 ng / mL to about 1000 ng / mL, about 100 ng / mL to about 1000 ng / mL, about 20 ng / mL to about 1000 ng / mL, about 300 ng / mL to about 1000 ng / mL, about 400 ng / mL to about 1000 ng / mL, about 500 ng / mL to about 1000 ng / mL, about 400 ng / mL to about 800 ng / mL, about 400 ng / mL to about 700 ng / mL, about 400 ng / mL to about 600 ng / mL, or about 500 ng / mL to about 600 ng / mL. In certain embodiments, the concentration of at least one SHH activator contacted with or exposed to the cells is about 400 ng / mL to about 600 ng / mL. In certain embodiments, the concentration of at least one SHH activator contacted 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 embodiments, the concentration of at least one SHH activator contacted or exposed to the cells is about 500 ng / mL.

[0162] In certain embodiments, the at least one SHH signaling activator comprises SHH C25II.

[0163] FGF activators The FGF family includes secreted signaling proteins (secreted FGFs) that signal via receptor tyrosine kinases. Phylogenetic analysis suggests that the 22 FGF genes can be organized into seven subfamilies, each containing two to four members. The branch length is proportional to the evolutionary distance between each gene.

[0164] In certain embodiments, the FGF activator is selected from FGF8a, FGF17, FGF18, FGF8b, FGF2, FGF4, and derivatives thereof. In certain embodiments, the FGF activator is selected from FGF8a, FGF17, FGF18, FGF2, FGF4, and derivatives thereof. In certain embodiments, the FGF activator is selected from FGF8a, FGF17, and FGF18.

[0165] The FGF8 subfamily consists of FGF8a, FGF8b, FGF17, and FGF18. Early patterning of the vertebrate midbrain and cerebellum is regulated by the midbrain / hindbrain organizer, which produces FGF8a, FGF8b, FGF17, and FGF18. FGF8b has been shown to function 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 r1 gene Gbx2, strongly activate the pathway inhibitor Spry1 / 2, and repress the midbrain gene Otx2 (Liu 2003). Furthermore, FGF8b extends the organizer along the junction between the induced Gbx2 domain and the remaining Otx2 region of the midbrain, which correlates with cerebellar development (Liu et al., Development. 2003 Dec;130(25):6175-85). 2003). In contrast, FGF8a, FGF17, and FGF18 cause midbrain expansion and upregulation of midbrain gene expression (Liu 2003).

[0166] In certain embodiments, the FGF activator can cause midbrain expansion and upregulate midbrain gene expression. In certain embodiments, the FGF activator is selected from FGF8a, FGF17, FGF18, FGF2, FGF4, derivatives thereof, and combinations thereof. In certain embodiments, the FGF activator includes or is FGF18.

[0167] In certain embodiments, cells are contacted with or exposed to at least one FGF activator for at least about 1 day, at least about 3 days, at least about 5 days, at least about 8 days, or at least about 10 days. In certain embodiments, cells are contacted with or exposed to at least one FGF activator for up to about 5 days, or up to about 10 days, or up to about 15 days, or up to about 20 days. In certain embodiments, cells are contacted with or exposed to at least one FGF activator for about 1 day to about 20 days, or about 1 day to about 15 days, or about 5 days to about 20 days, or about 5 days to about 15 days, or about 5 days to about 10 days, or about 10 days to about 20 days. In certain embodiments, cells are contacted with or exposed to at least one FGF activator for about 5 days to about 10 days. In certain embodiments, the cells are contacted with or exposed to at least one FGF activator for about 3 days, about 5 days, or about 8 days, hi certain embodiments, the cells are contacted with or exposed to at least one FGF activator for about 5 days.

[0168] In certain embodiments, the initial contact of the cells with or exposure of the cells to at least one FGF activator is at least about 5 days or at least about 10 days after the initial contact of the cells with or exposure of the cells to at least one SMAD inhibitor. In certain embodiments, the initial contact of the cells with or exposure of the cells to at least one FGF activator is within about 5 days, within about 10 days, or within about 15 days after the initial contact of the cells with or exposure of the cells to at least one SMAD inhibitor. In certain embodiments, the initial contact of the cells with or exposure of the cells to at least one FGF activator is between about 5 days and about 15 days, between about 5 days and about 10 days, or between about 10 days and about 15 days after the initial contact of the cells with or exposure of the cells to at least one SMAD inhibitor. In certain embodiments, the initial contact of the cells with or exposure of the cells to at least one FGF activator is between about 5 days and about 10 days after the initial contact of the cells with or exposure of the cells to at least one SMAD inhibitor. In certain embodiments, the initial contact of the cells with or exposure of the cells to at least one FGF activator is between about 10 days after the initial contact of the cells with or exposure of the cells to at least one SMAD inhibitor. In certain embodiments, the initial contact of the cells with or exposure of the cells to at least one FGF activator is between 9 days after the initial contact of the cells with or exposure of the cells to at least one SMAD inhibitor. In certain embodiments, the initial contact of the cells with or exposure of the cells to at least one FGF activator is between 10 days after the initial contact of the cells with or exposure of the cells to at least one SMAD inhibitor.In certain embodiments, the initial contact or exposure of the cells to the at least one FGF activator is 12 days after the initial contact or exposure of the cells to the at least one SMAD inhibitor.

[0169] In certain embodiments, the initial contact of the cells with or exposure of the cells to at least one FGF activator is about 5 days from the initial contact of the cells with or exposure of the cells to at least one SMAD inhibitor, and the cells are contacted with the FGF activator for at least 3 days. In certain embodiments, the initial contact of the cells with or exposure of the cells to at least one FGF activator is about 5 days from the initial contact of the cells with or exposure of the cells to at least one SMAD inhibitor, and the cells are contacted with the FGF activator for at least 5 days. In certain embodiments, the initial contact of the cells with or exposure of the cells to at least one FGF activator is about 10 days from the initial contact of the cells with or exposure of the cells to at least one SMAD inhibitor, and the cells are contacted with the FGF activator for at least 3 days. In certain embodiments, the initial contact of the cells with or exposure of the cells to the at least one FGF activator is about 10 days from the initial contact of the cells with or exposure of the cells to the at least one SMAD inhibitor, and the cells are contacted with the FGF activator for at least about 5 days. In certain embodiments, the initial contact of the cells with or exposure of the cells to the at least one FGF activator is 12 days from the initial contact of the cells with or exposure of the cells to the at least one SMAD inhibitor, and the cells are contacted with the FGF activator for at least 5 days.

[0170] In certain embodiments, the concentration of at least one FGF activator contacted with or exposed to the cells is about 10 ng / mL to about 500 ng / mL, about 50 ng / mL to about 500 ng / mL, about 100 ng / mL to about 500 ng / mL, about 100 ng / mL to about 400 ng / mL, about 100 ng / mL to about 300 ng / mL, about 100 ng / mL to about 200 ng / mL, or about 100 ng / mL to about 250 ng / mL. In certain embodiments, the concentration of at least one FGF activator contacted with or exposed to the cells is about 100 ng / mL to about 200 ng / mL. In certain embodiments, the concentration of at least one FGF activator contacted with or exposed to the cells is about 100 ng / mL. In certain embodiments, the concentration of at least one FGF activator contacted or exposed to the cells is about 200 ng / mL.

[0171] In certain embodiments, the at least one FGF activator comprises FGF18.

[0172] In certain non-limiting embodiments, stem cells are contacted or exposed to at least one TGFβ / Activin-Nodal inhibitor (e.g., SB431542, e.g., at a concentration of about 10 μM), at least one BMP inhibitor (e.g., LDN193189, e.g., at a concentration of about 250 nM), and at least one SHH activator (e.g., SHH C25II, e.g., at a concentration of about 500 ng / mL) for about 5 days (e.g., 7 days, e.g., from day 0 to day 6), and the cells are contacted with at least one Wnt activator (e.g., CHIR99021, e.g., at a concentration of about 0.7 μM for 5 days (e.g., 4 days, e.g., from day 0 to day 3), and at a concentration of about 7.5 μM for about 5 days (e.g., 6 days, e.g., from day 4 to day 9), and at a concentration of about 3 μM for about 2 days (e.g., from day 10 to day 11). The cells are contacted with at least The cells are contacted with or exposed to at least one FGF activator (e.g., FGF18, e.g., at a concentration of about 100 ng / ml), wherein the initial contact of the cells with the at least one FGF activator is about 10 days (e.g., 10 days or 12 days) after the initial contact of the cells with the at least one SMAD inhibitor, and the cells are contacted with the at least one FGF activator for about 5 days (e.g., 5 days (days 12-16) or 7 days (e.g., days 10-16)).

[0173] 5.2.6. Cell Culture Media In certain embodiments, the inhibitors and activators are added to cell culture media containing 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 Essential8® / Essential6® ("E8 / E6") medium, and combinations thereof. KSR medium, NB medium, N2 medium, B-27 medium, and E8 / E6 medium are commercially available. KSR medium is a defined, serum-free formulation optimized for the growth and maintenance of undifferentiated hESCs in culture.

[0174] In certain embodiments, the cell culture medium is KSR medium. The components of KSR medium are disclosed in WO 2011 / 149762. In certain embodiments, 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 μM 13-mercaptoethanol.

[0175] In certain embodiments, the cell culture medium is E8 / E6 medium. E8 / E6 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. Furthermore, E8 / E6 medium can be used as a base for formulating custom media for the culture of PSCs. An example of 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. An example of E8 / E6 medium is disclosed in WO 15 / 077648, which is incorporated by reference in its entirety. In certain embodiments, E8 / E6 cell culture medium comprises DMEM / F12, ascorbic acid, selenium, insulin, NaHCO3, transferrin, FGF2, and TGFβ. E8 / E6 medium differs from KSR medium in that E8 / E6 medium does not contain active BMP or Wnt components. Thus, in certain embodiments, when E8 / E6 medium is used to culture and differentiate a population of stem cells of the present disclosure into a population of proprioceptors, it is not necessary to add at least one SMAD signaling inhibitor (e.g., one that inhibits BMP) to the E8 / E6 medium.

[0176] 5.2.7. Differentiated cells In certain embodiments, the method includes obtaining a cell population of differentiated 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 indicative of mDA or mDA precursors. Non-limiting examples of markers indicative of mDA or mDA precursors include Engrailed-1 (EN1), Orthodenticle Homeobox 2 (OTX2), Tyrosine Hydroxylase (TH), Nuclear Receptor-Associated-1 Protein (NURR1), Forkhead Box Protein A2 (FOXA2), and LIM Homeobox Transcription Factor 1 Alpha (LMX1A), PITX3, LMO3, SNCA, ADCAP1, CHRNA4, and GIRK2.

[0177] In certain embodiments, the differentiated cells express at least one marker indicative of mDA or an mDA precursor for at least about 10 days (e.g., about 15 days (e.g., 16 days), about 20 days, about 30 days, about 40 days, or about 50 days) from initial contact of the cells with at least one SMAD inhibitor.

[0178] Treatment of cells with at least an FGF activator can result in sustained expression of EN1. EN1 is a survival factor for developing midbrain DA neurons and continues to exert neuroprotective and physiological functions in adult midbrain DA neurons. Thus, cells with sustained expression of EN1 can develop into functionally mature mDA neurons upon further development and maturation. In certain embodiments, differentiated cells have detectable levels of expression of EN1 for at least about 10 days, at least about 15 days, at least about 16 days, at least about 20 days, at least about 25 days, at least about 27 days, at least about 30 days, at least about 35 days, at least about 40 days, at least about 45 days, at least about 50 days, at least about 60 days, at least about 70 days, at least about 80 days, or at least about 90 days after initial contact of the stem cells with at least one SMAD inhibitor. In certain embodiments, differentiated cells have detectable levels of expression of EN1 for about 30 days after initial contact of the stem cells with at least one SMAD inhibitor. In certain embodiments, the differentiated cells have detectable levels of expression of EN1 for about 40 days from initial contact of the stem cells with at least one SMAD inhibitor.

[0179] In certain embodiments, differentiated cells derived from the methods of the present disclosure do not express or have low expression of at least one marker selected from PAX6, EMX2, LHX2, SMA, SIX1, PITX2, SIM1, POU4F1, PHOX2A, BARHL1, BARHL2, GBX2, HOXA2, HOXB2, POU5F1, NANOG, and combinations thereof.

[0180] In certain embodiments, the cells are contacted with the activators and inhibitors described herein at concentrations and for a time effective to increase the expression of a detectable level of at least one marker of DA neurons, e.g., EN1, or wherein the cells are type A9 neuronal cells.

[0181] In certain embodiments, cells are contacted with the activators and inhibitors described herein at concentrations and for periods of time effective to decrease the expression of SMA, SIX1, PITX2, SIM1, POU4F1 and / or PHOX2A.

[0182] 5.2.8. Differentiation of mDA precursors into mDA In certain embodiments, the cells are further contacted with DA neuron lineage-specific activators and inhibitors, such as L-glutamine, brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), cyclic adenosine monophosphate (cAMP), transforming growth factor beta (TGFβ, e.g., TGFβ3), ascorbic acid (AA), and DAPT (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, cells are contacted with the DA neuron lineage-specific activators and inhibitors for 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 7 days, at least about 8 days, at least about 9 days, or at least about 10 days or more, e.g., from about 2 to about 20 days, from about 3 to about 19 days, from about 4 to about 18 days, from about 5 to about 17 days, from about 6 to about 16 days, from about 7 to about 15 days, from about 8 to about 15 days, from about 9 to about 14 days, or from about 10 to about 13 days. In certain embodiments, cells are contacted with the DA neuron lineage-specific activators and inhibitors for up to about 2 days, 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, or up to about 10 days or more. In certain embodiments, the cells are contacted with the DA neuron lineage-specific activators and inhibitors for about 4 days, about 5 days, about 6 days, about 7 days, or about 8 days.

[0183] In certain embodiments, the cells are contacted with L-glutamine at a concentration of about 0.5 mM to about 5 mM, or about 1 mM to about 5 mM, or about 1.5 mM to about 2.5 mM, or about 1 mM to about 2 mM. In certain embodiments, the cells are contacted with L-glutamine at a concentration of about 2 mM.

[0184] In certain embodiments, cells are contacted with BDNF at a concentration of about 5 ng / mL to about 50 ng / mL, or about 10 ng / mL to about 50 ng / mL, or about 10 ng / mL to about 40 ng / mL, or about 20 ng / mL to about 50 ng / mL, or about 20 ng / mL to about 40 ng / mL, or about 10 ng / mL to about 30 ng / mL, or about 10 ng / mL to about 20 ng / mL, or about 20 ng / mL to about 30 ng / mL. In certain embodiments, cells are contacted with BDNF at a concentration of about 20 ng / mL.

[0185] In certain embodiments, the cells are contacted with ascorbic acid (AA) at a concentration of about 50 nM to about 500 nM, or about 100 nM to about 500 nM, or about 100 nM to about 400 nM, or about 200 nM to about 400 nM, or about 200 nM to about 300 nM, or about 100 nM to about 300 nM. In certain embodiments, the cells are contacted with AA at a concentration of about 200 nM.

[0186] In certain embodiments, cells are contacted with GDNF at a concentration of about 5 ng / ml to about 50 ng / ml, or about 10 ng / ml to about 50 ng / ml, or about 10 ng / ml to about 40 ng / ml, or about 20 ng / ml to about 50 ng / ml, or about 20 ng / ml to about 40 ng / ml, or about 10 ng / ml to about 30 ng / ml, or about 10 ng / ml to about 20 ng / ml, or about 20 ng / ml to about 30 ng / ml. In certain embodiments, cells are contacted with GDNF at a concentration of about 20 ng / ml.

[0187] In certain embodiments, the cells are contacted with cAMP at a concentration of about 200 nM to about 800 nM, or about 200 nM to about 700 nM, or about 300 nM to about 700 nM, or about 300 nM to about 600 nM, or about 400 nM to about 600 nM, or about 450 nM to about 550 nM. In certain embodiments, the cells are contacted with cAMP at a concentration of about 500 nM.

[0188] In certain embodiments, the cells are contacted with TGFβ3 at a concentration of about 0.01 ng / ml to about 5 ng / ml, or about 0.1 ng / ml to about 4 ng / ml, or about 0.5 ng / ml to about 5 ng / ml, or about 1 ng / ml to about 3 ng / ml, or about 1 ng / ml to about 2 ng / ml. In certain embodiments, the cells are contacted with TGFβ3 at a concentration of about 1 ng / ml.

[0189] In certain embodiments, differentiated midbrain DA precursors are further cultured as described in U.S. Patent Application Publication No. 2015 / 0010514, which is incorporated by reference in its entirety.

[0190] 5.3. Methods for Isolating Midbrain DA Neurons and Their Precursors The present disclosure provides a method for isolating mDA and its precursors based on at least one or at least two surface markers. In certain embodiments, the surface marker is a negative surface marker, and the cells do not express detectable levels of the negative surface marker. In certain embodiments, the cells express a reduced level of the negative surface marker compared to the average expression level of the negative surface marker in the population of cells from which the cells are isolated.

[0191] In certain embodiments, the surface marker is a positive surface marker and the cell expresses a detectable level of the positive surface marker, hi certain embodiments, the cell expresses an increased level of the positive surface marker compared to the average expression level of the positive surface marker of the population of cells from which the cell is isolated.

[0192] In certain embodiments, the disclosed methods for isolating mDA and its precursors from a population of cells include isolating cells that do not express a detectable level of at least one negative surface marker. In certain embodiments, the disclosed methods for isolating mDA and its precursors from a population of cells include isolating cells that do not express a detectable level of at least one negative surface marker or that express a reduced level of at least one negative surface marker compared to the average expression level of at least one negative surface marker in the population of cells. In certain embodiments, the disclosed methods for isolating mDA and its precursors from a population of cells include isolating cells that express a detectable level of at least one positive surface marker. In certain embodiments, the disclosed methods for isolating mDA and its precursors from a population of cells include isolating cells that express an increased level of at least one positive surface marker compared to the average expression level of at least one positive marker in the population of cells.

[0193] In certain embodiments, the disclosed methods for isolating mDA and its precursors from a population of cells comprise isolating cells that do not express a detectable level of at least one negative surface marker and that express a detectable level of at least one positive surface marker. In certain embodiments, the disclosed methods for isolating mDA and its precursors from a population of cells comprise isolating cells that (a) do not express a detectable level of at least one negative surface marker or express a reduced level of at least one negative surface marker compared to the average expression level of the at least one negative surface marker in the population of cells, and (b) have an elevated level of at least one positive surface marker compared to the average expression level of the at least one positive surface marker in the population of cells.

[0194] In certain embodiments, the negative surface markers are selected from CD49e (also known as integrin alpha 5), ​​CD99, CD340, and combinations thereof. In certain embodiments, the positive surface markers are selected from CD171, CD184, and combinations thereof.

[0195] In certain embodiments, the disclosed methods for isolating mDA and its precursors from a population of cells comprise isolating cells that do not express detectable levels of CD49e and express detectable levels of CD 184. In certain embodiments, the disclosed methods for isolating mDA and its precursors from a population of cells comprise isolating cells that do not express detectable levels of CD49e or express reduced levels of CD49e compared to the average expression level of CD49e in the population of cells and express increased levels of CD184 compared to the average expression level of CD184 in the population of cells.

[0196] Any surface marker-based cell isolation technique known in the art can be used in the disclosed methods, hi certain embodiments, flow cytometry is used in the disclosed isolation methods.

[0197] 5.4. Cell Populations and Compositions The present 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 indicative of mDA or mDA precursors. Non-limiting examples of markers indicative of mDA or mDA precursors include EN1, OTX2, TH, NURR1, FOXA2, LMX1A, PITX3, LMO3, SNCA, ADCAP1, CHRNA4, and GIRK2. The present disclosure also provides compositions comprising such cell populations. In certain embodiments, the in vitro differentiated cells are obtained by the differentiation methods described herein, for example, in Section 5.2.

[0198] 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, HOXA2, HOXB2, POU5F1, NANOG, and combinations thereof.

[0199] The present disclosure also 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 positive surface marker disclosed herein (e.g., Section 5.3) and do not express at least one negative surface marker disclosed herein (e.g., Section 5.3). The present disclosure also 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 an increased level of at least one positive surface marker disclosed herein (e.g., Section 5.3) relative to the average expression level of the at least one positive marker in the population of cells, and do not express a detectable level of, or express a reduced level of, at least one negative surface marker disclosed herein (e.g., Section 5.3) relative to the average expression level of the at least one negative surface marker in the population of cells.

[0200] In certain embodiments, at least about 50% of the cells (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%) do not express detectable levels of CD49e and express detectable levels of CD184. In certain embodiments, at least about 50% of the cells (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%) do not express detectable levels of CD49e or express a reduced level of CD49e compared to the average expression level of CD49e in the cell population, and express an increased level of CD184 compared to the average expression level of CD184 in the population of cells. Further, the present disclosure provides compositions comprising such cell populations.

[0201] In certain embodiments, the cells are contained in a composition that further comprises a biocompatible scaffold or matrix, such as a biocompatible three-dimensional scaffold that promotes tissue regeneration when the cells are implanted or transplanted into a subject. In certain embodiments, the biocompatible scaffold comprises extracellular matrix materials, 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 hydrogels. (See, e.g., U.S. Patent Application Publication Nos. 2015 / 0159135, 2011 / 0296542, 2009 / 0123433, and 2008 / 0268019, the contents of each of which are incorporated by reference in their entirety.) In certain embodiments, the composition further comprises growth factors to promote maturation of the implanted / transplanted cells into mesencephalic DA cells.

[0202] In certain embodiments, the composition comprises about 1×10 4 ~Approx. 1×10 10 , about 1×104 ~Approx. 1×10 5 , about 1×10 5 ~Approx. 1×10 9 , about 1×10 5 ~Approx. 1×10 6 , about 1×10 5 ~Approx. 1×10 7 , about 1×10 6 ~Approx. 1×10 7 , about 1×10 6 ~Approx. 1×10 8 , about 1×10 7 ~Approx. 1×10 8 , about 1×10 8 ~Approx. 1×10 9 , about 1×10 8 ~Approx. 1×10 10 , or approximately 1 × 10 9 ~Approx. 1×10 10 In certain embodiments, the cell population comprises about 1 x 10 cells and is administered to the subject. 5 pieces~approx. 1×10 7 The cells are administered to a subject.

[0203] In certain embodiments, the composition is frozen. In certain embodiments, the composition further comprises at least one cryoprotectant, such as, but not limited to, dimethyl sulfoxide (DMSO), glycerol, polyethylene glycol, sucrose, trehalose, dextrose, or a combination thereof.

[0204] In certain embodiments, the composition further comprises a biocompatible scaffold or matrix, e.g., a biocompatible three-dimensional scaffold that promotes tissue regeneration when cells are implanted or transplanted into a subject. In certain embodiments, the biocompatible scaffold comprises an extracellular matrix material, a synthetic polymer, a cytokine, a collagen, a polypeptide or protein, a polysaccharide including fibronectin, laminin, keratin, fibrin, fibrinogen, hyaluronic acid, heparin sulfate, chondroitin sulfate, agarose, or gelatin, and / or a hydrogel. (See, e.g., U.S. Patent Application Publication Nos. 2015 / 0159135, 2011 / 0296542, 2009 / 0123433, and 2008 / 0268019, the contents of each of which are incorporated by reference in their entirety.)

[0205] In certain embodiments, the composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier, and can be used to prevent and / or treat neurodegenerative disorders, including Parkinson's disease, Huntington's disease, Alzheimer's disease, and multiple sclerosis.

[0206] The presently disclosed subject matter also provides devices comprising the differentiated cells disclosed herein or compositions comprising same, non-limiting examples of which include syringes, fine glass tubes, stereotactic needles, and cannulas.

[0207] 5.5. Methods of Treating Neurodegenerative Disorders The cell populations and compositions disclosed herein (e.g., those disclosed in Section 5.4) can be used to treat neurodegenerative disorders. The presently disclosed subject matter provides methods of treating neurodegenerative disorders. In certain embodiments, the methods comprise administering an effective amount of the stem cell-derived mDA of the present disclosure, or a composition comprising same, to a subject suffering from a neurodegenerative disorder. In certain embodiments, the methods comprise administering an effective amount of in vitro differentiated cells, or a composition comprising such cells, that do not express detectable levels of at least one negative surface marker (e.g., CD49e) and express detectable levels of at least one positive surface marker (e.g., CD184), to a subject suffering from a neurodegenerative disorder. In certain embodiments, the method comprises administering to a subject suffering from a neurodegenerative disorder an effective amount of in vitro differentiated cells that do not express, or express a reduced level of, at least one negative surface marker (e.g., CD49e) relative to the average expression of the at least one negative surface marker of the population of cells from which the cells are isolated, and that express an increased level of at least one positive surface marker (e.g., CD184) relative to the average expression of the at least one positive surface marker of the population of cells from which the cells are isolated, or a composition comprising such cells. In certain embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

[0208] Non-limiting examples of neurodegenerative disorders include Parkinson's disease, Huntington's disease, Alzheimer's disease and multiple sclerosis.

[0209] In certain embodiments, the neurodegenerative disease is Parkinson's disease, whose primary motor symptoms include, but are not limited to, tremors in the hands, arms, legs, jaw, and face, bradykinesia or slowness of movement, stiffness or rigidity of the limbs and trunk, and postural instability or impaired balance and coordination.

[0210] In certain embodiments, the neurodegenerative disease is parkinsonism, which refers to a disease associated with a lack of dopamine in the basal ganglia, the part of the brain that controls movement.Symptoms include tremor, bradykinesia (extremely slow movement), flexed posture, postural instability, and rigidity.Non-limiting examples of parkinsonism include corticobasal degeneration, dementia with Lewy bodies, multiple system atrophy, and progressive supranuclear palsy.

[0211] The cells or compositions can be administered or provided systemically or directly to a subject to treat or prevent a neurodegenerative disorder. In certain embodiments, the cells or compositions are injected directly into an organ of interest (e.g., the central nervous system (CNS) or peripheral nervous system (PNS)). In certain embodiments, the cells or compositions are injected directly into the striatum.

[0212] The cells or compositions can be administered in any physiologically acceptable vehicle. The cells or compositions can be administered by local 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 by orthotopic (OT) injection.

[0213] Cells or compositions can be conveniently provided as sterile liquid preparations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions that can be buffered to a selected pH. Liquid preparations are typically easier to prepare than gels, other viscous compositions, and solid compositions. Furthermore, liquid compositions are somewhat more convenient to administer, particularly by injection. Viscous compositions, on the other hand, can be formulated within an appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can contain a carrier, which can be a solvent or dispersion medium containing, for example, water, saline, phosphate-buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof. Sterile injectable solutions can be prepared by incorporating a composition of the presently disclosed subject matter, such as a composition comprising the presently disclosed stem cell-derived progenitors, in the required amount of an appropriate solvent, along with various amounts of other ingredients, as needed. Such compositions can be mixed with an appropriate carrier, diluent, or excipient, such as, for example, sterile water, saline, glucose, dextrose, etc. The composition can also be lyophilized. Depending on the route of administration and the desired preparation, the compositions can contain auxiliary substances, such as wetting agents, dispersing or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or thickening additives, preservatives, flavorings, coloring agents, etc. 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.

[0214] Various additives can be added to enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, etc. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0215] If desired, the viscosity of the composition can be maintained at a selected level using a pharmaceutically acceptable thickening agent. Methylcellulose can be used because it is readily and economically available and easy to handle. Other suitable thickening agents include, for example, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, carbomer, etc. The concentration of the thickening agent can depend on the agent selected. The important point is to use an amount that achieves the selected viscosity. The selection of appropriate carriers and other additives depends on the exact route of administration and the nature of the particular dosage form, for example, a liquid dosage form (e.g., whether the composition is formulated into a solution, suspension, gel, or other liquid form, such as a time-release form or a liquid-filled form).

[0216] Those skilled in the art will recognize that the components of the composition should be selected to be chemically inert and not affect the viability or efficacy of the stem cell-derived precursors of the present disclosure, which will not present a problem to those skilled in the art of chemical and pharmaceutical principles, or which can be easily circumvented by reference to standard texts or by simple experimentation (without undue experimentation) from this disclosure and the literature cited herein.

[0217] One consideration regarding therapeutic use of cells is the quantity of cells needed to achieve an optimal effect, including, but not limited to, regrowth of CNS and / or PNS regions in a subject suffering from a neurodegenerative disorder and / or improved function of the CNS and / or PNS in a subject.

[0218] An "effective amount" (or "therapeutically effective amount") is an amount sufficient to affect a beneficial or desired clinical outcome upon treatment. An effective amount can be administered to a subject in at least one dose. In terms of treatment, an effective amount is an amount sufficient to palliate, ameliorate, stabilize, reverse, or slow the progression of a neurodegenerative disorder or pituitary disorder, or otherwise reduce the pathological consequences of a neurodegenerative disorder. An effective amount is generally determined by a physician on a case-by-case basis and is within the skill of one of ordinary skill in the art. When determining an appropriate dosage to achieve an effective amount, several factors are typically considered. These factors include the 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.

[0219] In certain embodiments, the effective amount of cells is sufficient to repopulate CNS and / or PNS regions in a subject suffering from a neurodegenerative disorder. In certain embodiments, the effective amount of cells is sufficient to improve CNS and / or PNS function in a subject suffering from a neurodegenerative disorder, e.g., improved function can be about 1%, 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 CNS and / or PNS function of a normal individual.

[0220] The amount of cells administered varies depending on the subject being treated. In certain embodiments, about 1 x 10 4 pieces~approx. 1×10 10 pieces, about 1×10 4 pieces~approx. 1×10 5 pieces, about 1×10 5 pieces~approx. 1×10 9 pieces, about 1×10 5 pieces~approx. 1×10 6 pieces, about 1×10 5 pieces~approx. 1×10 7 pieces, about 1×10 6 pieces~approx. 1×10 7 pieces, about 1×10 6 pieces~approx. 1×10 8 pieces, about 1×10 7 pieces~approx. 1×10 8 pieces, about 1×108 pieces~approx. 1×10 9 pieces, about 1×10 8 pieces~approx. 1×10 10 pieces, or approximately 1 x 10 9 pieces~approx. 1×10 10 In certain embodiments, about 1 x 10 cells are administered to a subject. 5 pieces~approx. 1×10 7 In certain embodiments, about 1 x 10 cells are administered to a subject suffering from a neurodegenerative disorder. 6 pieces~approx. 1×10 7 In certain embodiments, about 1 x 10 cells are administered to a subject suffering from a neurodegenerative disorder. 6 pieces~approx. 4×10 6 The cells are administered to a subject suffering from a neurodegenerative disorder. The precise determination of what is considered an effective dose may be based on factors individual to each subject, including the size, age, sex, weight, and condition of the particular subject. Dosage amounts can be readily ascertained by one skilled in the art from this disclosure and knowledge in the art.

[0221] 5.6.Kit The presently disclosed subject matter provides kits for inducing differentiation of stem cells into mDA or precursors thereof. In certain embodiments, the kits comprise (a) at least one SMAD signaling inhibitor, (b) at least one SHH signaling activator, (c) at least one Wnt signaling activator, and (d) at least one FGF signaling activator. In certain embodiments, the kits further comprise (e) instructions for inducing differentiation of the stem cells into a population of differentiated cells expressing at least one marker indicative of mDA or precursors thereof.

[0222] In certain embodiments, the instructions include contacting the stem cells with a specific sequence of inhibitor(s), activator(s), and molecule(s). The order in which the inhibitor(s), activator(s), and molecule(s) are contacted can be determined by the cell culture medium used to culture the stem cells.

[0223] In certain embodiments, the instructions include contacting the stem cells with inhibitor(s), activator(s), and molecule(s) as described by the methods of the present disclosure (see Section 5.2).

[0224] In certain embodiments, the present disclosure provides kits comprising an effective amount of a cell population or composition disclosed herein in a unit dosage form. In certain embodiments, the kits include a sterile container containing the therapeutic composition. Such a container may be a box, an ampoule, a bottle, a vial, a tube, a bag, a pouch, a blister pack, or other suitable container form known in the art. Such a container may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding pharmaceutical agents.

[0225] In certain embodiments, the kit includes instructions for administering the cell population or composition to a subject suffering from a neurodegenerative disorder. The instructions may include information regarding the use of the cells or composition to treat or prevent the neurodegenerative disorder. In certain embodiments, the instructions include at least one of: a description of the therapeutic agent, a dosing schedule and administration for treating or preventing the neurodegenerative disorder or its symptoms, precautions, warnings, indications, contraindications, overdose information, adverse reactions, animal pharmacology, clinical studies, and / or bibliographic references. The instructions may be printed directly on the container (if present), as a label affixed to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container. [Example]

[0226] 6. Working Example The presently disclosed subject matter will be better understood by reference to the following examples, which are provided by way of illustration of the presently disclosed subject matter, not by way of limitation.

[0227] Example 1: Optimization of midbrain DA neuron differentiation protocol Midbrain DA neurons and their precursors were derived from stem cells under a previously disclosed Wnt-Boost protocol (the "7.5 μM bump protocol (modified GMP V2B)" protocol disclosed in International Publication No. 2016 / 196661, which is incorporated by reference in its entirety), hereafter referred to as the "Wnt-Boost protocol" or "Boost protocol." EN1 expression was found to decrease starting from day 11 of differentiation with the Wnt-Boost protocol (see Figure 1). Maintaining EN1 expression in differentiated cells is important for generating mature, functional midbrain DA neurons. Therefore, to maintain EN1 expression, in this example, we tested adding FGF8b treatment to the Wnt-Boost protocol at the later stages of differentiation (see Figure 2). Cells were tested by contacting them with FGF8b in addition to the Wnt-Boost protocol from days 9 to 16, 10 to 16, 11 to 16, 12 to 16, 13 to 16, 14 to 16, or 15 to 16. Immunostaining of cells at day 16 of differentiation showed that EN1 protein expression was maintained in a FGF8b exposure duration-dependent manner (see Figure 3). EN1-positive cells also expressed FOXA2 and LMX1A. RNA expression measured in cells at day 30 of differentiation showed that expression levels of mDA or mDA precursor markers FOXA2, NURR1, LMX1A, OXT2, and TH were comparable across all conditions, indicating that EN1 mRNA expression was maintained in a FGF8b exposure duration-dependent manner (see Figures 4A-4B). However, mRNA levels of contaminating markers (non-mDA or non-mDA precursor markers), such as SMA and SIX1, were also induced in an FGF8b exposure duration-dependent manner (see Figure 4B). SIX1 immunostaining of cells at day 30 of differentiation confirmed the mRNA results (see Figure 5).

[0228] FGF8b has been used to induce midbrain DA neurons from pluripotent stem cells. FGF8, FGF17, and FGF18 are subfamily FGFs, and it has been demonstrated that FGF17b and FGF18 have distinct roles from FGF8b in midbrain development (Liu et al., Development. 2003 Dec;130(25):6175-85). FGF18 has also been shown to protect against 6-OHDA-induced midbrain dopamine neuron damage (Guo et al., Neuroscience. 2017 Jul 25;356:229-241). Furthermore, FGF8b (isthmus and rhombencephalon 1) elongates organizers along the junction between the induced Gbx2 domain and the remaining Otx2 region within the midbrain. FGF8a, FGF17, and FGF18 are responsible for midbrain expansion and upregulation of midbrain genes. FGF8b, FGF17 and FGF18 are all in the same FGF subgroup of paracrine FGFs to FGF8b.

[0229] FGF8b, FGF17, and FGF18 were tested by adding them to cell cultures at a concentration of 100 ng / ml from day 12 to day 16 under the WNT-Boost protocol. In cells on day 16 of differentiation, FGF18 induced mRNA expression levels of EN1 similar to those of FGF8b, but reduced mRNA expression levels of SMA were found (see Figures 6 and 7). EN1 protein expression was also maintained at a high level, as with FGF8b, in an FGF18 exposure duration-dependent manner.

[0230] At the maturation stage of differentiation, EN1 was still highly maintained under FGF8b and FGF18 treatment conditions (see Figure 8). Furthermore, both FGF18-treated and FGF8b-treated cells had reduced expression levels of PITX2 compared to cells differentiated by the WNT-Boost protocol, while FGF18-treated cells had reduced expression levels of SMA1 and SIX1 compared to FGF8b-treated cells (see Figure 8). These results indicate that FGF18 treatment results in sustained EN1 expression while minimizing or reducing the expression levels of non-mDA markers compared to FGF8b treatment.

[0231] We examined the in vivo survival of differentiated cells generated from the WNT-boost+FGF18 protocol. Cells generated from the WNT-boost and WNT-boost+FGF18 protocols were transplanted into intact mouse tissue. Cells generated from the WNT-boost+FGF18 protocol showed improved maintenance of EN1 expression in vivo compared with cells generated from the WNT-boost protocol (Figure 35A). Cells generated from the WNT-boost+FGF18 protocol also had better striatal innervation, with fibers already emerging from the graft core toward the periphery by 1 month after transplantation (Figure 35B).

[0232] Example 2: Purification of mDA using reporters NURR1 is a marker of postmitotic and immature midbrain DA neurons and is also expressed in mature midbrain DA neurons. It is a transcription factor and contributes to DA differentiation and maintenance.

[0233] To purify mDA from the cell population, we used endogenous NURR1::GFP reporter hPSCs (see Figure 9A). mDA were differentiated from the reporter cell line. Based on the fact that NURR1 mRNA expression was highly induced from day 20 of differentiation, FACS-based isolation of GFP-positive cells was performed on day 25 of differentiation (see Figures 9B-9C).

[0234] Single-cell qRT-PCR was performed on the NURR1:GFP-positive cell isolates on days 25 and 40 of differentiation. Nearly 100% of the NURR1:GFP-positive cells were found to express TH (a mature mDA marker), FOXA2, and LMX1A on day 40 of differentiation, indicating their mDA fate (see Figure 10A). Continuous culture of the isolated NURR1:GFP-positive cells up to day 60 showed that these cells expressed high levels of TH, indicating that these cells were highly pure mDA (see Figure 10B).

[0235] NURR1::GFP-positive midbrain DA neurons on day 25 of differentiation were transplanted into nude mice. Figure 11 shows that the transplanted cells survived in vivo and expressed TH, the human marker SC121, and GFP. Neurite outgrowth was observed in the cell transplantation area (see Figure 11).

[0236] NURR1:GFP hPSCs were then cultured under WNT-Boost and WNT-Boost+FGF18 (days 12-16) protocols. NURR1:GFP-positive cells were isolated on day 25 of differentiation and subsequently cultured continuously until day 40. At day 40 of differentiation, these midbrain DA neurons expressed high TH along with FOXA2 (see Figure 12).

[0237] mRNA expression analysis showed that mDAs derived from the WNT-Boost + FGF18 (days 12-16) protocol and sorted by NURR1:GFP had higher EN1 expression levels than mDAs derived from WNT-Boost and sorted by NURR1:GFP (see Figure 13A). These sorted cells were transplanted into immunodeficient mice. Both mDAs derived from the WNT-Boost protocol and the WNT-Boost + FGF18 (days 12-16) protocol showed excellent cell survival and expressed the markers SC121 and TH. However, FGF18-treated cells (WNT-Boost + FGF18 protocol) showed better neurite outgrowth from the transplanted area (see Figure 13B).

[0238] Example 3: Discovery of surface markers in purified DA neurons Published studies have shown that each iPSC line has variability in the derivation of specific cell types. It is difficult to generate the reported strains for each iPSC line used for midbrain DA cell purification. Furthermore, genetically engineered cells are not suitable for clinical use. This example used the NURR1::GFP reporter line to identify candidate surface markers, particularly those enriched in NURR1::GFP-positive cells but not in NURR1::GFP-negative cells, or vice versa.

[0239] In this example, 387 surface markers were examined in mDA differentiated cells at day 25 of differentiation derived from NURR1:GFP hPSCs (see Figure 14).

[0240] Two positive CD markers, CD171 and CD184, were enriched in the NURR1::GFP-positive population (see Figures 15A-15B), and three negative CD markers, CD49e, 99, and 340, were enriched in the NURR1::GFP-negative population (see Figures 16A-16B).

[0241] Cells were sorted by CD49e (negative and / or weak expression) on day 25 of differentiation under the WNT-Boost protocol or the WNT-boost + FGF18 (days 12-16) protocol and cultured for an additional 10 days. Cell morphology indicated that these cells were essentially pure mDA (see Figure 17). Sorted mDA on day 40 of differentiation (sorted on day 25 and cultured for an additional 15 days) had high TH immunostaining (see Figure 18).

[0242] To purify midbrain DA neurons, the CD49e marker was tested in another hPSC line, MEL1. Substantially pure mDA morphology was found in cells sorted for CD49e (negative and / or weak expression) on day 25 of differentiation and continuously cultured under the WNT-boost protocol and the WNT-boost + FGF18 (days 12-16) protocol for an additional 15 days (day 40 of differentiation) (see Figures 19-20). These sorted mDA neurons had high TH immunostaining (see Figure 21).

[0243] mRNA expression showed that CD49e-sorted cells differentiated under the WNT-boost+FGF18 (days 12-16) protocol had higher expression levels of EN1 and lower expression levels of PITX2 (glutamergic neurons, a subthalamic nucleus marker) than CD49e-sorted cells differentiated under the WNT-Boost protocol. Sorted cells differentiated under both protocols had little or no expression levels of non-mesencephalic DA markers (HOXA2, SMA1, and SIX1) (see Figure 22).

[0244] All three negative CD markers, CD49e, CD99, and CD340, were tested. A substantially pure neuronal morphology was observed in cells sorted for CD49e, CD99, or CD340 (negative and / or weakly expressing cells) at day 25 of differentiation and continuously cultured for an additional 15 days (day 40 of differentiation) (see Figure 23). However, mRNA expression of sorted cells showed increased expression of non-DA neuron markers, including PHOX2A, PITX2, POU4F1, and SIM1, suggesting that isolation based on CD49e, CD99, or CD340 did not exclude non-DA neurons (Figure 24).

[0245] It was thought that a double sorting strategy using CD184 could correct the lack of negative CD markers. Cxcr4 (CD184) is important for the migration and orientation of midbrain DA neurons during mouse midbrain development. FGF18-treated mDA neurons express CD184 + / CD49e - The A9 midbrain DA subtype can be enriched after selection with

[0246] FACS sorting was performed on midbrain DA cells at day 25 of differentiation derived from NURR1::GFP hPSCs using CD49E (PE) and CD171 (APC). Cells negatively sorted for single CD49e were found to have approximately 63% NURR1:GFP fraction. And CD171 positive sorting in combination with CD49e failed to enrich the NURR1:GFP population (see Figure 25).

[0247] However, sorting with CD49e for CD184 (positive expressing cells) can enrich the NURR1:GFP fraction by approximately 80% over single sorted cells (CD49e; 63%) (see Figure 26).

[0248] Then, FACS sorting was performed on cells at differentiation day 25 by CD49e and CD171 or CD49e and CD184. The morphology of cells cultured 2 days after sorting showed a pure neuronal shape except for cells sorted based on higher CD49e (see Figure 27).

[0249] mRNA expression is CD49e - / CD184 + We showed that (CD49e-negative / CD184-positive) cells had higher expression levels of mDA markers (NURR1, EN1, PITX3) and lower expression levels of non-mDA markers (PITX2, SIM1, POU4F1) than cells sorted by other methods (see Figure 28).

[0250] Next, we investigated whether CD49e and CD184 could strongly select mDA. As shown in Figure 29, a single CD49e- Sorting enriched the NURR1:GFP-positive population from approximately 20% to up to 43% in in vitro differentiated cells. As shown in Figure 30, the double CD49e - and CD184 + Sorting enriched the NURR1::GFP-positive fraction in in vitro differentiated cells to 74% and 85%.

[0251] After two weeks of in vitro culture, the sorted cells were found to be highly TH-positive, co-expressing FOXA2 and GFP. + mDA was shown, thereby confirming its identity (see Figure 31).

[0252] mRNA expression showed that dual CD marker-mediated sorted cells (CD49e- and CD184+) generally had higher expression of mDA markers (see Figure 32) than other CD sorted cells, while having lower expression levels of non-mDA markers (see Figure 33) at day 40 of differentiation sorted by CD markers at day 25.

[0253] We investigated the in vivo survival of differentiated cells sorted with the novel surface markers described herein. Differentiated cells were generated using the WNT-boost protocol, and CD49e-low, CD184-high cells were selected and transplanted into intact mouse brains. Compared to unsorted cells, tissues transplanted with sorted cells had enrichment for Th+ cells (Figure 34A), and reduced numbers of SOX2+ progenitor and KI67+ dividing cells at 1 month post-transplant (Figures 34B-34D).

[0254] While the subject matter of the present disclosure and its advantages have been described in detail, it should be understood that various changes, substitutions, and alterations can be made herein without departing from the spirit and scope of the present disclosure. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the processes, machines, manufacture, and compositions of matter, means, methods, and steps described herein. Those skilled in the art will readily appreciate from the present disclosure of the presently disclosed subject matter, processes, machines, manufacture, compositions of matter, means, methods, or steps that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein, can be utilized in accordance with the presently disclosed subject matter. Accordingly, it is intended that the appended claims include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps.

[0255] Various patents, patent applications, publications, product descriptions, protocols, and sequence accession numbers are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties for all purposes. In certain embodiments, for example, the following items are provided: (Item 1) 1. An in vitro method for inducing differentiation of stem cells, comprising: contacting the stem cells with at least one Small Mothers Against Decapentaplegic (SMAD) signaling inhibitor, at least one Sonic Hedgehog (SHH) signaling activator, and at least one Wingless (Wnt) signaling activator; and contacting the cells with at least one fibroblast growth factor (FGF) signaling activator to obtain a population of differentiated cells expressing at least one marker indicative of midbrain dopamine neurons (mDA) or their precursors, wherein the at least one FGF signaling activator is selected from FGF18, FGF17, FGF8a, and combinations thereof. (Item 2) 1. An in vitro method for inducing differentiation of stem cells, comprising: contacting the stem cells with at least one Small Mothers Against Decapentaplegic (SMAD) signaling inhibitor, at least one Sonic Hedgehog (SHH) signaling activator, and at least one Wingless (Wnt) signaling activator; and contacting the cells with at least one fibroblast growth factor (FGF) signaling activator to obtain a population of differentiated cells expressing at least one marker indicative of midbrain dopamine neurons (mDA) or precursors thereof, wherein the initial contacting of the cells with the at least one FGF signaling activator is at least about 5 days after the initial contacting of the cells with the at least one SMAD signaling inhibitor. (Item 3) 3. The method according to item 1 or 2, wherein said cells are contacted with said at least one FGF signalling activator for at least about 1 day. (Item 4) 4. The method according to any one of items 1 to 3, wherein said cells are contacted with said at least one activator of FGF signalling for up to about 15 days. (Item 5) 5. The method according to any one of items 1 to 4, wherein said cells are contacted with said at least one activator of FGF signalling for about 5 days. (Item 6) said initial contact of said cell with said at least one activator of FGF signaling results in at least one subsequent contact of said cell with said at least one inhibitor of SMAD signaling. After about 5 days, the method according to any one of items 1 to 5 is performed. (Item 7) 7. The method of any one of items 1 to 6, wherein the initial contacting of the cells with the at least one activator of FGF signaling is about 10 days after the initial contacting of the cells with the at least one inhibitor of SMAD signaling. (Item 8) 8. The method of any one of items 1 to 7, wherein the initial contacting of the cells with the at least one activator of FGF signaling is 12 days after the initial contacting of the cells with the at least one inhibitor of SMAD signaling. (Item 9) 9. The method of any one of items 1 to 8, wherein the cells are contacted with the at least one SMAD signaling inhibitor for about 5 days. (Item 10) 10. The method of any one of items 1 to 9, wherein the cells are contacted with the at least one SMAD signaling inhibitor for 7 days. (Item 11) 11. The method according to any one of items 1 to 10, wherein the cells are contacted with the at least one SHH signalling activator for about 5 days. (Item 12) 12. The method according to any one of items 1 to 11, wherein the cells are contacted with the at least one SHH signalling activator for 7 days. (Item 13) 13. The method according to any one of items 1 to 12, wherein said cells are contacted with said at least one activator of Wnt signalling for about 10 days. (Item 14) 14. The method according to any one of items 1 to 13, wherein said cells are contacted with said at least one activator of Wnt signalling for 12 days. (Item 15) 15. The method according to any one of items 1 to 14, wherein the concentration of said at least one activator of Wnt signalling is increased for about 4 days from its initial contact with said stem cells. (Item 16) Item 16. The method according to item 15, wherein the concentration of the at least one activator of Wnt signaling is increased by about 300% to about 1000% from the initial concentration of the at least one activator of Wnt signaling. (Item 17) 17. The method according to any one of items 15 to 16, wherein the concentration of the at least one activator of Wnt signalling is increased to a concentration of about 3 μM to 10 μM. (Item 18) 18. The method according to any one of items 15 to 17, wherein the concentration of the at least one activator of Wnt signalling is increased to a concentration of about 3 μM. (Item 19) 18. The method according to any one of items 15 to 17, wherein the concentration of the at least one activator of Wnt signalling is increased to a concentration of about 7.5 μM. (Item 20) 20. The method of any one of items 1 to 19, wherein the at least one FGF signaling activator comprises FGF18. (Item 21) 21. The method of any one of items 1 to 20, wherein the at least one SMAD signaling inhibitor is selected from a TGFβ / Activin-Nodal signaling inhibitor, a bone morphogenetic protein (BMP) signaling inhibitor, and a combination thereof. (Item 22) 22. The method of claim 21, wherein the at least one TGFβ / Activin-Nodal signaling inhibitor comprises an inhibitor of ALK5. (Item 23) 23. The method of claim 21 or 22, wherein the at least one TGFβ / Activin-Nodal signaling inhibitor comprises SB431542, or a derivative thereof, or a mixture thereof. (Item 24) Item 24. The method according to item 23, wherein the SB431542 derivative is A83-01. (Item 25) 25. The method of any one of items 217 to 24, wherein the at least one TGFβ / Activin-Nodal signaling inhibitor comprises SB431542. (Item 26) 22. The method of item 21, wherein the at least one BMP signaling inhibitor comprises LDN193189, noggin, dorsomorphin, derivatives thereof, or mixtures thereof. (Item 27) 27. The method of item 21 or 26, wherein the at least one BMP inhibitor comprises LDN-193189. (Item 28) 28. The method of any one of items 1 to 27, wherein the at least one Wnt signaling activator comprises a glycogen synthase kinase 3β (GSK3β) signaling inhibitor. (Item 29) 29. The method of item 28, wherein the at least one activator of Wnt signaling is selected from CHIR99021, Wnt3A, Wnt1, derivatives thereof, and mixtures thereof. (Item 30) 30. The method according to any one of items 1 to 29, wherein the at least one SHH signaling activator is selected from the group consisting of SHH protein, Smoothened agonist (SAG), derivatives thereof and mixtures thereof. (Item 31) 31. The method of claim 30, wherein the SHH protein comprises recombinant SHH, purified SHH, or a combination thereof. (Item 32) 32. The method of claim 31, wherein the recombinant SHH comprises a recombinant protein that is at least about 80% identical to an N-terminal fragment of mouse sonic hedgehog. (Item 33) 33. The method of item 31 or 32, wherein the recombinant SHH comprises SHH C25II. (Item 34) 31. The method of claim 30, wherein the SAG comprises palmorfamine. (Item 35) 35. The method of any one of items 1 to 34, wherein the at least one marker indicative of midbrain dopamine neurons or precursors thereof is selected from EN1, OTX2, TH, NURR1, FOXA2, PITX3, LMX1A, LMO3, SNCA, ADCAP1, CHRNA4, GIRK2, and combinations thereof. (Item 36) 36. The method of any one of items 1 to 35, wherein the differentiated cells have a detectable level of expression of the at least one marker indicative of midbrain dopamine neurons or precursors thereof at least about 10 days after the initial contact of the stem cells with the at least one SMAD signaling inhibitor. (Item 37) 37. The method of any one of items 1 to 36, wherein the differentiated cells have a detectable level of expression of EN1 about 30 days after the initial contact of the stem cells with the at least one SMAD signaling inhibitor. (Item 38) 37. The method of any one of items 1 to 36, wherein the differentiated cells have a detectable level of expression of EN1 about 40 days after the initial contact of the stem cell with the at least one SMAD signaling inhibitor. (Item 39) 39. The method of any one of items 1 to 38, wherein the differentiated cells do not express at least one marker selected from PAX6, EMX2, LHX2, SMA, SIX1, PITX2, SIM1, POU4F1, PHOX2A, BARHL1, BARHL2, GBX2, HOXA2, HOXB2, POU5F1, NANOG, and combinations thereof. (Item 40) 40. The method of any one of items 1 to 39, further comprising subjecting the population of differentiated cells to conditions favorable for differentiation of midbrain dopamine neuron precursors into midbrain dopamine neurons. (Item 41) 41. The method of item 40, wherein the conditions include exposing the cells to at least one of brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), cyclic adenosine monophosphate (cAMP), transforming growth factor beta 3 (TGFP3), ascorbic acid (AA), and DAPT. (Item 42) 42. The method according to any one of items 1 to 41, wherein the stem cells are selected from human, non-human primate or rodent non-embryonic stem cells; human, non-human primate or rodent embryonic stem cells; human, non-human primate or rodent induced pluripotent stem cells; and human, non-human primate or rodent recombinant pluripotent cells. (Item 43) 43. The method according to any one of items 1 to 42, wherein the stem cells are human stem cells. (Item 44) 44. The method according to any one of items 1 to 43, wherein the stem cells are pluripotent or multipotent stem cells. (Item 45) 45. The method according to any one of items 1 to 44, wherein the stem cells are pluripotent stem cells. (Item 46) 46. ​​The method according to any one of items 1 to 45, wherein the pluripotent stem cells are selected from embryonic stem cells, induced pluripotent stem cells, and combinations thereof. (Item 47) A cell population of in vitro differentiated cells, wherein the in vitro differentiated cells are obtainable by the method according to any one of items 1 to 46. (Item 48) A cell population of in vitro differentiated cells, wherein at least about 50% of the cells express at least one marker indicative of midbrain dopamine neurons or their precursors, and less than about 50% of the differentiated cells express at least one marker selected from PAX6, EMX2, LHX2, SMA, SIX1, PITX2, SIM1, POU4F1, PHOX2A, BARHL1, BARHL2, GBX2, HOXA2, HOXB2, POU5F1, NANOG, and combinations thereof. (Item 49) The at least one marker indicative of midbrain dopamine neurons or their precursors is EN1, OTX2, TH, NURR1, FOXA2, LMX1A, PITX3, LMO3, S 49. The cell population of item 48, wherein the target gene is selected from NCA, ADCAP1, CHRNA4, GIRK2, and combinations thereof. (Item 50) 50. A composition comprising the cell population according to item 48 or 49. (Item 51) 51. The composition of item 50, which is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier. (Item 52) A method for isolating midbrain dopamine neurons and their precursors from a population of cells, comprising isolating cells that do not express detectable levels of at least one negative surface marker and that express detectable levels of at least one positive surface marker. (Item 53) A method for isolating midbrain dopamine neurons and their precursors from a population of cells, comprising isolating cells that (a) do not express or express a reduced level of at least one negative surface marker, relative to the average expression level of the at least one negative surface marker, in the population of cells, and (b) have an elevated level of at least one positive surface marker, relative to the average expression level of the at least one positive surface marker in the population of cells. (Item 54) 54. The method of item 52 or 53, wherein the at least one negative surface marker is selected from CD171, CD184, and combinations thereof. (Item 55) 55. The method of any one of items 52 to 54, wherein the at least one negative surface marker comprises CD184. (Item 56) 56. The method of any one of items 52 to 55, wherein the at least one negative surface marker is selected from CD49e, CD99, CD340, and combinations thereof. (Item 57) 57. The method of any one of items 52 to 56, wherein the at least one negative surface marker comprises CD49e. (Item 58) 58. The method of any one of items 52 to 57, comprising isolating cells that do not express detectable levels of CD49e and that express detectable levels of CD184. (Item 59) 59. The method of any one of Items 52 to 58, comprising isolating cells that express no detectable level of CD49e or express a reduced level of CD49e relative to the average expression level of CD49e in the population of cells, and that express an increased level of CD184 relative to the average expression level of CD184 in the population of cells. (Item 60) A cell population of in vitro differentiated cells, wherein at least about 50% of the cells express a detectable level of at least one positive surface marker and do not express a detectable level of at least one negative surface marker. (Item 61) A cell population of in vitro differentiated cells, wherein at least about 50% of the cells express an increased level of at least one positive surface marker compared to the average expression level of the at least one positive surface marker in the population of cells, and do not express a detectable level of at least one negative surface marker or express a reduced level of at least one negative surface marker compared to the average expression level of the at least one negative surface marker in the population of cells. (Item 62) 62. The cell population of item 60 or 61, wherein the at least one negative surface marker is selected from CD171, CD184, and combinations thereof. (Item 63) 63. The cell population of any one of items 60 to 62, wherein the at least one negative surface marker comprises CD184. (Item 64) 64. The cell population of any one of items 60 to 63, wherein the at least one negative surface marker is selected from CD49e, CD99, CD340, and combinations thereof. (Item 65) 65. The cell population of any one of items 60 to 64, wherein the at least one negative surface marker comprises CD49e. (Item 66) 66. The cell population of any one of items 60 to 65, wherein the at least one negative surface marker comprises CD184 and the at least one negative surface marker comprises CD49e. (Item 67) A composition comprising the cell population according to any one of items 60 to 66. (Item 68) 68. The composition according to item 67, which is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier. (Item 69) A kit for inducing differentiation of stem cells into midbrain dopamine neurons or their precursors, comprising: (a) at least one SMAD signaling inhibitor; (b) at least one SHH signaling activator; (c) at least one activator of Wnt signaling; (d) at least one FGF signaling activator. (Item 70) (f) the kit of item 69, further comprising instructions for inducing differentiation of the stem cells into a population of differentiated cells expressing at least one midbrain DA progenitor marker. (Item 71) 1. A method of preventing and / or treating a neurodegenerative disorder in a subject, comprising administering to a subject an effective amount of: (a) the cell population according to any one of items 47 to 49 and 60 to 66, or (b) the composition according to any one of items 50, 51, 67 and 68 The method comprises administering to the subject one of: (Item 72) 69. The method of claim 68, wherein the neurodegenerative disorder is Parkinson's disease, Huntington's disease, Alzheimer's disease, or multiple sclerosis. (Item 73) 69. The cell population of any one of items 47 to 49 and 60 to 66 or the composition of any one of items 50, 51, 67 and 68 for use in preventing and / or treating a neurodegenerative disorder in a subject. (Item 74) 74. The cell population or composition for use according to item 73, wherein the neurodegenerative disorder is Parkinson's disease, Huntington's disease, Alzheimer's disease, or multiple sclerosis.

Claims

1. An in vitro method for inducing differentiation of cells expressing forkhead box protein A2 (FOXA2) and LIM homeobox transcription factor 1 alpha (LMX1A), comprising: and contacting the cells with at least one fibroblast growth factor (FGF) signaling activator to obtain a population of differentiated cells expressing at least one marker indicative of midbrain dopamine neurons (mDA) or precursors thereof, wherein the at least one FGF signaling activator is selected from FGF18, FGF17, and combinations thereof.

2. The method of claim 1, wherein the cells are contacted with the at least one FGF signaling activator for at least 1 day.

3. The method described in claim 1, wherein the cells are contacted with the at least one FGF signaling activator for up to 15 days.

4. The method described in claim 1, wherein the cells are contacted with the at least one FGF signaling activator for 5 days.

5. The method described in claim 1, wherein at least one FGF signaling activator comprises FGF18.

6. The method described in claim 1, wherein the at least one marker indicative of midbrain dopamine neurons or their precursors is selected from EN1, OTX2, TH, NURR1, PITX3, LMO3, SNCA, ADCAP1, CHRNA4, GIRK2, and combinations thereof.

7. The method described in claim 1, wherein the differentiated cells do not express at least one marker selected from PAX6, EMX2, LHX2, SMA, SIX1, PITX2, SIM1, POU4F1, PHOX2A, BARHL1, BARHL2, GBX2, HOXA2, HOXB2, POU5F1, NANOG, and combinations thereof.

8. The method of claim 1, further comprising subjecting the population of differentiated cells to conditions that favor differentiation of midbrain dopamine neuron precursors into midbrain dopamine neurons.

9. The method of claim 8, wherein the conditions include exposing the cells to at least one of brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), cyclic adenosine monophosphate (cAMP), transforming growth factor beta 3 (TGFβ3), ascorbic acid (AA), and DAPT.

10. The method of claim 1, wherein the cells are selected from non-embryonic stem cells of a human, non-human primate, or rodent; embryonic stem cells of a human, non-human primate, or rodent; induced pluripotent stem cells of a human, non-human primate, or rodent; and recombinant pluripotent cells of a human, non-human primate, or rodent.

11. The method of claim 1, wherein the cells are human stem cells.

12. The method of claim 1, wherein the cells are pluripotent or multipotent stem cells.

13. The method described in claim 12, wherein the cells are pluripotent stem cells.

14. The method described in claim 13, wherein the pluripotent stem cells are selected from embryonic stem cells, induced pluripotent stem cells, and combinations thereof.

15. A cell population of in vitro differentiated cells, wherein said in vitro differentiated cells are obtainable by the method of any one of claims 1 to 14.

16. A composition comprising the cell population of claim 15.

17. The composition of claim 16, which is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

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