Methods for production and isolation of midbrain dopamine neurons

Abstract

The present disclosure provides methods of generating midbrain dopamine neurons (mDAs) and their precursors, mDAs and their precursors produced by the methods, compositions comprising these cells, and uses thereof for preventing and / or treating neurological diseases. The present disclosure also provides methods of isolating mDAs and their precursors from a population of cells using novel surface markers.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 893,674, filed August 29, 2019, the contents of which are incorporated by reference in their entirety and priority to which is claimed. TECHNICAL FIELD

[0003] The present invention provides methods of generating midbrain dopamine neurons (mDAs) and their precursors, mDAs and their precursors generated by the methods, compositions comprising such cells, and uses of mDAs and their compositions for preventing and / or treating neurological diseases. The present invention also provides methods of isolating mDAs and their precursors from a population of cells using novel surface markers. BACKGROUND

[0004] Previously, embryonic and somatic stem cells have been used as therapeutic and model systems for neurodegenerative diseases. In the field of central nervous system (CNS) diseases, such as Huntington’s disease, Alzheimer’s disease, Parkinson’s disease, and multiple sclerosis, research and technological advances have emerged related to the directed differentiation of embryonic and somatic stem cells. However, the results of these studies have shown little ability to restore neuronal function in vivo and often lead to unwanted tumor growth in patients.

[0005] Accordingly, there remains a need for improved methods of generating midbrain dopamine neurons suitable for treating neurodegenerative diseases, such as Parkinson’s disease. SUMMARY

[0006] The present invention provides methods of generating midbrain dopamine neurons (mDAs) and their precursors, mDAs and their precursors generated by the methods, compositions comprising such cells, and uses of mDAs and their compositions for preventing and / or treating neurological diseases. The present invention also provides methods of isolating mDAs and their precursors from a population of cells using novel surface markers.

[0007] In certain embodiments, the present invention provides a method of in vitro inducing stem cells to differentiate, comprising: contacting stem cells with at least one inhibitor of Small Mothers Against Decapentaplegic (SMAD) signaling, at least one activator of Sonic Hedgehog (SHH) signaling, and at least one activator of wingless (Wnt) signaling; and contacting the cells with at least one activator of Fibroblast Growth Factor (FGF) signaling to obtain a population of differentiated cells expressing at least one marker indicative of midbrain dopamine neurons (mDAs) or their precursors, wherein the at least one FGF signaling activator is selected from the group consisting of FGF18, FGF17, FGF8a, and combinations thereof.

[0008] In certain embodiments, the application provides a method of differentiating stem cells in vitro, comprising: contacting stem cells with at least one inhibitor of Small Mothers Against Decapentaplegic (SMAD) signaling, at least one activator of Sonic Hedgehog (SHH) signaling, and at least one activator of wingless (Wnt) signaling; and contacting the cells with at least one activator of Fibroblast Growth Factor (FGF) signaling to obtain a population of differentiated cells expressing at least one marker indicative of midbrain dopamine neurons (mDAs) or precursors thereof, wherein the initial contacting of the cells with the at least one activator of FGF signaling is at least about 5 days from the initial contacting of the cells with the at least one inhibitor of SMAD signaling.

[0009] 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 certain embodiments, the cells are contacted with the at least one activator of FGF signaling for about 5 days.

[0010] In certain embodiments, the initial contacting of the cells with the at least one activator of FGF signaling is at least about 5 days from the initial contacting of the cells with the at least one inhibitor of SMAD signaling.

[0011] In certain embodiments, the initial contacting of the cells with the at least one activator of FGF signaling is about 10 days from the initial contacting of the cells with the at least one inhibitor of SMAD signaling. In certain embodiments, the initial contacting of the cells with the at least one activator of FGF signaling is 12 days from the initial contacting of the cells with the at least one inhibitor of SMAD signaling.

[0012] In certain embodiments, the cells are contacted with the at least one inhibitor of SMAD signaling for about 5 days. In certain embodiments, the cells are contacted with the at least one inhibitor of SMAD signaling for 7 days.

[0013] In certain embodiments, the cells are contacted with the at least one activator of SHH signaling for about 5 days. In certain embodiments, the cells are contacted with the at least one activator of SHH signaling for 7 days.

[0014] 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.

[0015] In certain embodiments, the concentration of the at least one activator of Wnt signaling is increased about 4 days from its initial contact with the stem cells. In certain embodiments, the concentration of the at least one activator of Wnt signaling is increased by about 300% to about 1000% of the initial concentration of the at least one activator of Wnt signaling. In certain embodiments, the concentration of the at least one activator of Wnt signaling is increased to a concentration of about 3 μΜ to 10 μΜ. In certain embodiments, the concentration of the at least one activator of Wnt signaling is increased to a concentration of about 3 μΜ. In certain embodiments, the concentration of the at least one activator of Wnt signaling is increased to a concentration of about 7.5 μΜ.

[0016] In certain embodiments, the at least one activator of FGF signaling comprises FGF18.

[0017] In certain embodiments, the at least one inhibitor of SMAD signaling is selected from the group consisting of an inhibitor of TGFβ / Activin-Nodal signaling, an inhibitor of bone morphogenetic protein (BMP) signaling, and combinations thereof.

[0018] In certain embodiments, the at least one inhibitor of TGFβ / Activin-Nodal signaling comprises an inhibitor of ALK5.

[0019] In certain embodiments, the at least one inhibitor of TGFβ / Activin-Nodal signaling comprises SB431542, or a derivative thereof, or a mixture thereof. In certain embodiments, the derivative of SB431542 is A83-01. In certain embodiments, the at least one inhibitor of TGFβ / Activin-Nodal signaling comprises SB431542.

[0020] In certain embodiments, the at least one inhibitor of BMP signaling comprises LDN193189, Noggin, dorsomorphin, a derivative thereof, or a mixture thereof. In certain embodiments, the at least one inhibitor of BMP comprises LDN-193189.

[0021] In certain embodiments, the at least one activator of Wnt signaling comprises an inhibitor of glycogen synthase kinase 3 beta (GSK3β) signaling.

[0022] In certain embodiments, the at least one activator of Wnt signaling is selected from the group consisting of CHIR99021, BIO, CHIR98014, lithium, 3F8, Wnt3A, Wntl, Wnt5a, derivatives thereof, and mixtures thereof. In certain embodiments, the at least one activator of Wnt signaling comprises CHIR99021.

[0023] In certain embodiments, the at least one activator of SHH signaling is selected from the group consisting of SHH protein, SMO agonist (SAG), derivatives thereof, and mixtures thereof. In certain embodiments, the SHH protein comprises recombinant SHH, purified SHH, or a combination of the foregoing.

[0024] In certain embodiments, the recombinant SHH comprises a recombinant protein having at least about 80% identity to a mouse Sonic Hedgehog N-terminal fragment. In certain embodiments, the recombinant SHH comprises SHH C25 II. In certain embodiments, the SAG comprises purmorphamine.

[0025] In certain embodiments, the at least one marker indicative of a midbrain dopamine neuron or a precursor thereof is selected from the group consisting of EN1, OTX2, TH, NURR1, FOXA2, PITX3, LMX1A, LMO3, SNCA, ADCAPl, CHRNA4, GIRK2, and combinations thereof.

[0026] In certain embodiments, the differentiated cell has a detectable level of expression of at least one marker indicative of a midbrain dopamine neuron or a precursor thereof about 10 days from the initial contact of the stem cell with the at least one inhibitor of SMAD signaling.

[0027] In certain embodiments, the differentiated cell has a detectable level of EN1 expression about 30 days from the initial contact of the stem cell with the at least one inhibitor of SMAD signaling. In certain embodiments, the differentiated cell has a detectable level of EN1 expression about 40 days from the initial contact of the stem cell with the at least one inhibitor of SMAD signaling.

[0028] In certain embodiments, the differentiated cell does not express at least one marker selected from the group consisting of PAX6, EMX2, LHX2, SMA, SIX1, PITX2, SIM1, POU4F1, PHOX2A, BARHL, BARHL2, GBX2, HOXA2, HOXB2, POU5F1, NANOG, and combinations thereof.

[0029] In certain embodiments, the method further comprises subjecting the population of differentiated cells to conditions that favor differentiation of the midbrain dopaminergic neuronal precursors into midbrain dopaminergic neurons.

[0030] In certain embodiments, the conditions comprise exposing the cells to at least one of: brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), cyclic adenosine monophosphate (cAMP), transforming growth factor beta 3 (TGFβ3), ascorbic acid (AA), and DAPT.

[0031] In certain embodiments, the stem cell is selected from the group consisting of a human, non-human primate, or rodent non-embryonic stem cell; a human, non-human primate, or rodent embryonic stem cell; a human, non-human primate, or rodent induced pluripotent stem cell; and a human, non-human primate, or rodent reprogrammed pluripotent stem cell. In certain embodiments, the stem cell is a human stem cell. In certain embodiments, the stem cell is a pluripotent or multipotent stem cell. In certain embodiments, the stem cell is a pluripotent stem cell. In certain embodiments, the pluripotent stem cell is selected from the group consisting of an embryonic stem cell, an induced pluripotent stem cell, and combinations thereof.

[0032] In another aspect, the application provides a population of cells comprising in vitro differentiated cells, wherein the in vitro differentiated cells are obtained by any of the foregoing methods.

[0033] In another aspect, the application provides a population of cells comprising in vitro differentiated cells, wherein at least about 50% of the cells express at least one marker indicative of a midbrain dopaminergic neuron or precursor thereof, and less than about 50% of the differentiated cells express at least one marker selected from the group consisting of 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 a midbrain dopaminergic neuron or precursor thereof is selected from the group consisting of EN1, OTX2, TH, NURR1, FOXA2, LMX1A, PITX3, LMO3, SNCA, ADCAPl, CHRNA4, GIRK2.

[0034] In another aspect, the application provides a composition comprising a population of cells as disclosed herein. In certain embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

[0035] In another aspect, the application provides a method of isolating midbrain dopaminergic neurons and precursors thereof from a population of cells, comprising isolating cells that do not express detectable levels of at least one negative surface marker and express detectable levels of at least one positive surface marker.

[0036] In another aspect, the present invention provides a method for isolating midbrain dopamine neurons and precursors thereof from a cell population, comprising isolating cells that: (a) do not express a detectable level of at least one negative surface marker or express at least one negative surface marker at a reduced level compared to the average expression level of at least one negative surface marker in the cell population; and (b) express at least one positive surface marker at an increased level compared to the average expression level of at least one positive marker in the cell population.

[0037] 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 but express detectable levels of CD184.

[0038] In certain embodiments, the method comprises 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 a cell population; and express increased levels of CD184 compared to the average expression level of CD184 in a cell population.

[0039] In another aspect, the present invention provides a cell population of in vitro differentiated cells, wherein at least about 50% of the cells express detectable levels of at least one positive surface marker and do not express detectable levels of at least one negative surface marker.

[0040] In another aspect, the present invention provides a cell population of in vitro differentiated cells, wherein at least about 50% of the cells express at least one positive surface marker at an increased level compared to the average expression level of at least one positive marker in the cell population; and do not express a detectable level of at least one negative surface marker or express at least one negative surface marker at a decreased level compared to the average expression level of at least one negative surface marker in the cell population.

[0041] In certain embodiments, the at least one positive surface marker is selected from the group consisting of 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 the group consisting of CD49e, CD99, CD340, and combinations thereof. In certain embodiments, the at least one negative surface marker comprises CD49e. In certain embodiments, the at least one positive surface marker comprises CD184 and the at least one negative surface marker comprises CD49e.

[0042] In another aspect, the present application provides a composition comprising a 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 application provides a kit for inducing differentiation of stem cells into midbrain dopamine neurons or precursors thereof, comprising: (a) at least one inhibitor of SMAD signaling; (b) at least one activator of SHH signaling; (c) at least one activator of Wnt signaling; and (d) at least one activator of FGF signaling.

[0043] In certain embodiments, the kit further comprises (f) instructions for inducing differentiation of stem cells into a population of differentiated cells expressing at least one midbrain DA precursor marker.

[0044] In another aspect, the present application provides a method of preventing and / or treating a neurodegenerative disease in a subject, comprising administering to the subject an effective amount of one of: (a) a cell population disclosed herein; or (b) a composition disclosed herein.

[0045] In certain embodiments, the neurodegenerative disease is Parkinson's disease, Huntington's disease, Alzheimer's disease, or multiple sclerosis.

[0046] In certain embodiments, a cell population disclosed herein or a composition disclosed herein is used to prevent and / or treat a neurodegenerative disease in a subject. In certain embodiments, the neurodegenerative disease is Parkinson's disease, Huntington's disease, Alzheimer's disease, or multiple sclerosis. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 EN1 expression in stem cell-derived mDAs and precursors using the Wnt-Boost protocol from day 3 to day 30 is shown.

[0048] Figure 2 Protocol depicting the use of FGF8b in combination with the Wnt-Boost protocol.

[0049] Figure 3EN1 protein is shown to be maintained in differentiated cells in an FGF8b contact duration-dependent manner. EN1 -expressing cells also express FOXA2 and LMX1A.

[0050] Figures 4A-4B mRNA expression levels of differentiated cells measured by qRT-PCR at day 30 are shown. Figure 4A mRNA expression levels of EN1 are shown to be maintained in an FGF8b contact duration-dependent manner, and expression levels of FOXA2, NURR1, and TH are comparable across all conditions. Figure 4B mRNA expression levels of non-mDA markers (e.g., SMA and SIX1) induced in an FGF8b contact duration-dependent manner are shown.

[0051] Figure 5 SIX1 immunostaining of FGF8b-treated cells at day 30 of differentiation is shown.

[0052] Figure 6 mRNA expression of markers in FGFs-treated cells at day 16 of differentiation is shown.

[0053] Figure 7 Immunostaining of markers in FGF8b and FGF18-treated cells at day 16 of differentiation is shown.

[0054] Figure 8 RNA expression of markers in FGF8b and FGF18-treated cells at day 27 and day 40 of differentiation is shown.

[0055] Figure 9A is a schematic showing the donor vector structure of NURR1 ::GFP reporter hPSCs.

[0056] Figure 9B NURR1 mRNA levels in cells differentiated from hPSCs using the Wnt-Boost protocol are shown.

[0057] Figure 9C FACS results of midbrain DA neurons differentiated from H9-hPSCs and NURR1 ::GFP hPSCs at day 25 of differentiation are shown.

[0058] Figure 10A Single cell qRT PCR results in NURR1 :GFP positive cells isolated at day 25 and day 40 of differentiation are shown.

[0059] Figure 10B Immunostaining of NURR1 sorted mDAs expressing TH, FOXA2, and NURR1 at day 60 of differentiation is shown. Cells were sorted at day 25 and then cultured continuously to day 60.

[0060] Figure 11 Immunostaining images of NURR1 ::GFP positive midbrain DA neurons transplanted in vivo 8 weeks after injection of cells into immunodeficient mice are provided.

[0061] Figure 12 Immunostaining images of NURR1 :GFP positive cells cultured according to the WNT-Boost protocol and the WNT-Boost + FGF18 (day 12 - day 16) protocol are provided. Cells were sorted on day 25 and then cultured continuously until day 40.

[0062] Figure 13A Expression of FOXA2, EN1 and NURR1 mRNA in NURR1 :GFP positive cells cultured according to the WNT-Boost protocol and the WNT-Boost + FGF18 (day 12 - day 16) protocol is provided. mRNA expression is compared for sorted and unsorted cells.

[0063] Figure 13B Sorted NURR1 :GFP positive cells cultured according to the WNT-Boost protocol and the WNT-Boost + FGF18 (day 12 - day 16) protocol are shown transplanted into immunodeficient mice. Neurite outgrowth and TH and SC121 expression are detected in ground cells.

[0064] Figure 14 390 surface markers are shown to be screened in mDAs at day 25 of differentiation from NURR1 ::GFP hPSCs using the Wnt-Boost protocol. Antibodies are conjugated to PE, FITC or APC.

[0065] Figure 15A Positive surface markers CD171 and CD184 are shown to be enriched in NURR1 + cells.

[0066] Figure 15B RNA expression of CD171 and CD184 in differentiated cells using the Wnt-Boost protocol is provided.

[0067] Figure 16A Negative surface markers CD49e, CD99 and CD340 are shown to be enriched in NURR1 + cells.

[0068] Figure 16B RNA expression of CD49e, CD99 and CD340 in differentiated cells using the Wnt-Boost protocol is provided.

[0069] Figure 17Cell morphology by CD49e weak or CD49e high sorting is provided. Cells were sorted at day 25 of in vitro differentiation under WNT-boost and WNT-boost + FGF18 protocols. After sorting, cells were cultured for an additional 15 days.

[0070] Figure 18 Immunostaining images of CD49e weak cells sorted at day 40 of in vitro differentiation under WNT-Boost protocol and WNT-Boost + FGF18 protocol are shown.

[0071] Figure 19 FACS sorting results based on CD49e purification of mDAs derived from hPSC cell line MEL1 are shown.

[0072] Figure 20 Morphology of MEL1-hPSC derived mDA cells sorted by CD49e weak or CD49e high is shown. Cells were sorted at day 25 of in vitro differentiation under WNT-Boost protocol and WNT-Boost + FGF18 protocol. After sorting, cells were cultured for an additional 15 days.

[0073] Figure 21 Immunostaining images of MEL1-hPSC derived CD49e weak mDAs at day 40 of in vitro differentiation under WNT-Boost protocol and WNT-Boost + FGF18 protocol are shown. CD49e weak cells were sorted at day 25.

[0074] Figure 22 Relative mRNA expression in MEL1-hPSC derived CD49e weak mDAs at day 40 of in vitro differentiation under WNT-boost protocol and WNT-boost + FGF18 protocol are shown. CD49e weak cells were sorted at day 25.

[0075] Figure 23 Morphology of CD49e, CD99 or CD340 sorted cells at day 40 of in vitro differentiation under WNT Boost + FGF18 protocol is shown. Cells were sorted at day 25 of in vitro differentiation.

[0076] Figure 24 Relative mRNA expression of CD49e, CD99 or CD340 sorted cells at day 40 of in vitro differentiation under WNT Boost + FGF18 protocol is shown. Cells were sorted at day 25 of in vitro differentiation.

[0077] Figure 25 FACS sorting results of NURR1+ cells sorted by 49E (PE) and 171 (APC) at day 25 of in vitro differentiation under WNT-Boost protocol are shown.

[0078] Figure 26 FACS sorting results of NURR1+ cells sorted on CD49e (PE) and CD184 (APC) at day 25 of in vitro differentiation under WNT-Boost protocol.

[0079] Figure 27 Cell morphology of cells sorted on CD49e, CD171, CD188. Cells were sorted at day 25 of in vitro differentiation under WNT-Boost protocol. Cells were cultured for an additional 2 days after sorting.

[0080] Figure 28 mRNA expression of cells sorted on CD49e, CD171, CD188. Cells were sorted at day 25 of in vitro differentiation under WNT-Boost protocol. Cells were cultured for an additional 2 days after sorting.

[0081] Figure 29 Enrichment of NURR1::GFP population in single CD49e weakly sorted cells at day 25 of in vitro differentiation.

[0082] Figure 30 Enrichment of NURR1::GFP population in CD49e weak CD184 high double sorted cells at day 25 of in vitro differentiation.

[0083] Figure 31 TH + Midbrain DA neurons co-express FOXA2 and GFP, suggesting midbrain DA neuron identity in the NURR1::GFP population enriched in the Figure 30 Midbrain DA neuron identity in the NURR1::GFP population enriched in the

[0084] Figure 32 Expression of midbrain DA marker mRNA in cells sorted on CD49e and CD184 at day 25 of in vitro differentiation. Cells were cultured for an additional 15 days in vitro after sorting.

[0085] Figure 33 Expression of non-midbrain DA mRNA in cells sorted on CD49e and CD184 at day 25 of in vitro differentiation. Cells were cultured for an additional 10 days in vitro after sorting.

[0086] Figures 34A-3 4D shows in vivo survival of transplanted cells sorted with CD markers of the disclosure (CD49e depleted and CD184 enriched) after in vitro differentiation under WNT-Boost protocol. Figure 34A Robust survival and enrichment of TH + cells in grafts of sorted cells compared to unsorted cells. Figure 34B SOX2 one month after transplantation+ Precursors and KI67 + The number of dividing cells was reduced. Figure 34C shows Figure 34B SOX2 in the middle + Quantification of stained cells. Figure 34D shows Figure 34B Ki67 in the middle + Quantification of cells.

[0087] Figures 35A-35B In vivo survival and EN1 expression of cells differentiated under WNT-boost and WNT-boost + FGF18 protocols are shown. Figure 35A Percentage of cells expressing EN1 is shown. Figure 35B Appearance of striatal innervation fibers at the graft site is shown. DETAILED DESCRIPTION

[0088] The present invention provides methods for generating mDAs and precursors thereof, mDAs and precursors thereof generated by such methods, compositions comprising such cells, and uses thereof for preventing and / or treating neurological diseases. Furthermore, the present invention provides methods for isolating mDAs and precursors thereof from a population of cells using novel surface markers.

[0089] The present invention is based at least on the finding that stem cell-derived mDAs produced by the methods of the present invention have sustained expression of EN1, e.g., expression of EN1 is maintained throughout development and maturation of the mDAs.

[0090] The present specification and examples describe non-limiting embodiments of the disclosed subject matter.

[0091] The detailed description is divided into the following subsections for the purpose of clarity without limitation:

[0092] 5.1. Definitions;

[0093] 5.2. Differentiation methods of stem cells;

[0094] 5.3. Methods of isolating midbrain DA neurons and precursors thereof;

[0095] 5.4. Compositions comprising midbrain DA neurons and precursors thereof;

[0096] 5.5. Methods of treating neurodegenerative diseases; and

[0097] 5.6. Kits

[0098] 5.1. Definitions

[0099] The terms used in this specification generally have their ordinary meanings in the art, in the context of the disclosure, and in the specific context of each term's usage in the specification. Some terms are discussed below, providing additional guidance on their meanings and use to those of skill in the art preparing or using compositions and methods relating to the present disclosure and how to make and use them.

[0100] The term“about” or“approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, limitations of the measurement system, etc. For example, “about” can mean within 3 or more standard deviations, per practices in the art. Or,“about” can mean ranges approximately 20% above and below a given value, e.g., approximately 10%, approximately 5%, or approximately 1% above and below the given value. Or, especially with regard to biological systems or processes, the term can mean within an order of magnitude, such as within 5-fold or 2-fold of a value.

[0101] As used herein, the term“signaling” when associated with“signaling proteins” refers to proteins that are activated or otherwise affected by ligands that bind to membrane receptor proteins or some other stimulatory factor. Examples of signaling proteins include, but are not limited to, SMADs, wingless (Wnt) complex proteins (including beta-catenin), NOTCH, transforming growth factor beta (TGF ), Activin, Nodal, glycogen synthase kinase 3 beta (GSK3 ) proteins, 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 linked to a cellular response. The ligand-activated receptor must first interact with other proteins inside the cell before the ligand produces an ultimate physiological effect on the behavior of the cell. Often, the behavior of a chain of several interacting cellular proteins is changed after receptor activation or inhibition. The entire cellular changes that result from receptor activation are called signaling mechanisms or signaling pathways.

[0102] As used herein, the term“signal” refers to internal and external factors that control changes in cellular structure and function. Their nature can be chemical or physical.

[0103] 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.

[0104] An "inhibitor" as used herein refers to a compound or molecule (e.g., a small molecule, a peptide, a peptidomimetic, a natural compound, an siRNA, an antisense nucleic acid, an aptamer, or an antibody) that interferes with (e.g., decreases, reduces, inhibits, eliminates, or blocks) the signaling function of a molecule or pathway. For example, an inhibitor can be any compound or molecule (e.g., including but not limited to a signaling molecule described herein) that alters any activity of a specified protein (e.g., a signaling molecule, any molecule associated with a specified signaling molecule, a specified associated molecule, e.g., glycogen synthase kinase 3 beta (GSK3β)), e.g., by directly contacting a SMAD signaling molecule, contacting a SMAD mRNA, causing a conformational change in a SMAD protein, reducing a SMAD protein level, or interfering with a SMAD interaction with a signaling partner (e.g., including those described herein), and affecting expression of a SMAD target gene (e.g., those described herein).

[0105] Inhibitors also include molecules that indirectly modulate a biological activity (e.g., a SMAD biological activity) by intercepting an upstream signaling molecule (e.g., within an extracellular domain, examples of signaling molecules and effectors include: Noggin, which sequesters bone morphogenetic proteins, inhibits activation of ALK receptors 1, 2, 3, and 6, thereby preventing downstream SMAD activation. Similarly, Chordin, Cerberus, Follistatin also similarly sequester extracellular activators of SMAD signaling. Bambi is a transmembrane protein that can also act as a pseudoreceptor to sequester extracellular TGFβ signaling molecules). Antibodies that block upstream or downstream proteins can be considered to inactivate extracellular activators of protein signaling, etc. Inhibitors are described as competitive (binding to the active site in a manner that excludes or reduces binding of another known binding compound) and allosteric (binding to the protein in a manner that interferes with binding of a compound to the active site of the protein, thereby altering the protein conformation) as well as inhibition caused by binding and affecting molecules upstream of a specified signaling molecule, thereby resulting in inhibition of the specified molecule. Inhibitors can be "direct inhibitors" that inhibit a signaling target or a signaling targeted pathway by actually contacting the signaling target.

[0106] An "activator" as used herein refers to a compound that increases, induces, stimulates, activates, promotes, or enhances the signaling function of a molecule or pathway (e.g., Wnt signaling, SHH signaling, etc.).

[0107] As used herein, the term "WNT" or "wingless" when referring to a ligand refers to a group of secreted proteins (i.e., integration 1 in humans) that are capable of interacting with WNT receptors, such as the Frizzled and LRP Derailed / RYK receptor family. As used herein, the term "WNT or wingless signaling pathway" refers to a signaling pathway composed of WnT family ligands and WnT family receptors (e.g., Frizzled and LRP Derailed / RYK receptors) with or without mediation by beta-catenin. In certain embodiments, the WNT signaling pathway includes mediation by beta-catenin (e.g., WNT / beta-catenin).

[0108] As used herein, the term "derivative" refers to a compound having a similar core structure.

[0109] 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 include at least about 10, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000 cells. The population can be a pure population comprising one cell type, such as a population of midbrain DA precursors or a population of undifferentiated stem cells. Alternatively, the population can include more than one cell type, such as a mixed cell population.

[0110] As used herein, the term "stem cell" refers to a cell that has the ability to divide indefinitely in culture and to give rise to specialized cells.

[0111] As used herein, the terms "embryonic stem cell" and "ESC" refer to a primitive (undifferentiated) cell derived from a pre-implantation stage embryo that is capable of dividing for long periods in culture without differentiating and is known to develop into cells and tissues of the three primary germ layers. Human embryonic stem cells refer to embryonic stem cells from a human embryo. As used herein, the term "human embryonic stem cell" or "hESC" refers to a pluripotent stem cell derived from an early human embryo up to and including the blastocyst stage that is capable of dividing for long periods in culture without differentiating and developing into cells and tissues of the three primary germ layers.

[0112] As used herein, the term "embryonic stem cell line" refers to a population of embryonic stem cells cultured under in vitro conditions that allow for proliferation without differentiation for days, months to years.

[0113] 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.

[0114] As used herein, the term "multipotent" refers to the ability to develop into more than one body cell type.

[0115] As used herein, the term "multipotent" refers to the ability to develop into more than one body cell type.

[0116] As used herein, the term "induced pluripotent stem cell" or "iPSC" refers to a type of pluripotent stem cell formed by introducing certain embryonic genes (such as, but not limited to, OCT4, SOX2, and KLF4 transgenes) into somatic cells (see, e.g., Takahashi and Yamanaka Cell 126, 663-676 (2006), incorporated herein by reference).

[0117] As used herein, the term "somatic cell" refers to any cell in the body other than a gamete (egg or sperm); sometimes referred to as an "adult" cell.

[0118] As used herein, the term "somatic (adult) stem cell" refers to a relatively rare undifferentiated cell found in many organs and differentiated tissues with limited self-renewal (in the laboratory) and differentiation capacity.

[0119] As used herein, the term "neuron" refers to a nerve cell, the main functional unit of the nervous system. A neuron consists of a cell body and its processes, one axon and at least one dendrite. Neurons transmit information to other neurons or cells by releasing neurotransmitters at synapses.

[0120] As used herein, the term "proliferation" refers to an increase in the number of cells.

[0121] As used herein, the term "undifferentiated" refers to a cell that has not yet developed into a specialized cell type.

[0122] As used herein, the term "differentiation" refers to the process by which an unspecialized embryonic cell acquires the characteristics of a specialized cell, such as a neuron, heart, liver, or muscle cell. Differentiation is controlled by the interaction between the cell's genes and physical and chemical conditions outside the cell, usually through signaling pathways involving proteins embedded in the cell surface.

[0123] As used herein, the term "directed differentiation" refers to the manipulation of stem cell culture conditions to induce differentiation into a specific (e.g., desired) cell type, such as neural, neural crest, cranial plate, and non-neural ectoderm precursors. In 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 more specialized cell fate.

[0124] As used herein, the term "induced differentiation" with respect to a cell refers to a change in the default cell type (genotype and / or phenotype) to a non-default cell type (genotype and / or phenotype). Thus, "inducing differentiation of a stem cell" refers to inducing a stem cell (e.g., a human stem cell) to divide into a progeny cell having characteristics different from the stem cell, e.g., genotype (e.g., changes in gene expression as determined by gene analysis (e.g., microarray)) and / or phenotype (e.g., changes in expression of protein markers of mDAs or their precursors, e.g., EN1, OTX2, TH, NURR1, FOXA2, LMX1A, PITX3, LMO3, SNCA, ADCAPl, CHRNA4, and GIRK2).

[0125] As used herein, the term "cell culture" refers to the in vitro growth of cells in an artificial medium for research or medical purposes.

[0126] As used herein, the term "culture medium" refers to the liquid that covers the cells in a culture vessel, e.g., a petri dish, multi-well plate, etc., and contains nutrients to nourish and support the cells. Growth factors can also be added to the culture medium to produce a desired change in the cells.

[0127] As used herein, the term "contacting" a compound (e.g., at least one inhibitor, activator, and / or inducer) with one or more cells refers to providing the compound in a location that allows the one or more cells to contact the compound. Contacting can be accomplished using any suitable method. For example, contacting can be achieved by adding the compound in concentrated form to the cells or cell population (e.g., in the context of a cell culture) to achieve the desired concentration. Contacting can also be achieved by including the compound as an ingredient of a formulation medium.

[0128] As used herein, the term "in vitro" refers to an artificial environment and processes or reactions that occur in an artificial environment. In vitro environments include, but are not limited to, test tubes and cell cultures.

[0129] As used herein, the term "in vivo" refers to a natural environment (e.g., an animal or a cell) and processes or reactions that occur in a natural environment, such as embryonic development, cell differentiation, neural tube formation, etc., as used herein.

[0130] As used herein, the term "expression" with respect to a gene or protein refers to the production of mRNA or protein that can be observed using an assay such as microarray analysis, antibody staining analysis, etc.

[0131] As used herein, the term "marker" or "cell marker" refers to a gene or protein that identifies a particular cell or cell type. A marker for a cell can not be limited to one marker, and a marker can refer to a "pattern" of markers such that a specified set of markers can distinguish one cell or cell type from another cell or cell type.

[0132] As used herein, the term "derived from" or "established from" or "differentiated from" when referring to any cell disclosed herein refers to a cell obtained from a final parent cell in a cell line (e.g., isolated, purified, etc.), a tissue (e.g., an isolated embryo, or a liquid using any manipulation, for example, but not limited to, single cell isolation, in vitro culture, treatment and / or mutagenesis (using, for example, morphogens, chemicals, radiation, viral infection, DNA sequence transfection, for example, using morphogens, etc.), selection for response to growth factors, cytokine treatment, selection progress, adhesion, lack of adhesion, sorting process, etc., of any cell contained in the cultured parent cell. The derived cell can be selected from a mixed population by response to growth factors, cytokine treatment, selection progress, adhesion, lack of adhesion, sorting process, etc.

[0133] An "individual" or "subject" herein is a vertebrate, such as a human or non-human animal, such as 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.

[0134] As used herein, the term "disease" refers to any symptom or condition that impairs or interferes with the normal functioning of a cell, tissue, or organ.

[0135] As used herein, the term "treatment" or "treat" refers to clinical intervention with the intent to alter the disease course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Therapeutic effects of treatment include, but are not limited to, preventing occurrence or reoccurrence of disease, alleviation of symptoms, reduction of any direct or indirect pathological consequences of disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and alleviation or improvement of prognosis. Treatment prevents worsening due to the condition or diagnosed subject or a subject suspected of having the condition by preventing progression of the disease or condition, and treatment also prevents onset of the condition or symptoms of the condition in a subject at risk for the condition or suspected of having the condition.

[0136] As used herein, the term "negative," "weak," or "-" when used in reference to any surface marker disclosed herein, means that the surface marker (e.g., CD49e) is not expressed at a detectable level, or is expressed at a reduced level in the cell as compared to the average expression of the surface marker in the population of cells from which the cell was selected or sorted. As used herein, the term "high," "strong," "+" "positive" when used in reference to any surface marker disclosed herein, means that the surface marker (e.g., CD184) is expressed at a detectable level or at an increased level as compared to the average expression of the surface marker in the population of cells.

[0137] In certain embodiments, cells are distinguished based on readily discernible differences in staining intensity according to the level of cell surface marker expression, as known to those of ordinary skill in the art. In certain embodiments, a cutoff can be set for designating a cell as a surface marker "weak," "negative," or "-" cell according to the distribution of staining intensity (e.g., fluorescence intensity) observed for all cells, wherein cells below about 50%, about 40%, about 30%, about 20%, about 10%, or about 5% of the staining intensity are designated as surface marker "weak," "negative," or "-" cells. In certain embodiments, a cutoff can be set for designating a cell as a surface marker "strong," "high," "+" cell according to the distribution of staining intensity (e.g., fluorescence intensity) observed for all cells, wherein cells above about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% of the staining intensity are designated as surface marker "strong," "high," "+" or "positive" cells. In certain embodiments, a frequency distribution of surface marker staining for all cells is obtained, and population curves are fitted to the higher staining and lower staining populations, and cells are assigned to the population to which they are most statistically likely to belong according to statistical analysis of the respective population distributions.

[0138] 5.2. Methods of Differentiating Stem Cells

[0139] The present application provides a method of inducing differentiation of stem cells, comprising contacting the stem cells with at least one inhibitor of Small Mothers Against Decapentaplegic (SMAD) signaling (referred to as a "SMAD inhibitor"), at least one activator of Sonic Hedgehog (SHH) signaling (referred to as a "SHH activator"), and at least one activator of wingless (Wnt) signaling (referred to as a "Wnt activator"); and further contacting the cells with at least one activator of Fibroblast Growth Factor (FGF) signaling (referred to as a "FGF activator"), to obtain a population of cells comprising differentiated cells expressing at least one marker indicative of mDAs or mDA precursors.

[0140] In certain embodiments, the at least one FGF activator is capable of promoting midbrain development. In certain embodiments, the at least one FGF activator is selected from the group consisting of FGF8a, FGF17, FGF18, FGF2, and FGF4. In certain embodiments, the at least one FGF activator is selected from the group consisting of FGF8a, FGF17, and FGF18. In certain embodiments, the at least one FGF activator comprises FGF18.

[0141] In certain embodiments, the initial contact of the cells with the at least one activator of FGF signaling is at least about 5 days from the initial contact of the cells with the at least one inhibitor of SMAD signaling. In certain embodiments, the initial exposure of the cells to the at least one FGF activator is at least about 10 days from the initial exposure of the stem cells to the at least one SMAD inhibitor. In certain embodiments, the FGF activator is selected from the group consisting of FGF8a, FGF17, FGF18, FGF8b, FGF2, and FGF4. Exposure of the cells to the at least one FGF activator can extend EN1 expression of the differentiated cells.

[0142] In certain embodiments, the at least one marker indicative of mDAs or mDA precursors is selected from the group consisting of EN1, FOX1A, LMX1A, OTX2, NURR1, TH, PITX3, LMO3, SNCA, ADCAPl, CHRNA4, and GIRK2.

[0143] In certain embodiments, the concentration of the at least one Wnt activator is increased during its exposure to the cells. In certain embodiments, the concentration of the at least one Wnt activator is increased beginning about 4 days from 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 having the increased concentration for at least about 7 days. In certain embodiments, at least one additional Wnt activator is added to increase the total concentration of Wnt activators.

[0144] In certain embodiments, the method further comprises contacting the cells with midbrain DA lineage specific activators and inhibitors (e.g., BDNF, GDNF, cAMP, TGF, ascorbic acid (AA), and / or DAPT) to induce differentiation of mDA precursors to mDAs.

[0145] 5.2.1. Stem Cells

[0146] The presently disclosed subject matter provides methods of in vitro inducing stem cell differentiation to produce mDAs and precursors thereof. In certain embodiments, the stem cell is a pluripotent stem cell. In certain embodiments, the pluripotent stem cell is selected from the group consisting of an embryonic stem cell (ESC), an induced pluripotent stem cell (IPSC), and combinations thereof. In certain embodiments, the stem cell is a multipotent stem cell. Non-limiting examples of stem cells that can be used with the presently disclosed methods 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 cell is a human stem cell. 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, ectodermal stem cells, F-class pluripotent stem cells, somatic stem cells, cancer stem cells, or any other cell capable of lineage-specific differentiation. In certain embodiments, the stem cell is a human embryonic stem cell (hESC). In certain embodiments, the stem cell is a human induced pluripotent stem cell (hiPSC).

[0147] In certain embodiments, the stem cell or progeny cell thereof comprises an introduced heterologous nucleic acid, wherein the nucleic acid can encode a desired nucleic acid or protein product or have informational value (see, e.g., U.S. Patent No. 6,312,911, incorporated by reference in its entirety). Non-limiting examples of protein products include markers that can be detected 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 proteins (EBFP, EBFP2, Azurite, mKalamal), cyan fluorescent proteins (ECFP, Cerulean, CyPet, mTurquooise2), and yellow fluorescent protein derivatives (YFP, Citrine, Venus, YPet, EYFP)), beta-galactosidase (LacZ), chloramphenicol acetyltransferase (cat), neomycin phosphotransferase (neo), enzymes such as 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 that encodes a protein that is readily detectable or readily analyzable, such as a colored protein, a fluorescent protein (e.g., GFP), or an enzyme (e.g., beta-galactosidase (lacZ gene)). In certain embodiments, the reporter can be driven by a recombinant promoter of a premature postmitotic midbrain DA neuron marker gene (e.g., NURR1).

[0148] 5.2.2. SMAD inhibitors

[0149] Non-limiting examples of SMAD inhibitors include inhibitors of transforming growth factor beta (TGF ) / Activin-Nodal signaling (referred to as“TGF / Activin-Nodal inhibitors”) and inhibitors of bone morphogenetic protein (BMP) signaling. In certain embodiments, the TGF / Activin-Nodal inhibitors can inactivate the ligands (including TGF, BMP, Nodal, and Activin) and / or block their signaling pathways by blocking receptors and downstream effectors. Non-limiting examples of TGF / Activin-Nodal inhibitors include those disclosed in WO / 2010 / 096496, WO / 2011 / 149762, WO / 2013 / 067362, WO / 2014 / 176606, WO / 2015 / 077648, Chambers et al., Nat Biotechnol. 2009 Mar;27(3):275-80, Kriks et al., Nature. 2011 Nov 6;480(7378):547-51, and Chambers et al., Nat Biotechnol. 2012 Jul 1;30(7):715-20 (2012), all of which are incorporated herein by reference for all purposes. In certain embodiments, the at least one TGF / Activin-Nodal inhibitor is selected from the group consisting of an ALK5 inhibitor, an ALK4 inhibitor, an ALK7 inhibitor, and combinations thereof. In certain embodiments, the TGF / Activin-Nodal inhibitor comprises an inhibitor of ALK5. In certain embodiments, the TGF / Activin-Nodal inhibitor is a small molecule selected from the group consisting of SB431542, derivatives thereof, and mixtures thereof. “SB431542” refers to the molecule with the number CAS 301836-41-9, the molecular formula of C 22 H 18 N4O3, named 4-[4-(l,3-benzodioxol-5-yl)-5-(2-pyridinyl)-lH-imidazol-2-yl]-benzamide, e.g., see the following structure:

[0150]

[0151] In certain embodiments, the TGF / Activin-Nodal inhibitor comprises SB431542. In certain embodiments, the TGF / Activin-Nodal inhibitor comprises a derivative of SB431542. In certain embodiments, the derivative of SB431542 is A83-01.

[0152] In certain embodiments, the at least one SMAD inhibitor comprises an inhibitor of BMP signaling (referred to as a "BMP inhibitor"). Non-limiting examples of BMP inhibitors include those disclosed in WO2011 / 149762, Chambers et al., Nat Biotechnol. 2009 Mar;27(3):275-80, Kriks et al., Nature. 2011 Nov 6;480(7378):547-51, and Chambers et al., Nat Biotechnol. 2012 Jul 1;30(7):715-20, all of which are incorporated by reference in their entirety. In certain embodiments, the BMP inhibitor is a small molecule selected from the group consisting of LDN193189, Noggin, Dorsomorphin, derivatives thereof, and mixtures thereof. "LDN193189" refers to the small molecule DM-3189, IUPAC name 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline, chemical formula C 25 H 22 N6, of the following formula.

[0153]

[0154] LDN193189 is capable of functioning as an inhibitor of SMAD signaling. LDN193189 is also a potent small molecule inhibitor of ALK2, ALK3, and ALK6 protein tyrosine kinases (PTKs), inhibiting signaling of the type I TGFbeta receptors ALK1 and ALK3 family members, thereby inhibiting the transmission of a variety of biological signals, including bone morphogenetic proteins (BMPs) BMP2, BMP4, BMP6, BMP7, and Activin cytokine signals, and 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 by reference herein).

[0155] In certain embodiments, the BMP inhibitor comprises LDN193189. In certain embodiments, the BMP inhibitor comprises Noggin.

[0156] In certain embodiments, the stem cells are exposed to one SMAD inhibitor, e.g., one TGFp / Activin inhibitor. In certain embodiments, the one TGFp / Activin-Nodal inhibitor is SB431542 or A83-01. In certain embodiments, the stem cells are exposed to two SMAD inhibitors. In certain embodiments, the two SMAD inhibitors are a TGFp / Activin-Nodal inhibitor and a BMP inhibitor. In certain embodiments, the stem cells are exposed to SB431542 or A83-01, and LDN193189 or Noggin. In certain embodiments, the stem cells are exposed to SB431542 and LDN193189.

[0157] In certain embodiments, the stem cells are contacted or exposed to at least one SMAD inhibitor for at least about 5 days, or at least about 10 days. In certain embodiments, the stem cells are contacted or exposed to at least one SMAD inhibitor for up to about 5 days or up to about 10 days. In certain embodiments, the stem cells are contacted or exposed to at least one SMAD inhibitor for about 5 days to about 10 days. In certain embodiments, the stem cells are contacted or exposed to at least one SMAD inhibitor for about 5 days. In certain embodiments, the stem cells are contacted or exposed to at least one SMAD inhibitor for 7 days. In certain embodiments, the cells are contacted 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 comprising 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 comprising the stem cells every day (daily) from day 0 to day 6.

[0158] In certain embodiments, the cells are contacted with or exposed to a TGFp / Activin inhibitor. In certain embodiments, the concentration of the TGFp / Activin-Nodal inhibitor contacted with or exposed to the cells is between about 1 mM to about 20 mM, between about 1 mM to about 10 mM, between about 1 mM to about 15 mM, between about 10 mM to about 15 mM, between about 5 mM to about 10 mM, between about 5 mM to about 15 mM, between about 5 mM to about 20 mM, or between about 15 mM to about 20 mM. In certain embodiments, the concentration of the TGFp / Activin-Nodal inhibitor contacted with or exposed to the cells is between about 1 mM to about 10 mM. In particular embodiments, the concentration of the TGFp / Activin-Nodal inhibitor contacted with or exposed to the cells is about 5 mM. In certain embodiments, the concentration of the TGFp / Activin-Nodal inhibitor contacted with or exposed to the cells is about 10 mM. In certain embodiments, the TGFp / Activin-Nodal inhibitor comprises SB431542 or a derivative thereof (e.g., A83-01). In certain embodiments, the TGFp / Activin-Nodal inhibitor comprises SB431542.

[0159] In certain embodiments, the cells are contacted with or exposed to a BMP inhibitor. In certain embodiments, the concentration of the BMP inhibitor contacted with or exposed to the cells is between about 50 nM and about 500 nM, or between about 100 nM and about 500 nM, or between about 200 nM and about 500 nM, or between about 200 nM and about 300 nM, or between about 200 nM and about 400 nM, or between about 100 nM and about 250 nM, or between about 100 nM and about 250 nM, or between about 200 nM and about 250 nM, or between about 250 nM and about 300 nM. In certain embodiments, the concentration of the BMP inhibitor contacted with or exposed to the cells is between about 200 nM and about 300 mM. In certain embodiments, the concentration of the BMP inhibitor contacted with or exposed to the cells is about 150 nM, about 200 nM, about 250 nM, about 300 nM, or about 350 nM. In certain embodiments, the concentration of the BMP inhibitor contacted with or exposed to the cells is about 250 nM. In certain embodiments, the BMP inhibitor comprises LDN193189 or a derivative thereof. In certain embodiments, the BMP inhibitor comprises LDN193189.

[0160] In certain embodiments, the cells are simultaneously contacted or exposed to a TGFp / Activin-Nodal inhibitor and a BMP inhibitor. In certain embodiments, the stem cells are contacted or exposed to a TGFp / Activin-Nodal inhibitor and a BMP inhibitor for 7 days. In certain embodiments, the cells are contacted or exposed to a TGFp / Activin-Nodal inhibitor and a BMP inhibitor from day 0 to day 6. In certain embodiments, the TGFp / Activin-Nodal inhibitor and the BMP inhibitor are added to the cell culture medium comprising stem cells every day or every other day from day 0 to day 6. In certain embodiments, the TGFp / Activin-Nodal inhibitor and the BMP inhibitor are added to the cell culture medium comprising stem cells every day (daily) from day 0 to day 6.

[0161] 5.2.3. Wnt activators

[0162] In certain embodiments, the at least one Wnt activator decreases GSK3P to activate Wnt signaling. Accordingly, in certain embodiments, the Wnt activator is a GSK3P inhibitor. GSK3P inhibitors are capable of activating the WNT signaling pathway, see, e.g., Cadigan et al., J Cell Sci 2006; 119: 395-402; Kikuchi et al., Cell Signaling. 2007; 19: 659-671, which are incorporated by reference in their entireties herein. As used herein, the term "glycogen synthase kinase 3P inhibitor" or "GSK3P inhibitor" refers to a compound that inhibits the glycogen synthase kinase 3P enzyme, see, e.g., Doble et al., J Cell Sci. 2003; 116: 1175-1186, which is incorporated by reference in its entirety herein.

[0163] Non-limiting examples of Wnt activators or GSK3 inhibitors include CHIR99021, Wnt3A, Wntl, Wnt5a, BIO ((3E)-6-bromo-3-[3-(hydroxylamino)indol-2-ylidene]-lH-indol-2-one), CHIR98014, lithium, 3F8, and those disclosed in WO2011 / 149762, W013 / 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 entirety. In certain embodiments, the at least one Wnt activator is a small molecule selected from CHIR99021, Wnt3A, Wntl, 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-methylpyrrol-2-ylmethylidene]-2-indolinone") refers to the IUPAC name 6-(2-(4-(2,4-dichlorophenyl)-5-(4-methyl-lH-imidazol-2-yl)pyrimidin-2-ylamino)ethylamino)nicotinonitrile, which is of the following formula.

[0164]

[0165] CHIR99021 has high selectivity, exhibiting nearly thousand-fold selectivity over a panel of related and unrelated kinases, with an IC50 = 6.7 nM for human GSK3beta, and nanomolar IC50 values for rodent GSK3beta homologs.

[0166] In certain embodiments, the cells are contacted 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 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 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 Wnt activator from day 0 to day 11. In certain embodiments, at least one Wnt activator is added to a cell culture medium containing the cells every day or every other day from day 0 to day 11. In certain embodiments, at least one Wnt activator is added to a cell culture medium containing the cells every day (daily) from day 0 to day 11.

[0167] In certain embodiments, the concentration of the at least one Wnt activator is increased during its exposure to the cell (also referred to as a "Wnt Boost"). In certain embodiments, the increase or Wnt Boost occurs for at least about 2 days, at least about 4 days, or at least about 5 days from the initial exposure of the cell to the at least one Wnt activator. In certain embodiments, the increase or Wnt Boost occurs for about 4 days from the initial exposure of the cell to the at least one Wnt activator.

[0168] In certain embodiments, the cells are contacted 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 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.

[0169] In certain embodiments, the cells are contacted or exposed to the increased post- concentration of the at least one Wnt activator for between about 5 days and about 15 days, or between about 5 days and about 10 days, or between about 10 days and about 15 days. In certain embodiments, the cells are contacted or exposed to the increased post-concentration of the at least one Wnt activator for between about 5 days and about 10 days. In certain embodiments, the cells are contacted or exposed to the increased post-concentration of the at least one Wnt activator for about 5 days, about 10 days, or about 15 days. In certain embodiments, the cells are contacted or exposed to the increased post-concentration of the at least one Wnt activator for about 5 days. In certain embodiments, the cells are contacted or exposed to the increased post-concentration of the at least one Wnt activator for 6 days. In certain embodiments, the cells are contacted or exposed to the increased post-concentration of the at least one Wnt activator from day 4 to day 9. In certain embodiments, the cells are contacted or exposed to the increased post-concentration of the at least one Wnt activator for about 10 days. In certain embodiments, the cells are contacted or exposed to the increased post-concentration of the at least one Wnt activator for 8 days. In certain embodiments, the cells are contacted or exposed to the increased post-concentration of the at least one Wnt activator from day 4 to day 11.

[0170] In certain embodiments, the initial concentration of the at least one Wnt activator with which the cells are contacted or exposed prior to the Wnt Boost is less than about 5 mM, less than about 3 mM, or less than about 1 mM, including but not limited to between about 0.01 mM and about 5 mM, between about 0.01 mM and about 3 mM, between about 0.05 mM and about 3 mM, between about 0.1 mM and about 3 mM, between about 0.5 mM and about 3 mM, between about 0.5 mM and about 2 mM, or between about 0.5 mM and about 1 mM. In certain embodiments, the initial concentration of the at least one Wnt activator with which the cells are contacted or exposed prior to the Wnt Boost is less than about 1 mM, e.g., about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, or about 1 mM. In certain embodiments, the initial concentration of the at least one Wnt activator with which the cells are contacted or exposed prior to the Wnt Boost is about 0.5 mM. In certain embodiments, the initial concentration of the at least one Wnt activator with which the cells are contacted or exposed prior to the Wnt Boost is about 0.7 mM.

[0171] In certain embodiments, the post-increase concentration of the at least one Wnt activator after Wnt Boost is about 3 μΜ or more, about 5 μΜ or more, about 10 μΜ or more, about 15 μΜ or more, or about 20 μΜ or more. In certain embodiments, the post-increase concentration of the at least one Wnt activator after Wnt Boost is between about 3 μΜ and about 15 μΜ, between about 3 μΜ and about 10 μΜ, or between about 5 μΜ and about 10 μΜ. In certain embodiments, the post-increase concentration of the at least one Wnt activator after Wnt Boost is about 3 μΜ, about 3.5 μΜ, about 4 μΜ, about 4.5 μΜ, about 5 μΜ, about 5.5 μΜ, about 6 μΜ, about 6.5 μΜ, about 7 μΜ, about 7.5 μΜ, about 8 μΜ, about 8.5 μΜ, about 9 μΜ, about 9.5 μΜ, or about 10 μΜ. In certain embodiments, the post-increase concentration of the at least one Wnt activator after Wnt Boost is about 3 μΜ. In certain embodiments, the post-increase concentration of the at least one Wnt activator after Wnt Boost is about 7 μΜ. In certain embodiments, the post-increase concentration of the at least one Wnt activator after Wnt Boost is about 7.5 μΜ.

[0172] In certain embodiments of the foregoing methods, the concentration of the at least one Wnt activator is increased between about 50% and about 2000% of the initial concentration contacted with or exposed to the cells, or between about 100% and about 1500%, or between about 150% and about 1500%, or between about 200% and about 1500%, or between about 250% and about 1500%, or between about 300% and about 1500%, or between about 300% and about 1000%, or between about 300% and about 400%, or between about 500% and about 1000%, or between about 800% and about 1000%, or between about 900% and about 1000%, or between about 950% and about 1000%. In certain embodiments, the concentration of the at least one Wnt activator is increased between about 300% and about 1000% of the initial concentration contacted with or exposed to the cells. In certain embodiments, the concentration of the at least one Wnt activator is increased about 300% to about 400% of the initial concentration contacted with or exposed to the cells. In certain embodiments, the concentration of the at least one Wnt activator is increased about 900% to about 1000% of the initial concentration contacted with or exposed to the cells. In certain embodiments, the concentration of the at least one Wnt activator is increased about 300%, about 350%, about 400%, about 450%, about 500%, about 550%, about 600%, about 650%, about 700%, about 750%, about 800%, about 850%, about 900%, about 950%, about 1000%, about 1050%, or about 1100% of the initial concentration contacted with or exposed to the cells. In certain embodiments, the concentration of the at least one Wnt activator is increased about 300% of the initial concentration contacted with or exposed to the cells. In certain embodiments, the concentration of the at least one Wnt activator is increased about 350% of the initial concentration contacted with or exposed to the cells. In certain embodiments, the concentration of the at least one Wnt activator is increased about 950% of the initial concentration contacted with or exposed to the cells. In certain embodiments, the concentration of the at least one Wnt activator is increased about 1000% of the initial concentration contacted with or exposed to the cells.

[0173] In certain embodiments, the at least one Wnt activator comprises a GSK3 inhibitor. In certain embodiments, the at least one Wnt activator comprises CHIR99021 or a derivative thereof. In certain embodiments, the at least one Wnt activator comprises CHIR99021.

[0174] 5.2.4. SHH activators

[0175] As used herein, the term "sonic hedgehog," "SHH," or "Shh" refers to a protein of one of at least three proteins in the mammalian signaling pathway family known as hedgehog factors, the other being desert hedgehog (DHH) and the third being Indian hedgehog (IHH). SHH interacts with at least two transmembrane proteins through interaction with the transmembrane molecule Patched (PTC) and Smoothened (SMO). SHH typically binds to PTC, which allows SMO to function as a signal sensor to activate. In the absence of SHH, PTC normally inhibits SMO, which in turn activates transcriptional repressors, so certain genes are not transcribed. When SHH is present and binds to PTC, PTC cannot interfere with the function of SMO. With SMO uninhibited, certain proteins are able to enter the nucleus and act as transcription factors, thereby activating certain genes (see Gilbert, 2000 Developmental Biology (Sunderland, Mass., Sinauer Associates, Inc., Publishers). In certain embodiments, the SHH activator refers to any molecule or compound that is capable of activating the SHH signaling pathway, including molecules or compounds that are capable of binding to PTC or SMO. In certain embodiments, the SHH activator is selected from the group consisting of a molecule that binds PCT, a molecule that binds SMO, and combinations thereof. Non-limiting examples of SHH activators include 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 a SHH protein, a SMO agonist, or a combination thereof. In certain embodiments, the SHH protein comprises a recombinant SHH, a 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 a mouse SHH N-terminal fragment. In certain embodiments, the recombinant SHH comprises SHH C25 II. In certain embodiments, the SMO agonist comprises a morphinan.

[0176] In certain embodiments, the cells are contacted or exposed to the at least one SHH activator for at least about 5 days or at least about 10 days. In certain embodiments, the cells are contacted or exposed to the at least one SHH activator for up to about 5 days or up to about 10 days. In certain embodiments, the cells are contacted or exposed to the at least one SHH activator for between about 5 days and about 10 days. In certain embodiments, the cells are contacted or exposed to the at least one SHH activator for about 5 days. In certain embodiments, the cells are contacted or exposed to the at least one SHH activator for 7 days. In certain embodiments, the cells are contacted or exposed to the at least one SHH activator from day 0 to day 6. In certain embodiments, the at least one SHH activator is added to the cell culture medium comprising the cells every day or every other day from day 0 to day 6. In certain embodiments, the at least one SHH activator is added to the cell culture medium comprising the cells every day (daily) from day 0 to day 6.

[0177] In certain embodiments, the at least one SHH activator contacted or exposed to the cells is at a concentration of between 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 at least one SHH activator contacted or exposed to the cells is at a concentration of between about 400 ng / mL to about 600 ng / mL. In certain embodiments, the at least one SHH activator contacted or exposed to the cells is at a concentration of 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 at least one SHH activator contacted or exposed to the cells is at a concentration of about 500 ng / mL.

[0178] In certain embodiments, the at least one activator of SHH signaling comprises SHH C25II.

[0179] 5.2.5. FGF activators

[0180] The FGF family includes secreted signaling proteins (secreted FGFs) that signal to receptor tyrosine kinases. Phylogenetic analysis indicates that the 22 Fgf genes can be divided into seven subfamilies, each containing two to four members. Branch lengths are proportional to evolutionary distance between each gene.

[0181] In certain embodiments, the FGF activator is selected from the group consisting of FGF8a, FGF17, FGF18, FGF8b, FGF2, FGF4, and derivatives thereof. In certain embodiments, the FGF activator is selected from the group consisting of FGF8a, FGF17, FGF18, FGF2, FGF4, and derivatives thereof. In certain embodiments, the FGF activator is selected from the group consisting of FGF8a, FGF17, FGF18.

[0182] The FGF8 subfamily consists of FGF8a, FGF8b, FGF17, and FGF18. Early patterning of the midbrain and cerebellum in vertebrates is regulated by mid / hindbrain tissue organizers that produce FGF8a, FGF8b, FGF17, and FGF18. Studies have shown that FGF8b functions differently than FGF8a, FGF17, and FGF18 (Liu et al., Development. 2003 Dec; 130(25):6175-85). FGF8b is the only protein that can induce the rl gene Gbx2 and strongly activate the pathway inhibitor Spry 1 / 2, as well as repress the midbrain gene Otx2 (Liu 2003). In addition, FGF8b extends the organizer along the junction between the induced Gbx2 domain and the remaining Otx2 region in the midbrain, which is associated with cerebellar development (Liu 2003). In contrast, FGF8a, FGF17, and FGF18 cause midbrain expansion and upregulate midbrain gene expression (Liu 2003).

[0183] In certain embodiments, the FGF activator is capable of causing midbrain expansion and upregulating midbrain gene expression. In certain embodiments, the FGF activator is selected from the group consisting of FGF8a, FGF17, FGF18, FGF2, FGF4, derivatives thereof, and combinations thereof. In certain embodiments, the FGF activator comprises or is FGF18.

[0184] In certain embodiments, the cells are contacted or exposed to the 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, the cells are contacted or exposed to the 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, the cells are contacted or exposed to the at least one FGF activator for about 1 day to about 20 days, 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, the cells are contacted or exposed to the at least one FGF activator for about 5 days to about 10 days. In certain embodiments, the cells are contacted or exposed to the at least one FGF activator for about 3 days, about 5 days, or about 8 days. In certain embodiments, the cells are contacted or exposed to the at least one FGF activator for about 5 days.

[0185] In certain embodiments, the initial contact or initial exposure of the cells to the at least one FGF activator is at least about 5 days or at least about 10 days from the initial contact or initial exposure of the cells to the at least one SMAD inhibitor. In certain embodiments, the initial contact or initial exposure of the cells to the at least one FGF activator is no later than about 5 days, no later than about 10 days, or no later than about 15 days from the initial contact or initial exposure of the cells to the at least one SMAD inhibitor. In certain embodiments, the initial contact or initial exposure of the cells to the 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 from the initial contact or initial exposure of the cells to the at least one SMAD inhibitor. In certain embodiments, the initial contact or initial exposure of the cells to the at least one FGF activator is between about 5 days and about 10 days from the initial contact or initial exposure of the cells to the at least one SMAD inhibitor. In certain embodiments, the initial contact or initial exposure of the cells to the at least one FGF activator is about 10 days from the initial contact or initial exposure of the cells to the at least one SMAD inhibitor. In certain embodiments, the initial contact or initial exposure of the cells to the at least one FGF activator is about 9 days from the initial contact or initial exposure of the cells to the at least one SMAD inhibitor. In certain embodiments, the initial contact or initial exposure of the cells to the at least one FGF activator is 10 days from the initial contact or initial exposure of the cells to the at least one SMAD inhibitor. In certain embodiments, the initial contact or initial exposure of the cells to the at least one FGF activator is 12 days from the initial contact or initial exposure of the cells to the at least one SMAD inhibitor.

[0186] In certain embodiments, the initial contact or exposure of the cells with the at least one FGF activator is about 5 days from the initial contact or exposure of the cells with the at least one SMAD inhibitor, and the cells are contacted with the at least one FGF activator for about 3 days. In certain embodiments, the initial contact or exposure of the cells with the at least one FGF activator is about 5 days from the initial contact or exposure 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. In certain embodiments, the initial contact or exposure of the cells with the at least one FGF activator is about 10 days from the initial contact or exposure of the cells with the at least one SMAD inhibitor, and the cells are contacted with the at least one FGF activator for about 3 days. In certain embodiments, the initial contact or exposure of the cells with the at least one FGF activator is about 10 days from the initial contact or exposure 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. In certain embodiments, the initial contact or exposure of the cells with the at least one FGF activator is 12 days from the initial contact or exposure of the cells with the at least one SMAD inhibitor, and the cells are contacted with the at least one FGF activator for 5 days.

[0187] In certain embodiments, the concentration of the at least one FGF activator contacted with or exposed to the cells is between about 10 ng / mL 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, between about 100 ng / mL to about 250 ng / mL. In certain embodiments, the concentration of the at least one FGF activator contacted with or exposed to the cells is between about 100 ng / mL to about 200 ng / mL, the concentration of the at least one FGF activator contacted with or exposed to the cells is about 100 ng / mL. In certain embodiments, the concentration of the at least one FGF activator contacted with or exposed to the cells is about 200 ng / mL.

[0188] In certain embodiments, the at least one FGF activator comprises FGF18.

[0189] In certain non-limiting embodiments, the stem cells are contacted or exposed to at least one TGFp / Activin-Nodal inhibitor (e.g., SB431542, e.g., at a concentration of about 10 mM), 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 C25 II, 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 the at least one Wnt activator (e.g., CHIR99021, e.g., for 5 days (e.g., 4 days, e.g., from day 0 to day 3) at a concentration of about 0.7 mM, and for about 5 days (e.g., 6 days, e.g., from day 4 to day 9) at a concentration of about 7.5 mM, and for about 2 days (e.g., from day 10 to day 11) at a concentration of about 3 mM). The cells are contacted 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) from 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 (from day 12 to day 16) or 7 days (e.g., from day 10 to day 16).

[0190] 5.2.6. Cell Culture Media

[0191] In certain embodiments, the above-described inhibitors and activators are added to a cell culture medium comprising the cells. Suitable cell culture media include, but are not limited to, Serum Replacement (“KSR”) medium, Medium (NB), N2 Medium, B-27 Medium, and E8 / E6 Medium, and combinations thereof. KSR medium, NB medium, N2 medium, B-27 medium, and E8 / E6 medium are each commercially available. KSR medium is an optimally defined serum-free formulation for growing and maintaining hESCs in culture.

[0192] In certain embodiments, the cell culture medium is KSR medium. The ingredients of KSR medium are disclosed in WO 2011 / 149762. In certain embodiments, the 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 Knockout DMEM, 150 mL Knockout Serum Replacement, 10 mL 200 mM L-glutamine, 10 mL Pen / Strep, 10 mL 10 mM MEM, and 55 mM 13-mercaptoethanol.

[0193] 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 shown to support somatic cell reprogramming. In addition, E8 / E6 medium can be used as a PSC culture self-defined medium formulation base. An exemplary 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 exemplary E8 / E6 medium is disclosed in WO 15 / 077648, which is incorporated by reference in its entirety. In certain embodiments, the E8 / E6 cell culture medium comprises DMEM / F12, ascorbic acid, selenium, insulin, NaHC03, transferrin, FGF2, and TGFp. E8 / E6 medium differs from KSR medium in that E8 / E6 medium does not comprise active BMP or Wnt components. Thus, in certain embodiments, when culturing the stem cell population of the present disclosure to differentiate into a proprioceptor population using E8 / E6 medium, it is not necessary to add at least one inhibitor of SMAD signaling (e.g., an inhibitor of BMP) to the E8 / E6 medium.

[0194] 5.2.7. Differentiated cells

[0195] In certain embodiments, the method comprises obtaining a 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 an mDA or mDA precursor. Non-limiting examples of markers indicative of an mDA or mDA precursor include engrailed-1 (EN1), orthodenticle homeobox 2 (OTX2), tyrosine hydroxylase (TH), nuclear receptor related-1 protein (NURR1), forkhead box protein A2 (FOXA2), and LIM homeobox transcription factor 1 alpha (LMX1A), PITX3, LMO3, SNCA, ADCAPl, CHRNA4, and GIRK2.

[0196] In certain embodiments, the differentiated cells express the at least one marker indicative of an mDA or mDA precursor is 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 the initial contact of the cells with the at least one SMAD inhibitor.

[0197] Treatment of the cells with the at least one FGF activator can result in sustained expression of EN1. EN1 is a survival factor for midbrain DA neurons during development and continues to exert neuroprotective and physiological functions in adult midbrain DA neurons. Thus, cells that sustain expression of EN1 can develop into functionally mature mDAs upon further development and maturation. In certain embodiments, the differentiated cells have detectable levels of EN1 expression 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 from the initial contact of the stem cells with the at least one SMAD inhibitor. In certain embodiments, the differentiated cells have detectable levels of EN1 expression about 30 days from the initial contact of the stem cells with the at least one SMAD inhibitor. In certain embodiments, the differentiated cells have detectable levels of EN1 expression about 40 days from the initial contact of the stem cells with the at least one SMAD inhibitor.

[0198] In certain embodiments, the differentiated cells derived from the methods of the present disclosure do not express or have low expression of at least one marker selected from the group consisting of PAX6, EMX2, LHX2, SMA, SIX1, PITX2, SIM1, POU4F1, PHOX2A, BARHL1, BARHL2, GBX2, HOXA2, HOXB2, POU5F1, NANOG, and combinations thereof.

[0199] In certain embodiments, the cells are contacted with the activators and inhibitors described herein at a concentration and for a time effective to increase expression of at least one DA neuron marker (e.g., EN1) to a detectable level, e.g., wherein the cells are A9-type neuronal cells.

[0200] In certain embodiments, the cells are contacted with the activators and inhibitors described herein at a concentration and for a time effective to decrease expression of SMA, SIX1, PITX2, SIM1, POU4F1, and / or PHOX2A.

[0201] 5.2.8. Differentiation of mDA Precursors into mDAs

[0202] In certain embodiments, the cells are further contacted with DA neuron lineage-specific activators and inhibitors, e.g., 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-1,1-dimethylethyl ester, LY-374973, N-[N-(3,5-difluorophenylacetyl)-L-alanyl]-S-phenylglycine tert-butyl ester; or N-[N-(3,5-difluorophenylacetyl)-L-alanyl]-S-phenylglycine tert-butyl ester). In certain embodiments, the cells are contacted with the aforementioned 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 longer, for example, between about 2 days and about 20 days, between about 3 days and about 19 days, between about 4 days and about 18 days, between about 5 days and about 17 days, between about 6 days and about 16 days, between about 7 days and about 15 days, between about 8 days and about 15 days, between about 9 days and about 14 days, or between about 10 days and about 13 days. In certain embodiments, the cells are contacted with the aforementioned 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 longer. In certain embodiments, the cells are contacted with the aforementioned DA neuron lineage-specific activators and inhibitors for about 4 days, about 5 days, about 6 days, about 7 days, or about 8 days.

[0203] 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.

[0204] In certain embodiments, the 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, the cells are contacted with BDNF at a concentration of about 20 ng / ml.

[0205] 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.

[0206] In certain embodiments, the 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, the cells are contacted with GDNF at a concentration of about 20 ng / ml.

[0207] 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.

[0208] In certain embodiments, the cells are contacted with TGFβ3 at a concentration between 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.

[0209] In certain embodiments, the differentiated midbrain DA precursors are further cultured as described in U.S. Publication No. 2015 / 0010514, which is incorporated by reference in its entirety.

[0210] 5.3. Methods of isolating midbrain DA neurons and precursors thereof

[0211] The present disclosure provides methods of isolating mDAs and precursors thereof based on at least one or at least two surface markers. In certain embodiments, the surface marker is a negative surface marker, wherein the cells do not express detectable levels of the negative surface marker. In certain embodiments, the cells express reduced levels of the negative surface marker as compared to the average expression level of the negative surface marker of the cell population from which the cells are isolated.

[0212] In certain embodiments, the surface marker is a positive surface marker, wherein the cells express detectable levels of the positive surface marker. In certain embodiments, the cells express increased levels of the positive surface marker as compared to the average expression level of the positive surface marker of the cell population from which the cells are isolated.

[0213] In certain embodiments, the methods for isolating mDAs and precursors thereof from a cell population of the present disclosure comprise isolating cells that do not express detectable levels of at least one negative surface marker. In certain embodiments, the methods for isolating mDAs and precursors thereof from a cell population of the present disclosure comprise isolating cells that do not express detectable levels of at least one negative surface marker or express reduced levels of at least one negative surface marker as compared to the average expression level of the at least one negative surface marker. In certain embodiments, the methods for isolating mDAs and precursors thereof from a cell population of the present disclosure comprise isolating cells that express detectable levels of at least one positive surface marker. In certain embodiments, the methods for isolating mDAs and precursors thereof from a cell population of the present disclosure comprise isolating cells that express increased levels of at least one positive surface marker as compared to the average expression level of the at least one positive marker of the cell population.

[0214] In certain embodiments, the methods of the present disclosure for isolating mDAs and their precursors from a population of cells comprise isolating cells that do not express detectable levels of at least one negative surface marker and express detectable levels of at least one positive surface marker. In certain embodiments, the methods of the present disclosure for isolating mDAs and their precursors from a population of cells comprise isolating cells that: (a) do not express detectable levels of at least one negative surface marker or express decreased levels of at least one negative surface marker as compared to the average expression level of the at least one negative surface marker in the population of cells; and (b) express increased levels of at least one positive surface marker as compared to the average expression level of the at least one positive marker in the population of cells.

[0215] In certain embodiments, the negative surface marker is selected from the group consisting of CD49e (also known as integrin alpha 5), CD99, CD340, and combinations thereof. In certain embodiments, the positive surface marker is selected from the group consisting of CD171, CD184, and combinations thereof.

[0216] In certain embodiments, the methods of the present disclosure for isolating mDAs and their precursors from a population of cells comprise isolating cells that do not express detectable levels of CD49e and express detectable levels of CD184. In certain embodiments, the methods of the present disclosure for isolating mDAs and their precursors from a population of cells comprise isolating cells that do not express detectable levels of CD49e or express decreased levels of CD49e as compared to the average expression level of CD49e in the population of cells; and express increased levels of CD184 as compared to the average expression level of CD184 in the population of cells.

[0217] Any surface marker-based cell isolation technique known in the art can be used in the methods of the present disclosure. In certain embodiments, flow cytometry is used in the isolation methods of the present disclosure.

[0218] 5.4. Cell Populations and Compositions

[0219] The present disclosure provides a population of in vitro differentiated cells in which 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 mDAs or mDA precursors. Non-limiting examples of markers indicative of mDAs or mDA precursors include EN1, OTX2, TH, NURR1, FOXA2, LMX1A, PITX3, LMO3, SNCA, ADCAPl, CHRNA4, and GIRK2. The present disclosure also provides compositions comprising such populations of cells. In certain embodiments, the in vitro differentiated cells are obtained by the differentiation methods described herein (e.g., in Section 5.2).

[0220] 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 the group consisting of PAX6, EMX2, LHX2, SMA, SIX1, PITX2, SIM1, POU4F1, PHOX2A, BARHL1, BARHL2, GBX2, HOXA2, HOXB2, POU5F1, NANOG, and combinations thereof.

[0221] The present application 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., in Section 5.3) and do not express at least one negative surface marker disclosed herein (e.g., in Section 5.3). The present application 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., in Section 5.3) at an increased level compared to the average expression level of the at least one positive surface marker in the cell population; and do not express at a detectable level or express at a decreased level compared to the average expression level of the at least one negative surface marker in the cell population at least one negative surface marker disclosed herein (e.g., in Section 5.3).

[0222] In certain embodiments, 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 do not express a detectable level of CD49e and express a detectable level of CD184. In certain embodiments, 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 do not express a detectable level of CD49e or express a reduced level of CD49e as compared to the average expression level of CD49e in the population of cells; and express an increased level of CD184 as compared to the average expression level of CD184 in the population of cells. Further, the present disclosure provides compositions comprising such a population of cells.

[0223] In certain embodiments, the cells are comprised in a composition further comprising a biocompatible scaffold or matrix, e.g., 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 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. 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 a growth factor for promoting maturation of the implanted / transplanted cells into midbrain DA cells.

[0224] In certain embodiments, the composition administered to the subject comprises about 1 x 105 4 to about 1 x 105 10 , about 1 x 105 4 to about 1 x 105 5 , about 1 x 105 5 to about 1 x 105 9 , about 1 x 105 5 to about 1 x 105 6 , about 1 x 105 5 to about 1 x 105 7 , about 1 x 105 6 to about 1 x 105 7 , about 1 x 105 6 to about 1 x 105 8 , about 1 x 105 7 to about 1 x 105 8about 1 x 105 8 about 1 x 105 9 about 1 x 105 8 about 1 x 105 10 about 1 x 105 9 about 1 x 105 10 a population of cells. In certain embodiments, about 1 x 105 5 about 1 x 105 7 cells thereof are administered to the subject.

[0225] 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 combinations thereof.

[0226] In certain embodiments, the composition further comprises a biocompatible scaffold or matrix, such as a biocompatible three-dimensional scaffold that facilitates tissue regeneration when the cells are implanted or transplanted into a subject. In certain embodiments, the biocompatible scaffold comprises extracellular matrix material, synthetic polymers, cytokines, collagens, polypeptides or proteins, polysaccharides, including fibronectin, laminin, keratin, fibrin, fibrillin, hyaluronic acid, heparin sulfate, chondroitin sulfate, agarose or gelatin, and / or hydrogels. (See, e.g., U.S. Publication Nos. 2015 / 0159135, 2011 / 0296542, 2009 / 0123433, and 2008 / 0268019, the contents of each of which are incorporated by reference in their entirety).

[0227] In certain embodiments, the composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier. The composition can be used to prevent and / or treat neurodegenerative diseases, including Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, and multiple sclerosis.

[0228] The presently disclosed subject matter also provides a device comprising differentiated cells or a composition comprising differentiated cells, as disclosed herein. Non-limiting examples of devices include syringes, fine glass tubes, stereotactic needles, and cannulas.

[0229] 5.5. Methods of treating neurodegenerative diseases

[0230] The cell populations and compositions disclosed herein (e.g., those disclosed in Section 5.4) can be used to treat neurodegenerative diseases. The subject matter of the present disclosure provides methods for treating neurodegenerative diseases. In certain embodiments, the methods comprise administering to a subject suffering from a neurodegenerative disease an effective amount of a stem cell-derived mDA of the present disclosure, or a composition comprising the same. In certain embodiments, the methods comprise administering to a subject suffering from a neurodegenerative disease an effective amount of in vitro differentiated cells that do not express detectable levels of at least one negative surface marker (e.g., CD49e) but express detectable levels of at least one positive surface marker (e.g., CD184), or a composition comprising such cells. In certain embodiments, the method comprises administering to a subject suffering from a neurodegenerative disease an effective amount of in vitro differentiated cells that do not express detectable levels of at least one negative surface marker (e.g., CD49e) or express at least one negative surface marker (e.g., CD49e) at a reduced level compared to the average expression of at least one negative surface marker in the cell population from which the cells were isolated, and express at least one positive surface marker (e.g., CD184) at an increased level compared to the average expression of at least one positive surface marker in the cell population from which the cells were isolated; or a composition comprising these cells. In certain embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

[0231] Non-limiting examples of neurodegenerative diseases include Parkinson's disease, Huntington's disease, Alzheimer's disease, and multiple sclerosis.

[0232] In certain embodiments, the neurodegenerative disease is Parkinson's disease. The main motor symptoms of Parkinson's disease include, but are not limited to, tremors in the hands, arms, legs, jaw, and face, slow or bradykinesia, stiffness or soreness in the limbs and trunk, postural instability, or impaired balance and coordination.

[0233] In certain embodiments, the neurodegenerative disease is Parkinson's disease, which refers to a disease associated with a lack of dopamine in the basal ganglia, a part of the brain that controls movement. Symptoms include tremors, bradykinesia (extremely slow movements), hunched posture, postural instability, and rigidity. Non-limiting examples of Parkinson's disease include corticobasal degeneration, dementia with Lewy bodies, multiple system atrophy, and progressive supranuclear palsy.

[0234] The cell or composition can be administered or provided to a subject systematically or directly to treat or prevent neurodegenerative diseases. In some embodiments, the cell or composition is injected directly into a target organ (e.g., central nervous system (CNS) or peripheral nervous system (PNS)). In some embodiments, the cell or composition is injected directly into the striatum.

[0235] The cells or compositions can be administered in any physiologically acceptable carrier. 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 having a neurodegenerative disease by orthotopic (OT) injection.

[0236] The cells or compositions can conveniently be presented as a sterile liquid preparation, for example, as an isotonic aqueous solution, suspension, emulsion, dispersion, or viscous composition, which can be buffered to a selected pH. Liquid preparations are generally easier to prepare than gels, other viscous compositions, and solid compositions. Moreover, liquid compositions are more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within an appropriate viscosity range to provide longer contact time with particular tissues. Liquid or viscous compositions can include a carrier, which can be a solvent or dispersion medium containing, for example, water, saline, phosphate buffered saline, polyhydric alcohol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof. A sterile injectable solution of the subject matter of the present disclosure (e.g., a composition comprising stem cell-derived precursors of the present disclosure) can be prepared by incorporating the subject matter of the present disclosure in the required amount in the appropriate solvent with the other ingredients as desired. Such compositions can be mixed with suitable carriers, diluents, or excipients (e.g., sterile water, physiological saline, glucose, dextrose, and the like). The compositions can also be lyophilized. The compositions can include auxiliary substances such as wetting or dispersing agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, coloring agents, and the like, depending on the route of administration and the desired formulation. Reference can be made to standard texts for formulating appropriate dosage formulations, such as “REMINGTON’S PHARMACEUTICAL SCIENCE”, 17thEdition, 1985, incorporated herein by reference, without undue experimentation.

[0237] Various additives that enhance the stability and sterility of the compositions can be added, including antibacterial preservatives, antioxidants, chelating agents, and buffers. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, and the like). Prolonged absorption of injectable pharmaceutical forms can be brought about by the use of agents delaying absorption (e.g., aluminum monostearate and gelatin).

[0238] 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 available and economical, and easy to use. Other suitable thickening agents include, for example, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, carbomer, and the like. The concentration of the thickening agent will depend on the pharmaceutical agent selected. It is important that the amount used be such that the selected viscosity is achieved. The selection of suitable carriers and other additives will depend on the particular route of administration and the nature of the particular dosage form, e.g., liquid dosage forms (e.g., whether the composition will be formulated as a solution, suspension, gel, or other liquid form, e.g., a time release form or a liquid filled form).

[0239] Those skilled in the art will recognize that the components of the composition should be chosen to be chemically inert and not affect the activity or efficacy of the stem cell-derived precursors of the disclosure. This presents no problem to one of skill in the chemical and pharmaceutical arts, or can be readily avoided from the disclosure and documents cited herein by reference to standard texts or by simple experimentation (not involving undue experimentation).

[0240] One consideration with respect to the cellular therapeutic use is the number of cells needed to achieve optimal effect. Optimal effect includes, but is not limited to, re-population of a CNS and / or PNS region of a subject having a neurodegenerative disease, and / or improvement in CNS and / or PNS function of a subject.

[0241] An "effective amount" (or "therapeutically effective amount") refers to an amount that is sufficient to affect a beneficial or intended clinical outcome following treatment. An effective amount can be administered to a subject in one or more doses. In terms of treatment, an effective amount refers to an amount that is sufficient to reduce, ameliorate, stabilize, reverse, or slow the progression of a neurodegenerative disease or pituitary disease, or otherwise reduce the pathological consequences of a neurodegenerative disease. An effective amount is generally determined by a physician, depending on the particular circumstances of the case, and is within the skill of the art. Several factors are typically taken into account in determining the appropriate dosage for achieving an effective amount. These factors include the age, sex, and weight of the subject, the disease being treated, the severity of the disease, and the form and effective concentration of the cells being administered.

[0242] In certain embodiments, an effective amount of the cells is an amount sufficient to re-populate a CNS and / or PNS region of a subject having a neurodegenerative disease. In certain embodiments, an effective amount of the cells is an amount sufficient to improve the function of a CNS and / or PNS of a subject having a neurodegenerative disease, e.g., the 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 normal human CNS and / or PNS function.

[0243] The number of cells to be administered varies depending on the subject being treated. In certain embodiments, from about 1 x 105 4 to about 1 x 105 10 , from about 1 x 105 4 to about 1 x 105 5 , from about 1 x 105 5 to about 1 x 105 9 , from about 1 x 105 5 to about 1 x 105 6 , from about 1 x 105 5 to about 1 x 105 7 , from about 1 x 105 6 to about 1 x 105 7 , from about 1 x 105 6 to about 1 x 105 8 , from about 1 x 105 7 to about 1 x 105 8 , from about 1 x 105 8 to about 1 x 105 9 , from about 1 x 105 8 to about 1 x 105 10 , or from about 1 x 105 9 to about 1 x 105 10 cells are administered to a subject having a neurodegenerative disease. In certain embodiments, from about 1 x 105 5 to about 1 x 105 7 cells are administered to a subject having a neurodegenerative disease. In certain embodiments, from about 1 x 105 6 to about 1 x 105 7 cells are administered to a subject having a neurodegenerative disease. In certain embodiments, from about 1 x 105 6 to about 4 x 105 6 cells are administered to a subject having a neurodegenerative disease. The precise determination of an effective dosage can depend on individual factors for each subject, including their size, age, sex, weight, and the condition of the particular subject. One of skill in the art can readily determine a dosage in accordance with the present application and the knowledge in the art.

[0244] 5.6. Kits

[0245] The presently disclosed subject matter provides a kit for inducing stem cells to differentiate into mDAs or precursors thereof. In certain embodiments, the kit comprises (a) at least one inhibitor of SMAD signaling, (b) at least one activator of Wnt signaling, (c) at least one activator of SHH signaling, and (d) at least one activator of FGF signaling. In certain embodiments, the kit further comprises (e) instructions for inducing stem cells to differentiate into a population of differentiated cells expressing at least one marker indicative of mDAs or precursors thereof.

[0246] In certain embodiments, the instructions comprise contacting the stem cells with the inhibitors, activators, and molecules in a particular order. The order of contacting the inhibitors, activators, and molecules can be determined by the cell culture medium used to culture the stem cells.

[0247] In certain embodiments, the instructions comprise contacting the stem cells with the inhibitors, activators, and molecules described in the methods of the disclosure (see Section 5.2).

[0248] In certain embodiments, the disclosure provides a kit comprising an effective amount of a unit dosage form of the cell population or composition disclosed herein. In certain embodiments, the kit comprises a sterile container containing the therapeutic composition; these containers can be boxes, ampules, bottles, vials, tubes, packets, bags, blister-packs, or other suitable container forms known in the art. These containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.

[0249] In certain embodiments, the kit comprises instructions for administering the cell population or composition to a subject having a neurodegenerative disease. The instructions can include information about using the cells or composition to treat or prevent a neurodegenerative disease. In certain embodiments, the instructions comprise at least one of the following: a description of the therapeutic agent; a dosage schedule and administration for treating or preventing a neurodegenerative disease or symptoms thereof; precautions; warnings; indications; contraindications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and / or references. The instructions can be printed directly on the container, if any, or applied as a label to the container, or as a separate sheet, pamphlet, card, or folder supplied with the container.

[0250] 6. EMBODIMENT

[0251] The presently disclosed subject matter will be better understood by reference to the following examples, which are offered by way of illustration and not by way of limitation.

[0252] Example 1: Optimization of a midbrain DA neuron differentiation protocol

[0253] Midbrain DA neurons and their precursors were derived from stem cells under the previously disclosed Wnt-Boost protocol (the “7.5 mM bump protocol (modification of GMP V2B)” protocol disclosed in International Publication No. WO 2016 / 196661 (i.e., Comparative Document 1, US2018094242A1), which is incorporated by reference in its entirety) (hereinafter the “Wnt-Boost protocol” or “Boost protocol”). It was discovered that EN1 expression decreased starting at day 11 of differentiation using the Wnt-Boost protocol (seeFigure 1 ). Maintaining EN1 expression in the differentiating cells is important for generating mature and functional midbrain DA neurons. Therefore, to maintain EN1 expression, this example tested the addition of FGF8b treatment to the Wnt-Boost protocol at later stages of differentiation (see Figure 2 ). Cells were also tested for FGF8b exposure from day 9 to day 16, from day 10 to day 16, from day 11 to day 16, from day 12 to day 16, from day 13 to day 16, from day 14 to day 16, or from day 15 to day 16 in addition to the Wnt-Boost protocol. Immunostaining of cells at day 16 of differentiation showed that EN1 protein expression was maintained in a duration of FGF8b exposure dependent manner (see Figure 3 ). EN1 positive cells also expressed FOXA2 and LMX1A. RNA expression measured in cells at day 30 of differentiation indicated that the expression levels of mDA or mDA precursor markers FOXA2, NURR1, LMX1A, OXT2, and TH were comparable across all conditions, and mRNA expression of EN1 was maintained in a duration of FGF8b exposure dependent manner (see Figures 4A-4B ). However, mRNA levels of contaminant markers (non-mDA or non-mDA precursor markers) such as SMA and SIX1 were also induced in a duration of FGF8b exposure dependent manner (see Figure 4B ). SIX1 immunostaining of cells at day 30 of differentiation confirmed the mRNA results (see Figure 5 ).

[0254] FGF8b has been used to derive midbrain DA neurons from pluripotent stem cells. FGF8, FGF17, and FGF18 are a subfamily of FGFs, and it has been demonstrated that FGF17b, FGF18 have different roles in midbrain development than FGF8b (Liu et al., Development. 2003 Dec; 130(25):6175-85). Studies have also shown that FGF18 can prevent midbrain dopamine neuron injury caused by 6-OHDA (Guo et al., Neuroscience, 2017 Jul 25; 356:229-241). Furthermore, FGF8b (isthmic and rhombencephalic primary node 1) extends organizer along the junction between the induced Gbx2 domain and the remaining Otx2 region in the midbrain. FGF8a, FGF17, and FGF18 are responsible for the expansion of the midbrain and upregulation of midbrain genes. FGF8b, FGF17, and FGF18 are all paracrine FGFs to the same FGF subpopulation of FGF8b.

[0255] Under the WNT-Boost protocol, FGF8b, FGF17 and FGF18 were tested by adding them to the cell culture at a concentration of 100 ng / ml from day 12 to day 16. The study found that in cells at day 16 of differentiation, FGF18 induced mRNA expression levels of EN1 similar to FGF8b, but reduced mRNA expression levels of SMA (see Figure 6 and Figure 7 ). As with FGF8b, expression of EN1 protein was also highly maintained in a duration of FGF18 contact-dependent manner.

[0256] At the mature stage of differentiation, EN1 was still highly maintained under FGF8b and FGF18 treatment (see Figure 8 ). In addition, PITX2 expression levels were reduced in both FGF18 and FGF8b treated cells compared to cells differentiated by the WNT-Boost protocol, while SMA1 and SIX1 expression levels were lower in FGF18 treated cells than in FGF8b treated cells (see Figure 8 ). These results indicate that FGF18 treatment results in sustained expression of EN1 compared to FGF8b treatment, while minimizing or reducing expression levels of non-mDA markers.

[0257] In vivo survival of differentiated cells produced by the WNT-boost+FGF18 protocol was examined. Cells produced by the WNT-boost and WNT-boost+FGF18 protocols were transplanted into intact mice. Cells produced by the WNT-boost+FGF18 protocol had improved maintenance of EN1 expression in vivo compared to cells produced by the WNT-boost protocol ( Figure 35A ). Cells produced by the WNTboost+FGF18 protocol also had better striatal innervation 1 month post-transplantation, with fibers having extended from the core of the graft to the periphery ( Figure 35B ).

[0258] Example 2: Purification of mDAs using a reporter

[0259] NURR1 is a marker of post-mitotic and immature midbrain DA neurons, and is also expressed in mature midbrain DA neurons. It is a transcription factor that contributes to differentiation and maintenance of DA.

[0260] To purify mDAs from a cell population, this example used an endogenous NURR1 ::GFP reporter hPSC (see Figure 9A ). mDAs were differentiated from the reporter cell line. Based on the high induction of MURR1 mRNA expression at day 20 of differentiation, GFP positive cells were isolated based on FACS at day 25 of differentiation (see Figures 9B-9C ).

[0261] Single cell qRT-PCR was performed on NURR1:GFP positive cells isolated at day 25 and day 40 of differentiation. It was found that at day 40 of differentiation, nearly about 100% of NURR1:GFP positive cells expressed TH (a mature mDA marker), FOXA2 and LMX1A, indicating mDA fate (see, Figure 10A ). The isolated NURR1:GFP positive cells were cultured on for day 60, and the results showed that these cells expressed high levels of TH, indicating that these cells were high purity mDAs (see Figure 10B ).

[0262] At day 25 of differentiation, NURR1::GFP positive midbrain DA neurons were transplanted into nude mice. Figure 11 The transplanted cells were shown to survive in vivo and express TH, the human marker SC121 and GFP. Neurite outgrowth was found at the cell transplantation area (see Figure 11 ).

[0263] NURR1:GFP hPSCs were cultured under WNT-Boost and WNT-Boost+FGF18 protocols (day 12 to day 16). At day 25 of differentiation, NURR1:GFP positive cells were isolated and then cultured on until day 40. At day 40 of differentiation, these midbrain DA neurons expressed high TH and FOXA2 (see, Figure 12 ).

[0264] mRNA expression analysis showed that mDAs derived from WNT-Boost+FGF18 (day 12-day 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 in vivo. Both mDAs derived from WNT-Boost protocol and WNT-Boost+FGF18 (day 12 to day 16) protocol showed excellent cell survival and expressed the marker SC121 and TH. However, the cells treated with FGF18 (WNT-Boost+FGF18 protocol) showed better neurite outgrowth at the transplantation area (see Figure 13B ).

[0265] Example 3: Surface markers found in purified DA neurons

[0266] Published papers have shown that each iPSC line is different in terms of the specific cell types induced. It is difficult to generate a reporter line for each iPSC line for the purification of midbrain DA cells. In addition, genetically engineered cells are not suitable for clinical use. This example uses the NURR1 ::GFP reporter cell line to identify candidate surface markers, especially surface markers that are enriched in NURR1 ::GFP positive cells but not in NURR1 ::GFP negative cells, and vice versa.

[0267] This example tested 387 surface markers in mDA differentiated cells derived from NURR1 ::GFP hPSCs at day 25 of differentiation (see Figure 14 ).

[0268] 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 ).

[0269] Under the WNT-Boost protocol or the WNT-Boost + FGF18 (day 12-day 16) protocol, cells were sorted by CD49e (negative and / or weak expression) at day 25 of differentiation and cultured for another 10 days. Cell morphology showed that these cells were essentially pure mDAs (see Figure 17 ). The sorted mDAs at day 40 of differentiation (sorted at day 25 and cultured for another 15 days) had high TH immunostaining Figure 18 ).

[0270] The CD49e marker was tested in another hPSC line, MEL1, to purify midbrain DA neurons. At day 25 of differentiation, essentially pure mDA morphology was found in cells sorted by CD49e (negative and / or weak expression) and cultured for another 15 days (day 40 of differentiation) under the WNT-boost protocol and the WNT-boost + FGF18 (day 12-day 16) protocol (see Figures 19-20 ). These sorted mDAs had high TH immunostaining (see Figure 21 ).

[0271] mRNA expression showed that CD49e sorted cells differentiated under the WNT-boost + FGF18 (days 12-16) protocol had higher levels of EN1 expression and lower levels of PITX2 (a glutamatergic neuron (subthalamic nucleus marker)) expression compared to CD49e sorted cells differentiated under the WNT-boost protocol. Sorted cells differentiated under both protocols had little to no expression levels of non-mesencephalic DA markers (HOXA2, SMA1, and SIX1) (see Figure 22 ).

[0272] All three negative CD markers, CD49e, CD99, and CD340 were detected. At day 25 of differentiation and continuing for an additional 15 days (day 40 of differentiation), essentially pure neuronal morphology was observed in cells sorted with CD49e, CD99, or CD340 (negative and / or weakly expressing cells) (see Figure 23 ). However, mRNA expression of sorted cells showed increased expression of non-DA neuronal markers including PHOX2A, PITX2, POU4F1, and SIM1, suggesting that CD49e, CD99, or CD340 based isolation does not exclude non-DA neurons Figure 24 ).

[0273] It was thought that a double sorting strategy using CD184 could resolve the deficiency of negative CD markers. Cxcr4 (CD184) plays an important role in the migration and orientation of mesencephalic DA neurons during mouse mesencephalic development. FGF18 treated mDAs can be enriched for the A9 mesencephalic DA subtype after CD184 + / CD49e - sorting.

[0274] At day 25 of differentiation, mesencephalic DAs derived from NURR1 ::GFP hPSCs were FACS sorted using CD49E (PE) and CD171 (APC). It was found that single CD49e negative sorted cells were approximately 63% NURR1 ::GFP positive. CD171 positive sorting did not enrich the NURR1 ::GFP population combined with CD49e (see Figure 25 ).

[0275] However, sorting with CD49e and CD184 (positively expressing cells) can enrich the NURR1 ::GFP population to approximately 80% compared to single sorted cells (CD49e; 63%) (see Figure 26 ).

[0276] FACS sorting in cells by CD49e and CD171 or CD49e and CD184 on day 25 of differentiation. Cell morphology showed pure neuronal shape 2 days after sorting, except for cells sorted based on higher CD49e (see Figure 27 ).

[0277] mRNA expression showed that CD49e - / CD184 + (CD49e negative / CD184 positive) sorted cells had higher levels of mDA marker (NURR1, EN1, PITX3) expression and lower levels of non-mDA marker (PITX2, SIM1 and POU4F1) expression compared to other CD sorted cells (see Figure 28 ).

[0278] Next, it was investigated whether CD49e and CD184 could reliably sort mDA. As shown in Figure 29 , single CD49e - sorting enriched the NURR1:GFP positive cell population from ~20% to up to 43% in in vitro differentiated cells. As shown in Figure 30 , double CD49e - and CD184 + sorting enriched the NURR1:GFP positive fraction to 74% and 85% in in vitro differentiated cells.

[0279] After 2 weeks of in vitro culture, sorted cells showed high TH + mDA co-expressing FOXA2 and GFP, confirming the identity of mDA (see Figure 31 ).

[0280] mRNA expression showed that double CD marker mediated sorted cells (CD49e - and CD184 + ) generally had higher levels of mDA marker expression (see Figure 32 ) and lower levels of non-mDA marker expression (see Figure 33 ) compared to other CD sorted cells at day 40 of differentiation.

[0281] In vivo survival of differentiated cells sorted with novel surface markers of the present disclosure was tested. Differentiated cells were generated according to the WNT-boost protocol, sorted for CD49e low CD184 high cells and transplanted into the intact mouse brain. Tissues transplanted with sorted cells were enriched in Th+ cells ( Figure 34A ) compared to unsorted cells, and the number of SOX2+ precursors and KI67+ dividing cells was reduced ( Figures 34B-3 4D) one month after transplantation.

[0282] While the subject matter of the present disclosure and the advantages thereof 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 disclosure. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, and composition of matter, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the disclosure, processes, machines, manufacture, compositions of matter, means, methods, or steps, presently existing or later to be developed, that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein can be utilized according to the present subject matter. Accordingly, the appended claims are intended to include within their scope all such processes, machines, manufacture, compositions of matter, means, methods, or steps.

[0283] Various patents, patent publications, publications, product descriptions, protocols and sequences are cited throughout this application, the disclosures of each of which are incorporated herein by reference in their entireties for all purposes.

Claims

1. An in vitro method for inducing stem cell differentiation, comprising: The stem cells are contacted with at least one inhibitor of Small Mothers Against Decapentaplegic (SMAD) signaling, at least one activator of sonic hedgehog (SHH) signaling, and at least one activator of wingless (Wnt) signaling; and the cells are contacted with at least one activator of fibroblast growth factor (FGF) signaling to obtain a differentiated cell population expressing at least one marker indicative of midbrain dopamine neurons (mDA) or precursors thereof, wherein the at least one activator of FGF signaling is selected from FGF18, FGF17, and combinations thereof.

2. An in vitro method for inducing stem cell differentiation, comprising: contacting the stem cells with at least one inhibitor of Small Mothers Against Decapentaplegic (SMAD) signaling, at least one activator of Sonic Hedgehog (SHH) signaling, and at least one activator of wingless (Wnt) signaling; and contacting the cells with at least one activator of fibroblast growth factor (FGF) signaling 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 activator of FGF signaling is at least 5 days from the initial contacting of the cells with the at least one inhibitor of SMAD signaling, and wherein the at least one activator of FGF signaling is selected from FGF18, FGF17, and combinations thereof.

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

4. The method of claim 1 or 2, wherein the cells are contacted with the at least one activator of FGF signaling for up to 15 days.

5. The method of claim 1 or 2, wherein the cells are contacted with the at least one activator of FGF signaling for 5 days.

6. The method of claim 1 or 2, wherein the initial contacting of the cells with the at least one activator of FGF signaling is 10 days from the initial contacting of the cells with the at least one inhibitor of SMAD signaling.

7. The method of claim 1 or 2, wherein the initial contacting of the cells with the at least one activator of FGF signaling is 12 days from the initial contacting of the cells with the at least one inhibitor of SMAD signaling.

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

9. The method of claim 1 or 2, wherein the cells are contacted with the at least one inhibitor of SMAD signaling for 7 days.

10. The method of claim 1 or 2, wherein the cells are contacted with the at least one activator of SHH signaling for 5 days.

11. The method of claim 1 or 2, wherein the cells are contacted with the at least one activator of SHH signaling for 7 days.

12. The method of claim 1 or 2, wherein the cells are contacted with the at least one activator of Wnt signaling for 10 days.

13. The method of claim 1 or 2, wherein the cells are contacted with the at least one activator of Wnt signaling for 12 days.

14. The method of claim 1 or 2, wherein the concentration of the at least one activator of Wnt signaling is increased 4 days from its initial contact with the stem cells.

15. The method of claim 14, wherein the concentration of the at least one activator of Wnt signaling is increased by 300% to 1000% compared to the initial concentration of the at least one activator of Wnt signaling.

16. The method of claim 15, wherein the concentration of the at least one activator of Wnt signaling is increased to a concentration of 3 μM to 10 μM.

17. The method of claim 15, wherein the concentration of the at least one activator of Wnt signaling is increased to a concentration of 3 μM.

18. The method of claim 15, wherein the concentration of the at least one activator of Wnt signaling is increased to a concentration of 7.5 μM.

19. The method of claim 1 or 2, wherein the at least one activator of FGF signaling comprises FGF18.

20. The method of claim 1 or 2, wherein the at least one inhibitor of SMAD signaling is selected from the group consisting of inhibitors of TGFβ / Activin-Nodal signaling, inhibitors of bone morphogenetic protein (BMP) signaling, and combinations thereof.

21. The method of claim 20, wherein the at least one inhibitor of TGFβ / Activin-Nodal signaling comprises an inhibitor of ALK5.

22. The method of claim 21, wherein the at least one inhibitor of TGF / Activin-Nodal signaling comprises SB431542, or a derivative thereof, or a mixture thereof.

23. The method of claim 22, wherein the derivative of SB431542 is A83-01.

24. The method of claim 20, wherein the at least one inhibitor of TGFβ / Activin-Nodal signaling comprises SB431542.

25. The method of claim 20, wherein the at least one inhibitor of BMP signaling comprises LDN193189, Noggin, doxormorphine, a derivative thereof, or a mixture thereof.

26. The method of claim 25, wherein the inhibitor of at least one BMP comprises LDN-193189.

27. The method of claim 1 or 2, wherein the at least one activator of Wnt signaling comprises an inhibitor of glycogen synthase kinase 3β (GSK3β) signaling.

28. The method of claim 27, wherein the at least one activator of Wnt signaling is selected from the group consisting of CHIR99021, Wnt3A, Wnt1, derivatives thereof, and mixtures thereof.

29. The method of claim 1 or 2, wherein the at least one activator of SHH signaling is selected from the group consisting of SHH protein, smoothened agonist (SAG), derivatives thereof, and mixtures thereof.

30. The method of claim 29, wherein the SHH protein comprises recombinant SHH, purified SHH, or a combination thereof.

31. The method of claim 30, wherein the recombinant SHH comprises a recombinant protein identical to the N-terminal fragment of mouse sonic hedgehog.

32. The method of claim 30, wherein the recombinant SHH comprises SHH C25II.

33. The method of claim 29, wherein the SAG comprises purmorphamine.

34. The method of claim 1 or 2, 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.

35. The method of claim 1 or 2, wherein the differentiated cells have detectable levels of expression of at least one marker indicative of midbrain dopamine neurons or precursors thereof at least 10 days from the initial contacting of the stem cells with the at least one inhibitor of SMAD signaling.

36. The method of claim 1 or 2, wherein the differentiated cells have detectable levels of expression of EN1 30 days from the initial contacting of the stem cells with the at least one inhibitor of SMAD signaling.

37. The method of claim 1 or 2, wherein the differentiated cells have detectable levels of expression of EN1 40 days from the initial contacting of the stem cells with the at least one inhibitor of SMAD signaling.

38. The method of claim 1 or 2, wherein the differentiated cells do not express at least one marker selected from the group consisting of: PAX6, EMX2, LHX2, SMA, SIX1, PITX2, SIM1, POU4F1, PHOX2A, BARHL1, BARHL2, GBX2, HOXA2, HOXB2, POU5F1, NANOG, and combinations thereof.

39. The method of claim 1 or 2, further comprising subjecting the differentiated cell population to conditions that favor differentiation of midbrain dopamine neuron precursors into midbrain dopamine neurons.

40. The method of claim 39, wherein the conditions comprise 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 β3 (TGFβ3), ascorbic acid (AA), and DAPT.

41. The method of claim 1 or 2, wherein the stem cell is 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.

42. The method of claim 1 or 2, wherein the stem cells are human stem cells.

43. The method of claim 1 or 2, wherein the stem cells are multipotent or pluripotent stem cells.

44. The method of claim 43, wherein the stem cells are pluripotent stem cells.

45. The method of claim 44, wherein the pluripotent stem cells are selected from the group consisting of embryonic stem cells, induced pluripotent stem cells, and combinations thereof.

46. ​​A cell population of in vitro differentiated cells, wherein the in vitro differentiated cells are obtained by the method of any one of claims 1-45.

47. A composition comprising the cell population of claim 46.

48. The composition according to claim 47, which is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

49. A kit for inducing stem cells to differentiate into midbrain dopamine neurons or their precursors, comprising: (a) at least one inhibitor of SMAD signaling; (b) at least one activator of SHH signaling; (c) at least one activator of Wnt signaling; and (d) at least one activator of FGF signaling; wherein the at least one activator of FGF signaling is selected from FGF18, FGF17, and combinations thereof.

50. The kit of claim 49, further comprising (f) instructions for inducing differentiation of the stem cells into a differentiated cell population expressing at least one midbrain DA precursor marker.

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